Researcher Profile and Settings

Master

Affiliation (Master)

• Faculty of Medicine　Pathological Science　Department of Microbiology and Immunology

Affiliation (Master)

• Faculty of Medicine　Pathological Science　Department of Microbiology and Immunology

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Profile and Settings

Affiliation

• Hokkaido University, Graduate School of Medicine, Professor

Profile and Settings

• Name (Japanese)

  Kobayashi

• Name (Kana)

  Koichi S

• Name

  201801006158765434

Alternate Names

https://hokudaiimmunology.wixsite.com/kobayashi

Affiliation

• Hokkaido University, Graduate School of Medicine, Professor

Achievement

Research Interests

• Innate Immunity, NLR proteins, TLRs, NOD2, NLRC5, MHC class I, cancer immunity, SARS-CoV-2   

Research Areas

• Life sciences / Immunology

Research Experience

• 2022/10 - Today Hokkaido University Vaccine Development Center Professor
• 2020 - Today テキサスA&M大学 Health Science Center (Adjunct Professor) 兼任
• 2017 - Today Hokkaido Unviersity School of Medicine, Professor
• 2017 - 2020 テキサスA&M大学 Health Science Center, Research Professor
• 2012 - 2017 Texas A&M University School of Medicine, Professor
• 2011 - 2012 Harvard University/Dana-Farber Cancer Institute Associate Professor
• 2004 - 2011 Harvard University/Dana-Farber Cancer Institute Department of Pathology/Department of Cancer Immunology and AIDS Assistant Professor
• 2002 - 2004 Yale University School of Medicine, Associate Research Scientist
• 1998 - 2002 Yale University School of Medicine, Postdoctoral Fellow
• 1993 - 1994 Asahi General Hospital, Chief Resident
• 1992 - 1994 Asahi General Hospital, Internal Medicine
• 1991 - 1992 Chiba University Hospital, Resident

Education

• 1998 -   Chiba University Graduate School of Medicine, PhD
• 1991 -   Chiba University School of Medicine

Awards

• 2022 Hokkaido University, Faculty of Medicine Excellent Research Award
  
• 2018 武田科学振興財団 武田報彰研究助成
  
• 2009 Haartman Seminar, Haartman Institute, University of Helsinki
  
• 2006/10 Cancer Research Institute Investigator Award
   

  受賞者: Koichi S Kobayashi
• 2005 The Claudia Adams Barr Investigator
  
• 2005 Fellow, Arthritis National Research Foundation
  
• 2004/07 Arthritis National Research Foundation Fellow
   

  受賞者: Koichi S Kobayashi
• 1999/06 Cancer Research Institute Postdoctoral Fellowship
   

  受賞者: Koichi S Kobayashi
• 1998/03 The Naito Foundation The Naito Foundation Scholarship
   

  受賞者: Koichi S Kobayashi

Published Papers

• NLRC5/MHC class I transactivator: A key target for immune escape by SARS-CoV-2.

  Baohui Zhu, Ryota Ouda, Yusuke Kasuga, Paul de Figueiredo, Koichi S Kobayashi

  BioEssays : news and reviews in molecular, cellular and developmental biology 46 (4) e2300109  2024/04 

  Antigen presentation to CD8+ T cells by MHC class I molecules is essential for host defense against viral infections. Various mechanisms have evolved in multiple viruses to escape immune surveillance and defense to support viral proliferation in host cells. Through in vitro SARS-CoV-2 infection studies and analysis of COVID-19 patient samples, we found that SARS-CoV-2 suppresses the induction of the MHC class I pathway by inhibiting the expression and function of NLRC5, a major transcriptional regulator of MHC class I genes. In this review, we discuss the molecular mechanisms for suppression of the MHC class I pathway and clinical implications for COVID-19.
• Targeted demethylation and activation of NLRC5 augment cancer immunogenicity through MHC class I.

  Xin Sun, Toshiyuki Watanabe, Yoshitaka Oda, Weidong Shen, Alaa Ahmad, Ryota Ouda, Paul de Figueiredo, Hidemitsu Kitamura, Shinya Tanaka, Koichi S Kobayashi

  Proceedings of the National Academy of Sciences of the United States of America 121 (6) e2310821121  2024/02/06 

  Impaired expression of MHC (major histocompatibility complex) class I in cancers constitutes a major mechanism of immune evasion. It has been well documented that the low level of MHC class I is associated with poor prognosis and resistance to checkpoint blockade therapies. However, there is lmited approaches to specifically induce MHC class I to date. Here, we show an approach for robust and specific induction of MHC class I by targeting an MHC class I transactivator (CITA)/NLRC5, using a CRISPR/Cas9-based gene-specific system, designated TRED-I (Targeted reactivation and demethylation for MHC-I). The TRED-I system specifically recruits a demethylating enzyme and transcriptional activators on the NLRC5 promoter, driving increased MHC class I antigen presentation and accelerated CD8+ T cell activation. Introduction of the TRED-I system in an animal cancer model exhibited tumor-suppressive effects accompanied with increased infiltration and activation of CD8+ T cells. Moreover, this approach boosted the efficacy of checkpoint blockade therapy using anti-PD1 (programmed cell death protein) antibody. Therefore, targeting NLRC5 by this strategy provides an attractive therapeutic approach for cancer.
• FBXO11 constitutes a major negative regulator of MHC class II through ubiquitin-dependent proteasomal degradation of CIITA.

  Yusuke Kasuga, Ryota Ouda, Masashi Watanabe, Xin Sun, Miki Kimura, Shigetsugu Hatakeyama, Koichi S Kobayashi

  Proceedings of the National Academy of Sciences of the United States of America 120 (24) e2218955120  2023/06/13 

  Major histocompatibility complex (MHC) class I and II molecules play critical roles in the activation and regulation of adaptive immunity through antigen presentation to CD8+ and CD4+ T cells, respectively. Strict regulation of MHC expression is critical for proper immune responses. CIITA (MHC class II transactivator), an NLR (nucleotide-binding domain, leucine-rich-repeat containing) protein, is a master regulator of MHC class II (MHC-II) gene transcription. Although it has been known that CIITA activity is regulated at the transcriptional and protein levels, the mechanism to determine CIITA protein level has not been elucidated. Here, we show that FBXO11 is a bona fide E3 ligase of CIITA and regulates CIITA protein level through ubiquitination-mediated degradation. A nonbiased proteomic approach for CIITA-binding protein identified FBXO11, a member of the Skp1-Cullin-1-F-box E3 ligase complex, as a binding partner of CIITA but not MHC class I transactivator, NLRC5. The cycloheximide chase assay showed that the half-life of CIITA is mainly regulated by FBXO11 via the ubiquitin-proteasome system. The expression of FBXO11 led to the reduced MHC-II at the promoter activity level, transcriptional level, and surface expression level through downregulation of CIITA. Moreover, human and mouse FBXO11-deficient cells display increased levels of MHC-II and related genes. In normal and cancer tissues, FBXO11 expression level is negatively correlated with MHC-II. Interestingly, the expression of FBXO11, along with CIITA, is associated with prognosis of cancer patients. Therefore, FBXO11 is a critical regulator to determine the level of MHC-II, and its expression may serve as a biomarker for cancer.
• Regulation of MHC Class I Expression in Lung Epithelial Cells during Inflammation.

  Justine Mathé, Mohamed Benhammadi, Koichi S Kobayashi, Sylvie Brochu, Claude Perreault

  Journal of immunology (Baltimore, Md. : 1950) 208 (5) 1021 - 1033 2022/03/01 

  Lung infections are a perennial leading cause of death worldwide. The lung epithelium comprises three main cell types: alveolar type I (AT1), alveolar type II (AT2), and bronchiolar cells. Constitutively, these three cell types express extremely low amounts of surface MHC class I (MHC I) molecules, that is, <1% of levels found on medullary thymic epithelial cells (ECs). We report that inhalation of the TLR4 ligand LPS upregulates cell surface MHC I by ∼25-fold on the three subtypes of mouse lung ECs. This upregulation is dependent on Nlrc5, Stat1, and Stat2 and caused by a concerted production of the three IFN families. It is nevertheless hampered, particularly in AT1 cells, by the limited expression of genes instrumental in the peptide loading of MHC I molecules. Genes involved in production and response to cytokines and chemokines were selectively induced in AT1 cells. However, discrete gene subsets were selectively downregulated in AT2 or bronchiolar cells following LPS inhalation. Genes downregulated in AT2 cells were linked to cell differentiation and cell proliferation, and those repressed in bronchiolar cells were primarily involved in cilium function. Our study shows a delicate balance between the expression of transcripts maintaining lung epithelium integrity and transcripts involved in Ag presentation in primary lung ECs.
• Metabolomics of Acute vs. Chronic Spinach Intake in an Apc–Mutant Genetic Background: Linoleate and Butanoate Metabolites Targeting HDAC Activity and IFN–γ Signaling

  Ying-Shiuan Chen, Jia Li, Sultan Neja, Sabeeta Kapoor, Jorge Tovar Perez, Chakrapani Tripathi, Rani Menon, Arul Jayaraman, Kyongbum Lee, Wan Dashwood, Shan Wang, Ke Zhang, Koichi Kobayashi, Praveen Rajendran, Roderick Dashwood

  Cells 11 (3) 573 - 573 2022/02/07 

  There is growing interest in the crosstalk between the gut microbiome, host metabolomic features, and disease pathogenesis. The current investigation compared long–term (26 week) and acute (3 day) dietary spinach intake in a genetic model of colorectal cancer. Metabolomic analyses in the polyposis in rat colon (Pirc) model and in wild–type animals corroborated key contributions to anticancer outcomes by spinach–derived linoleate bioactives and a butanoate metabolite linked to increased α–diversity of the gut microbiome. Combining linoleate and butanoate metabolites in human colon cancer cells revealed enhanced apoptosis and reduced cell viability, paralleling the apoptosis induction in colon tumors from rats given long–term spinach treatment. Mechanistic studies in cell–based assays and in vivo implicated the linoleate and butanoate metabolites in targeting histone deacetylase (HDAC) activity and the interferon–γ (IFN–γ) signaling axis. Clinical translation of these findings to at–risk patients might provide valuable quality–of–life benefits by delaying surgical interventions and drug therapies with adverse side effects.
• Live attenuated bacterium limits cancer resistance to CAR-T therapy by remodeling the tumor microenvironment.

  Fengguang Guo, Jugal K Das, Koichi S Kobayashi, Qing-Ming Qin, Thomas A Ficht, Robert C Alaniz, Jianxun Song, Paul De Figueiredo

  Journal for immunotherapy of cancer 10 (1) 2022/01 

  The tumor microenvironment (TME) is characterized by the activation of immune checkpoints, which limit the ability of immune cells to attack the growing cancer. To overcome immune suppression in the clinic, antigen-expressing viruses and bacteria have been developed to induce antitumor immunity. However, the safety and targeting specificity are the main concerns of using bacteria in clinical practice as antitumor agents. In our previous studies, we have developed an attenuated bacterial strain (Brucella melitensis 16M ∆vjbR, henceforth Bm∆vjbR) for clinical use, which is safe in all tested animal models and has been removed from the select agent list by the Centers for Disease Control and Prevention. In this study, we demonstrated that Bm∆vjbR homed to tumor tissue and improved the TME in a murine model of solid cancer. In addition, live Bm∆vjbR promoted proinflammatory M1 polarization of tumor macrophages and increased the number and activity of CD8+ T cells in the tumor. In a murine colon adenocarcinoma model, when combined with adoptive transfer of tumor-specific carcinoembryonic antigen chimeric antigen receptor CD8+ T cells, tumor cell growth and proliferation was almost completely abrogated, and host survival was 100%. Taken together, these findings demonstrate that the live attenuated bacterial treatment can defeat cancer resistance to chimeric antigen receptor T-cell therapy by remodeling the TME to promote macrophage and T cell-mediated antitumor immunity.
• SARS-CoV-2 inhibits induction of the MHC class I pathway by targeting the STAT1-IRF1-NLRC5 axis.

  Ji-Seung Yoo, Michihito Sasaki, Steven X Cho, Yusuke Kasuga, Baohui Zhu, Ryota Ouda, Yasuko Orba, Paul de Figueiredo, Hirofumi Sawa, Koichi S Kobayashi

  Nature communications 12 (1) 6602 - 6602 2021/11/15 

  The MHC class I-mediated antigen presentation pathway plays a critical role in antiviral immunity. Here we show that the MHC class I pathway is targeted by SARS-CoV-2. Analysis of the gene expression profile from COVID-19 patients as well as SARS-CoV-2 infected epithelial cell lines reveals that the induction of the MHC class I pathway is inhibited by SARS-CoV-2 infection. We show that NLRC5, an MHC class I transactivator, is suppressed both transcriptionally and functionally by the SARS-CoV-2 ORF6 protein, providing a mechanistic link. SARS-CoV-2 ORF6 hampers type II interferon-mediated STAT1 signaling, resulting in diminished upregulation of NLRC5 and IRF1 gene expression. Moreover, SARS-CoV-2 ORF6 inhibits NLRC5 function via blocking karyopherin complex-dependent nuclear import of NLRC5. Collectively, our study uncovers an immune evasion mechanism of SARS-CoV-2 that targets the function of key MHC class I transcriptional regulators, STAT1-IRF1-NLRC5.
• MHC class I transactivator NLRC5 in host immunity, cancer and beyond.

  Steven X Cho, Saptha Vijayan, Ji-Seung Yoo, Toshiyuki Watanabe, Ryota Ouda, Ning An, Koichi S Kobayashi

  Immunology 162 (3) 252 - 261 2021/03 

  The presentation of antigenic peptides by major histocompatibility complex (MHC) class I and class II molecules is crucial for activation of the adaptive immune system. The nucleotide-binding domain and leucine-rich repeat receptor family members CIITA and NLRC5 function as the major transcriptional activators of MHC class II and class I gene expression, respectively. Since the identification of NLRC5 as the master regulator of MHC class I and class-I-related genes, there have been major advances in understanding the function of NLRC5 in infectious diseases and cancer. Here, we discuss the biological significance and mechanism of NLRC5-dependent MHC class I expression.
• NLRC5/CITA expression correlates with efficient response to checkpoint blockade immunotherapy.

  Sayuri Yoshihama, Steven X Cho, Jason Yeung, Xuedong Pan, Gregory Lizee, Kranti Konganti, Valen E Johnson, Koichi S Kobayashi

  Scientific reports 11 (1) 3258 - 3258 2021/02/05 

  Checkpoint blockade-mediated immunotherapy is emerging as an effective treatment modality for multiple cancer types. However, cancer cells frequently evade the immune system, compromising the effectiveness of immunotherapy. It is crucial to develop screening methods to identify the patients who would most benefit from these therapies because of the risk of the side effects and the high cost of treatment. Here we show that expression of the MHC class I transactivator (CITA), NLRC5, is important for efficient responses to anti-CTLA-4 and anti-PD1 checkpoint blockade therapies. Melanoma tumors derived from patients responding to immunotherapy exhibited significantly higher expression of NLRC5 and MHC class I-related genes compared to non-responding patients. In addition, multivariate analysis that included the number of tumor-associated non-synonymous mutations, predicted neo-antigen load and PD-L2 expression was capable of further stratifying responders and non-responders to anti-CTLA4 therapy. Moreover, expression or methylation of NLRC5 together with total somatic mutation number were significantly correlated with increased patient survival. These results suggest that NLRC5 tumor expression, alone or together with tumor mutation load constitutes a valuable predictive biomarker for both prognosis and response to anti-CTLA-4 and potentially anti-PD1 blockade immunotherapy in melanoma patients.
• IFN-λ Enhances Constitutive Expression of MHC Class I Molecules on Thymic Epithelial Cells.

