Agnieszka D. Truax

Inflammasome-independent role of AIM2 in suppressing colon tumorigenesis via DNA-PK and Akt.

The inflammasome activates caspase-1 and the release of interleukin-1β (IL-1β) and IL-18, and several inflammasomes protect against intestinal inflammation and colitis-associated colon cancer (CAC) in animal models. The absent in melanoma 2 (AIM2) inflammasome is activated by double-stranded DNA, and AIM2 expression is reduced in several types of cancer, but the mechanism by which AIM2 restricts tumor growth remains unclear. We found that Aim2-deficient mice had greater tumor load than Asc-deficient mice in the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colorectal cancer. Tumor burden was also higher in Aim2−/−/ApcMin/+ than in APCMin/+ mice. The effects of AIM2 on CAC were independent of inflammasome activation and IL-1β and were primarily mediated by a non–bone marrow source of AIM2. In resting cells, AIM2 physically interacted with and limited activation of DNA-dependent protein kinase (DNA-PK), a PI3K-related family member that promotes Akt phosphorylation, whereas loss of AIM2 promoted DNA-PK–mediated Akt activation. AIM2 reduced Akt activation and tumor burden in colorectal cancer models, while an Akt inhibitor reduced tumor load in Aim2−/− mice. These findings suggest that Akt inhibitors could be used to treat AIM2-deficient human cancers.

Regulation of Class I Major Histocompatibility Complex (MHC) by Nucleotide-binding Domain, Leucine-rich Repeat-containing (NLR) Proteins

Background: Multiple functions have been ascribed to NLRC5 including MHC-I transcription and cytokine responses. Results: We generated Nlrc5−/− mice and showed that Nlrc5 increases classical and nonclassical MHC-I and causes removal of the gene-silencing H3K27me3 histone modification on MHC-I promoter. Conclusion: Nlrc5 regulates MHC-I expression. Significance: NLRC5, with CIITA, constitutes an NLR subfamily that regulates MHC. Most of the nucleotide-binding domain, leucine-rich repeat (NLR) proteins regulate responses to microbial and damage-associated products. Class II transactivator (CIITA) has a distinct function as the master regulator of class II major histocompatibility complex (MHC-II) transcription. Recently, human NLRC5 was found to regulate MHC-I in cell lines; however, a host of conflicting positive and negative functions has been attributed to this protein. To address the function of NLRC5 in a physiologic setting, we generated an Nlrc5−/− strain that contains a deletion in the exon that encodes the nucleotide-binding domain. We have not detected a role for this protein in cytokine induction by pathogen-associated molecular patterns and viruses. However, Nlrc5−/− cells showed a dramatic decrease of classical (H-2K) and nonclassical (Tla) MHC-I expression by T/B lymphocytes, natural killer (NK) cells, and myeloid-monocytic lineages. As a comparison, CIITA did not affect mouse MHC-I expression. Nlrc5−/− splenocytes and bone marrow-derived macrophages were able to up-regulate MHC-I in response to IFN-γ; however, the absolute levels of MHC-I expression were significantly lower than WT controls. Chromatin immunoprecipitation of IFN-γ-treated cells indicates that Nlrc5 reduced the silencing H3K27me3 histone modification, but did not affect the activating AcH3 modification on a MHC-I promoter. In summary, we conclude that Nlrc5 is important in the regulation of MHC-I expression by reducing H3K27me3 on MHC-I promoter and joins CIITA as an NLR subfamily that controls MHC gene transcription.

Regulation of Acetylation at the Major Histocompatibility Complex Class II Proximal Promoter by the 19S Proteasomal ATPase Sug1

ABSTRACT Recent studies have made evident the fact that the 19S regulatory component of the proteasome has functions that extend beyond degradation, particularly in the regulation of transcription. Although 19S ATPases facilitate chromatin remodeling and acetylation events in yeast (Saccharomyces cerevisiae), it is unclear if they play similar roles in mammalian cells. We have recently shown that the 19S ATPase Sug1 positively regulates the transcription of the critical inflammatory gene for major histocompatibility complex class II (MHC-II) by stabilizing enhanceosome assembly at the proximal promoter. We now show that Sug1 is crucial for regulating histone H3 acetylation at the MHC-II proximal promoter. Sug1 binds to acetylated histone H3 and, in the absence of Sug1, histone H3 acetylation is dramatically decreased at the proximal promoter, with a preferential loss of acetylation at H3 lysine 18. Sug1 also binds to the MHC-II histone acetyltransferase CREB-binding protein (CBP) and is critical for the recruitment of CBP to the MHC-II proximal promoter. Our current study strongly implicates the 19S ATPase Sug1 in modifying histones to initiate MHC-II transcription and provides novel insights into the role of the proteasome in the regulation of mammalian transcription.

