ABSTRACT
Moraxella catarrhalis is an important human respiratory pathogen and a major causative agent of otitis media and chronic obstructive pulmonary disease. Toll-like receptors contribute to, but cannot fully account for, the complexity of the immune response seen in M. catarrhalis infection. Using primary mouse bone marrow-derived macrophages to examine the host response to M. catarrhalis infection, our global transcriptomic and targeted cytokine analyses revealed activation of immune signalling pathways by both membrane-bound and cytosolic pattern-recognition receptors. We show that M. catarrhalis and its outer membrane vesicles or lipooligosaccharide (LOS) can activate the cytosolic innate immune sensor caspase-4/11, gasdermin-D-dependent pyroptosis, and the NLRP3 inflammasome in human and mouse macrophages. This pathway is initiated by type I interferon signalling and guanylate-binding proteins (GBPs). We also show that inflammasomes and GBPs, particularly GBP2, are required for the host defence against M. catarrhalis in mice. Overall, our results reveal an essential role for the interferon-inflammasome axis in cytosolic recognition and immunity against M. catarrhalis, providing new molecular targets that may be used to mitigate pathological inflammation triggered by this pathogen.
Subject(s)
Caspases , Inflammasomes , Mice , Humans , Animals , Caspases/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Moraxella catarrhalis/metabolism , Carrier Proteins , Immunity, InnateABSTRACT
Inflammasome signaling is a central pillar of innate immunity triggering inflammation and cell death in response to microbes and danger signals. Here, we show that two virulence factors from the human bacterial pathogen Clostridium perfringens are nonredundant activators of the NLRP3 inflammasome in mice and humans. C. perfringens lecithinase (also known as phospolipase C) and C. perfringens perfringolysin O induce distinct mechanisms of activation. Lecithinase enters LAMP1+ vesicular structures and induces lysosomal membrane destabilization. Furthermore, lecithinase induces the release of the inflammasome-dependent cytokines IL-1ß and IL-18, and the induction of cell death independently of the pore-forming proteins gasdermin D, MLKL and the cell death effector protein ninjurin-1 or NINJ1. We also show that lecithinase triggers inflammation via the NLRP3 inflammasome in vivo and that pharmacological blockade of NLRP3 using MCC950 partially prevents lecithinase-induced lethality. Together, these findings reveal that lecithinase activates an alternative pathway to induce inflammation during C. perfringens infection and that this mode of action can be similarly exploited for sensing by a single inflammasome.
Subject(s)
Inflammasomes , NLR Family, Pyrin Domain-Containing 3 Protein , Humans , Mice , Animals , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Clostridium perfringens/metabolism , Virulence Factors , Inflammation , Interleukin-1beta/metabolism , Nerve Growth Factors , Cell Adhesion Molecules, NeuronalABSTRACT
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.
Subject(s)
Colitis/metabolism , Colitis/therapy , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/therapy , Inflammasomes/metabolism , Animals , Colitis/microbiology , Colorectal Neoplasms/microbiology , Gastrointestinal Microbiome , HumansABSTRACT
Preclinical Research The antidiabetic drug, metformin, can inhibit the release of inflammatory mediators in several disease conditions. The present study was carried out to evaluate the efficacy of metformin in ameliorating edema formation, oxidative stress, mediator release and vascular changes associated with acute inflammation in the rat carrageenan model. Metformin dose-dependently inhibited paw swelling induced by carrageenan and normalized the tissue levels of the inflammatory markers myeloperoxidase and nitrite. It also maintained oxidative homeostasis as indicated by near normal levels of the oxidative stress markers glutathione, thiobarbituric acid reactive substances, catalase and superoxide dismutase. The histopathology of the paw tissue in metformin-treated animals was similar to that in normal paw and had similar effects to diclofenac. In a rat peritonitis model, metformin reduced vascular permeability and cellular infiltration. In conclusion, this study shows that metformin has a potential for use in treating various inflammatory conditions.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Inflammation/drug therapy , Metformin/pharmacology , Oxidative Stress/drug effects , Acute Disease , Animals , Carrageenan , Catalase/metabolism , Diclofenac/pharmacology , Disease Models, Animal , Edema/drug therapy , Female , Glutathione/metabolism , Hypoglycemic Agents/pharmacology , Inflammation/pathology , Inflammation Mediators/metabolism , Male , Rats , Rats, Wistar , Superoxide Dismutase/metabolism , Thiobarbituric Acid Reactive Substances/metabolismABSTRACT
