Bradley N. Martin

Caspase-8 as an Effector and Regulator of NLRP3 Inflammasome Signaling

Background: NLRP3 inflammasomes regulate caspase-1-dependent IL-1β release and pyroptotic death in dendritic cells (DC) and macrophages. Results: Caspase-8 mediates IL-1β production and apoptosis by NLRP3 inflammasomes in caspase-1-deficient DC and facilitates pyroptosis in wild type DC. Conclusion: Caspase-8 is activated within NLRP3 inflammasome signaling platforms. Significance: In addition to caspase-1, NLRP3 inflammasomes engage caspase-8 as an important effector of innate immune signaling responses. We recently described the induction of noncanonical IL-1β processing via caspase-8 recruited to ripoptosome signaling platforms in myeloid leukocytes. Here, we demonstrate that activated NLRP3·ASC inflammasomes recruit caspase-8 to drive IL-1β processing in murine bone marrow-derived dendritic cells (BMDC) independent of caspase-1 and -11. Sustained stimulation (>2 h) of LPS-primed caspase-1-deficient (Casp1/11−/−) BMDC with the canonical NLRP3 inflammasome agonist nigericin results in release of bioactive IL-1β in conjunction with robust caspase-8 activation. This IL-1β processing and caspase-8 activation do not proceed in Nlrp3−/− or Asc−/− BMDC and are suppressed by pharmacological inhibition of caspase-8, indicating that caspase-8 can act as a direct IL-1β-converting enzyme during NLRP3 inflammasome activation. In contrast to the rapid caspase-1-mediated death of wild type (WT) BMDC via NLRP3-dependent pyroptosis, nigericin-stimulated Casp1/11−/− BMDC exhibit markedly delayed cell death via NLRP3-dependent apoptosis. Biochemical analyses of WT and Casp1/11−/− BMDC indicated that caspase-8 is proteolytically processed within detergent-insoluble ASC-enriched protein complexes prior to extracellular export during nigericin treatment. Although nigericin-stimulated caspase-1 activation and activity are only modestly attenuated in caspase-8-deficient (Casp8−/−Rip3−/−) BMDC, these cells do not exhibit the rapid loss of viability of WT cells. These results support a contribution of caspase-8 to both IL-1β production and regulated death signaling via NLRP3 inflammasomes. In the absence of caspase-1, NLRP3 inflammasomes directly utilize caspase-8 as both a pro-apoptotic initiator and major IL-1β-converting protease. In the presence of caspase-1, caspase-8 acts as a positive modulator of the NLRP3-dependent caspase-1 signaling cascades that drive both IL-1β production and pyroptotic death.

The psoriasis-associated D10N variant of the adaptor Act1 with impaired regulation by the molecular chaperone hsp90

Act1 is an essential adaptor molecule in IL-17-mediated signaling and is recruited to the IL-17 receptor upon IL-17 stimulation. Here, we report that Act1 is a client protein of the molecular chaperone, Hsp90. The Act1 variant (D10N) linked to psoriasis susceptibility is defective in its interaction with Hsp90, resulting in a global loss of Act1 function. Act1-/- mice modeled the mechanistic link between Act1 loss of function and psoriasis susceptibility. Although Act1 is necessary for IL-17-mediated inflammation, Act1-/- mice exhibited a hyper TH17 response and developed spontaneous IL-22-dependent skin inflammation. In the absence of IL-17-signaling, IL-22 is the main contributor to skin inflammation, providing a molecular mechanism for the association of Act1 (D10N) with psoriasis susceptibility.Act1 is an essential adaptor in interleukin 17 (IL-17)-mediated signaling and is recruited to the receptor for IL-17 after stimulation with IL-17. Here we found that Act1 was a client protein of the molecular chaperone hsp90. The D10N variant of Act1 (Act1(D10N)) that is linked to susceptibility to psoriasis was defective in its interaction with hsp90, which resulted in a global loss of Act1 function. Act1-deficient mice modeled the mechanistic link between loss of Act1 function and susceptibility to psoriasis. Although Act1 was necessary for IL-17-mediated inflammation, Act1-deficient mice had a hyperactive response of the TH17 subset of helper T cells and developed spontaneous IL-22-dependent skin inflammation. In the absence of IL-17 signaling, IL-22 was the main contributor to skin inflammation, which provides a molecular mechanism for the association of Act1(D10N) with psoriasis susceptibility.

