RORγt-Raftlin1 complex regulates the pathogenicity of Th17 cells and colonic inflammation.

Th17 cells that produce Interleukin IL-17 are pathogenic in many human diseases, including inflammatory bowel disease, but are, paradoxically, essential for maintaining the integrity of the intestinal barrier in a non-inflammatory state. However, the intracellular mechanisms that regulate distinct transcriptional profiles and functional diversity of Th17 cells remain unclear. Here we show Raftlin1, a lipid raft protein, specifically upregulates and forms a complex with RORγt in pathogenic Th17 cells. Disruption of the RORγt-Raftlin1 complex results in the reduction of pathogenic Th17 cells in response to Citrobacter rodentium; however, there is no effect on nonpathogenic Th17 cells in response to commensal segmented filamentous bacteria. Mechanistically, we show that Raftlin1 recruits distinct phospholipids to RORγt and promotes the pathogenicity of Th17 cells. Thus, we have identified a mechanism that drives the pathogenic function of Th17 cells, which could provide a platform for advanced therapeutic strategies to dampen Th17-mediated inflammatory diseases.

Possible role of IL-23 inhibitor in autoimmune hemolytic anemia.

Rituximab and prednisone are commonly used treatments for autoimmune hemolytic anemia (AIHA), where the body's immune system attacks and destroys its red blood cells. However, some AIHA patients may become refractory to rituximab treatment, and this can result in continued hemolysis and persistent anemia, making it challenging for affected individuals to manage their symptoms. The underlying causes of rituximab refractoriness in AIHA patients can be complex and vary from patient to patient. Herein, we present a case of newly diagnosed warm and cold AIHA that remained in remission with an interleukin-23 inhibitor.

Inner mitochondrial membrane protein Prohibitin 1 mediates Nix-induced, Parkin-independent mitophagy.

Autophagy of damaged mitochondria, called mitophagy, is an important organelle quality control process involved in the pathogenesis of inflammation, cancer, aging, and age-associated diseases. Many of these disorders are associated with altered expression of the inner mitochondrial membrane (IMM) protein Prohibitin 1. The mechanisms whereby dysfunction occurring internally at the IMM and matrix activate events at the outer mitochondrial membrane (OMM) to induce mitophagy are not fully elucidated. Using the gastrointestinal epithelium as a model system highly susceptible to autophagy inhibition, we reveal a specific role of Prohibitin-induced mitophagy in maintaining intestinal homeostasis. We demonstrate that Prohibitin 1 induces mitophagy in response to increased mitochondrial reactive oxygen species (ROS) through binding to mitophagy receptor Nix/Bnip3L and independently of Parkin. Prohibitin 1 is required for ROS-induced Nix localization to mitochondria and maintaining homeostasis of epithelial cells highly susceptible to mitochondrial dysfunction.

Pak2-mediated phosphorylation promotes RORγt ubiquitination and inhibits colonic inflammation.

Dysregulated interleukin-17 (IL-17) expression and its downstream signaling is strongly linked to inflammatory bowel diseases (IBDs). However, the molecular mechanisms by which the function of RORγt, the transcription factor of IL-17, is regulated remains elusive. By a mass spectrometry-based approach, we identify that Pak2, a serine (S)/threonine (T) kinase, directly associates with RORγt. Pak2 recognizes a conserved KRLS motif within RORγt and phosphorylates the S-316 within this motif. Genetic deletion of Pak2 in Th17 cells reduces RORγt phosphorylation, increases IL-17 expression, and induces severe colitis upon adoptive transfer to Rag1-/- mice. Similarly, reconstitution of RORγt-S316A mutant in Rorc-/- Th17 cells enhances IL-17 expression and colitis severity. Mechanistically, we demonstrate that Pak2-mediated phosphorylation causes a conformational change resulting in exposure of the ubiquitin ligase Itch interacting PPLY motif and degradation of RORγt. Thus, we have uncovered a mechanism by which the activity of RORγt is regulated that can be exploited therapeutically.