  Mohamed Benhammadi, Justine Mathé, Maude Dumont-Lagacé, Koichi S Kobayashi, Louis Gaboury, Sylvie Brochu, Claude Perreault

  Journal of immunology (Baltimore, Md. : 1950) 205 (5) 1268 - 1280 2020/09/01 

  Regulation of MHC class I (MHC I) expression has been studied almost exclusively in hematolymphoid cells. We report that thymic epithelial cells (TECs), particularly the medullary TECs, constitutively express up to 100-fold more cell surface MHC I proteins than epithelial cells (ECs) from the skin, colon, and lung. Differential abundance of cell surface MHC I in primary ECs is regulated via transcription of MHC I and of genes implicated in the generation of MHC I-binding peptides. Superior MHC I expression in TECs is unaffected by deletion of Ifnar1 or Ifngr1, but is lessened by deletion of Aire, Ifnlr1, Stat1, or Nlrc5, and is driven mainly by type III IFN produced by medullary TECs. Ifnlr1 -/- mice show impaired negative selection of CD8 thymocytes and, at 9 mo of age, present autoimmune manifestations. Our study shows unanticipated variation in MHC I expression by ECs from various sites and provides compelling evidence that superior expression of MHC I in TECs is crucial for proper thymocyte education.
• Green Tea Polyphenols Modify the Gut Microbiome in db/db Mice as Co-Abundance Groups Correlating with the Blood Glucose Lowering Effect.

  Tingting Chen, Anna B Liu, Shili Sun, Nadim J Ajami, Matthew C Ross, Hong Wang, Le Zhang, Kenneth Reuhl, Koichi Kobayashi, Janet C Onishi, Liping Zhao, Chung S Yang

  Molecular nutrition & food research 63 (8) e1801064  2019/04 

  SCOPE: The effects of green tea polyphenols, Polyphenon E (PPE), and black tea polyphenols, theaflavins (TFs), on gut microbiota and development of diabetes in db/db mice are investigated and compared. METHODS AND RESULTS: Supplementation of PPE (0.1%) in the diet of female db/db mice for 7 weeks decreases fasting blood glucose levels and mesenteric fat while increasing the serum level of insulin, possibly through protection against β-cell damage. However, TFs are less or not effective. Microbiome analysis through 16S rRNA gene sequencing shows that PPE and TFs treatments significantly alter the bacterial community structure in the cecum and colon, but not in the ileum. The key bacterial phylotypes responding to the treatments are then clustered into 11 co-abundance groups (CAGs). CAGs 6 and 7, significantly increased by PPE but not by TFs, are negatively associated with blood glucose levels. The operational taxonomic units in these CAGs are from two different phyla, Firmicutes and Bacteroidetes. CAG 10, decreased by PPE and TFs, is positively associated with blood glucose levels. CONCLUSION: Gut microbiota respond to tea polyphenol treatments as CAGs instead of taxa. Some of the CAGs associated with the blood glucose lowering effect are enriched by PPE, but not TFs.
• Class I transactivator, NLRC5: a central player in the MHC class I pathway and cancer immune surveillance.

  Vijayan S, Sidiq T, Yousuf S, van den Elsen PJ, Kobayashi KS

  Immunogenetics 71 (3) 273 - 282 0093-7711 2019/03 [Refereed][Not invited]
   

  Major histocompatibility complex (MHC) class I and class II molecules play critical roles in the activation of the adaptive immune system by presenting antigens to CD8+ and CD4+ T cells, respectively. Although it has been well known that CIITA (MHC class II transactivator), an NLR (nucleotide-binding domain, leucine-rich-repeat containing) protein, as a master regulator of MHC class II gene expression, the mechanism of MHC class I gene transactivation was unclear. Recently, another NLR protein, NLRC5 (NLR family, CARD domain-containing 5), was identified as an MHC class I transactivator (CITA). NLRC5 is a critical regulator for the transcriptional activation of MHC class I genes and other genes involved in the MHC class I antigen presentation pathway. CITA/NLRC5 plays a crucial role in human cancer immunity through the recruitment and activation of tumor killing CD8+ T cells. Here, we discuss the molecular function and mechanism of CITA/NLRC5 in the MHC class I pathway and its role in cancer.
• The IL-33-PIN1-IRAK-M axis is critical for type 2 immunity in IL-33-induced allergic airway inflammation.

  Nechama M, Kwon J, Wei S, Kyi AT, Welner RS, Ben-Dov IZ, Arredouani MS, Asara JM, Chen CH, Tsai CY, Nelson KF, Kobayashi KS, Israel E, Zhou XZ, Nicholson LK, Lu KP

  Nat Commun. 23;9(1):1603. (1) 1603 - 1603 2018/04 [Refereed][Not invited]
   

  Interleukin 33 (IL-33) is among the earliest-released cytokines in response to allergens that orchestrate type 2 immunity. The prolyl cis-trans isomerase PIN1 is known to induce cytokines for eosinophil survival and activation by stabilizing cytokines mRNAs, but the function of PIN1 in upstream signaling pathways in asthma is unknown. Here we show that interleukin receptor associated kinase M (IRAK-M) is a PIN1 target critical for IL-33 signaling in allergic asthma. NMR analysis and docking simulations suggest that PIN1 might regulate IRAK-M conformation and function in IL-33 signaling. Upon IL-33-induced airway inflammation, PIN1 is activated for binding with and isomerization of IRAK-M, resulting in IRAK-M nuclear translocation and induction of selected proinflammatory genes in dendritic cells. Thus, the IL-33-PIN1-IRAK-M is an axis critical for dendritic cell activation, type 2 immunity and IL-33 induced airway inflammation.
• Quiescent Tissue Stem Cells Evade Immune Surveillance

  Judith Agudo, Eun Sook Park, Samuel A. Rose, Eziwoma Alibo, Robert Sweeney, Maxime Dhainaut, Koichi S. Kobayashi, Ravi Sachidanandam, Alessia Baccarini, Miriam Merad, Brian D. Brown

  Immunity 48 (2) 271 - 285.e5 1097-4180 2018/02/20 [Refereed][Not invited]
   

  Stem cells are critical for the maintenance of many tissues, but whether their integrity is maintained in the face of immunity is unclear. Here we found that cycling epithelial stem cells, including Lgr5+ intestinal stem cells, as well as ovary and mammary stem cells, were eliminated by activated T cells, but quiescent stem cells in the hair follicle and muscle were resistant to T cell killing. Immune evasion was an intrinsic property of the quiescent stem cells resulting from systemic downregulation of the antigen presentation machinery, including MHC class I and TAP proteins, and is mediated by the transactivator NLRC5. This process was reversed upon stem cell entry into the cell cycle. These studies identify a link between stem cell quiescence, antigen presentation, and immune evasion. As cancer-initiating cells can derive from stem cells, these findings may help explain how the earliest cancer cells evade immune surveillance. Agudo et al. find that cycling tissue stem cells are subject to immune clearance, but quiescent stem cells downregulate the antigen presentation machinery and evade immune surveillance.
• E3 Ubiquitin ligase ZNRF4 negatively regulates NOD2 signalling and induces tolerance to MDP (vol 8, 15865, 2017)

  Pradeep Bist, Wan Shoo Cheong, Aylwin Ng, Neha Dikshit, Bae Hoon Kim, Niyas Kudukkil Pulloor, Hanif Javanmard Khameneh, Matija Hedl, Avinash R. Shenoy, Vanniarajan Balamuralidhar, Najib Bin Abdul Malik, Michelle Hong, Albert Neutzner, Keh-Chuang Chin, Koichi S. Kobayashi, Antonio Bertoletti, Alessandra Mortellaro, Clara Abraham, John D. MacMicking, Ramnik J. Xavier, Bindu Sukumaran

  NATURE COMMUNICATIONS 8 16141 - 16141 2041-1723 2017/08 [Refereed][Not invited]
   

  This corrects the article DOI: 10.1038/ncomms15865.
• E3 Ubiquitin ligase ZNRF4 negatively regulates NOD2 signalling and induces tolerance to MDP

  Pradeep Bist, Wan Shoo Cheong, Aylwin Ng, Neha Dikshit, Bae-Hoon Kim, Niyas Kudukkil Pulloor, Hanif Javanmard Khameneh, Matija Hedl, Avinash R. Shenoy, Vanniarajan Balamuralidhar, Najib Bin Abdul Malik, Michelle Hong, Albert Neutzner, Keh-Chuang Chin, Koichi S. Kobayashi, Antonio Bertoletti, Alessandra Mortellaro, Clara Abraham, John D. MacMicking, Ramnik J. Xavier, Bindu Sukumaran

  NATURE COMMUNICATIONS 8 15865 - 15865 2041-1723 2017/06 [Refereed][Not invited]
   

  Optimal regulation of the innate immune receptor nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is essential for controlling bacterial infections and inflammatory disorders. Chronic NOD2 stimulation induces non-responsiveness to restimulation, termed NOD2-induced tolerance. Although the levels of the NOD2 adaptor, RIP2, are reported to regulate both acute and chronic NOD2 signalling, how RIP2 levels are modulated is unclear. Here we show that ZNRF4 induces K48-linked ubiquitination of RIP2 and promotes RIP2 degradation. A fraction of RIP2 localizes to the endoplasmic reticulum (ER), where it interacts with ZNRF4 under either 55 unstimulated and muramyl dipeptide-stimulated conditions. Znrf4 knockdown monocytes have sustained nuclear factor kappalight- chain-enhancer of activated B cells (NF-kappa B) activation, and Znrf4 knockdown mice have reduced NOD2-induced tolerance and more effective control of Listeria monocytogenes infection. Our results thus demonstrate E3-ubiquitin ligase ZNRF4-mediated RIP2 degradation as a negative regulatory mechanism of NOD2-induced NF-kappa B, cytokine and anti-bacterial responses in vitro and in vivo, and identify a ZNRF4-RIP2 axis of fine-tuning NOD2 signalling to promote protective host immunity.
• Global Reprogramming of Host Kinase Signaling in Response to Fungal Infection

  Aseem Pandey, Sheng Li Ding, Qing-Ming Qin, Rahul Gupta, Gabriel Gomez, Furong Lin, Xuehuan Feng, Luciana Fachini da Costa, Sankar P. Chaki, Madhu Katepalli, Elizabeth D. Case, Erin J. van Schaik, Tabasum Sidiq, Omar Khalaf, Angela Arenas, Koichi S. Kobayashi, James E. Samuel, Gonzalo M. Rivera, Robert C. Alaniz, Sing-Hoi Sze, Xiaoning Qian, William J. Brown, Allison Rice-Ficht, William K. Russell, Thomas A. Ficht, Paul de Figueiredo

  CELL HOST & MICROBE 21 (5) 637 - + 1931-3128 2017/05 [Refereed][Not invited]
   

  Cryptococcus neoformans (Cn) is a deadly fungal pathogen whose intracellular lifestyle is important for virulence. Host mechanisms controlling fungal phagocytosis and replication remain obscure. Here, we perform a global phosphoproteomic analysis of the host response to Cryptococcus infection. Our analysis reveals numerous and diverse host proteins that are differentially phosphorylated following fungal ingestion by macrophages, thereby indicating global reprogramming of host kinase signaling. Notably, phagocytosis of the pathogen activates the host autophagy initiation complex (AIC) and the upstream regulatory components LKB1 and AMPK alpha, which regulate autophagy induction through their kinase activities. Deletion of Prkaa1, the gene encoding AMPK alpha 1, in monocytes results in resistance to fungal colonization of mice. Finally, the recruitment of AIC components to nascent Cryptococcus-containing vacuoles (CnCVs) regulates the intracellular trafficking and replication of the pathogen. These findings demonstrate that host AIC regulatory networks confer susceptibility to infection and establish a proteomic resource for elucidating host mechanisms that regulate fungal intracellular parasitism.
• NLRC5/CITA: A Key Player in Cancer Immune Surveillance

  Sayuri Yoshihama, Saptha Vijayan, Tabasum Sidiq, Koichi S. Kobayashi

  Trends in Cancer 3 (1) 28 - 38 2405-8033 2017/01/01 [Refereed][Not invited]
   

  Cancer cells need to escape immune surveillance for successful tumor growth. Loss of MHC class I has been described as a major immune evasion strategy in many cancers. MHC class I transactivator (CITA), NLRC5 [nucleotide-binding domain and leucine-rich repeats containing (NLR) family, caspase activation and recruitment domain (CARD) domain containing 5], is a key transcription coactivator of MHC class I genes. Recent genetic studies have revealed that NLRC5 is a major target for cancer immune evasion mechanisms. The reduced expression or activity of NLRC5 caused by promoter methylation, copy number loss, or somatic mutations is associated with defective MHC class I expression, impaired cytotoxic T cell activation, and poor patient prognosis. Here, we review the role of NLRC5 in cancer immune evasion and the future prospects for cancer research.
• Nod2: A Critical Regulator of Ileal Microbiota and Crohn's Disease

  Tabasum Sidiq, Sayuri Yoshihama, Isaac Downs, Koichi S. Kobayashi

  FRONTIERS IN IMMUNOLOGY 7 367 - 367 1664-3224 2016/09 [Refereed][Not invited]
   

  The human intestinal tract harbors large bacterial community consisting of commensal, symbiotic, and pathogenic strains, which are constantly interacting with the intestinal immune system. This interaction elicits a non-pathological basal level of immune responses and contributes to shaping both the intestinal immune system and bacterial community. Recent studies on human microbiota are revealing the critical role of intestinal bacterial community in the pathogenesis of both systemic and intestinal diseases, including Crohn's disease (CD). NOD2 plays a key role in the regulation of microbiota in the small intestine. NOD2 is highly expressed in ileal Paneth cells that provide critical mechanism for the regulation of ileal microbiota through the secretion of anti-bacterial compounds. Genome mapping of CD patients revealed that loss of function mutations in NOD2 are associated with ileal CD. Genome-wide association studies further demonstrated that NOD2 is one of the most critical genetic factor linked to ileal CD. The bacterial community in the ileum is indeed dysregulated in Nod2-deficient mice. Nod2-deficient ileal epithelia exhibit impaired ability of killing bacteria. Thus, altered interactions between ileal microbiota and mucosal immunity through NOD2 mutations play significant roles in the disease susceptibility and pathogenesis in CD patients, thereby depicting NOD2 as a critical regulator of ileal microbiota and CD.
• CITA/NLRC5: A critical transcriptional regulator of MHC class I gene expression

  Isaac Downs, Saptha Vijayan, Tabasum Sidiq, Koichi S. Kobayashi

  BIOFACTORS 42 (4) 349 - 357 0951-6433 2016/07 [Refereed][Not invited]
   

  Major histocompatibility complex (MHC) class I and class II molecules play essential roles in the development and activation of the human adaptive immune system. An NLR protein, CIITA (MHC class II transactivator) has been recognized as a master regulator of MHC class II gene expression, albeit knowledge about the regulatory mechanism of MHC class I gene expression had been limited. Recently identified MHC class I transactivator (CITA), or NLRC5, also belongs to the NLR protein family and constitutes a critical regulator for the transcriptional activation of MHC class I genes. In addition to MHC class I genes, CITA/NLRC5 induces the expression of (2)-microglobulin, TAP1 and LMP2, essential components of the MHC class I antigen presentation pathway. Therefore, CITA/NLRC5 and CIITA are transcriptional regulators that orchestrate the concerted expression of critical components in the MHC class I and class II pathways, respectively. (c) 2016 BioFactors, 42(4):349-357, 2016
• NLRC5/MHC class I transactivator is a target for immune evasion in cancer

  Sayuri Yoshihama, Jason Roszik, Isaac Downs, Torsten B. Meissner, Saptha Vijayan, Bjoern Chapuy, Tabasum Sidiq, Margaret A. Shipp, Gregory A. Lizee, Koichi S. Kobayashi

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 113 (21) 5999 - 6004 0027-8424 2016/05 [Refereed][Not invited]
   