IKKα negatively regulates ASC-dependent inflammasome activation

The inflammasomes are multiprotein complexes that activate caspase-1 in response to infections and stress, resulting in the secretion of pro-inflammatory cytokines. Here we report that IKKα is a critical negative regulator of ASC-dependent inflammasomes. IKKα controls the inflammasome at the level of the adaptor ASC, which interacts with IKKα in the nucleus of resting macrophages in an IKKα kinase-dependent manner. Loss of IKKα kinase activity results in inflammasome hyperactivation. Mechanistically, the downstream nuclear effector IKKi facilitates translocation of ASC from the nucleus to the perinuclear area during inflammasome activation. ASC remains under the control of IKKα in the perinuclear area following translocation of the ASC/IKKα complex. Signal 2 of NLRP3 activation leads to inhibition of IKKα kinase activity through the recruitment of PP2A, allowing ASC to participate in NLRP3 inflammasome assembly. Taken together, these findings reveal a IKKi-IKKα-ASC axis that serves as a common regulatory mechanism for ASC-dependent inflammasomes.

T cell-intrinsic ASC critically promotes TH17-mediated experimental autoimmune encephalomyelitis

Interleukin 1β (IL-1β) is critical for the in vivo survival, expansion and effector function of IL-17–producing helper T (TH17) cells during autoimmune responses, including experimental autoimmune encephalomyelitis (EAE). However, the spatiotemporal role and cellular source of IL-1β during EAE pathogenesis are poorly defined. In the present study, we uncovered a T cell–intrinsic inflammasome that drives IL-1β production during TH17-mediated EAE pathogenesis. Activation of T cell antigen receptors induced expression of pro-IL-1β, whereas ATP stimulation triggered T cell production of IL-1β via ASC-NLRP3–dependent caspase-8 activation. IL-1R was detected on TH17 cells but not on type 1 helper T (TH1) cells, and ATP-treated TH17 cells showed enhanced survival compared with ATP-treated TH1 cells, suggesting autocrine action of TH17-derived IL-1β. Together these data reveal a critical role for IL-1β produced by a TH17 cell–intrinsic ASC–NLRP3–caspase-8 inflammasome during inflammation of the central nervous system.

A novel IL-17 signaling pathway controlling keratinocyte proliferation and tumorigenesis via the TRAF4-ERK5 axis.

Wu et al. report a novel IL-17–mediated cascade via the IL-17R–TRAF4–ERK5 axis that directly stimulates keratinocyte proliferation and skin tumor formation in mice.

IRAKM-Mincle axis links cell death to inflammation: Pathophysiological implications for chronic alcoholic liver disease

Lipopolysaccharide (LPS)‐mediated activation of Toll‐like receptors (TLRs) in hepatic macrophages and injury to hepatocytes are major contributors to the pathogenesis of alcoholic liver disease. However, the mechanisms by which TLR‐dependent inflammatory responses and alcohol‐induced hepatocellular damage coordinately lead to alcoholic liver disease are not completely understood. In this study, we found that mice deficient in interleukin‐1 receptor‐associated kinase M (IRAKM), a proximal TLR pathway molecule typically associated with inhibition of TLR signaling, were actually protected from chronic ethanol‐induced liver injury. In bone marrow‐derived macrophages challenged with low concentrations of LPS, which reflect the relevant pathophysiological levels of LPS in both alcoholic patients and ethanol‐fed mice, the IRAKM Myddosome was preferentially formed. Further, the IRAKM Myddosome mediated the up‐regulation of Mincle, a sensor for cell death. Mincle‐deficient mice were also protected from ethanol‐induced liver injury. The endogenous Mincle ligand spliceosome‐associated protein 130 (SAP130) is a danger signal released by damaged cells; culture of hepatocytes with ethanol increased the release of SAP130. Ex vivo studies in bone marrow‐derived macrophages suggested that SAP130 and LPS synergistically activated inflammatory responses, including inflammasome activation. Conclusion: This study reveals a novel IRAKM‐Mincle axis that contributes to the pathogenesis of ethanol‐induced liver injury. (Hepatology 2016;64:1978‐1993).