RORγt protein modifications and IL-17-mediated inflammation.

RORγt, the master transcription factor for cytokine interleukin (IL)-17, is expressed explicitly in Th17 cells, γδT cells, and type 3 innate lymphoid cells in mice and humans. Since dysregulated IL-17 expression is strongly linked to several human inflammatory diseases, the RORγt-IL-17 axis has been the focus of intense research. Recently, several studies have shown that RORγt is modified by multiple post-translational mechanisms, including ubiquitination, acetylation, SUMOylation, and phosphorylation. This review discusses how post-translational modifications modulate RORγt function and its turnover to regulate IL-17-driven inflammation. Broad knowledge of these pathways is crucial for a clear understanding of the pathogenic role of RORγt+IL-17+ cells and for the development of putative therapeutic strategies to target IL-17-driven diseases such as multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease.

Targeting Mitochondrial Damage as a Therapeutic for Ileal Crohn's Disease.

Paneth cell defects in Crohn's disease (CD) patients (called the Type I phenotype) are associated with worse clinical outcomes. Recent studies have implicated mitochondrial dysfunction in Paneth cells as a mediator of ileitis in mice. We hypothesized that CD Paneth cells exhibit impaired mitochondrial health and that mitochondrial-targeted therapeutics may provide a novel strategy for ileal CD. Terminal ileal mucosal biopsies from adult CD and non-IBD patients were characterized for Paneth cell phenotyping and mitochondrial damage. To demonstrate the response of mitochondrial-targeted therapeutics in CD, biopsies were treated with vehicle or Mito-Tempo, a mitochondrial-targeted antioxidant, and RNA transcriptome was analyzed. During active CD inflammation, the epithelium exhibited mitochondrial damage evident in Paneth cells, goblet cells, and enterocytes. Independent of inflammation, Paneth cells in Type I CD patients exhibited mitochondrial damage. Mito-Tempo normalized the expression of interleukin (IL)-17/IL-23, lipid metabolism, and apoptotic gene signatures in CD patients to non-IBD levels. When stratified by Paneth cell phenotype, the global tissue response to Mito-Tempo in Type I patients was associated with innate immune, lipid metabolism, and G protein-coupled receptor (GPCR) gene signatures. Targeting impaired mitochondria as an underlying contributor to inflammation provides a novel treatment approach for CD.

NLRP6 in host defense and intestinal inflammation.

NLRP6 is a member of the NLR (nucleotide-oligomerization domain-like receptor) family of proteins that recognize pathogen-derived factors and damage-associated molecular patterns in the cytosol. The function of NLRP6 has been attributed to the maintenance of epithelial integrity and host defense against microbial infections. Under some physiological conditions, NLRP6 forms a complex with ASC and caspase-1 or caspase-11 to form an inflammasome complex cleaving pro-interleukin-1ß (IL-1ß) and IL-18 into their biologically active forms. Here, we summarize recent advances in the understanding of the mechanisms of activation of the NLRP6 inflammasome and discuss its relevance to human disease.

Cutting Edge: Hypoxia-Induced Ubc9 Promoter Hypermethylation Regulates IL-17 Expression in Ulcerative Colitis.

Dysregulated IL-17 expression is central to the pathogenesis of several inflammatory disorders, including ulcerative colitis. We have shown earlier that SUMOylation of ROR-γt, the transcription factor for IL-17, regulates colonic inflammation. In this study, we show that the expression of Ubc9, the E2 enzyme that targets ROR-γt for SUMOylation, is significantly reduced in the colonic mucosa of ulcerative colitis patients. Mechanistically, we demonstrate that hypoxia-inducible factor 1α (HIF-1α) binds to a CpG island within the Ubc9 gene promoter, resulting in its hypermethylation and reduced Ubc9 expression. CRISPR-Cas9-mediated inhibition of HIF-1α normalized Ubc9 and attenuated IL-17 expression in Th17 cells and reduced diseases severity in Rag1 -/- mice upon adoptive transfer. Collectively, our study reveals a novel epigenetic mechanism of regulation of ROR-γt that could be exploited in inflammatory diseases.