The innate immune response contributes to the development or attenuation of acute and chronic diseases, including cancer. Microbial DNA and mislocalized DNA from damaged host cells can activate different host responses that shape disease outcomes. Here, we show that mice and humans lacking a single allele of the DNA repair protein Ku70 had increased susceptibility to the development of intestinal cancer. Mechanistically, Ku70 translocates from the nucleus into the cytoplasm where it binds to cytosolic DNA and interacts with the GTPase Ras and the kinase Raf, forming a tripartite protein complex and docking at Rab5+Rab7+ early-late endosomes. This Ku70-Ras-Raf signalosome activates the MEK-ERK pathways, leading to impaired activation of cell cycle proteins Cdc25A and CDK1, reducing cell proliferation and tumorigenesis. We also identified the domains of Ku70, Ras, and Raf involved in activating the Ku70 signaling pathway. Therapeutics targeting components of the Ku70 signalosome could improve the treatment outcomes in cancer.
Subject(s)
Neoplasms , Signal Transduction , Animals , Humans , Mice , Cell Proliferation , DNA , MAP Kinase Signaling System , Neoplasms/geneticsABSTRACT
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.
Subject(s)
Colitis, Ulcerative , Fecal Microbiota Transplantation , Animals , Female , Humans , Mice , Feces , GTP-Binding Proteins , Intestinal Mucosa , Treatment OutcomeABSTRACT
Intestinal fibroblasts maintain homeostasis and contribute to inflammatory responses and the development of cancer. Intestinal fibroblasts express pattern recognition receptors which can mount an immune response. Since intestinal fibroblasts interact with diverse immune and nonimmune cells, further insights into the biology of intestinal fibroblasts could expand our knowledge of the development, homeostasis, and pathophysiology of the intestine. Here, we describe a simple protocol for the isolation, cultivation, and maintenance of primary fibroblasts from the mouse colon. These cells express α-smooth muscle actin, a characteristic of specialized contractile fibroblasts called myofibroblasts. We also outline the use of these colonic fibroblasts for immunoblotting and immunofluorescence assays with or without stimulation with a growth factor.
Subject(s)
Fibroblasts , Intestines , Mice , Animals , Fibroblasts/metabolism , Myofibroblasts/metabolism , Colon/metabolism , Actins/metabolismABSTRACT
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 ß-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 αß and γδ T cells, even in the absence of transforming growth factor-ß. 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.
Subject(s)
Haploinsufficiency , Lectins, C-Type , Animals , Humans , Mice , Autoimmunity , CTLA-4 Antigen/genetics , Lectins, C-Type/geneticsABSTRACT
Waldenström macroglobulinemia (WM) is characterized by bone marrow infiltration with malignant lymphoplasmacytic cells (LPCs), a smaller population of plasma cells (PCs), and hypersecretion of IgM monoclonal protein. Here, we show that CD45low, CD38+, and CD138+ PCs and CD45high, CD38-, CD138-, CD19+, and CD20+ LPCs carry a heterozygous L265P mutation in the Toll-like receptor signaling adaptor MYD88. Both PCs and LPCs express the same auto-reactive IgHV sequences, suggesting a similar clonal origin and role for auto-antigens in WM cell survival. PCs are primarily responsible for IgM production even without substantial cell proliferation. When cultured in isolation, LPCs give rise to more differentiated PCs and secrete less IgM. Our analyses suggest that malignant PCs arise from the clonal LPC population, and are primarily responsible for IgM secretion in WM. Targeting malignant PCs may have therapeutic benefits in the treatment of WM and improve the duration of response and potentially, survival.