  Cancer cells develop under immune surveillance, thus necessitating immune escape for successful growth. Loss of MHC class I expression provides a key immune evasion strategy in many cancers, although the molecular mechanisms remain elusive. MHC class I transactivator (CITA), known as "NLRC5" [NOD-like receptor (NLR) family, caspase recruitment (CARD) domain containing 5], has recently been identified as a critical transcriptional coactivator of MHC class I gene expression. Here we show that the MHC class I transactivation pathway mediated by CITA/NLRC5 constitutes a target for cancer immune evasion. In all the 21 tumor types we examined, NLRC5 expression was highly correlated with the expression of MHC class I, with cytotoxic T-cell markers, and with genes in the MHC class I antigen-presentation pathway, including LMP2/LMP7, TAP1, and beta 2-microglobulin. Epigenetic and genetic alterations in cancers, including promoter methylation, copy number loss, and somatic mutations, were most prevalent in NLRC5 among all MHC class I-related genes and were associated with the impaired expression of components of the MHC class I pathway. Strikingly, NLRC5 expression was significantly associated with the activation of CD8(+) cytotoxic T cells and patient survival in multiple cancer types. Thus, NLRC5 constitutes a novel prognostic biomarker and potential therapeutic target of cancers.
• Gastric LTi cells promote lymphoid follicle formation but are limited by IRAK-M and do not alter microbial growth

  J. Shiu, M. B. Piazuelo, H. Ding, S. J. Czinn, M. L. Drakes, A. Banerjee, N. Basappa, K. S. Kobayashi, W. F. Fricke, T. G. Blanchard

  MUCOSAL IMMUNOLOGY 8 (5) 1047 - 1059 1933-0219 2015/09 [Refereed][Not invited]
   

  Lymphoid tissue inducer (LTi) cells are activated by accessory cell IL-23, and promote lymphoid tissue genesis and antibacterial peptide production by the mucosal epithelium. We investigated the role of LTi cells in the gastric mucosa in the context of microbial infection. Mice deficient in IRAK-M, a negative regulator of TLR signaling, were investigated for increased LTi cell activity, and antibody mediated LTi cell depletion was used to analyze LTi cell dependent antimicrobial activity. H. pylori infected IRAK-M deficient mice developed increased gastric IL-17 and lymphoid follicles compared to wild type mice. LTi cells were present in naive and infected mice, with increased numbers in IRAK-M deficient mice by two weeks. Helicobacter and Candida infection of LTi cell depleted rag1(-/-) mice demonstrated LTi-dependent increases in calprotectin but not RegIII proteins. However, pathogen and commensal microbiota populations remained unchanged in the presence or absence of LTi cell function. These data demonstrate LTi cells are present in the stomach and promote lymphoid follicle formation in response to infection, but are limited by IRAK-M expression. Additionally, LTi cell mediated antimicrobial peptide production at the gastric epithelium is less efficacious at protecting against microbial pathogens than has been reported for other tissues.
• Microbiota regulates type 1 diabetes through Toll-like receptors

  Michael P. Burrows, Pavel Volchkov, Koichi S. Kobayashi, Alexander V. Chervonsky

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 112 (32) 9973 - 9977 0027-8424 2015/08 [Refereed][Not invited]
   

  Deletion of the innate immune adaptor myeloid differentiation primary response gene 88 (MyD88) in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D) results in microbiota-dependent protection from the disease: MyD88-negative mice in germfree (GF), but not in specific pathogen-free conditions develop the disease. These results could be explained by expansion of particular protective bacteria ("specific lineage hypothesis") or by dominance of negative (tolerizing) signaling over proinflammatory signaling ("balanced signal hypothesis") in mutant mice. Here we found that colonization of GF mice with a variety of intestinal bacteria was capable of reducing T1D in MyD88-negative (but not wild-type NOD mice), favoring the balanced signal hypothesis. However, the receptors and signaling pathways involved in prevention or facilitation of the disease remained unknown. The protective signals triggered by the microbiota were revealed by testing NOD mice lacking MyD88 in combination with knockouts of several critical components of innate immune sensing for development of T1D. Only MyD88-and TIR-domain containing adapter inducing IFN beta (TRIF) double deficient NOD mice developed the disease. Thus, TRIF signaling (likely downstream of Toll-like receptor 4, TLR4) serves as one of the microbiota-induced tolerizing pathways. At the same time another TLR (TLR2) provided prodiabetic signaling by controlling the microbiota, as reduction in T1D incidence caused by TLR2 deletion was reversed in GF TLR2-negative mice. Our results support the balanced signal hypothesis, in which microbes provide signals that both promote and inhibit autoimmunity by signaling through different receptors, including receptors of the TLR family.
• Ubiquitination-mediated regulation of RIP2 in NOD2 signaling and MDP tolerance induction

  Bindu Sukumaran, Pradeep Bist, Aywin Ng, Bae Hoon Kim, Niyas Kudukkil Pulloor, Hanif Khameneh, Matija Hedi, Neha Dikshit, Avinash Shenoy, Vanniarajan Balamuralidhar, Michelle Hong, Albert Neutzner, Keh-Chuang Chin, Koichi Kobayashi, Antonio Bertoletti, Alessandra Mortellaro, Clara Abraham, John MacMicking, Ramnik Xavier

  JOURNAL OF IMMUNOLOGY 194 0022-1767 2015/05 [Not refereed][Not invited]
  
• NOD2 Signaling Contributes to Host Defense in the Lungs against Escherichia coli Infection (Retraction of vol 80, pg 2558, 2012)

  Balamayooran Theivanthiran, Sanjay Batra, Gayathriy Balamayooran, Shanshan Cai, Koichi Kobayashi, Richard A. Flavell, Samithamby Jeyaseelan

  INFECTION AND IMMUNITY 83 (5) 2199 - 2199 0019-9567 2015/05 [Not refereed][Not invited]
  
• Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

  Masanori Miyata, Ji-Yun Lee, Seiko Susuki-Miyata, Wenzhuo Y. Wang, Haidong Xu, Hirofumi Kai, Koichi S. Kobayashi, Richard A. Flavell, Jian-Dong Li

  NATURE COMMUNICATIONS 6 6062 - 6062 2041-1723 2015/01 [Refereed][Not invited]
   

  Glucocorticoids are among the most commonly used anti-inflammatory agents. Despite the enormous efforts in elucidating the glucocorticoid-mediated anti-inflammatory actions, how glucocorticoids tightly control overactive inflammatory response is not fully understood. Here we show that glucocorticoids suppress bacteria-induced inflammation by enhancing IRAK-M, a central negative regulator of Toll-like receptor signalling. The ability of glucocorticoids to suppress pulmonary inflammation induced by non-typeable Haemophilus influenzae is significantly attenuated in IRAK-M-deficient mice. Glucocorticoids improve the survival rate after a lethal non-typeable Haemophilus influenzae infection in wild-type mice, but not in IRAK-M-deficient mice. Moreover, we show that glucocorticoids and non-typeable Haemophilus influenzae synergistically upregulate IRAK-M expression via mutually and synergistically enhancing p65 and glucocorticoid receptor binding to the IRAK-M promoter. Together, our studies unveil a mechanism by which glucocorticoids tightly control the inflammatory response and host defense via the induction of IRAK-M and may lead to further development of anti-inflammatory therapeutic strategies.
• Macrophage Phenotype Controls Long-Term AKI Outcomes-Kidney Regeneration versus Atrophy

  Maciej Lech, Regina Groebmayr, Mi Ryu, Georg Lorenz, Ingo Hartter, Shrikant R. Mulay, Heni Eka Susanti, Koichi S. Kobayashi, Richard A. Flavell, Hans-Joachim Anders

  JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 25 (2) 292 - 304 1046-6673 2014/02 [Refereed][Not invited]
   

  The mechanisms that determine full recovery versus subsequent progressive CKD after AKI are largely unknown. Because macrophages regulate inflammation as well as epithelial recovery, we investigated whether macrophage activation influences AKI outcomes. IL-1 receptor-associated kinase-M (IRAK-M) is a macrophage-specific inhibitor of Toll-like receptor (TLR) and IL-1 receptor signaling that prevents polarization toward a proinflammatory phenotype. In postischemic kidneys of wild-type mice, IRAK-M expression increased for 3 weeks after AKI and declined thereafter. However, genetic depletion of IRAK-M did not affect immunopathology and renal dysfunction during early postischemic AKI. Regarding long-term outcomes, wild-type kidneys regenerated completely within 5 weeks after AKI. In contrast, IRAK-M-/- kidneys progressively lost up to two-thirds of their original mass due to tubule loss, leaving atubular glomeruli and interstitial scarring. Moreover, M1 macrophages accumulated in the renal interstitial compartment, coincident with increased expression of proinflammatory cytokines and chemokines. Injection of bacterial CpG DNA induced the same effects in wild-type mice, and TNF-alpha blockade with etanercept partially prevented renal atrophy in IRAK-M-/- mice. These results suggest that IRAK-M induction during the healing phase of AKI supports the resolution of M1 macrophage- and TNF-alpha-dependent renal inflammation, allowing structural regeneration and functional recovery of the injured kidney. Conversely, IRAK-M loss-of-function mutations or transient exposure to bacterial DNA may drive persistent inflammatory mononuclear phagocyte infiltrates, which impair kidney regeneration and promote CKD. Overall, these results support a novel role for IRAK-M in the regulation of wound healing and tissue regeneration.
• NOD2 Signaling Contributes to Host Defense in the Lungs against Escherichia coli Infection (vol 80, pg 2558, 2012)

  Balamayooran Theivanthiran, Sanjay Batra, Gayathriy Balamayooran, Shanshan Cai, Koichi Kobayashi, Richard A. Flavell, Samithamby Jeyaseelan

  INFECTION AND IMMUNITY 81 (11) 4324 - 4324 0019-9567 2013/11 [Not refereed][Not invited]
  
• IRAK-M Expression Limits Dendritic Cell Activation and Proinflammatory Cytokine Production in Response to Helicobacter pylori

  Jessica Shiu, Steven J. Czinn, Koichi S. Kobayashi, Yezhou Sun, Thomas G. Blanchard

  PLOS ONE 8 (6) e66914  1932-6203 2013/06 [Refereed][Not invited]
   

  Helicobacter pylori (H. pylori) infects the gastric mucosa and persists for the life of the host. Bacterial persistence may be due to the induction of regulatory T cells (T-regs) which may have protective effects against other diseases such as asthma. It has been shown that H. pylori modulates the T cell response through dendritic cell reprogramming but the molecular pathways involved are relatively unknown. The goal of this study was to identify critical elements of dendritic cell (DC) activation and evaluate potential influence on immune activation. Microarray analysis was used to demonstrate limited gene expression changes in H. pylori stimulated bone marrow derived DCs (BMDCs) compared to the BMDCs stimulated with E. coli. IRAK-M, a negative regulator of TLR signaling, was upregulated and we selectedit for investigation of its role in modulating the DC and T cell responses. IRAK-M-/- and wild type BMDC were compared for their response to H. pylori. Cells lacking IRAK-M produced significantly greater amounts of proinflammatory MIP-2 and reduced amounts of immunomodulatory IL-10 than wild type BMDC. IRAK-M-/- cells also demonstrated increased MHC II expression upon activation. However, IRAK-M-/- BMDCs were comparable to wild type BMDCs in inducing T-helper 17 (T(H)17) and T-reg responses as demonstrated in vitro using BMDC CD4+ T cells co-culture assays, and in vivo though the adoptive transfer of CD4(+) FoxP3-GFP T cells into H. pylori infected IRAK-M-/- mice. These results suggest that H. pylori infection leads to the upregulation of anti-inflammatory molecules like IRAK-M and that IRAK-M has a direct impact on innate functions in DCs such as cytokine and costimulation molecule upregulation but may not affect T cell skewing.
• IRAK-M Deficiency Leads to Increased Gastric Lymphoid Aggregate Formation and Antibody Responses During H. pylori Infection

  Jessica Shiu, Hua Ding, Koichi S. Kobayashi, Steven J. Czinn, Tom G. Blanchard

  GASTROENTEROLOGY 144 (5) S313 - S313 0016-5085 2013/05 [Not refereed][Not invited]
  
• IRAK-M IN RENAL FIBROSIS UPON UNILATERAL URETERAL OBSTRUCTION

  Maciej Lech, Roman Guenthner, Georg Lorenz, Mi Ryu, Regina Groebmayr, Heni Susanti, Koichi S. Kobayashi, Richard A. Flavell, Hans-Joachim Anders

  NEPHROLOGY DIALYSIS TRANSPLANTATION 28 190 - 190 0931-0509 2013/05 [Not refereed][Not invited]
  
• Altered Gut Microbiota Promotes Colitis-Associated Cancer in IL-1 Receptor-Associated Kinase M-Deficient Mice

  Klara Klimesova, Miloslav Kverka, Zuzana Zakostelska, Tomas Hudcovic, Tomas Hrncir, Renata Stepankova, Pavel Rossmann, Jakub Ridl, Martin Kostovcik, Jakub Mrazek, Jan Kopecny, Koichi S. Kobayashi, Helena Tlaskalova-Hogenova

  INFLAMMATORY BOWEL DISEASES 19 (6) 1266 - 1277 1078-0998 2013/05 [Refereed][Not invited]
   

  Background:Microbial sensing by Toll-like receptors (TLR) and its negative regulation have an important role in the pathogenesis of inflammation-related cancer. In this study, we investigated the role of negative regulation of Toll-like receptors signaling and gut microbiota in the development of colitis-associated cancer in mouse model.Methods:Colitis-associated cancer was induced by azoxymethane and dextran sodium sulfate in wild-type and in interleukin-1 receptor-associated kinase M (IRAK-M)-deficient mice with or without antibiotic (ATB) treatment. Local cytokine production was analyzed by multiplex cytokine assay or enzyme-linked immunosorbent assay, and regulatory T cells were analyzed by flow cytometry. Changes in microbiota composition during tumorigenesis were analyzed by pyrosequencing, and -glucuronidase activity was measured in intestinal content by fluorescence assay.Results:ATB treatment of wild-type mice reduced the incidence and severity of tumors. Compared with nontreated mice, ATB-treated mice had significantly lower numbers of regulatory T cells in colon, altered gut microbiota composition, and decreased -glucuronidase activity. However, the -glucuronidase activity was not as low as in germ-free mice. IRAK-M-deficient mice not only developed invasive tumors, but ATB-induced decrease in -glucuronidase activity did not rescue them from severe carcinogenesis phenotype. Furthermore, IRAK-M-deficient mice had significantly increased levels of proinflammatory cytokines in the tumor tissue.Conclusions:We conclude that gut microbiota promotes tumorigenesis by increasing the exposure of gut epithelium to carcinogens and that IRAK-M-negative regulation is essential for colon cancer resistance even in conditions of altered microbiota. Therefore, gut microbiota and its metabolic activity could be potential targets for colitis-associated cancer therapy.
• Regulation of intestinal microbiota by the NLR protein family

  Amlan Biswas, Koichi S. Kobayashi

  INTERNATIONAL IMMUNOLOGY 25 (4) 207 - 214 0953-8178 2013/04 [Refereed][Not invited]
   

  Regulation of intestinal microbiota by NLR proteins.The human intestine harbors a diverse microbial community consisting of a large number of bacteria and other micro-organisms that have co-evolved with the host intestinal immune system. During this process, microbiota and the host immune system shape one another by various mechanisms to achieve a successful symbiotic relationship. An increasing amount of evidence suggests that dysbiosisuthe breakdown of such harmonized colonizationumay result in infectious and inflammatory disorders, and recent advances in our studies indicate that receptors such as Toll-like receptors and NLR (nucleotide-binding oligomerization domain-like receptor; or nucleotide-binding domain- and leucine-rich repeat-containing receptor) proteins that detect micro-organisms and their products play a critical role in maintaining intestinal homeostasis. In this review, we summarize the role of NLR proteins in the regulation of intestinal microbiota. NLR proteins belong to a diverse family of cytoplasmic microbial sensors, mutations of which are involved in various disorders, including inflammatory bowel diseases. Understanding of the different roles of NLR family proteins in the intestine is, therefore, an important step towards the development of therapeutics against digestive diseases.
• Shaping Intestinal Bacterial Community by TLR and NLR Signaling