Deletion of Cbl-b inhibits CD8+ T-cell exhaustion and promotes CAR T-cell function.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy is an emerging option for cancer treatment, but its efficacy is limited, especially in solid tumors. This is partly because the CAR T cells become dysfunctional and exhausted in the tumor microenvironment. However, the key pathways responsible for impaired function of exhausted cells remain unclear, which is essential to overcome CAR T-cell exhaustion. METHODS: Analysis of RNA-sequencing data from CD8+ tumor-infiltrating lymphocytes (TILs) led to identification of Cbl-b as a potential target. The sequencing data were validated using a syngeneic MC38 colon cancer model. To analyze the in vivo role of Cbl-b in T-cell exhaustion, tumor growth, % PD1+Tim3+ cells, and expression of effector cytokines were analyzed in cbl-b+/+ and cbl-b-/- mice. To evaluate the therapeutic potential of Cbl-b depletion, we generated a new CAR construct, hCEAscFv-CD28-CD3ζ.GFP, that recognizes human carcinoembryonic antigen (CEA). cbl-b+/+ and cbl-b-/- CEA-CAR T cells were generated by retroviral transduction. Rag-/- mice bearing MC38-CEA cells were injected with cbl-b+/+ and cbl-b-/- ; CEA-CAR T cells, tumor growth, % PD1+Tim3+ cells and expression of effector cytokines were analyzed. RESULTS: Our results show that the E3 ubiquitin ligase Cbl-b is upregulated in exhausted (PD1+Tim3+) CD8+ TILs. CRISPR-Cas9-mediated inhibition of Cbl-b restores the effector function of exhausted CD8+ TILs. Importantly, the reduced growth of syngeneic MC38 tumors in cbl-b-/- mice was associated with a marked reduction of PD1+Tim3+ CD8+ TILs. Depletion of Cbl-b inhibited CAR T-cell exhaustion, resulting in reduced MC38-CEA tumor growth, reduced PD1+Tim3+ cells and increased expression of interferon gamma, tumor necrosis factor alpha, and increased tumor cell killing. CONCLUSION: Our studies demonstrate that deficiency of Cbl-b overcomes endogenous CD8+ T-cell exhaustion, and deletion of Cbl-b in CAR T cells renders them resistant to exhaustion. Our results could facilitate the development of efficient CAR T-cell therapy for solid tumors by targeting Cbl-b.

Nuclear partitioning of Prohibitin 1 inhibits Wnt/ß-catenin-dependent intestinal tumorigenesis.

The Wnt/ß-catenin signaling pathway is aberrantly activated in the majority of colorectal cancer cases due to somatic mutations in the adenomatous polyposis coli (APC) gene. Prohibitin 1 (PHB1) serves pleiotropic cellular functions with dynamic subcellular trafficking, facilitating signaling crosstalk between organelles. Nuclear-localized PHB1 is an important regulator of gene transcription. Using mice with inducible intestinal epithelial cell (IEC)-specific deletion of Phb1 (Phb1iΔIEC) and mice with IEC-specific overexpression of Phb1 (Phb1Tg), we demonstrate that IEC-specific PHB1 combats intestinal tumorigenesis in the ApcMin/+ mouse model by inhibiting Wnt/ß-catenin signaling. Forced nuclear accumulation of PHB1 in human RKO or SW48 CRC cell lines increased AXIN1 expression and decreased cell viability. PHB1 deficiency in CRC cells decreased AXIN1 expression and increased ß-catenin activation that was abolished by XAV939, a pharmacological AXIN stabilizer. These results define a role of PHB1 in inhibiting the Wnt/ß-catenin pathway to influence the development of intestinal tumorigenesis. Induction of nuclear PHB1 trafficking provides a novel therapeutic option to influence AXIN1 expression and the ß-catenin destruction complex in Wnt-driven intestinal tumorigenesis.

