Jenny P.Y. Ting

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.

Characterization of NLRP12 during the In Vivo Host Immune Response to Klebsiella pneumoniae and Mycobacterium tuberculosis

The majority of nucleotide binding domain leucine rich repeats-containing (NLR) family members has yet to be functionally characterized. Of the described NLRs, most are considered to be proinflammatory and facilitate IL-1β production. However, a newly defined sub-group of NLRs that function as negative regulators of inflammation have been identified based on their abilities to attenuate NF-κB signaling. NLRP12 (Monarch-1) is a prototypical member of this sub-group that negatively regulates both canonical and noncanonical NF-κB signaling in biochemical assays and in colitis and colon cancer models. The role of NLRP12 in infectious diseases has not been extensively studied. Here, we characterized the innate immune response of Nlrp12−/− mice following airway exposure to LPS, Klebsiella pneumoniae and Mycobacterium tuberculosis. In response to E. coli LPS, Nlrp12−/− mice showed a slight decrease in IL-1β and increase in IL-6 production, but these levels were not statistically significant. During K. pneumoniae infection, we observed subtle differences in cytokine levels and significantly reduced numbers of monocytes and lymphocytes in Nlrp12−/− mice. However, the physiological relevance of these findings is unclear as no overt differences in the development of lung disease were observed in the Nlrp12−/− mice. Likewise, Nlrp12−/− mice demonstrated pathologies similar to those observed in the wild type mice following M. tuberculosis infection. Together, these data suggest that NLRP12 does not significantly contribute to the in vivo host innate immune response to LPS stimulation, Klebsiella pneumonia infection or Mycobacterium tuberculosis.

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.

MAVS-dependent host species range and pathogenicity of human hepatitis A virus

Hepatotropic viruses are important causes of human disease, but the intrahepatic immune response to hepatitis viruses is poorly understood because of a lack of tractable small- animal models. We describe a murine model of hepatitis A virus (HAV) infection that recapitulates critical features of type A hepatitis in humans. We demonstrate that the capacity of HAV to evade MAVS-mediated type I interferon responses defines its host species range. HAV-induced liver injury was associated with interferon-independent intrinsic hepatocellular apoptosis and hepatic inflammation that unexpectedly resulted from MAVS and IRF3/7 signaling. This murine model thus reveals a previously undefined link between innate immune responses to virus infection and acute liver injury, providing a new paradigm for viral pathogenesis in the liver.

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.

G Protein signaling modulator-3 inhibits the inflammasome activity of NLRP3.

Background: NLRP3 is a key regulator of innate inflammation and is linked to inflammatory diseases. Results: GPSM3 associates with NLRP3 and inhibits its function. Conclusion: GPSM3 specifically inhibits NLRP3-dependent inflammasome activity by interacting with its leucine-rich repeat domain. Significance: This association uncovers a putative new mechanism of NLRP3 control, linking a G protein modulator to NLRP3-dependent inflammatory diseases. Inflammasomes are multi-protein complexes that regulate maturation of the interleukin 1β-related cytokines IL-1β and IL-18 through activation of the cysteine proteinase caspase-1. NOD-like receptor family, pyrin domain containing 3 (NLRP3) protein is a key component of inflammasomes that assemble in response to a wide variety of endogenous and pathogen-derived danger signals. Activation of the NLRP3-inflammasome and subsequent secretion of IL-1β is highly regulated by at least three processes: transcriptional activation of both NLRP3 and pro-IL-1β genes, non-transcriptional priming of NLRP3, and final activation of NLRP3. NLRP3 is predominantly expressed in cells of the hematopoietic lineage. Using a yeast two-hybrid screen, we identified the hematopoietic-restricted protein, G protein signaling modulator-3 (GPSM3), as a NLRP3-interacting protein and a negative regulator of IL-1β production triggered by NLRP3-dependent inflammasome activators. In monocytes, GPSM3 associates with the C-terminal leucine-rich repeat domain of NLRP3. Bone marrow-derived macrophages lacking GPSM3 expression exhibit an increase in NLRP3-dependent IL-1β, but not TNF-α, secretion. Furthermore, GPSM3-null mice have enhanced serum and peritoneal IL-1β production following Alum-induced peritonitis. Our findings suggest that GPSM3 acts as a direct negative regulator of NLRP3 function.