  Koichi S. Kobayashi

  PROBIOTIC BACTERIA AND THEIR EFFECT ON HUMAN HEALTH AND WELL-BEING 107 32 - 42 0084-2230 2013 [Refereed][Not invited]
   

  Human intestines harbor a diverse microbial community composed of a large number of bacteria and other microorganisms. These intestinal microbiota have evolved to achieve a symbiotic relationship with the host. In addition to aiding host metabolic pathways by breaking down foods and supplying nutrients to their host, microbiota play an important role in the development and maintenance of the host immune system. At the same time, the detection of microorganisms and their products by innate immune receptors such as TLRs (Toll-like receptors) and NLR (nucleotide-binding domain, leucine-rich repeats, or NOD-like receptor) proteins are critical for maintaining intestinal homeostasis by shaping microbial communities. This review summarizes recent progress about the role of TLRs and NLRs in the regulation of intestinal microbiota. Accumulating evidence suggest that intestinal microbiota have a large impact on both intestinal and systemic diseases. Therefore, understanding the mechanism of microbial regulation by TLRs and NLRs is important for the advancement of therapeutic interventions against digestive and other diseases. Copyright (c) 2013 S. Karger AG, Basel
• NLRC5: a key regulator of MHC class I-dependent immune responses

  Koichi S. Kobayashi, Peter J. van den Elsen

  NATURE REVIEWS IMMUNOLOGY 12 (12) 813 - 820 1474-1733 2012/12 [Refereed][Not invited]
   

  The expression of MHC class I molecules is crucial for the initiation and regulation of adaptive immune responses against pathogens. NOD-, LRR- and CARD-containing 5 (NLRC5) was recently identified as a specific transactivator of MHC class I genes (CITA). NLRC5 and the master regulator for MHC class II genes, class II transactivator (CIITA), interact with similar MHC promoter-bound factors. Here, we provide a broad overview of the molecular mechanisms behind MHC class I transcription and the role of the class I transactivator NLRC5 in MHC class I-dependent immune responses.
• Receptor interacting protein-2 contributes to host defense against Anaplasma phagocytophilum infection

  Bindu Sukumaran, Yasunori Ogura, Joao H. F. Pedra, Koichi S. Kobayashi, Richard A. Flavell, Erol Fikrig

  FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY 66 (2) 211 - 219 0928-8244 2012/11 [Refereed][Not invited]
   

  The Gram-negative obligate intracellular bacterium Anaplasma phagocytophilum is the causative agent of human granulocytic anaplasmosis (HGA), an emerging tick-borne infectious disease occurring worldwide. HGA is generally self-limiting; however, the underlying mechanisms, particularly the innate immune pathways that mediate the immune clearance of A. phagocytophilum, are less understood. We herein report an unexpected role for Receptor interacting protein-2 (Rip2), the adaptor protein for the Nod-like receptors (NLRs), Nod1/Nod2, in the host immune response against A. phagocytophilum infection. Although A. phagocytophilum genome is reported to lack the genes encoding the known ligands of Nod1 and Nod2, its infection upregulated the transcription of Rip2 in human primary neutrophils. Our results revealed that Rip2-deficient mice had significantly higher bacterial load than wild-type controls throughout the infection period. In addition, the Rip2-deficient mice took strikingly longer duration to clear A. phagocytophilum infection. Detailed analysis identified that interferon gamma (IFN?) and interleukin (IL)-18 but not IL-12, macrophage inflammatory protein-2, and KC response were diminished in A. phagocytophilum-challenged Rip2-deficient mice. Together, these results revealed that Rip2 plays important roles in the immune control of A. phagocytophilum and may contribute to our understanding of the host response to Rickettsiales.
• Proteasomal Degradation of Nod2 Protein Mediates Tolerance to Bacterial Cell Wall Components

  Kyoung-Hee Lee, Amlan Biswas, Yuen-Joyce Liu, Koichi S. Kobayashi

  JOURNAL OF BIOLOGICAL CHEMISTRY 287 (47) 39800 - 39811 0021-9258 2012/11 [Refereed][Not invited]
   

  The innate immune system serves as the first line of defense by detecting microbes and initiating inflammatory responses. Although both Toll-like receptor (TLR) and nucleotide binding domain and leucine-rich repeat (NLR) proteins are important for this process, their excessive activation is hazardous to hosts; thus, tight regulation is required. Endotoxin tolerance is refractory to repeated lipopolysaccharide (LPS) stimulation and serves as a host defense mechanism against septic shock caused by an excessive TLR4 response during Gram-negative bacterial infection. Gram-positive bacteria as well as their cell wall components also induce shock. However, the mechanism underlying tolerance is not understood. Here, we show that activation of Nod2 by its ligand, muramyl dipeptide (MDP) in the bacterial cell wall, induces rapid degradation of Nod2, which confers MDP tolerance in vitro and in vivo. Nod2 is constitutively associated with a chaperone protein, Hsp90, which is required for Nod2 stability and protects Nod2 from degradation. Upon MDP stimulation, Hsp90 rapidly dissociates from Nod2, which subsequently undergoes ubiquitination and proteasomal degradation. The SOCS-3 protein induced by Nod2 activation further facilitates this degradation process. Therefore, Nod2 protein stability is a key factor in determining responsiveness to MDP stimulation. This indicates that TLRs and NLRs induce a tolerant state through distinct molecular mechanisms that protect the host from septic shock.
• NOD2 Signaling Contributes to Host Defense in the Lungs against Escherichia coli Infection (Retracted article. See vol. 83, pg. 2199, 2015)

  Balamayooran Theivanthiran, Sanjay Batra, Gayathriy Balamayooran, Shanshan Cai, Koichi Kobayashi, Richard A. Flavell, Samithamby Jeyaseelan

  INFECTION AND IMMUNITY 80 (7) 2558 - 2569 0019-9567 2012/07 [Refereed][Not invited]
   

  Bacterial pneumonia remains a significant cause of mortality in the United States. The innate immune response is the first line of defense against invading bacteria. Neutrophil recruitment to the lungs is the first step in a multistep sequence leading to bacterial clearance. Ligand interaction with pattern-recognizing receptors (PRRs) leads to chemokine production, which drives neutrophils to the site of infection. Although we demonstrated that RIP2 is important for host defense in the lungs against Escherichia coli, the individual roles of NODI and NOD2(-/-) in pulmonary defense have not been addressed. Here, we explored the role of NOD2 in neutrophil-mediated host defense against an extracellular pathogen, E. coli. We found enhanced bacterial burden and reduced neutrophil and cytokine/chemokine levels in the lungs of NOD2(-/-) mice following E. coli infection. Furthermore, we observed reduced activation of NF-kappa B and mitogen-activated protein kinases (MAPKs) in the lungs of NOD2(-/-) mice upon E. coli challenge. Moreover, NOD2(-/-) neutrophils show impaired intracellular bacterial killing. Using NOD2/RIP2(-/-) mice, we observed bacterial burden and neutrophil accumulation in the lungs similar to those seen with NOD2(-/-) mice. In addition, bone marrow-derived macrophages obtained from NOD2/RIP2(-/-) mice demonstrate a reduction in activation of NF-kappa B and MAPKs similar to that seen with NOD2(-/-) mice in response to E. coli. These findings unveil a previously unrecognized role of the NOD2-RIP2 axis for host defense against extracellular Gram-negative bacteria. This pathway may represent a novel target for the treatment of lung infection/inflammation.
• Cutting Edge: Impaired MHC Class I Expression in Mice Deficient for Nlrc5/Class I Transactivator

  Amlan Biswas, Torsten B. Meissner, Taro Kawai, Koichi S. Kobayashi

  JOURNAL OF IMMUNOLOGY 189 (2) 516 - 520 0022-1767 2012/07 [Refereed][Not invited]
   

  MHC class I and class II are crucial for the adaptive immune system. Although regulation of MHC class II expression by CIITA has long been recognized, the mechanism of MHC class I transactivation has been largely unknown until the recent discovery of NLRC5/class I transactivator. In this study, we show using Nlrc5-deficient mice that NLRC5 is required for both constitutive and inducible MHC class I expression. Loss of Nlrc5 resulted in severe reduction in the expression of MHC class I and related genes such as beta(2)-microglobulin, Tap1, or Lmp2, but did not affect MHC class II levels. IFN-gamma stimulation could not overcome the impaired MHC class I expression in Nlrc5-deficient cells. Upon infection with Listeria monocyogenes, Nlrc5-deficient mice displayed impaired CD8(+) T cell activation, accompanied with increased bacterial loads. These findings illustrate critical roles of NLRC5/class I transactivator in MHC class I gene regulation and host defense by CD8(+) T cell responses. The Journal of Immunology, 2012, 189: 516-520.
• Interleukin-1 Receptor-Associated Kinase M-Deficient Mice Demonstrate an Improved Host Defense during Gram-negative Pneumonia

  Jacobien J. Hoogerwerf, Gerritje J. W. van der Windt, Dana C. Blok, Arie J. Hoogendijk, Alex F. de Vos, Cornelis van 't Veer, Sandrine Florquin, Koichi S. Kobayashi, Richard A. Flavell, Tom van der Poll

  MOLECULAR MEDICINE 18 (7) 1067 - 1075 1076-1551 2012/07 [Refereed][Not invited]
   

  Pneumonia is a common cause of morbidity and mortality and the most frequent source of sepsis. Bacteria that try to invade normally sterile body sites are recognized by innate immune cells through pattern recognition receptors, among which toll-like receptors (TLRs) feature prominently. Interleukin-1 receptor (IL-1R)-associated kinase (IRAK)-M is a proximal inhibitor of TLR signaling expressed by epithelial cells and macrophages in the lung. To determine the role of IRAK-M in host defense against bacterial pneumonia, IRAK-M-deficient (IRAK-M-/-) and normal wild-type (WT) mice were infected intranasally with Klebsiella pneumoniae. IRAK-M mRNA was upregulated in lungs of WT mice with Klebsiella pneumonia, and the absence of IRAK-M resulted in a strongly improved host defense as reflected by reduced bacterial growth in the lungs, diminished dissemination to distant body sites, less peripheral tissue injury and better survival rates. Although IRAK-M-/- alveolar macrophages displayed enhanced responsiveness toward intact K. pneumoniae and Klebsiella lipopolysaccharide (LPS) in vitro, IRAK-M-/- mice did not show increased cytokine or chemokine levels in their lungs after infection in vivo. The extent of lung inflammation was increased in IRAK-M-/- mice shortly after K. pneumoniae infection, as determined by semiquantitative scoring of specific components of the inflammatory response in lung tissue slides. These data indicate that IRAK-M impairs host defense during pneumonia caused by a common gram-negative respiratory pathogen. Online address: http://www.molmed.org doi: 10.2119/molmed.2011.00450
• NLRC5: a newly discovered MHC class I transactivator (CITA)

  Torsten B. Meissner, Amy Li, Koichi S. Kobayashi

  MICROBES AND INFECTION 14 (6) 477 - 484 1286-4579 2012/06 [Refereed][Not invited]
   

  Major histocompatibility complex (MHC) class I and class II are crucial for the function of the human adaptive immune system. An NLR protein, CIITA (MHC class II transactivator), is a master regulator of MHC class 11 gene expression as well as of some of the genes involved in MHC class II antigen presentation. It has recently been discovered that another member of the NI:.,R protein family, NLRC5, transcriptionally activates MHC class I genes, and thus acts as "CITA" (MHC class I transactivator), a counterpart to CIITA. In addition to MHC class I genes, NLRC5 can induce the expression of beta 2M, TAP1 and LMP2, essential components of MHC class I antigen presentation. These findings indicate that NLRC5 and CIITA are transcriptional regulators that orchestrate the concerted expression of critical components in the MHC class I and MHC class 11 pathways, respectively. (C) 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
• Interleukin 1 Receptor-Associated Kinase M Impairs Host Defense During Pneumococcal Pneumonia

  Gerritje J. W. van der Windt, Dana C. Blok, Jacobien J. Hoogerwerf, Adriana J. J. Lammers, Alex F. de Vos, Cornelis van't Veer, Sandrine Florquin, Koichi S. Kobayashi, Richard A. Flavell, Tom van der Poll

  JOURNAL OF INFECTIOUS DISEASES 205 (12) 1849 - 1857 0022-1899 2012/06 [Refereed][Not invited]
   

  Background. Streptococcus pneumoniae is the most common causative organism in community-acquired pneumonia. Pneumococci that try to invade the lower airways are recognized by innate immune cells through pattern recognition receptors, including Toll-like receptors 2, 4, and 9. Interleukin 1 (IL-1) receptor-associated kinase (IRAK)-M is a proximal inhibitor of Toll-like receptor signaling. Methods. To determine the role of IRAK-M in host defense during pneumococcal pneumonia, IRAK-M-deficient and wild-type mice were intranasally infected with S. pneumoniae. Results. IRAK-M-deficient mice demonstrated a reduced lethality after infection with S. pneumoniae via the airways. Whereas bacterial burdens were similar in IRAK-M-deficient and wild-type mice early (3 hours) after infection, from 24 hours onward the number of pneumococci recovered from lungs and distant body sites were 10-100-fold lower in the former mouse strain. The diminished bacterial growth and dissemination in IRAK-M-deficient mice were preceded by an increased early influx of neutrophils into lung tissue and elevated pulmonary levels of IL-1 beta and CXCL1. IRAK-M deficiency did not influence bacterial growth after intravenous administration of S. pneumoniae. Conclusions. These data suggest that IRAK-M impairs host defense during pneumococcal pneumonia at the primary site of infection at least in part by inhibiting the early immune response.
• Intracellular Invasion of Orientia tsutsugamushi Activates Inflammasome in ASC-Dependent Manner

  Jung-Eun Koo, Hye-Jin Hong, Andrea Dearth, Koichi S. Kobayashi, Young-Sang Koh

  PLOS ONE 7 (6) e39042  1932-6203 2012/06 [Refereed][Not invited]
   

  Orientia tsutsugamushi, a causative agent of scrub typhus, is an obligate intracellular bacterium, which escapes from the endo/phagosome and replicates in the host cytoplasm. O. tsutsugamushi infection induces production of pro-inflammatory mediators including interleukin-1 beta (IL-1 beta), which is secreted mainly from macrophages upon cytosolic stimuli by activating cysteine protease caspase-1 within a complex called the inflammasome, and is a key player in initiating and maintaining the inflammatory response. However, the mechanism for IL-1 beta maturation upon O. tsutsugamushi infection has not been identified. In this study, we show that IL-1 receptor signaling is required for efficient host protection from O. tsutsugamushi infection. Live Orientia, but not heat-or UV-inactivated Orientia, activates the inflammasome through active bacterial uptake and endo/phagosomal maturation. Furthermore, Orientia-stimulated secretion of IL-1 beta and activation of caspase-1 are ASC- and caspase-1- dependent since IL-1 beta production was impaired in Asc- and caspase-1-deficient macrophages but not in Nlrp3-, Nlrc4- and Aim2-deficient macrophages. Therefore, live O. tsutsugamushi triggers ASC inflammasome activation leading to IL-1 beta production, which is a critical innate immune response for effective host defense.
• NLRC5 Cooperates with the RFX Transcription Factor Complex To Induce MHC Class I Gene Expression

  Torsten B. Meissner, Yuen-Joyce Liu, Kyoung-Hee Lee, Amy Li, Amlan Biswas, Marja C. J. A. van Eggermond, Peter J. van den Elsen, Koichi S. Kobayashi

  JOURNAL OF IMMUNOLOGY 188 (10) 4951 - 4958 0022-1767 2012/05 [Refereed][Not invited]
   