The Synthetic Small Molecule FL3 Combats Intestinal Tumorigenesis via Axin1-Mediated Inhibition of Wnt/ß-Catenin Signaling.

Colorectal cancer exhibits aberrant activation of Wnt/ß-catenin signaling. Many inhibitors of the Wnt/ß-catenin pathway have been tested for Wnt-dependent cancers including colorectal cancer, but are unsuccessful due to severe adverse reactions. FL3 is a synthetic derivative of natural products called flavaglines, which exhibit anti-inflammatory and cytoprotective properties in intestinal epithelial cells, but has not been previously tested in cell or preclinical models of intestinal tumorigenesis. In vitro studies suggest that flavaglines target prohibitin 1 (PHB1) as a ligand, but this has not been established in the intestine. PHB1 is a highly conserved protein with diverse functions that depend on its posttranslational modifications and subcellular localization. Here, we demonstrate that FL3 combats intestinal tumorigenesis in the azoxymethane-dextran sodium sulfate and ApcMin/+ mouse models and in human colorectal cancer tumor organoids (tumoroids) by inhibiting Wnt/ß-catenin signaling via induction of Axin1 expression. FL3 exhibited no change in cell viability in normal intestinal epithelial cells or human matched-normal colonoids. FL3 response was diminished in colorectal cancer cell lines and human colorectal cancer tumoroids harboring a mutation at S45 of ß-catenin. PHB1 deficiency in mice or in human colorectal cancer tumoroids abolished FL3-induced expression of Axin1 and drove tumoroid death. In colorectal cancer cells, FL3 treatment blocked phosphorylation of PHB1 at Thr258, resulting in its nuclear translocation and binding to the Axin1 promoter. These results suggest that FL3 inhibits Wnt/ß-catenin signaling via PHB1-dependent activation of Axin1. FL3, therefore, represents a novel compound that combats Wnt pathway-dependent cancers, such as colorectal cancer. SIGNIFICANCE: Targeting of PHB1 by FL3 provides a novel mechanism to combat Wnt-driven cancers, with limited intestinal toxicity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/17/3519/F1.large.jpg.

Deubiquitination of NLRP6 inflammasome by Cyld critically regulates intestinal inflammation.

The inflammasome NLRP6 plays a crucial role in regulating inflammation and host defense against microorganisms in the intestine. However, the molecular mechanisms by which NLRP6 function is inhibited to prevent excessive inflammation remain unclear. Here, we demonstrate that the deubiquitinase Cyld prevents excessive interleukin 18 (IL-18) production in the colonic mucosa by deubiquitinating NLRP6. We show that deubiquitination inhibited the NLRP6-ASC inflammasome complex and regulated the maturation of IL-18. Cyld deficiency in mice resulted in elevated levels of active IL-18 and severe colonic inflammation following Citrobacter rodentium infection. Further, in patients with ulcerative colitis, the concentration of active IL-18 was inversely correlated with CYLD expression. Thus, we have identified a novel regulatory mechanism that inhibits the NLRP6-IL-18 pathway in intestinal inflammation.

Mitochondrial dysfunction during loss of prohibitin 1 triggers Paneth cell defects and ileitis.

