Bacillus cereus non-haemolytic enterotoxin activates the NLRP3 inflammasome

Inflammasomes are important for host defence against pathogens and homeostasis with commensal microbes. Here, we show non-haemolytic enterotoxin (NHE) from the neglected human foodborne pathogen Bacillus cereus is an activator of the NLRP3 inflammasome and pyroptosis. NHE is a non-redundant toxin to haemolysin BL (HBL) despite having a similar mechanism of action. Via a putative transmembrane region, subunit C of NHE initiates binding to the plasma membrane, leading to the recruitment of subunit B and subunit A, thus forming a tripartite lytic pore that is permissive to efflux of potassium. NHE mediates killing of cells from multiple lineages and hosts, highlighting a versatile functional repertoire in different host species. These data indicate that NHE and HBL operate synergistically to induce inflammation and show that multiple virulence factors from the same pathogen with conserved function and mechanism of action can be exploited for sensing by a single inflammasome

DECTIN-1: A modifier protein in CTLA-4 haploinsufficiency.

Autosomal dominant loss-of-function (LoF) variants in cytotoxic T-lymphocyte associated protein 4 (CTLA4) cause immune dysregulation with autoimmunity, immunodeficiency and lymphoproliferation (IDAIL). Incomplete penetrance and variable expressivity are characteristic of IDAIL caused by CTLA-4 haploinsufficiency (CTLA-4h), pointing to a role for genetic modifiers. Here, we describe an IDAIL proband carrying a maternally inherited pathogenic CTLA4 variant and a paternally inherited rare LoF missense variant in CLEC7A, which encodes for the β-glucan pattern recognition receptor DECTIN-1. The CLEC7A variant led to a loss of DECTIN-1 dimerization and surface expression. Notably, DECTIN-1 stimulation promoted human and mouse regulatory T cell (Treg) differentiation from naïve αβ and γδ T cells, even in the absence of transforming growth factor-β. Consistent with DECTIN-1's Treg-boosting ability, partial DECTIN-1 deficiency exacerbated the Treg defect conferred by CTL4-4h. DECTIN-1/CLEC7A emerges as a modifier gene in CTLA-4h, increasing expressivity of CTLA4 variants and acting in functional epistasis with CTLA-4 to maintain immune homeostasis and tolerance.S

The role of cytosolic DNA sensors in colitis and intestinal cancer

Background and Aim: The innate immune response contributes to the development or attenuation of cancer. In the gastrointestinal tract, DNA is often liberated from microbes or damaged host cells and can activate protective responses against the development of cancer. Patients with colorectal cancer whose tissues express a higher level of the DNA sensor Ku70 survive longer than those with a lower expression. In addition, the gene encoding Ku70 is frequently mutated in tumour samples from patients with colorectal cancer, suggesting a putative role of Ku70 in the development and/or progression of colorectal cancer. Ku70 has an established role in DNA repair and has been implicated in controlling intestinal homeostasis in mice. This PhD thesis investigates the role of Ku70 in colitis-associated colorectal cancer and genetically-predisposed intestinal cancer in mice and characterises the mechanisms of action of the Ku70 signalling pathway in tumorigenesis. Methods: Two mouse models of intestinal cancer are used. In the colitis and colitis-associated colorectal cancer model, littermate wild-type (WT) mice and mice lacking one allele of Ku70 (Ku70-Het mice) or both alleles of Ku70 (Ku70-KO mice) were treated with the procarcinogen azoxymethane and the inflammatory agent dextran sodium sulfate. In the genetically-predisposed intestinal cancer model, WT and Ku70-Het mice were crossed with Apc-Het mice carrying a mutation in the gene encoding the tumour suppressor protein APC, which predisposes mice to the development of spontaneous intestinal cancer. In both models, tumour numbers were enumerated in the intestinal tissue of mice. To identify the signalling pathways controlled by Ku70, I performed a phosphoproteomic screen using mass spectrometry. Further, I used histology, immunohistochemistry, immunoblotting, ELISA, qRT-PCR, overexpression system and immunoprecipitation techniques to gain insights into the molecular changes during the development of colitis and intestinal cancer. Results: I found that Ku70-Het mice lost more body weight during the development of colitis and colitis-associated colorectal cancer and had a greater tumour burden compared with littermate WT mice. Ku70-KO mice were highly susceptible to colitis and did not survive the duration of the colitis-associated colorectal cancer experiment. Furthermore, Apc-Het-Ku70-Het mice had enhanced tumour burden in the small and large intestine compared to littermate Apc-Het mice. The effects of Ku70 on attenuating colitis and intestinal cancer were independent of the role of Ku70 in DNA repair, and the production of inflammatory markers and interferons. Instead, Ku70 interacted with the GTPase Ras and the kinase Raf, forming a tripartite protein complex that activated the kinases MEK and ERK. I show that the alpha/beta-domain and the C-terminal domain of Ku70 interacted with the GTPase domain of Ras, whereas the C-terminal domain of Ku70 interacted with the conserved region 2 of Raf. The Ku70-Ras-Raf complex induced MEK-ERK activation, leading to reduced activation of the phosphatase Cdc25A and cell-cycle kinase CDK1, and reduced tumour burden. The Ku70-Ras-Raf complex interacted with endosomal markers Rab5 and Rab7, suggesting possible docking at the endosomal membrane. Moreover, my preliminary data indicate that Ku70 may migrate from the nucleus to the cytoplasm during the development of colitis, presumably for the sensing of cytoplasmic DNA. Conclusions: In this thesis, I uncovered that Ku70 induces the formation of a novel signalosome complex, composed of Ku70, Ras and Raf. This signalosome docks at the endosomal membrane and induces activation of the MEK-ERK signalling pathway during tumorigenesis, potentially attenuating the development of intestinal cancer. These findings may facilitate the design of therapeutics targeting components of the Ku70 signallin