NLRP12 attenuates colon inflammation by maintaining colonic microbial diversity and promoting protective commensal bacterial growth

Inflammatory bowel diseases involve the dynamic interaction of host genetics, the microbiome and inflammatory responses. Here we found lower expression of NLRP12 (which encodes a negative regulator of innate immunity) in human ulcerative colitis, by comparing monozygotic twins and other patient cohorts. In parallel, Nlrp12 deficiency in mice caused increased basal colonic inflammation, which led to a less-diverse microbiome and loss of protective gut commensal strains (of the family Lachnospiraceae) and a greater abundance of colitogenic strains (of the family Erysipelotrichaceae). Dysbiosis and susceptibility to colitis associated with Nlrp12 deficency were reversed equally by treatment with antibodies targeting inflammatory cytokines and by the administration of beneficial commensal Lachnospiraceae isolates. Fecal transplants from mice reared in specific-pathogen-free conditions into germ-free Nlrp12-deficient mice showed that NLRP12 and the microbiome each contributed to immunological signaling that culminated in colon inflammation. These findings reveal a feed-forward loop in which NLRP12 promotes specific commensals that can reverse gut inflammation, while cytokine blockade during NLRP12 deficiency can reverse dysbiosis.

Expression profile of innate immune receptors, NLRs and AIM2, in human colorectal cancer: correlation with cancer stages and inflammasome components

NLRs (nucleotide-binding domain leucine-rich repeat proteins or NOD-like receptors) are regulators of inflammation and immunity. A subgroup of NLRs and the innate immune receptor, AIM2 (absent-in-melanoma 2), can induce the assembly of a large caspase-1 activating complex called the inflammasome. Other NLRs regulate key signaling pathways such as NF-kB and MAPK. Since inflammation is a central component of colorectal cancer (CRC), this work was undertaken to analyze NLR and AIM2 expression in human CRC by combining bioinformatics analysis and experimental verification using clinical tissue samples. Additional experiments analyzed the association of (i) gene expression and cancer staging, and (ii) gene expression among inflammasome components. Ten public CRC datasets from the Oncomine® Platform were analyzed. Genes analyzed include NLRP1, NLRP3, NLRP6, NLRP12, NLRC3, NLRC4, NLRC5, NOD1, NOD2 and AIM2. Additionally, forty case-matched cancer samples and adjacent healthy control tissues isolated from a cohort of Chinese CRC patients were profiled. Three patterns of gene expression in CRC are shown. The expression of NLRC3, a checkpoint of inflammation, and the inflammasome components NLRP1, NLRP3, NLRC4 and AIM2 were reduced in CRC. NOD1 and NOD2 expression was increased in CRC, while NLRC5, NLRP6 and NLRP12 showed little difference compared to controls. Reduced expression of NLRC3 in CRC was verified in all available databases analyzed and confirmed with our patient cohort. Furthermore, the extent of NLRC3 and AIM2 gene reduction was correlated with cancer progression. This report reveals the potential value of NLR and AIM2 genes as biomarkers of CRC and cancer progression.

Early epigenetic events regulate the adaptive immune response gene CIITA

Precise regulation of Major Histocompatibility class II (MHC II) genes plays important roles in initiation, propagation and termination of adaptive immune responses by controlling antigen presentation to CD4+ T cells. MHC II genes are constitutively expressed in only a few cell types and are inducibly expressed by the inflammatory response cytokine interferon gamma (IFNγ) in all nucleated cells. The regulation of MHC II is tightly controlled by a Master Regulator, the class II transactivator (CIITA), which is a general regulator of both constitutive and inducible MHC II expression. Although much is known about the transcription factors necessary for CIITA expression, less is known about the epigenetic modifications and the requisite enzymes needed to provide these transcription factors access to DNA. We show here that multiple epigenetic changes occur at the IFNγ inducible CIITA promoter within 20’ of IFNγ stimulation and that these changes correlate with the opening of the promoter and the initiation of transcription. Our study links these rapidly occurring epigenetic events at the inducible CIITA promoter to decreased promoter binding of the histone methyltransferase EZH2, and shows that decreased promoter binding of EZH2 transforms this previously tightly regulated and cytokine inducible promoter into a constitutively active and dysregulated gene.

Phosphorylation and Ubiquitination of Degron Proximal Residues Are Essential for Class II Transactivator (CIITA) Transactivation and Major Histocompatibility Class II Expression

Major histocompatibility (MHC) class II molecules are cell surface glycoproteins that present extracellular antigens to CD4+ T cells and are essential for initiation of the adaptive immune response. MHC class II expression requires recruitment of a master regulator, the class II transactivator (CIITA), to the MHC class II promoter. Post-translational modifications to CIITA play important roles in modulating CIITA mediated transcription of various genes in different cell types. We have previously linked regulation of CIITA to the Ubiquitin Proteasome System (UPS), and we and others have demonstrated that mono-ubiquitination of CIITA dramatically increases its transactivity whereas poly-ubiquitination leads to CIITA degradation. Here we identify three degron proximal lysine residues, Lys-315, Lys-330, and Lys-333, and a phosphorylation site, Ser-280, located within the CIITA degron, that regulate CIITA ubiquitination, stability, and MHC class II expression. Together, these findings contribute to the developing post-translational modification code for CIITA.