  Tight regulation of MHC class I gene expression is critical for CD8 T cell activation and host adaptive-immune responses. The promoters of MHC class I genes contain a well-conserved core module, the W/S-X-Y motif, which assembles a nucleoprotein complex termed MHC enhanceosome. A member of the nucleotide-binding domain, leucine-rich repeat (NLR) protein family, NLRC5, is a newly identified transcriptional regulator of MHC class I genes. NLRC5 associates with and transactivates the proximal promoters of MHC class I genes, although the molecular mechanism of transactivation has not been understood. In this article, we show that NLRC5-mediated MHC class I gene induction requires the W/S and X1, X2 cis-regulatory elements. The transcription factors RFX5, RFXAP, and RFXANK/B, which compose the RFX protein complex and associate with the X1 box, cooperate with NLRC5 for MHC class I expression. Coimmunoprecipitation experiments revealed that NLRC5 specifically interacts with the RFX subunit RFXANK/B via its ankyrin repeats. In addition, we show that NLRC5 can cooperate with ATF1 and the transcriptional coactivators CBP/p300 and general control nonderepressible 5, which display histone acetyltransferase activity. Taken together, our data suggest that NLRC5 participates in an MHC class I-specific enhanceosome, which assembles on the conserved W/S-X-Y core module of the MHC class I proximal promoters, including the RFX factor components and CREB/ATF1 family transcription factors, to promote MHC class I gene expression. The Journal of Immunology, 2012,188: 4951-4958.
• Control of NOD2 and Rip2-dependent innate immune activation by GEF-H1

  Yun Zhao, Carmen Alonso, Isabel Ballester, Joo Hye Song, Sun Young Chang, Bayasi Guleng, Seiji Arihiro, Peter J. Murray, Ramnik Xavier, Koichi S. Kobayashi, Hans-Christian Reinecker

  INFLAMMATORY BOWEL DISEASES 18 (4) 603 - 612 1078-0998 2012/04 [Refereed][Not invited]
   

  Background: Genetic variants of nucleotide-binding oligomerization domain 2 (NOD2) lead to aberrant microbial recognition and can cause chronic inflammatory diseases in patients with Crohn's disease (CD). Methods: We utilized gene-specific siRNA mediated knockdown and expression of guanine nucleotide exchange factor H1 (GEF-H1) in wildtype, Rip2-, and Nod2-deficient macrophages, HCT-116 and HEK 293 cells to determine the role of GEF-H1 in NOD2 and Rip2-mediated NF kappa B- dependent induction of proinflammatory cytokine expression. Confocal microscopy was used to determine subcellular distribution of GEFH1, Rip2, and NOD2. Results: We identified GEF-H1 as an unexpected component of innate immune regulation during microbial pattern recognition by NOD2. Surprisingly, GEF-H1-mediated the activation of Rip2 during signaling by NOD2, but not in the presence of the 3020insC variant of NOD2 associated with CD. GEF-H1 functioned downstream of NOD2 as part of Rip2-containing signaling complexes and was responsible for phosphorylation of Rip2 by Src tyrosine kinase. Rip2 variants lacking the tyrosine target of GEF-H1-mediated phosphorylation were unable to mediate NF-kappa B activation in Rip2-deficient macrophages and failed to transduce NOD2 signaling. GEF-H1 is required downstream of NOD2 as part of Rip2-containing signaling complexes for the activation of innate immune responses. Conclusions: GEF-H1 connects tyrosine kinase function to NOD-like receptor signaling and is fundamental to the regulation of microbial recognition by ubiquitous innate immune mechanisms mediated by Rip2 kinase.
• The nucleotide-binding domain of NLRC5 is critical for nuclear import and transactivation activity

  Torsten B. Meissner, Amy Li, Yuen-Joyce Liu, Etienne Gagnon, Koichi S. Kobayashi

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 418 (4) 786 - 791 0006-291X 2012/02 [Refereed][Not invited]
   

  Major histocompatibility complex (MHC) class I and class II are crucial for the function of the human adaptive immune system. A member of the NLR (nucleotide-binding domain, leucine-rich repeat) protein family, NLRC5, has recently been identified as a transcriptional regulator of MHC class land related genes. While a 'master regulator' of MHC class II genes, CIITA, has long been known, NLRC5 specifically associates with and transactivates the proximal promoters of MHC class I genes. In this study, we analyzed the molecular requirements of NLRC5 nuclear import and transactivation activity. We show that NLRC5-mediated MHC class I gene induction requires an intact nuclear localization signal and nuclear distribution of NLRC5. In addition, we find that the nucleotide-binding domain (NBD) of NLRC5 is critical not only for nuclear translocation but also for the transactivation of MHC class I genes. Changing the cellular localization of NLRC5 is likely to immediately impact MHC class I expression as well as MHC class I-mediated antigen presentation. NLRC5 may thus provide a promising target for the modulation of MHC class I antigen presentation, especially in the setting of transplant medicine. (C) 2012 Elsevier Inc. All rights reserved.
• Molecular mechanism of downregualtion of MHC class I molecules in cancer

  Koichi S. Kobayashi

  INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 30 S5 - S5 1107-3756 2012 [Not refereed][Not invited]
  
• TANK-binding kinase 1 (TBK1) controls cell survival through PAI-2/serpinB2 and transglutaminase 2 (Proceedings of the National Academy of Sciences of the United States of America (2012) 109, 4 (E177-E186))

  Delhase, M., Kim, S.-Y., Lee, H., Naiki-Ito, A., Chen, Y., Ahn, E.-R., Murata, K., Kim, S.-J., Lautsch, N., Kobayashi, K.S., Shirai, T., Karin, M., Nakanishi, M.

  Proceedings of the National Academy of Sciences of the United States of America 109 (11) 4332 - 4335 0027-8424 2012 [Refereed][Not invited]
  
• Nod2: a key regulator linking microbiota to intestinal mucosal immunity

  Amlan Biswas, Tanja Petnicki-Ocwieja, Koichi S. Kobayashi

  JOURNAL OF MOLECULAR MEDICINE-JMM 90 (1) 15 - 24 0946-2716 2012/01 [Refereed][Not invited]
   

  The human intestine harbors a large number of bacteria that are constantly interacting with the intestinal immune system, eliciting non-pathological basal level immune responses. Increasing evidence points to dysbiosis of microbiota in the intestine as an underlying factor in inflammatory bowel disease susceptibility. Loss-of-function mutations in NOD2 are among the stronger genetic factors linked to ileal Crohn's disease. Indeed, Nod2 is a key regulator of microbiota in the intestine, as microflora in the terminal ileum is dysregulated in Nod2-deficient mice. Nod2 is highly expressed in Paneth cells, which are responsible for the regulation of ileal microflora by antimicrobial compounds, and Nod2-deficient ileal intestinal epithelia are unable to kill bacteria efficiently. It is therefore likely that NOD2 mutations in Crohn's disease may increase disease susceptibility by altering interactions between ileal microbiota and mucosal immunity.
• TANK-binding kinase 1 (TBK1) controls cell survival through PAI-2/serpinB2 and transglutaminase 2.

  Delhase M, Kim SY, Lee H, Naiki-Ito A, Chen Y, Ahn ER, Murata K, Kim SJ, Lautsch N, Kobayashi KS, Shirai T, Karin M, Nakanishi M

  Proceedings of the National Academy of Sciences of the United States of America 109 (4) E177 - 86 0027-8424 2012/01 [Refereed][Not invited]
  
• NLRC5/CITA: a novel regulator of class I major histocompatibility complex genes.

  Kobayashi KS

  Journal of immunodeficiency & disorders 1 (1) 2012 [Refereed][Not invited]
  
• Interleukin-1 receptor-associated kinase-M suppresses systemic lupus erythematosus

  Maciej Lech, Claudia Kantner, Onkar P. Kulkarni, Mi Ryu, Ekaterina Vlasova, Juergen Heesemann, David Anz, Stefan Endres, Koichi S. Kobayashi, Richard A. Flavell, Javier Martin, Hans-Joachim Anders

  ANNALS OF THE RHEUMATIC DISEASES 70 (12) 2207 - 2217 0003-4967 2011/12 [Refereed][Not invited]
   

  Objectives Interleukin-1 receptor-associated kinase (IRAK)-M suppresses Toll-like receptor (TLR)-mediated activation of innate immunity during infection. A similar role was hypothesised for IRAK-M in autoimmunity. Methods Irak-m-deficient mice were crossed with autoimmune C57BL/6-lpr/lpr mice and detailed phenotype analysis was performed. Results Irak-m deficiency converted the mild autoimmune phenotype of C57BL/6-lpr/lpr mice into a massive lymphoproliferative syndrome with lethal autoimmune lung disease and lupus nephritis. Irak-m deficiency induced a number of interferon-related genes, cytokines and plasma cell survival factors in spleen cells of these mice. Irak-m-deficient C57BL/6-lpr/lpr mice showed expansion of autoreactive T cells, dysfunctional regulatory T cells and plasma cells which was associated with increased lupus autoantibody production. TLR7 antagonism almost completely abrogated this phenotype consistent with IRAK-M-mediated suppression of TLR7 signalling in vitro. Conclusions These data identify a previously unknown function of IRAK-M-namely, suppression of TLR7-mediated autoimmunity-and mutant IRAK-M as a previously unknown genetic risk for murine SLE.
• Intrinsic expression of Nod2 in CD4(+) T lymphocytes is not necessary for the development of cell-mediated immunity and host resistance to Toxoplasma gondii

  Braulia C. Caetano, Amlan Biswas, Djalma S. Lima-Junior, Luciana Benevides, Tiago W. P. Mineo, Catarina V. Horta, Kyoung-Hee Lee, Joao S. Silva, Ricardo T. Gazzinelli, Dario S. Zamboni, Koichi S. Kobayashi

  EUROPEAN JOURNAL OF IMMUNOLOGY 41 (12) 3627 - 3631 0014-2980 2011/12 [Refereed][Not invited]
   

  Nod2 belongs to the nucleotide-binding domain leucine-rich repeat family of proteins and senses bacterial cell wall components to initiate innate immune responses against various pathogens. Recently, it has been reported that T-cell-intrinsic expression of Nod2 promotes host defense against Toxoplasma gondii infection by inducing type 1 immunity. Here, we present results that demonstrate that Nod2 does not play a role in the defense against T. gondii infection. Nod2-deficient mice were fully capable of inducing Th1 immune responses and did not show enhanced susceptibility to infection. Upon TCR stimulation in vitro, Nod2-deficient CD4(+) T cells showed normal activation, IL-2 production, proliferation, and Th1/2 differentiation. Nod2 mRNA and protein were expressed in CD4(+) T and CD8(+) T cells at substantial levels. Therefore, Nod2, although expressed in CD4(+) T cells, does not have an intrinsic function in T-cell activation and differentiation.
• Receptor-Interacting Protein 2 Controls Pulmonary Host Defense to Escherichia coli Infection via the Regulation of Interleukin-17A

  Theivanthiran Balamayooran, Sanjay Batra, Gayathriy Balamayooran, Shanshan Cai, Koichi S. Kobayashi, Richard A. Flavell, Samithamby Jeyaseelan

  INFECTION AND IMMUNITY 79 (11) 4588 - 4599 0019-9567 2011/11 [Refereed][Not invited]
   

  Recognition of microbial patterns by host receptors is the first step in a multistep sequence leading to neutrophil-dependent host resistance. Although the role of membrane-bound sensors in bacterial recognition has been examined in detail, the importance of cytosolic sensors in the lungs is largely unexplored. In this context, there is a major lack of understanding related to the downstream signaling mediators, such as cells and/or molecules, during acute extracellular Gram-negative bacterial pneumonia. In order to determine the role of NOD-like receptors (NLRs), we used an experimental Escherichia coli infection model using mice deficient in the gene coding for the NLR adaptor, receptor-interacting protein 2 (RIP2). RIP2(-/-) mice with E. coli infection displayed higher bacterial burden and reduced neutrophil recruitment and tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), macrophage inflammatory protein 2 (MIP-2), and CXCL5/LIX expression, along with attenuated histopathological changes in the lungs. Decreased IL-17A levels were observed, along with lower numbers of IL-17A-producing T cells, in RIP2(-/-) mice after infection. RIP2(-/-) mice also show reduced IL-6 and IL-23 levels in the lungs, along with decreased activation of STAT3 after infection. Furthermore, activation of NF-kappa B and mitogen-activated protein kinases (MAPKs) and expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in the lungs of infected RIP2(-/-) mice were attenuated following infection. Although neutrophil mobilization to the blood was impaired in RIP2(-/-) mice following infection, the expression of CD62P, CD11a/18, CD11b, and CXCR2 on blood and lung neutrophils was not altered between infected wild-type (WT) and RIP2(-/-) mice. Thus, RIP2 contributes to neutrophil-dependent host defense against an extracellular Gram-negative pathogen via (i) IL-17A regulation and (ii) neutrophil mobilization to the blood.
• Muramyl dipeptide and its derivatives: peptide adjuvant in immunological disorders and cancer therapy.

  Ogawa C, Liu YJ, Kobayashi KS

  Current bioactive compounds 7 (3) 180 - 197 1573-4072 2011/09 [Refereed][Not invited]
  
• Control of RIP2 Dependent Innate Immune Activation by GEF-111

  Yun Zhao, Joo Hye Song, Carmen Alonso, Isabel Ballaster, Bayasi Guleng, Seiji Arihiro, Peter Murray, Ramnik J. Xavier, Koichi S. Kobayashi, Hans-Christian Reinecker

  GASTROENTEROLOGY 140 (5) S497 - S497 0016-5085 2011/05 [Not refereed][Not invited]
  
• A Novel Aminosaccharide Compound Blocks Immune Responses by Toll-like Receptors and Nucleotide-binding Domain, Leucine-rich Repeat Proteins

  Kyoung-Hee Lee, Yuen-Joyce Liu, Amlan Biswas, Chikako Ogawa, Koichi S. Kobayashi

  JOURNAL OF BIOLOGICAL CHEMISTRY 286 (7) 5727 - 5735 0021-9258 2011/02 [Refereed][Not invited]
   

  Toll-like receptors (TLRs) and nucleotide-binding domain, leucine-rich repeat (NLR) proteins are two major forms of innate immune receptors that trigger inflammatory responses by various biological mechanisms such as cytokine production, recruitment of inflammatory cells, or activation of adaptive immunity. Although the innate immune system is designed to fight against infectious pathogens, excessive activation of TLR or NLR signaling pathways may lead to unwarranted inflammation with hazardous outcomes, including septic shock or inflammatory diseases. As part of the search for effective therapeutics to regulate these responses, here we show that a novel aminosaccharide compound, named DFK1012, inhibits immune responses caused by TLR and NLR activation. Treatment with DFK1012, but not its derivatives DFK845 or DFK846, strongly inhibited pro-inflammatory cytokine production upon stimulation via either TLR or NLR proteins in macrophages. Importantly, we have not observed cytotoxicity in any range of its working concentration. Treatment with DFK1012 did not interfere with TLR- or NLR-induced activation of p38 and JNK, phosphorylation/degradation of I kappa B, and subsequent nuclear translocation of NF-kappa B subunit p65, suggesting that the inhibitory activity of DFK1012 is not due to the suppression of downstream signaling. Indeed, DFK1012 did not impair transcription of pro-inflammatory cytokine genes but rather promoted post-translational degradation of pro-inflammatory cytokines. Therefore, DFK1012 is a novel anti-inflammatory compound that drives proteolysis of proinflammatory cytokines induced by TLR and NLR stimulation. DFK1012 may represent a novel class of potential therapeutic agents aimed at the treatment of inflammatory disorders.
• Nod2 Suppresses Borrelia burgdorferi Mediated Murine Lyme Arthritis and Carditis through the Induction of Tolerance

  Tanja Petnicki-Ocwieja, Alicia S. DeFrancesco, Erin Chung, Courtney T. Darcy, Roderick T. Bronson, Koichi S. Kobayashi, Linden T. Hu

  PLOS ONE 6 (2) e17414  1932-6203 2011/02 [Refereed][Not invited]
   