OBJECTIVE: Although perturbations in mitochondrial function and structure have been described in the intestinal epithelium of Crohn's disease and ulcerative colitis patients, the role of epithelial mitochondrial stress in the pathophysiology of inflammatory bowel diseases (IBD) is not well elucidated. Prohibitin 1 (PHB1), a major component protein of the inner mitochondrial membrane crucial for optimal respiratory chain assembly and function, is decreased during IBD. DESIGN: Male and female mice with inducible intestinal epithelial cell deletion of Phb1 (Phb1iΔIEC ) or Paneth cell-specific deletion of Phb1 (Phb1ΔPC ) and Phb1fl/fl control mice were housed up to 20 weeks to characterise the impact of PHB1 deletion on intestinal homeostasis. To suppress mitochondrial reactive oxygen species, a mitochondrial-targeted antioxidant, Mito-Tempo, was administered. To examine epithelial cell-intrinsic responses, intestinal enteroids were generated from crypts of Phb1iΔIEC or Phb1ΔPC mice. RESULTS: Phb1iΔIEC mice exhibited spontaneous ileal inflammation that was preceded by mitochondrial dysfunction in all IECs and early abnormalities in Paneth cells. Mito-Tempo ameliorated mitochondrial dysfunction, Paneth cell abnormalities and ileitis in Phb1iΔIEC ileum. Deletion of Phb1 specifically in Paneth cells (Phb1ΔPC ) was sufficient to cause ileitis. Intestinal enteroids generated from crypts of Phb1iΔIEC or Phb1ΔPC mice exhibited decreased viability and Paneth cell defects that were improved by Mito-Tempo. CONCLUSION: Our results identify Paneth cells as highly susceptible to mitochondrial dysfunction and central to the pathogenesis of ileitis, with translational implications for the subset of Crohn's disease patients exhibiting Paneth cell defects.

Gut Microbiota Contributes to Spontaneous Colitis in E3 Ligase Itch-Deficient Mice.

Inflammatory bowel diseases are associated with complex shifts in microbiota composition. However, it remains unclear whether specific subsets of commensal bacteria induce inflammatory bowel diseases in genetically susceptible hosts. In this study, we found that deficiency of the E3 ligase Itch, which leads to spontaneous colitis and rectal prolapse, is associated with alteration of the gut microbiota. 16S rRNA sequencing showed expansion of colitogenic Bacteroides sp. in Itch-/- mice. Treatment with broad-spectrum antibiotics substantially reduced colonic inflammation in Itch-/- mice. Microbiota of Itch-/- mice failed to induce spontaneous colitis upon transfer to Itch+/+ mice but aggravated chemically induced colitis. Furthermore, we found that Bacteroides vulgatus, which is expanded in Itch-/- mice, was sufficient to induce colon inflammation in Itch-/- mice.

SCGN deficiency results in colitis susceptibility.

Inflammatory bowel disease (IBD) affects 1.5-3.0 million people in the United States. IBD is genetically determined and many common risk alleles have been identified. Yet, a large proportion of genetic predisposition remains unexplained. In this study, we report the identification of an ultr arare missense variant (NM_006998.3:c.230G > A;p.Arg77His) in the SCGN gene causing Mendelian early-onset ulcerative colitis. SCGN encodes a calcium sensor that is exclusively expressed in neuroendocrine lineages, including enteroendocrine cells and gut neurons. SCGN interacts with the SNARE complex, which is required for vesicle fusion with the plasma membrane. We show that the SCGN mutation identified impacted the localization of the SNARE complex partner, SNAP25, leading to impaired hormone release. Finally, we show that mouse models of Scgn deficiency recapitulate impaired hormone release and susceptibility to DSS-induced colitis. Altogether, these studies demonstrate that functional deficiency in SCGN can result in intestinal inflammation and implicates the neuroendocrine cellular compartment in IBD.

SUMOylation of ROR-γt inhibits IL-17 expression and inflammation via HDAC2.

Dysregulated ROR-γt-mediated IL-17 transcription is central to the pathogenesis of several inflammatory disorders, yet the molecular mechanisms that govern the transcription factor activity of ROR-γt in the regulation of IL-17 are not fully defined. Here we show that SUMO-conjugating enzyme Ubc9 interacts with a conserved GKAE motif in ROR-γt to induce SUMOylation of ROR-γt and suppress IL-17 expression. Th17 cells expressing SUMOylation-defective ROR-γt are highly colitogenic upon transfer to Rag1-/- mice. Mechanistically, SUMOylation of ROR-γt facilitates the binding of HDAC2 to the IL-17 promoter and represses IL-17 transcription. Mice with conditional deletion of HDAC2 in CD4+ T cells have elevated IL-17 expression and severe colitis. The identification of the Ubc9/ROR-γt/HDAC2 axis that governs IL-17 expression may open new venues for the development of therapeutic measures for inflammatory disorders.