The Innate Immune Receptor NLRX1 Functions as a Tumor Suppressor by Reducing Colon Tumorigenesis and Key Tumor-Promoting Signals.

SUMMARY NOD-like receptor (NLR) proteins are intracellular innate immune sensors/receptors that regulate immunity. This work shows that NLRX1 serves as a tumor suppressor in colitis-associated cancer (CAC) and sporadic colon cancer by keeping key tumor promoting pathways in check. Nlrx1−/− mice were highly susceptible to CAC, showing increases in key cancer-promoting pathways including nuclear factor κB (NF-κB), mitogen-activated protein kinase (MAPK), signal transducer and activator of transcription 3 (STAT3), and interleukin 6 (IL-6). The tumor-suppressive function of NLRX1 originated primarily from the non-hematopoietic compartment. This prompted an analysis of NLRX1 function in the Apcmin/+ genetic model of sporadic gastrointestinal cancer. NLRX1 attenuated Apcmin/+ colon tumorigenesis, cellular proliferation, NF-κB, MAPK, STAT3 activation, and IL-6 levels. Application of anti-interleukin 6 receptor (IL6R) antibody therapy reduced tumor burden, increased survival, and reduced STAT3 activation in Nlrx1−/−Apcmin/+ mice. As an important clinical correlate, human colon cancer samples expressed lower levels of NLRX1 than healthy controls in multiple patient cohorts. These data implicate anti-IL6R as a potential personalized therapy for colon cancers with reduced NLRX1.

MicroRNA-141 regulates the expression level of ICAM-1 on endothelium to decrease myocardial ischemia-reperfusion injury

A growing number of studies have suggested microRNAs (miRNAs) are involved in the modulation of myocardial ischemia-reperfusion (MI/R) injury; however, the role of endogenous miRNAs targeting endothelial cells (ECs) and its interaction with ICAM-1 in the setting of MI/R remain poorly understood. Our microarray results showed that miR-146a, miR-146b-5p, miR-155*, miR-155, miR-497, and miR-451 were significantly upregulated, whereas, miR-141 and miR-564 were significantly downregulated in the ECs challenged with TNF-α for 6 h. Real-time PCR analyses additionally validated that the expression levels of miR-146a, miR-155*, and miR-141 were consistent with the microarray results. Then, ICAM-1 was identified as a novel target of miR-141 by Target Scan software and the reporter gene system. Further functional experiments showed that elevated levels of miR-141 inhibited ICAM-1 expression and diminished leukocytes adhesion to ECs in vitro. In an in vivo murine model of MI/R injury, pretreatment with miR-141 mimics through the tail vein downregulated the expression level of ICAM-1 in heart and attenuated MI/R injury as evidenced by decreased infarct size and decline of serum cardial troponin I (cTnI) and lactate dehydrogenase (LDH) concentration. The cardioprotective effects of miR-141 mimics may be attributed to the decreased infiltration of CD11b(+) cells and F4/80(+) macrophages into ischemic myocardium tissue. In conclusion, our results demonstrate that miR-141, as a novel repressor of ICAM-1, is involved in the attenuation of MI/R injury via antithetical regulation of ICAM-1 and inflammatory cells infiltration. Thus miR-141 may constitute a new therapeutic target in the setting of ischemic heart disease.

Association of the 19S proteasomal ATPases with the ATPase-binding domain of CIITA is essential for CIITA stability and MHC class II expression.

Major histocompatibility class II (MHC class II) molecules are glycoproteins that present extracellular antigens to CD4+ T cells and are essential for initiation of adaptive immune responses. MHC class II expression requires recruitment of a master regulator, the class II transactivator (CIITA), to the MHC class II promoter. Others and we have earlier linked CIITA to the ubiquitin‐proteasome system by showing that mono‐ubiquitination of CIITA increases its transactivity, whereas poly‐ubiquitination of CIITA leads to its degradation. We have further shown that the 26S proteasome also has non‐proteolytic functions in MHC class II transcription, as 19S ATPase subunits of the 26S proteasome positively regulate MHC class II transcription and are necessary for stable promoter binding of CIITA. Although these basic requirements of the proteasome to initiate MHC class II transcription are known, how CIITA is recruited, stabilized, and degraded remains unclear. Here, we identify a novel N‐terminal 19S ATPase‐binding domain of CIITA. The ATPase‐binding domain lies within the proline/serine/threonine‐rich region of CIITA and encompasses a majority of the CIITA degron sequence. Absence of the ATPase‐binding domain increases the half‐life of CIITA, but blocks MHC class II surface expression, indicating that CIITA requires interaction with the 19S ATPases for both appropriate deployment and destruction.