Inflammasomes in Colitis and Colorectal Cancer: Mechanism of Action and Therapies

Colorectal cancer is a multifactorial disease and a leading cause of cancer-related deaths worldwide. Inflammation is a driver across multiple stages in the development of colorectal cancer. The inflammasome is a cytosolic multiprotein complex of the innate immune system central to the regulation of inflammation, pyroptosis, and other cellular processes important for maintaining gut homeostasis. Studies using mouse models of colitis and colitis-associated colorectal cancer have highlighted diverse and sometimes contrasting roles of inflammasomes in maintaining a balance between intestinal barrier function and the gut microbiota. In addition, persistent and/or dysregulated stimulation of inflammasome sensors finetune inflammation and tumorigenesis in the intestine. This review highlights the emerging role of inflammasome signaling in colitis and colitis-associated colorectal cancer. We also review the key mechanisms by which inflammasome signaling modulate inflammation and tumor development. Finally, we speculate the importance of using more tightly regulated experimental approaches to examine the role of gut microbiota in colorectal cancer.A.P. and C.S. are supported by a John Curtin School of Medical Research International Ph.D. scholarship. S.M.M. is supported by the Australian National University, The Gretel and Gordon Bootes Medical Research Foundation, and the National Health and Medical Research Council of Australia under Project Grants (APP1141504, APP1146864, APP1162103, and APP1163358) and the R.D. Wright Career Development Fellowship (APP1162025). The authors apologize to researchers whose work was not cited or cited through reviews owing to space limitation

Gasdermins deliver a deadly punch to cancer

The pore-forming gasdermin proteins mediate a lytic and proinflammatory form of cell death called pyroptosis and have been linked to the host defense against infection. Two recent studies published in Nature revealed that induction of pyroptosis in tumor cells promotes anti-tumor activity, highlighting gasdermins as potential new targets in cancer immunotherapy

Profiling the colonic mucosal response to fecal microbiota transplantation identifies a role for GBP5 in colitis in humans and mice

Host molecular responses to fecal microbiota transplantation (FMT) in ulcerative colitis are not well understood. Here, we profile the human colonic mucosal transcriptome prior to and following FMT or placebo to identify molecules regulated during disease remission. FMT alters the transcriptome above the effect of placebo (n = 75 vs 3 genes, q < 0.05), including modulation of structural, metabolic and inflammatory pathways. This response is attributed to responders with no consistency observed in non-responders. Regulated pathways in responders include tight junctions, calcium signalling and xenobiotic metabolism. Genes significantly regulated longitudinally in responders post-FMT could discriminate them from responders and non-responders at baseline and non-responders post-FMT, with GBP5 and IRF4 downregulation being associated with remission. Female mice with a deletion of GBP5 are more resistant to developing colitis than their wild-type littermates, showing higher colonic IRF4 phosphorylation. The colonic mucosal response discriminates UC remission following FMT, with GBP5 playing a detrimental role in colitis