Elevated d-2-hydroxyglutarate during colitis drives progression to colorectal cancer.

d-2-hydroxyglutarate (D2HG) is produced in the tricarboxylic acid cycle and is quickly converted to α-ketoglutarate by d-2-hydroxyglutarate dehydrogenase (D2HGDH). In a mouse model of colitis-associated colon cancer (CAC), urine level of D2HG during colitis correlates positively with subsequent polyp counts and severity of dysplasia. The i.p. injection of D2HG results in delayed recovery from colitis and severe tumorigenesis. The colonic expression of D2HGDH is decreased in ulcerative colitis (UC) patients at baseline who progress to cancer. Hypoxia-inducible factor (Hif)-1α is a key regulator of D2HGDH transcription. Our study identifies urine D2HG and tissue D2HGDH expression as biomarkers to identify patients at risk for progressing from colitis to cancer. The D2HG/D2HGDH pathway provides potential therapeutic targets for the treatment of CAC.

IL-17-driven intestinal fibrosis is inhibited by Itch-mediated ubiquitination of HIC-5.

Intestinal fibrosis is a major complication in inflammatory bowel diseases, but the regulatory mechanism that inhibits fibrosis remains unclear. Here we demonstrate that Itch-/-myofibroblasts express increased amounts of profibrotic collagen type I and α-SMA in response to IL-17. Mechanistically, we demonstrate that Itch directly binds to HIC-5 and targets it for K63-linked ubiquitination to inhibit IL-17-driven intestinal fibrosis. Reconstitution of Itch-/- myofibroblasts with wild-type Itch but not the Itch-C830A mutant normalized the expression of profibrotic genes. Similarly, shRNA-mediated inhibition of HIC-5 normalized the expression of profibrotic gene expression. Thus, we have uncovered a novel mechanism by which Itch negatively regulates intestinal fibrosis.

TIEG1 modulates ß-catenin sub-cellular localization and enhances Wnt signaling in bone.

We have previously demonstrated that TGFß Inducible Early Gene-1 (TIEG1), also known as KLF10, plays important roles in mediating skeletal development and homeostasis in mice. TIEG1 has also been identified in clinical studies as one of a handful of genes whose altered expression levels or allelic variations are associated with decreased bone mass and osteoporosis in humans. Here, we provide evidence for the first time that TIEG1 is involved in regulating the canonical Wnt signaling pathway in bone through multiple mechanisms of action. Decreased Wnt signaling in the absence of TIEG1 expression is shown to be in part due to impaired ß-catenin nuclear localization resulting from alterations in the activity of AKT and GSK-3ß. We also provide evidence that TIEG1 interacts with, and serves as a transcriptional co-activator for, Lef1 and ß-catenin. Changes in Wnt signaling in the setting of altered TIEG1 expression and/or activity may in part explain the observed osteopenic phenotype of TIEG1 KO mice as well as the known links between TIEG1 expression levels/allelic variations and patients with osteoporosis.

Itch inhibits IL-17-mediated colon inflammation and tumorigenesis by ROR-γt ubiquitination.

Dysregulated expression of interleukin 17 (IL-17) in the colonic mucosa is associated with colonic inflammation and cancer. However, the cell-intrinsic molecular mechanisms by which IL-17 expression is regulated remain unclear. We found that deficiency in the ubiquitin ligase Itch led to spontaneous colitis and increased susceptibility to colon cancer. Itch deficiency in the TH17 subset of helper T cells, innate lymphoid cells and γδ T cells resulted in the production of elevated amounts of IL-17 in the colonic mucosa. Mechanistically, Itch bound to the transcription factor ROR-γt and targeted ROR-γt for ubiquitination. Inhibition or genetic inactivation of ROR-γt attenuated IL-17 expression and reduced spontaneous colonic inflammation in Itch(-/-) mice. Thus, we have identified a previously unknown role for Itch in regulating IL-17-mediated colonic inflammation and carcinogenesis.