  The internalization of Borrelia burgdorferi, the causative agent of Lyme disease, by phagocytes is essential for an effective activation of the immune response to this pathogen. The intracellular, cytosolic receptor Nod2 has been shown to play varying roles in either enhancing or attenuating inflammation in response to different infectious agents. We examined the role of Nod2 in responses to B. burgdorferi. In vitro stimulation of Nod2 deficient bone marrow derived macrophages (BMDM) resulted in decreased induction of multiple cytokines, interferons and interferon regulated genes compared with wild-type cells. However, B. burgdorferi infection of Nod2 deficient mice resulted in increased rather than decreased arthritis and carditis compared to control mice. We explored multiple potential mechanisms for the paradoxical response in in vivo versus in vitro systems and found that prolonged stimulation with a Nod2 ligand, muramyl dipeptide (MDP), resulted in tolerance to stimulation by B. burgdorferi. This tolerance was lost with stimulation of Nod2 deficient cells that cannot respond to MDP. Cytokine patterns in the tolerance model closely paralleled cytokine profiles in infected Nod2 deficient mice. We propose a model where Nod2 has an enhancing role in activating inflammation in early infection, but moderates inflammation after prolonged exposure to the organism through induction of tolerance.
• Negative regulation of Toll-like receptor signaling plays an essential role in homeostasis of the intestine

  Amlan Biswas, Jeanette Wilmanski, Huamei Forsman, Tomas Hrncir, Liming Hao, Helena Tlaskalova-Hogenova, Koichi S. Kobayashi

  EUROPEAN JOURNAL OF IMMUNOLOGY 41 (1) 182 - 194 0014-2980 2011/01 [Refereed][Not invited]
   

  A healthy intestinal tract is characterized by controlled homeostasis due to the balanced interaction between commensal bacteria and the host mucosal immune system. Human and animal model studies have supported the hypothesis that breakdown of this homeostasis may underlie the pathogenesis of inflammatory bowel diseases. However, it is not well understood how intestinal microflora stimulate the intestinal mucosal immune system and how such activation is regulated. Using a spontaneous, commensal bacteria-dependent colitis model in IL-10-deficient mice, we investigated the role of TLR and their negative regulation in intestinal homeostasis. In addition to IL-10(-/-)MyD88(-/-) mice, IL-10(-/-)TLR4(-/-) mice exhibited reduced colitis compared to IL-10(-/-) mice, indicating that TLR4 signaling plays an important role in inducing colitis. Interestingly, the expression of IRAK-M, a negative regulator of TLR signaling, is dependent on intestinal commensal flora, as IRAK-M expression was reduced in mice re-derived into a germ-free environment, and introduction of commensal bacteria into germ-free mice induced IRAK-M expression. IL-10(-/-)IRAK-M(-/-) mice exhibited exacerbated colitis with increased inflammatory cytokine gene expression. Therefore, this study indicates that intestinal microflora stimulate the colitogenic immune system through TLR and negative regulation of TLR signaling is essential in maintaining intestinal homeostasis.
• The pattern recognition receptors Nod1 and Nod2 account for neutrophil recruitment to the lungs of mice infected with Legionella pneumophila

  Mariana S. Frutuoso, Juliana I. Hori, Marcelo S. F. Pereira, Djalma S. L. Junior, Fabiane Sonego, Koichi S. Kobayashi, Richard A. Flavell, Fernando Q. Cunha, Dario S. Zamboni

  MICROBES AND INFECTION 12 (11) 819 - 827 1286-4579 2010/10 [Refereed][Not invited]
   

  The intracellular bacterium Legionella pneumophila induces a severe form of pneumonia called Legionnaires diseases, which is characterized by a strong neutrophil (NE) infiltrate to the lungs of infected individuals. Although the participation of pattern recognition receptors, such as Toll-like receptors, was recently demonstrated, there is no information on the role of nod-like receptors (NLRs) for bacterial recognition in vivo and for NE recruitment to the lungs. Here, we employed a murine model of Legionnaires disease to evaluate host and bacterial factors involved in NE recruitment to the mice lungs. We found that L. pneumophila type four secretion system, known as Dot/Icm, was required for NE recruitment as dot/icm mutants fail to trigger NE recruitment in a process independent of bacterial multiplication. By using mice deficient for Nod1, Nod2, and Rip2, we found that these receptors accounted for NE recruitment to the lungs of infected mice. In addition, Rip2-dependent responses were important for cytokine production and bacterial clearance. Collectively, these studies show that Nod1, Nod2, and Rip2 account for generation of innate immune responses in vivo, which are important for NE recruitment and bacterial clearance in a murine model of Legionnaires diseases. (C) 2010 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
• IL-1 Receptor-associated Kinase M Downregulates DSS-induced Colitis

  Martin Berglund, Silvia Melgar, Koichi S. Kobayashi, Richard A. Flavell, Elisabeth Hultgren Hornquist, Olof H. Hultgren

  INFLAMMATORY BOWEL DISEASES 16 (10) 1778 - 1786 1078-0998 2010/10 [Refereed][Not invited]
   

  Background: Ulcerative colitis is associated with increased colon permeability resulting in bacterial translocation into the lamina propria. We investigate the importance of the Toll-like receptor (TLR) regulating protein IL-1 receptor-associated kinase M (IRAK-M) using the erosive dextran sulfate sodium (DSS)-induced model of colitis. Methods: IRAK-M-competent and -incompetent mice were treated with 3% DSS for 5 days followed by 2 days of regular drinking water. Clinical signs of disease were followed for 7 days. At day 7 the mice were sacrificed and plasma and tissue were collected for histopathological examination and analyses of the production of cytokines and chemokines as well as expression of T-cell transcription factors. Results: At day 7 IRAK-M-deficient mice display a reduced total body weight (77.1 +/- 2.1 versus 88.5 +/- 2.0, *P=0.002) and an increased macroscopical (2.7 +/- 0.2 versus 1.6 +/- 0.1, *P 0.002) and histopathological (6.0 +/- 0 versus 3.3 +/- 60.5, *P < 0.001) colon score compared to wildtype mice. Furthermore, IRAK-M-deficient mice have increased colon mRNA expression of proinflammatory cytokines and increased tumor necrosis factor concentrations (41.1 +/- 13.5 versus 12.8 +/- 2.0 pg/mL, *P = 0.010) in plasma. Conclusions: This is the first report examining the role of IRAK-M in colitis. We find that IRAK-M is of critical importance in downregulating induction and progression of DSS colitis, and thereby suggesting that IRAK-M might be a target for future interventional therapies. (Inflamm Bowel Dis 2010; 16: 1778-1786)
• NLR family member NLRC5 is a transcriptional regulator of MHC class I genes

  Torsten B. Meissner, Amy Li, Amlan Biswas, Kyoung-Hee Lee, Yuen-Joyce Liu, Erkan Bayir, Dimitrios Iliopoulos, Peter J. van den Elsen, Koichi S. Kobayashi

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 107 (31) 13794 - 13799 0027-8424 2010/08 [Refereed][Not invited]
   

  MHC class I plays a critical role in the immune defense against viruses and tumors by presenting antigens to CD8 T cells. An NLR protein, class II transactivator (CIITA), is a key regulator of MHC class II gene expression that associates and cooperates with transcription factors in the MHC class II promoter. Although CIITA also transactivates MHC class I gene promoters, loss of CIITA in humans and mice results in the severe reduction of only MHC class II expression, suggesting that additional mechanisms regulate the expression of MHC class I. Here, we identify another member of the NLR protein family, NLRC5, as a transcriptional regulator of MHC class I genes. Similar to CIITA, NLRC5 is an IFN-gamma-inducible nuclear protein, and the expression of NLRC5 resulted in enhanced MHC class I expression in lymphoid as well as epithelial cell lines. Using chromatin immunoprecipitation and reporter gene assays, we show that NLRC5 associates with and activates the promoters of MHC class I genes. Furthermore, we show that the IFN-gamma-induced up-regulation of MHC class I requires NLRC5, because knockdown of NLRC5 specifically impaired the expression of MHC class I. In addition to MHC class I genes, NLRC5 also induced the expression of beta 2-microglobulin, transporter associated with antigen processing, and large multifunctional protease, which are essential for MHC class I antigen presentation. Our results suggest that NLRC5 is a transcriptional regulator, orchestrating the concerted expression of critical components in the MHC class I pathway.
• Induction and rescue of Nod2-dependent Th1-driven granulomatous inflammation of the ileum

  Amlan Biswas, Yuen-Joyce Liu, Liming Hao, Atsushi Mizoguchi, Nita H. Salzman, Charles L. Bevins, Koichi S. Kobayashi

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 107 (33) 14739 - 14744 0027-8424 2010/08 [Refereed][Not invited]
   

  Mutations in the NOD2 gene are strong genetic risk factors for ileal Crohn's disease. However, the mechanism by which these mutations predispose to intestinal inflammation remains a subject of controversy. We report that Nod2-deficient mice inoculated with Helicobacter hepaticus, an opportunistic pathogenic bacterium, developed granulomatous inflammation of the ileum, characterized by an increased expression of Th1-related genes and inflammatory cytokines. The Peyer's patches and mesenteric lymph nodes were markedly enlarged with expansion of IFN-gamma-producing CD4 and CD8 T cells. Rip2-deficient mice exhibited a similar phenotype, suggesting that Nod2 function likely depends on the Rip2 kinase in this model. Transferring wild-type bone marrow cells into irradiated Nod2-deficient mice did not rescue the phenotype. However, restoring crypt antimicrobial function of Nod2-deficient mice by transgenic expression of alpha-defensin in Paneth cells rescued the Th1 inflammatory phenotype. Therefore, through the regulation of intestinal microbes, Nod2 function in nonhematopoietic cells of the small intestinal crypts is critical for protecting mice from a Th1-driven granulomatous inflammation in the ileum. The model may provide insight into Nod2 function relevant to inflammation of ileal Crohn's disease.
• MyD88-Dependent Signaling Contributes to Host Defense against Ehrlichial Infection

  Young-Sang Koh, Jung-Eun Koo, Amlan Biswas, Koichi S. Kobayashi

  PLOS ONE 5 (7) e11758  1932-6203 2010/07 [Refereed][Not invited]
   

  The ehrlichiae are small Gram-negative obligate intracellular bacteria in the family Anaplasmataceae. Ehrlichial infection in an accidental host may result in fatal diseases such as human monocytotropic ehrlichiosis, an emerging, tick-borne disease. Although the role of adaptive immune responses in the protection against ehrlichiosis has been well studied, the mechanism by which the innate immune system is activated is not fully understood. Using Ehrlichia muris as a model organism, we show here that MyD88-dependent signaling pathways play a pivotal role in the host defense against ehrlichial infection. Upon E. muris infection, MyD88-deficient mice had significantly impaired clearance of E. muris, as well as decreased inflammation, characterized by reduced splenomegaly and recruitment of macrophages and neutrophils. Furthermore, MyD88-deficient mice produced markedly lower levels of IL-12, which correlated well with an impaired Th1 immune response. In vitro, dendritic cells, but not macrophages, efficiently produced IL-12 upon E. muris infection through a MyD88-dependent mechanism. Therefore, MyD88-dependent signaling is required for controlling ehrlichial infection by playing an essential role in the immediate activation of the innate immune system and inflammatory cytokine production, as well as in the activation of the adaptive immune system at a later stage by providing for optimal Th1 immune responses.
• Cooperation between Multiple Microbial Pattern Recognition Systems Is Important for Host Protection against the Intracellular Pathogen Legionella pneumophila

  Kristina A. Archer, Florence Ader, Koichi S. Kobayashi, Richard A. Flavell, Craig R. Roy

  INFECTION AND IMMUNITY 78 (6) 2477 - 2487 0019-9567 2010/06 [Refereed][Not invited]
   

  Multiple pattern recognition systems have been shown to initiate innate immune responses to microbial pathogens. The degree to which these detection systems cooperate with each other to provide host protection is unknown. Here, we investigated the importance of several immune surveillance pathways in protecting mice against lethal infection by the intracellular pathogen Legionella pneumophila, the causative agent of a severe pneumonia called Legionnaires' disease. Rip2 and Naip5/NLRC4 signaling was found to contribute to the innate immune response generated against L. pneumophila in the lung. Elimination of Rip2 or Naip5/NLRC4 signaling in MyD88-deficient mice resulted in increased replication and dissemination of L. pneumophila and higher rates of mortality. Irradiated wild-type mice receiving bone marrow cells from pattern recognition receptor-deficient mice displayed L. pneumophila infection phenotypes similar to those of donor mice. Rip2 and Naip5/NLRC4 signaling provided additive effects in protecting MyD88-deficient mice from lethal infection by L. pneumophila, with the contribution of Naip5/NLRC4 being slightly greater than that of Rip2. Thus, activation of the Rip2, MyD88, and Naip5/NLRC4 signaling pathways triggers a coordinated and synergistic response that protects the host against lethal infection by L. pneumophila. These data provide new insight into how different pattern recognition systems interact functionally to generate innate immune responses that protect the host from lethal infection by activating cellular pathways that restrict intracellular replication of L. pneumophila and by recruiting to the site of infection additional phagocytes that eliminate extracellular bacteria.
• Identification of Drosophila Yin and PEPT2 as Evolutionarily Conserved Phagosome-associated Muramyl Dipeptide Transporters

  Guillaume M. Charriere, W. K. Eddie Ip, Stephanie Dejardin, Laurent Boyer, Anna Sokolovska, Michael P. Cappillino, Bobby J. Cherayil, Daniel K. Podolsky, Koichi S. Kobayashi, Neal Silverman, Adam Lacy-Hulbert, Lynda M. Stuart

  JOURNAL OF BIOLOGICAL CHEMISTRY 285 (26) 20147 - 20154 0021-9258 2010/06 [Refereed][Not invited]
   

  NOD2 (nucleotide-binding oligomerization domain containing 2) is an important cytosolic pattern recognition receptor that activates NF-kappa B and other immune effector pathways such as autophagy and antigen presentation. Despite its intracellular localization, NOD2 participates in sensing of extracellular microbes such as Staphylococcus aureus. NOD2 ligands similar to the minimal synthetic ligand muramyl dipeptide (MDP) are generated by internalization and processing of bacteria in hydrolytic phagolysosomes. However, how these derived ligands exit this organelle and access the cytosol to activate NOD2 is poorly understood. Here, we address how phagosome-derived NOD2 ligands access the cytosol in human phagocytes. Drawing on data from Drosophila phagosomes, we identify an evolutionarily conserved role of SLC15A transporters, Drosophila Yin and PEPT2, as MDP transporters in fly and human phagocytes, respectively. We show that PEPT2 is highly expressed by human myeloid cells. Ectopic expression of both Yin and PEPT2 increases the sensitivity of NOD2-dependent NF-kappa B activation. Additionally, we show that PEPT2 associates with phagosome membranes. Together, these data identify Drosophila Yin and PEPT2 as evolutionarily conserved phagosome-associated transporters that are likely to be of particular importance in delivery of bacteria-derived ligands generated in phagosomes to cytosolic sensors recruited to the vicinity of these organelles.
• A Role for IL-1 Receptor-Associated Kinase-M in Prostaglandin E-2-Induced Immunosuppression Post-Bone Marrow Transplantation

  Leah L. N. Hubbard, Megan N. Ballinger, Peedikayil E. Thomas, Carol A. Wilke, Theodore J. Standiford, Koichi S. Kobayashi, Richard A. Flavell, Bethany B. Moore

  JOURNAL OF IMMUNOLOGY 184 (11) 6299 - 6308 0022-1767 2010/06 [Refereed][Not invited]
   