A Novel IL-25 Signaling Pathway through STAT5

IL-25 is a member of the IL-17 family of cytokines that promotes Th2 cell–mediated inflammatory responses. IL-25 signals through a heterodimeric receptor (IL-25R) composed of IL-17RA and IL-17RB, which recruits the adaptor molecule Act1 for downstream signaling. Although the role of IL-25 in potentiating type 2 inflammation is well characterized by its ability to activate the epithelium as well as T cells, the components of its signaling cascade remain largely unknown. In this study, we found that IL-25 can directly activate STAT5 independently of Act1. Furthermore, conditional STAT5 deletion in T cells or epithelial cells led to a defective IL-25–initiated Th2 polarization as well as defective IL-25 enhancement of Th2 responses. Finally, we found that STAT5 is recruited to the IL-25R in a ligand-dependent manner through unique tyrosine residues on IL-17RB. Together, these findings reveal a novel Act1-independent IL-25 signaling pathway through STAT5 activation.

Human Colon Tumors Express a Dominant-Negative Form of SIGIRR That Promotes Inflammation and Colitis-Associated Colon Cancer in Mice

BACKGROUND & AIMSnSingle immunoglobulin and toll-interleukin 1 receptor (SIGIRR), a negative regulator of the Toll-like and interleukin-1 receptor (IL-1R) signaling pathways, controls intestinal inflammation and suppresses colon tumorigenesis in mice. However, the importance of SIGIRR in human colorectal cancer development has not been determined. We investigated the role of SIGIRR in development of human colorectal cancer.nnnMETHODSnWe performed RNA sequence analyses of pairs of colon tumor and nontumor tissues, each collected from 68 patients. Immunoblot and immunofluorescence analyses were used to determine levels of SIGIRR protein in primary human colonic epithelial cells, tumor tissues, and colon cancer cell lines. We expressed SIGIRR and mutant forms of the protein in Vaco cell lines. We created and analyzed mice that expressed full-length (control) or a mutant form of Sigirr (encoding SIGIRR(N86/102S), which is not glycosylated) specifically in the intestinal epithelium. Some mice were given azoxymethane (AOM) and dextran sulfate sodium to induce colitis-associated cancer. Intestinal tissues were collected and analyzed by immunohistochemical and gene expression profile analyses.nnnRESULTSnRNA sequence analyses revealed increased expression of a SIGIRR mRNA isoform, SIGIRR(ΔE8), in colorectalxa0cancer tissues compared to paired nontumor tissues. SIGIRR(ΔE8) is not modified by complex glycans and is therefore retained in the cytoplasm-it cannot localize to the cell membrane or reduce IL1R signaling. SIGIRR(ΔE8) interacts with and has a dominant-negative effect on SIGIRR, reducing its glycosylation, localization to the cell surface, and function. Most SIGIRR detected in human colon cancer tissues was cytoplasmic, whereas in nontumor tissues it was found at the cell membrane. Mice that expressed SIGIRR(N86/102S) developed more inflammation and formed larger tumors after administration of azoxymethane and dextran sulfate sodium than control mice; colon tissues from these mutant mice expressed higher levels of the inflammatory cytokines IL-17A and IL-6 had activation of the transcription factors STAT3 and NFκB. SIGIRR(N86/102S) expressed in colons of mice did not localize to the epithelial cell surface.nnnCONCLUSIONnLevels of SIGIRR are lower in human colorectal tumors, compared with nontumor tissues; tumors contain the dominant-negative isoform SIGIRR(ΔE8). This mutant protein blocks localization of full-lengthxa0SIGIRR to the surface of colon epithelial cells and itsxa0ability to downregulate IL1R signaling. Expression of SIGIRR(N86/102S) in the colonic epithelium of mice increases expression of inflammatory cytokines and formation and size of colitis-associated tumors.