Type 2 innate lymphoid cells treat and prevent acute gastrointestinal graft-versus-host disease

Acute graft-versus-host disease (aGVHD) is the most common complication for patients undergoing allogeneic stem cell transplantation. Despite extremely aggressive therapy targeting donor T cells, patients with grade III or greater aGVHD of the lower GI tract, who do not respond to therapy with corticosteroids, have a dismal prognosis. Thus, efforts to improve understanding of the function of local immune and non-immune cells in regulating the inflammatory process in the GI tract during aGVHD are needed. Here, we demonstrate, using murine models of allogeneic BMT, that type 2 innate lymphoid cells (ILC2s) in the lower GI tract are sensitive to conditioning therapy and show very limited ability to repopulate from donor bone marrow. Infusion of donor ILC2s was effective in reducing the lethality of aGVHD and in treating lower GI tract disease. ILC2 infusion was associated with reduced donor proinflammatory Th1 and Th17 cells, accumulation of donor myeloid-derived suppressor cells (MDSCs) mediated by ILC2 production of IL-13, improved GI tract barrier function, and a preserved graft-versus-leukemia (GVL) response. Collectively, these findings suggest that infusion of donor ILC2s to restore gastrointestinal tract homeostasis may improve treatment of severe lower GI tract aGVHD.

NLRX1 suppresses tumorigenesis and attenuates histiocytic sarcoma through the negative regulation of NF-κB signaling

Histiocytic sarcoma is an uncommon malignancy in both humans and veterinary species. Research exploring the pathogenesis of this disease is scarce; thus, diagnostic and therapeutic options for patients are limited. Recent publications have suggested a role for the NLR, NLRX1, in acting as a tumor suppressor. Based on these prior findings, we hypothesized that NLRX1 would function to inhibit tumorigenesis and thus the development of histiocytic sarcoma. To test this, we utilized Nlrx1−/− mice and a model of urethane-induced tumorigenesis. Nlrx1−/− mice exposed to urethane developed splenic histiocytic sarcoma that was associated with significant up-regulation of the NF-λB signaling pathway. Additionally, development of these tumors was also significantly associated with the increased regulation of genes associated with AKT signaling, cell death and autophagy. Together, these data show that NLRX1 suppresses tumorigenesis and reveals new genetic pathways involved in the pathobiology of histiocytic sarcoma.

Prion‐like MAVS aggregation in lupus patients associates with increased interferon‐I

Increased levels of type I interferon (IFN) and type I IFN–regulated genes are found in patients with systemic lupus erythematosus (SLE) and may be central to its pathogenesis. Mitochondrial antiviral signaling protein (MAVS) is a key regulator of type I IFN that undergoes a dramatic prion‐like aggregation and self propagates the activation signal from viral RNA to amplify downstream IFN production. We undertook this study to determine whether such MAVS aggregates might play a role in the sustained increased production of type I IFN in SLE.

NLRP12 provides a critical checkpoint for osteoclast differentiation.

Significance Members of the nucleotide-binding leucine-rich repeat-containing receptor (NLR) family are generally thought of as initiators of inflammation and are important in a number of inflammatory diseases. However, recent evidence has started to emerge that several NLRs can serve as checkpoint proteins against specific inflammatory pathways. Although checkpoint proteins are well accepted for their importance in adaptive immunity, their roles in innate immunity are still nascent. Receptor activator of nuclear factor kappa B ligand (RANKL), a tumor necrosis factor family cytokine responsible for basal and most forms of pathologic osteoclastogenesis, sends important differentiation signals through the alternative nuclear factor kappa B pathway. This report shows that an NLR member, nucleotide-binding leucine-rich repeat and pyrin domain-containing receptor 12, provides a brake on the activity of RANKL even in noninflammatory settings, extending the role for this type of NLR beyond inflammation-related disease. The alternative or noncanonical nuclear factor kappa B (NF-κB) pathway regulates the osteoclast (OC) response to receptor activator of nuclear factor kappa B ligand (RANKL) and thus bone metabolism. Although several lines of evidence support the emerging concept that nucleotide-binding leucine-rich repeat and pyrin domain-containing receptor 12 (NLRP12) impedes alternative NF-κB activation in innate immune cells, a functional role for NLRP12 outside an inflammatory disease model has yet to be reported. Our study demonstrates that NLRP12 has a protective role in bone via suppression of alternative NF-κB–induced osteoclastogenesis and is down-modulated in response to osteoclastogenic stimuli. Here, we show that retroviral overexpression of NLRP12 suppressed RelB nuclear translocation and OC formation. Conversely, genetic ablation of NLRP12 promoted NIK stabilization, RelB nuclear translocation, and increased osteoclastogenesis in vitro. Using radiation chimeras, we demonstrated these in vitro observations dovetail with our in vivo findings that NLRP12 deficiency leads to enhanced OC numbers accompanied by a significant decline in bone mass under physiological conditions. Consistent with the basal bone phenotype, we also observed an enhanced osteolytic response following RANKL injection over the calvaria of NLRP12-deficient chimeric mice compared with wild-type control mice. Thus, modulation of NLRP12 levels controls alternative NF-κB signaling in OC precursors, altering bone homeostasis and osteolytic responses.