  Following immune reconstitution, hematopoietic stem cell transplant patients often display reduced immune function and are especially susceptible to lung infections. In a mouse model of syngeneic bone marrow transplantation (BMT), we previously reported that PGE(2) is overproduced in lungs of BMT mice, significantly impairing host defense against Pseudomonas aeruginosa. This impairment in host defense post-BMT is also marked by diminished alveolar macrophage (AM) phagocytosis, bacterial killing, and production of TNF-alpha and cysteinyl leukotrienes. However, a mechanism by which overproduction of PGE(2) suppresses pulmonary host defense post-BMT is unknown. As IL-1R-associated kinase (IRAK)-M is a known inhibitor of MyD88-dependent IL-1R/TLR signaling and macrophage function, we sought to determine whether IRAK-M is involved in PGE(2)-induced immunosuppression post-BMT. We found that IRAK-M expression is elevated 3.5-fold in BMT AMs relative to control AMs, and this is related to AM overproduction of PGE(2). Furthermore, genetic ablation of IRAK-M in the bone marrow of BMT mice restores host defense against P. aeruginosa. Despite AM overproduction of PGE(2) and elevated E prostanoid 2 receptor expression, AM phagocytosis, killing, and production of cysteinyl leukotrienes and TNF-alpha are restored in the absence of IRAK-M post-BMT. Also, treatment with PGE(2) does not inhibit AM phagocytosis in the absence of IRAK-M. These data suggest that the absence of IRAK-M in the hematopoietic compartment post-BMT enhances pulmonary host defense and mitigates AM sensitivity to the inhibitory effects of PGE(2). Therefore, strategies to limit IRAK-M elevation post-BMT may be efficacious in reducing patient susceptibility to infection. The Journal of Immunology, 2010, 184: 6299-6308.
• Cutting Edge: Nucleotide-Binding Oligomerization Domain 1-Dependent Responses Account for Murine Resistance against Trypanosoma cruzi Infection

  Grace K. Silva, Fredy R. S. Gutierrez, Paulo M. M. Guedes, Catarina V. Horta, Larissa D. Cunha, Tiago W. P. Mineo, Juliana Santiago-Silva, Koichi S. Kobayashi, Richard A. Flavell, Joao S. Silva, Dario S. Zamboni

  JOURNAL OF IMMUNOLOGY 184 (3) 1148 - 1152 0022-1767 2010/02 [Refereed][Not invited]
   

  An effective innate immune recognition of the intracellular protozoan parasite Trypanosoma cruzi is critical for host resistance against Chagas disease, a severe and chronic illness that affects millions of people in Latin America. In this study, we evaluated the participation of nucleotide-binding oligomerization domain (Nod)like receptor proteins in host response to T cruzi infection and found that Nod1-dependent, but not Nod2-dependent, responses are required for host resistance against infection. Bone marrow-derived macrophages from Nod1(-/-) mice showed an impaired induction of NF-kappa B-dependent products in response to infection and failed to restrict T cruzi infection in presence of IFN-gamma. Despite normal cytokine production in the sera, Nod1(-/-) mice were highly susceptible to T cruzi infection, in a similar manner to MyD88(-/-) and NO synthase 2(-/-) mice. These studies indicate that Nod1-dependent responses account for host resistance against T cruzi infection by mechanisms independent of cytokine production. The Journal of Immunology, 2010, 184: 1148-1152.
• Nod2 is required for the regulation of commensal microbiota in the intestine

  Tanja Petnicki-Ocwieja, Tomas Hrncir, Yuen-Joyce Liu, Amlan Biswas, Tomas Hudcovic, Helena Tlaskalova-Hogenova, Koichi S. Kobayashi

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 106 (37) 15813 - 15818 0027-8424 2009/09 [Refereed][Not invited]
   

  Mutations in the Nod2 gene are among the strongest genetic risk factors in the pathogenesis of ileal Crohn's disease, but the exact contributions of Nod2 to intestinal mucosal homeostasis are not understood. Here we show that Nod2 plays an essential role in controlling commensal bacterial flora in the intestine. Analysis of intestinal bacteria from the terminal ilea of Nod2-deficient mice showed that they harbor an increased load of commensal resident bacteria. Furthermore, Nod2-deficient mice had a diminished ability to prevent intestinal colonization of pathogenic bacteria. In vitro, intestinal crypts isolated from terminal ilea of Nod2-deficient mice were unable to kill bacteria effectively, suggesting an important role of Nod2 signaling in crypt function. Interestingly, the expression of Nod2 is dependent on the presence of commensal bacteria, because mice re-derived into germ-free conditions expressed significantly less Nod2 in their terminal ilea, and complementation of commensal bacteria into germ-free mice induced Nod2 expression. Therefore, Nod2 and intestinal commensal bacterial flora maintain a balance by regulating each other through a feedback mechanism. Dysfunction of Nod2 results in a break-down of this homeostasis.
• Pharmacological postconditioning effect of muramyl dipeptide is mediated through RIP2 and TAK1

  Pierre Sicard, Sebastien Jacquet, Koichi S. Kobayashi, Richard A. Flavell, Michael S. Marber

  CARDIOVASCULAR RESEARCH 83 (2) 277 - 284 0008-6363 2009/07 [Refereed][Not invited]
   

  Despite their ability to cause septic shock and myocardial dysfunction, components of Gram-negative bacterial cell walls, like lipopolysaccharide, have been shown in numerous studies to induce myocardial protection during ischaemia-reperfusion injury. Muramyl dipeptide (MDP) is another such component recognized by an intracellular receptor, nucleotide-binding oligomerization domain 2. Receptor activation leads to intracellular signals through receptor interacting protein-2 (RIP2) and tumour growth factor-beta-activated kinase-1 (TAK1). However, little is known about the RIP2/TAK1 pathway in the heart. The aim of this study was to determine whether the RIP2/TAK1 pathway has a cardioprotective role in a mouse model of myocardial infarction. We isolated and subjected wild-type (WT) and RIP2(-/-) mouse hearts to 30 min of global ischaemia and 120 min of reperfusion with or without perfusion of MDP (10 mu g/mL) before or after the ischaemic period and determined the infarct size. We examined activation of the TAK1/nuclear factor kappa B (NF kappa B) signalling pathway. The effect of TAK1 inhibition on MDP-induced cardioprotection was also evaluated. Exposure to MDP during reperfusion significantly reduced infarct size in WT hearts (from 51.7 +/- 5.6% in control to 38.1 +/- 6.7%, P < 0.05), but not in RIP2(-/-) hearts or in WT hearts with coincident pharmacological inhibition of TAK1. MDP treatment significantly increased the levels of p-TAK1 and p-JNK (Jun N-terminal kinase) and led to NF kappa B activation via phosphorylation and degradation of IkappaB in the WT, but not in the RIP2(-/-), myocardium. These results indicate that MDP at reperfusion induced cardioprotection through an RIP2/TAK1-dependent mechanism.
• Requirement for AHNAK1-mediated calcium signaling during T lymphocyte cytolysis

  Didi Matza, Abdallah Badou, Mithilesh K. Jha, Tim Willinger, Andrey Antov, Shomyseh Sanjabi, Koichi S. Kobayashi, Vincent T. Marchesi, Richard A. Flavell

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 106 (24) 9785 - 9790 0027-8424 2009/06 [Refereed][Not invited]
   

  Cytolytic CD8(+) T cells (CTLs) kill virally infected cells, tumor cells, or other potentially autoreactive T cells in a calcium-dependent manner. To date, the molecular mechanism that leads to calcium intake during CTL differentiation and function has remained unresolved. We demonstrate that desmoyokin (AHNAK1) is expressed in mature CTLs, but not in naive CD8(+) T cells, and is critical for calcium entry required for their proper function during immune response. We show that mature AHNAK1-deficient CTLs exhibit reduced Ca(v)1.1 alpha 1 subunit expression (also referred to as L-type calcium channels or alpha 1S pore-forming subunits), which recently were suggested to play a role in calcium entry into CD4(+) T cells. AHNAK1-deficient CTLs show marked reduction in granzyme-B production, cytolytic activity, and IFN-gamma secretion after T cell receptor stimulation. Our results demonstrate an AHNAK1-dependent mechanism controlling calcium entry during CTL effector function.
• Activation of nucleotide oligomerization domain 2 exacerbates a murine model of proteoglycan-induced arthritis

  H. L. Rosenzweig, M. M. Jann, T. T. Glant, T. M. Martin, S. R. Planck, W. van Eden, P. J. S. van Kooten, R. A. Flavell, K. S. Kobayashi, J. T. Rosenbaum, M. P. Davey

  JOURNAL OF LEUKOCYTE BIOLOGY 85 (4) 711 - 718 0741-5400 2009/04 [Refereed][Not invited]
   

  In addition to its role in innate immunity, nucleotide oligomerization domain 2 (NOD2) has been shown to play a suppressive role in models of colitis. Notably, mutations in NOD2 cause the inherited granulomatous disease of the joints called Blau syndrome, thereby linking NOD2 with joint disease as well. However, the role of NOD2 in joint inflammation has not been clarified. We demonstrate here that NOD2 is functional within the mouse joint and promotes inflammation, as locally or systemically administered muramyl dipeptide (MDP; the NOD2 agonist) resulted in significant joint inflammation that was abolished in NOD2-deficient mice. We then sought to investigate the role of NOD2 in a mouse model of inflammatory arthritis dependent on adaptive immunity using TCR-transgenic mice whose T cells recognized the dominant epitope of proteoglycan (PG). Mice immunized with PG in the presence of MDP developed a more severe inflammatory arthritis and histopathology within the joints. Antigen-specific activation of splenocytes was enhanced by MDP with respect to IFN-gamma production, which would be consistent with the Th1-mediated disease in vivo. Intriguingly, NOD2 deficiency did not alter the PG-induced arthritis, indicating that NOD2 does not play an essential role in this model of joint disease when it is not activated by MDP. In conclusion, we demonstrate that in a model of inflammatory arthritis dependent on T and B cell priming, NOD2 activation potentiates disease. However, the absence of NOD2 does not alter the course of inflammatory arthritis, in contrast to models of intestinal inflammation. J. Leukoc. Biol. 85: 711-718; 2009.
• NOD2-Deficient Mice Have Impaired Resistance to Mycobacterium tuberculosis Infection through Defective Innate and Adaptive Immunity

  Maziar Divangahi, Serge Mostowy, Francois Coulombe, Robert Kozak, Loic Guillot, Frederic Veyrier, Koichi S. Kobayashi, Richard A. Flavell, Philippe Gros, Marcel A. Behr

  JOURNAL OF IMMUNOLOGY 181 (10) 7157 - 7165 0022-1767 2008/11 [Refereed][Not invited]
   

  NOD2/CARD15 mediates innate immune responses to mycobacterial infection. However, its role in the regulation of adaptive immunity has remained unknown. In this study, we examined host defense, T cell responses, and tissue pathology in two models of pulmonary mycobacterial infection, using wild-type and Nod2-deficient mice. During the early phase of aerosol infection with Mycobacterium tuberculosis, Nod2(-/-) mice had similar bacterial counts but reduced inflammatory response on histopathology at 4 and 8 wk postchallenge compared with wild-type animals. These findings were confirmed upon intratracheal infection of mice with attenuated Mycobacterium bovis bacillus Calmette-Guerin. Analysis of the lungs 4 wk after bacillus Calmette-Guerin infection demonstrated that Nod2(-/-) mice had decreased production of type 1 cytokines; and reduced recruitment of CD8(+) and CD4(+) T cells. Ag-specific T cell responses in both the spleens and thoracic lymph nodes were diminished in Nod2(-/-) mice, indicating impaired adaptive antimycobacterial immunity. The immune regulatory role of NOD2 was not restricted to the lung since Nod2 disruption also led to reduced type 1 T cell activation following i.m. bacillus Calmette-Guerin infection. To determine the importance of diminished innate and adaptive immunity, we measured bacterial burden 6 mo after aerosol infection with M. tuberculosis and followed a second infected group for assessment of survival. Nod2(-/-) mice had a higher bacterial burden in the lungs 6 mo after infection and succumbed sooner than did wild-type controls. Taken together, these data indicate that NOD2 mediates resistance to mycobacterial infection via both innate and adaptive immunity. The Journal of Immunology, 2008, 181: 7157-7165.
• Type IV Secretion-Dependent Activation of Host MAP Kinases Induces an Increased Proinflammatory Cytokine Response to Legionella pneumophila

  Sunny Shin, Christopher L. Case, Kristina A. Archer, Catarina V. Nogueira, Koichi S. Kobayashi, Richard A. Flavell, Craig R. Roy, Dario S. Zamboni

  PLOS PATHOGENS 4 (11) e1000220  1553-7366 2008/11 [Refereed][Not invited]
   

  The immune system must discriminate between pathogenic and nonpathogenic microbes in order to initiate an appropriate response. Toll-like receptors (TLRs) detect microbial components common to both pathogenic and nonpathogenic bacteria, whereas Nod-like receptors (NLRs) sense microbial components introduced into the host cytosol by the specialized secretion systems or pore-forming toxins of bacterial pathogens. The host signaling pathways that respond to bacterial secretion systems remain poorly understood. Infection with the pathogen Legionella pneumophila, which utilizes a type IV secretion system (T4SS), induced an increased proinflammatory cytokine response compared to avirulent bacteria in which the T4SS was inactivated. This enhanced response involved NF-kappa B activation by TLR signaling as well as Nod1 and Nod2 detection of type IV secretion. Furthermore, a TLR- and RIP2-independent pathway leading to p38 and SAPK/JNK MAPK activation was found to play an equally important role in the host response to virulent L. pneumophila. Activation of this MAPK pathway was T4SS-dependent and coordinated with TLR signaling to mount a robust proinflammatory cytokine response to virulent L. pneumophila. These findings define a previously uncharacterized host response to bacterial type IV secretion that activates MAPK signaling and demonstrate that coincident detection of multiple bacterial components enables immune discrimination between virulent and avirulent bacteria.
• Role of the protein kinase TBK1 in TNF alpha-induced apoptosis

  Mireille Delhase, Takashi Yagi, Koichi S. Kobayashi, Makoto Nakanishi

  CYTOKINE 43 (3) 326 - 326 1043-4666 2008/09 [Not refereed][Not invited]
  
• The role of RIP2 in p38 MAPK activation in the stressed heart

  Sebastien Jacquet, Yasuhiro Nishino, Sarawut Kumphune, Pierre Sicard, James E. Clark, Koichi S. Kobayashi, Richard A. Flavell, Jan Eickhoff, Matt Cotten, Michael S. Marber

  JOURNAL OF BIOLOGICAL CHEMISTRY 283 (18) 11964 - 11971 0021-9258 2008/05 [Refereed][Not invited]
   

  The activation of p38 MAPK by dual phosphorylation aggravates myocardial ischemic injury and depresses cardiac contractile function. SB203580, an ATP-competitive inhibitor of p38 MAPK and other kinases, prevents this dual phosphorylation during ischemia. Studies in non-cardiac tissue have shown receptor-interacting protein 2 (RIP2) lies upstream of p38 MAPK, is SB203580-sensitive and ischemia-responsive, and aggravates ischemic injury. We therefore examined the RIP2-p38 MAPK signaling axis in the heart. Adenovirus-driven expression of wild-type RIP2 in adult rat ventricular myocytes caused robust, SB203580-sensitive dual phosphorylation of p38 MAPK associated with activation of p38 MAPK kinases MKK3, MKK4, and MKK6. The effect of SB203580 was recapitulated by unrelated inhibitors of RIP2 or the downstream MAPK kinase kinase, TAK1. However, overexpression of wild-type, kinase-dead, caspase recruitment domain-deleted, or kinase-dead and caspase recruitment domain-deleted forms of RIP2 had no effect on the activating dual phosphorylation of p38 MAPK during simulated ischemia. Similarly, p38 MAPK activation and myocardial infarction size in response to true ischemia did not differ between hearts from wild-type and RIP2 null mice. However, both p38 MAPK activation and the contractile depression caused by the endotoxin component muramyl dipeptide were attenuated by SB203580 and in RIP2 null hearts. Although RIP2 can cause myocardial p38 MAPK dual phosphorylation in the heart under some circumstances, it is not responsible for the SB203580-sensitive pattern of activation during ischemia.
• NLR proteins: integral members of innate immunity and mediators of inflammatory diseases

  Jeanette M. Wilmanski, Tanja Petnicki-Ocwieja, Koichi S. Kobayashi

  JOURNAL OF LEUKOCYTE BIOLOGY 83 (1) 13 - 30 0741-5400 2008/01 [Refereed][Not invited]
   