The flavonoid cyanidin blocks binding of the cytokine interleukin-17A to the IL-17RA subunit to alleviate inflammation in vivo

A molecule from red berries blocks the activity of a proinflammatory cytokine and alleviates inflammation in mice. How red berries reduce inflammation Members of the interleukin-17 (IL-17) family of proinflammatory cytokines are important in the immune response to infections; however, excessive IL-17 signaling is associated with autoimmune inflammatory diseases, such as asthma, psoriasis, and rheumatoid arthritis. Through a screen of small molecules, Liu et al. found that cyanidin, a flavonoid found in red berries and other fruits, bound to the IL-17 receptor (IL-17RA) in a manner that blocked the binding of IL-17A. In several mouse models of inflammatory disease, cyanidin alleviated inflammation induced by T cells that produce IL-17A. Together, these data suggest that cyanidin should be further developed as a small-molecule inhibitor of IL-17A–dependent inflammatory diseases. Cyanidin, a key flavonoid that is present in red berries and other fruits, attenuates the development of several diseases, including asthma, diabetes, atherosclerosis, and cancer, through its anti-inflammatory effects. We investigated the molecular basis of cyanidin action. Through a structure-based search for small molecules that inhibit signaling by the proinflammatory cytokine interleukin-17A (IL-17A), we found that cyanidin specifically recognizes an IL-17A binding site in the IL-17A receptor subunit (IL-17RA) and inhibits the IL-17A/IL-17RA interaction. Experiments with mice demonstrated that cyanidin inhibited IL-17A–induced skin hyperplasia, attenuated inflammation induced by IL-17–producing T helper 17 (TH17) cells (but not that induced by TH1 or TH2 cells), and alleviated airway hyperreactivity in models of steroid-resistant and severe asthma. Our findings uncover a previously uncharacterized molecular mechanism of action of cyanidin, which may inform its further development into an effective small-molecule drug for the treatment of IL-17A–dependent inflammatory diseases and cancer.

IL-17 induced NOTCH1 activation in oligodendrocyte progenitor cells enhances proliferation and inflammatory gene expression.

NOTCH1 signalling contributes to defective remyelination by impairing differentiation of oligodendrocyte progenitor cells (OPCs). Here we report that IL-17 stimulation induces NOTCH1 activation in OPCs, contributing to Th17-mediated demyelinating disease. Mechanistically, IL-17R interacts with NOTCH1 via the extracellular domain, which facilitates the cleavage of NOTHC1 intracellular domain (NICD1). IL-17-induced NOTCH1 activation results in the interaction of IL-17R adaptor Act1 with NICD1, followed by the translocation of the Act1–NICD1 complex into the nucleus. Act1–NICD1 are recruited to the promoters of several NOTCH1 target genes (including STEAP4, a metalloreductase important for inflammation and cell proliferation) that are specifically induced in the spinal cord by Th17 cells. A decoy peptide disrupting the IL-17RA–NOTCH1 interaction inhibits IL-17-induced NOTCH1 activation and attenuates Th17-mediated experimental autoimmune encephalitis (EAE). Taken together, these findings demonstrate critical crosstalk between the IL-17 and NOTCH1 pathway, regulating Th17-induced inflammatory and proliferative genes to promote demyelinating disease.