  The innate immune system is the first line of defense against microorganisms and is conserved in plants and animals. The nucleotide-binding domain, leucine rich containing (NLR) protein family is a recent addition to the members of innate immunity effector molecules. These proteins are characterized by a central oligomerization domain, termed nucleotide-binding domain (NBD) and a protein interaction domain, leucine-rich repeats (LRRs) at the C terminus. It has been shown that NLR proteins are localized to the cytoplasm and recognize microbial products. To date, it is known that Nod1 and Nod2 detect bacterial cell wall components, whereas Ipaf and Naip detect bacterial flagellin, and NACHT/LRR/Pyrin 1 has been shown to detect anthrax lethal toxin. NLR proteins comprise a diverse protein family ( over 20 in humans), indicating that NLRs have evolved to acquire specificity to various pathogenic microorganisms, thereby controlling host-pathogen interactions. Activation of NLR proteins results in inflammatory responses mediated by NF-kappa B, MAPK, or Caspase-1 activation, accompanied by subsequent secretion of proinflammatory cytokines. Mutations in several members of the NLR protein family have been linked to inflammatory diseases, suggesting these molecules play important roles in maintaining host-pathogen interactions and inflammatory responses. Therefore, understanding NLR signaling is important for the therapeutic intervention of various infectious and inflammatory diseases.
• A scaffold protein, AHNAK1, is required for calcium signaling during T cell activation

  Didi Matza, Abdallah Badou, Koichi S. Kobayashi, Karen Goldsmith-Pestana, Yutaka Masuda, Akihiko Komuro, Diane McMahon-Pratt, Vincent T. Marchesi, Richard A. Flavell

  IMMUNITY 28 (1) 64 - 74 1074-7613 2008/01 [Refereed][Not invited]
   

  Engagement of the T cell antigen receptor (TCR) during antigen presentation initiates a coordinated action of a large number of signaling proteins and ion channels. AHNAK1 is a scaffold protein, highly expressed by CD4(+) T cells, and is a critical component for calcium signaling. We showed that AHNAK1-deficient mice were highly susceptible to Leishmania major infection. AHNAK1-deficient CD4(+) T cells responded poorly to TCR stimulation in vitro with low proliferation and low Interleukin-2 production. Furthermore, AHNAK1 deficiency resulted in a reduced calcium influx upon TCR crosslinking and subsequent poor activation of the transcription factor NFAT. AHNAK1 was required for plasma membrane expression of L-type calcium channels alpha 1S (Ca(v)1.1), probably through its interaction with the beta regulatory subunit. Thus, AHNAK1 plays an essential role in T cell Ca2+ signaling through Ca(v)1 channels, triggered via TCR activation; therefore, AHNAK1 is a potential target for therapeutic intervention.
• MDP-induced interleukin-1 beta processing requires Nod2 and CIAS1/NALP3

  Qilin Pan, John Mathison, Colleen Fearns, Vladimir V. Kravchenko, Jean Da Silva Correia, Hal M. Hoffman, Koichi S. Kobayashi, John Bertin, Ethan P. Grant, Anthony J. Coyle, Fayyaz S. Sutterwala, Yasunori Ogura, Richard A. Flavell, Richard J. Ulevitch

  JOURNAL OF LEUKOCYTE BIOLOGY 82 (1) 177 - 183 0741-5400 2007/07 [Refereed][Not invited]
   

  Nucleotide-binding oligomerization domain (Nod)2 is a sensor of muramyl dipeptides (AIDP) derived from bacterial peptidoglycan. Nod2 also plays a role in some autoinflammatory diseases. Cold-induced autoinflammatory syndrome I (CIAS1)/NACHT domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NALP3) has been suggested to be sufficient for MDP-dependent release of mature IL-1 beta, but the role of Nod2 in this process is unclear. Using mice bearing selective gene deletions, we provide in vitro and in vivo data showing that AIDP-induced IL-1 beta release requires Nod2 and CIASI/NALP3 as well as receptor-interacting protein-2 (Rip2), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1. In contrast, AIDP-dependent IL-6 production only requires Nod2 and Rip2. Together, our data provide a new understanding of this important pathway of IL-I beta production and allow for further studies of the role of these proteins within the broader context of inflammatory disease.
• Osteopontin expression is essential for interferon-alpha production by plasmacytoid dendritic cells

  ML Shinohara, LR Lu, J Bu, MBF Werneck, KS Kobayashi, LH Glimcher, H Cantor

  NATURE IMMUNOLOGY 7 (5) 498 - 506 1529-2908 2006/05 [Refereed][Not invited]
   

  The observation that the T-bet transcription factor allows tissue-specific upregulation of intracellular osteopontin (Opn-i) in plasmacytoid dendritic cells (pDCs) suggests that Opn might contribute to the expression of interferon-alpha (IFN-alpha) in those cells. Here we show that Opn deficiency substantially reduced Toll-like receptor 9 (TLR9)-dependent IFN-alpha responses but spared expression of transcription factor NF-kappa B-dependent proinflammatory cytokines. Shortly after TLR9 engagement, colocalization of Opn-i and the adaptor molecule MyD88 was associated with induction of transcription factor IRF7-dependent IFN-alpha gene expression, whereas deficient expression of Opn-i was associated with defective nuclear translocation of IRF7 in pDCs. The importance of the Opn-IFN-alpha pathway was emphasized by its essential involvement in cross-presentation in vitro and in anti herpes simplex virus 1 IFN-alpha response in vivo. The finding that Opn-i selectively coupled TLR9 signaling to expression of IFN-alpha but not to that of other proinflammatory cytokines provides new molecular insight into the biology of pDCs.
• The Birc1e cytosolic pattern-recognition receptor contributes to the detection and control of Legionella pneumophila infection

  DS Zamboni, KS Kobayashi, T Kohlsdorf, Y Ogura, EM Long, RE Vance, K Kuida, S Mariathasan, VM Dixit, RA Flavell, WF Dietrich, CR Roy

  NATURE IMMUNOLOGY 7 (3) 318 - 325 1529-2908 2006/03 [Refereed][Not invited]
   

  Baculovirus inhibitor of apoptosis repeat-containing 1 (Birc1) proteins have homology to several germline-encoded receptors of the innate immune system. However, their function in immune surveillance is not clear. Here we describe a Birc1e-dependent signaling pathway that restricted replication of the intracellular pathogen Legionella pneumophila in mouse macrophages. Translocation of bacterial products into host-cell cytosol was essential for Birc1e-mediated control of bacterial replication. Caspase-1 was required for Birc1e-dependent antibacterial responses ex vivo in macrophages and in a mouse model of Legionnaires' disease. The interleukin 1 beta converting enzyme-protease-activating factor was necessary for L. pneumophila growth restriction, but interleukin 1 beta was not required. These results establish Birc1e as a nucleotide-binding oligomerization-leucine-rich repeat protein involved in the detection and control of intracellular L. pneumophila.
• IL-1-receptor-associated kinase M is a central regulator of osteoclast differentiation and activation

  HM Li, E Cuartas, WG Cui, YW Choi, TD Crawford, HZ Ke, KS Kobayashi, RA Flavell, A Vignery

  JOURNAL OF EXPERIMENTAL MEDICINE 201 (7) 1169 - 1177 0022-1007 2005/04 [Refereed][Not invited]
   

  Osteoporosis is a serious problem worldwide; it is characterized by bone fractures in response to relatively mild trauma. Osteoclasts originate from the fusion of macrophages and they play a central role in bone development and remodeling via the resorption of bone. Therefore, osteoclasts are important mediators of bone loss that leads, for example, to osteoporosis. Interleukin (IL)-1 receptor (IL-1R)-associated kinase M (IRAK-M) is only expressed in cells of the myeloid lineage and it inhibits signaling downstream of IL-1R and Toll-like receptors (TLRs). However, it lacks a functional catalytic site and, thus, cannot function as a kinase. IRAK-M associates with, and prevents the dissociation of, IRAK-IRAK-4-TNF receptor-associated factor 6 from the TLR signaling complex, with resultant disruption of downstream signaling. Thus, IRAK-M acts as a dominant negative IRAK. We show here that mice that lack IRAK-M develop severe osteoporosis, which is associated with the accelerated differentiation of osteoclasts, an increase in the half-life of osteoclasts, and their activation. Ligation of IL-1R or TLRs results in hyperactivation of NF-kappa B and mitogen-activated protein kinase signaling pathways, which are essential for osteoclast differentiation. Thus, IRAK-M is a key regulator of the bone loss that is due to osteoclastic resorption of bone.
• Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract

  KS Kobayashi, M Chamaillard, Y Ogura, O Henegariu, N Inohara, G Nunez, RA Flavell

  SCIENCE 307 (5710) 731 - 734 0036-8075 2005/02 [Refereed][Not invited]
   

  The gene encoding the Nod2 protein is frequently mutated in Crohn's disease (CD) patients, although the physiological function of Nod2 in the intestine remains elusive. Here we show that protective immunity mediated by Nod2 recognition of bacterial muramyl dipeptide is abolished in Nod2-deficient mice. These animals are susceptible to bacterial infection via the oral route but not through intravenous or peritoneal delivery. Nod2 is required for the expression of a subgroup of intestinal anti-microbial peptides, known as cryptdins. The Nod2 protein is thus a critical regulator of bacterial immunity within the intestine, providing a possible mechanism for Nod2 mutations in CD.
• Shielding the double-edged sword: negative regulation of the innate immune system

  KS Kobayashi, RA Flavell

  JOURNAL OF LEUKOCYTE BIOLOGY 75 (3) 428 - 433 0741-5400 2004/03 [Refereed][Not invited]
   

  The innate immune system is evolutionarily conserved among all multicellular organisms and is the first line of defense against microorganisms. It enables the host not only to combat pathogenic organisms but also to cohabit with nonpathogenic microorganisms by balancing the host-microorganism interaction. The innate immune response is activated rapidly (within hours) compared with adaptive immunity. Activation of the innate immune system allows the activation of the adaptive immune response by production of proinflammatory cytokines and by providing stimulatory signals via major histocompatibility complex molecules and costimulatory molecules such as CD40, CD80, or CD86; together, these lead to the full activation of both immune systems to fight against pathogenic microorganisms. Activation of the innate immune system, however, can be a double-edged sword for the host. Proinflammatory cytokines mediate a positive feedback loop on the innate immune system, and overproduction of cytokines, if unchecked, is hazardous to the host and may cause severe outcomes such as hyperthermia, organ failure, and even death in extreme cases. Moreover, if the overproduction of proinflammatory cytokines persists, it may cause chronic inflammatory diseases. During evolution, the innate immune system has acquired complicated regulatory systems to control itself so that this "sword" will not kill the host. Various mechanisms including inhibition of Toll-like receptor signaling by interleukin-1 receptor-associated kinase-M have evolved for this purpose and are important not only to fight against pathogenic microorganisms efficiently but also are critical for the peaceful coexistence with commensal bacterial flora.
• Impaired membrane resealing and autoimmune myositis in synaptotagmin VII-deficient mice

  S Chakrabarti, KS Kobayashi, RA Flavell, CB Marks, K Miyake, DR Liston, KT Fowler, FS Gorelick, NW Andrews

  JOURNAL OF CELL BIOLOGY 162 (4) 543 - 549 0021-9525 2003/08 [Refereed][Not invited]
   

  Members of the synaptotagmin family have been proposed to function as Ca2+ sensors in membrane fusion. Syt VII is a ubiquitously expressed synaptotagmin previously implicated in plasma membrane repair and Trypanosoma cruzi invasion, events which are mediated by the Ca2+-regulated exocytosis of lysosomes. Here, we show that embryonic fibroblasts from Syt VII-deficient mice are less susceptible to trypanosome invasion, and defective in lysosomal exocytosis and resealing after wounding. Examination of mutant mouse tissues revealed extensive fibrosis in the skin and skeletal muscle. inflammatory myopathy, with muscle fiber invasion by leukocytes and endomysial collagen deposition, was associated with elevated creatine kinase release and progressive muscle weakness. Interestingly, similar to what is observed in human polymyositis/dermatomyositis, the mice developed a strong antinuclear antibody response, characteristic of autoimmune disorders. Thus, defective plasma membrane repair in tissues under mechanical stress may favor the development of inflammatory autoimmune disease.
• Intracellular debugging

  KS Kobayashi, EE Eynon, RA Flavell

  NATURE IMMUNOLOGY 4 (7) 652 - 654 1529-2908 2003/07 [Refereed][Not invited]
   

  A family of intracellular molecules called NODs has been proposed to sense bacteria within the cell. New data now show that NOD1 senses a distinct bacterial component and may thus be important in responding to bacteria with this molecular signature.

MISC

• NLRC5のがんと免疫療法における役割

  應田涼太, 吉濱小百合, 春日優介, 坐間のゆり, 小林弘一  77-  (1)  114  -120  2022/01
• 細菌パターン認識受容体NOD2の腸管免疫における役割

  應田涼太, 尾野純也, 吉濱小百合, 小林弘一  臨床免疫・アレルギー科  70-  (5)  497  -502  2018
• クローン病の病態理解に役立つNOD2の基礎

  吉濱小百合, 小林弘一  11-  (1)  13  -18  2017/11
• NLRC5/CITA : the role in cancer immune evasion

  吉濱小百合, 小林弘一  臨床免疫・アレルギー科  66-  (6)  400  -406  2016/10

Books etc

• 体の中の”小さな生態系”腸内フローラによる免疫ホメオスタシスNOD2による消化管恒常性維持機構

  小林弘一 (Single work)
  2011/11
• 自然免疫TLRのネガティブフィードバック調節機構

  小林弘一, 佐藤あやの 
  2003/12

Association Memberships

• Texas A&M Immunology Consortirum   American Society for Biochemistry and Molecular Biology   American Association of Advanced Science   American Association of Immunologists   北海道医学会   日本ウイルス学会   日本免疫学会   

Research Projects

• エピゲノム編集を用いた新規肺癌免疫療法の開発

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2023/06 -2025/03 

  Author : 小林 弘一, 田中 努
• Development of novel anti-viral therapeuticing an advanced screening technologys us

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2022/10 -2025/03 

  Author : 小林 弘一, 田村 友和, 福原 崇介, 應田 涼太
• The role of MHC class I transactivator NLRC5 in COVID-19

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2022/04 -2025/03 

  Author : 小林 弘一, 澤 洋文, 福原 崇介, 應田 涼太
• プロテオーム解析とCROP-seq技術を用いた新規MHC-I遺伝子誘導因子の同定

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2020/07 -2022/03 

  Author : 小林 弘一, 畠山 鎮次, 應田 涼太
• Novel biomarkers for the immune checkpoint inhibitors for cancer

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2018/08 -2020/03 

  Author : Kobayashi Koichi

   

  We recently found that NLRC5/MHC class I transactivator (CITA) is a mastery transcriptional coactivator of MHC class I genes as well as genes involved in the MHC-I dependent antigen presentation pathway. Moreover, we discovered that NLRC5-mediated MHC class I transactivation pathway constitutes a major mechanism for immune evasion in multiple cancers and NLRC5 expression was significantly associated with activation of CD8 cytotoxic T cells and patient survival in melanoma and other cancers. In this proposal, we analyzed the effectiveness of NLRC5 as a prognostic and predictive biomarker in melanoma. We have analyzed the melanoma patient cohort who received anti-CTLA4 therapy or anti-PD1 therapy. Our data showed that the expression level is associated with the response to checkpoint blocking therapies. This study suggests that NLRC5 expression would be an useful biomarker to determine the appropriate therapy for the cancer patients.

Industrial Property Rights

• 特願2003/0157539:A novel target to regulate bone mass  

  Kobayashi K, Vignery A, Flavell RA
• 特願WO/2003/060153:IRAK-M is a negative regulators of Toll-like receptor signaling  

  Kobayashi K, Medzhitov R, Flavell RA
• 特願WO/2003/059285:RIP2: A mediator of signaling in the innate and adaptive immune systems  

  Kobayashi K, Medzhitov R, Flavell RA