ABSTRACT
Inflammasomes are cytosolic signaling complexes capable of sensing microbial ligands to trigger inflammation and cell death responses. Here, we show that guanylate-binding proteins (GBPs) mediate pathogen-selective inflammasome activation. We show that mouse GBP1 and GBP3 are specifically required for inflammasome activation during infection with the cytosolic bacterium Francisella novicida. We show that the selectivity of mouse GBP1 and GBP3 derives from a region within the N-terminal domain containing charged and hydrophobic amino acids, which binds to and facilitates direct killing of F. novicida and Neisseria meningitidis, but not other bacteria or mammalian cells. This pathogen-selective recognition by this region of mouse GBP1 and GBP3 leads to pathogen membrane rupture and release of intracellular content for inflammasome sensing. Our results imply that GBPs discriminate between pathogens, confer activation of innate immunity, and provide a host-inspired roadmap for the design of synthetic antimicrobial peptides that may be of use against emerging and re-emerging pathogens.
Subject(s)
Carrier Proteins , Inflammasomes , Animals , Bacteria/metabolism , Carrier Proteins/metabolism , Cytosol/metabolism , GTP-Binding Proteins/metabolism , Immunity, Innate , Inflammasomes/metabolism , Mammals/metabolism , Mice , Signal TransductionABSTRACT
OBJECTIVES: Roxithromycin, a macrolide antibiotic, has been shown to ameliorate acetic acid induced colitis in rats by suppressing inflammation and oxidative stress. The aim of this study was to evaluate the effect of roxithromycin on small intestinal transit and cholinergic responsiveness of the colonic smooth muscles of colitic rats. METHODS: Colitis was induced in rats by acetic acid and the small intestinal transit was determined by measuring the distance traversed by charcoal meal from the gastro-duodenal junction in 1 h. The test drug roxithromycin, reference drug mesalazine and anti-inflammatory drug diclofenac were administered orally before inducing colitis and their effect on intestinal transit was compared with colitic control group. The effect on cholinergic responsiveness of colonic smooth muscles was evaluated in vitro by plotting a dose-response curve using different concentrations of acetylcholine. The concentration producing 50% of maximal response (EC50) was calculated for all the treatment groups. RESULTS: The small intestinal transit was enhanced in colitic rats as compared to normal rats (86.00 ± 1.36 vs. 57.00 ± 1.34 cm; p<0.001). Like mesalazine, roxithromycin normalized intestinal transit while diclofenac was ineffective. The results of in vitro experiment show that colitis increased cholinergic responsiveness of the colonic smooth muscles that was not affected by roxithromycin and mesalazine while diclofenac significantly decreased it. CONCLUSIONS: This study shows that like mesalazine, roxithromycin affords protection in colitis mainly by normalizing propulsive movement of the small intestine than by affecting cholinergic responsiveness of the colonic smooth muscles.
Subject(s)
Colitis , Roxithromycin , Animals , Colitis/drug therapy , Colon , Inflammation/drug therapy , Muscle, Smooth , Rats , Roxithromycin/adverse effectsABSTRACT
Organelles are critical structures in mediating the assembly and activation of inflammasomes in mammalian cells, resulting in inflammation and cell death. Assembly of inflammasomes can occur at the mitochondria, endoplasmic reticulum, nucleus, trans-Golgi network, or pathogen surface, facilitated by the overarching architecture of the cytoskeleton. NLRP3 and Pyrin inflammasome sensors may form smaller speckles and converge on a single larger speck at the microtubule-organizing center (MTOC). This signaling hub activates multiple mammalian inflammatory and apoptotic caspases, cytokine substrates, the pore-forming protein gasdermin D, and the plasma membrane rupture protein ninjurin-1 (NINJ1), allowing pyroptosis, cellular disintegration, and inflammation to ensue. In this review, we highlight the role of mammalian cell types and organellar architectures in executing inflammasome responses.
Subject(s)
Inflammasomes , NLR Family, Pyrin Domain-Containing 3 Protein , Animals , Caspases/metabolism , Cell Adhesion Molecules, Neuronal , Humans , Inflammasomes/metabolism , Inflammation , Mammals/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Nerve Growth Factors , PyroptosisABSTRACT
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.
Subject(s)
Bacillus cereus/pathogenicity , Enterotoxins/toxicity , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Animals , Bacterial Proteins/toxicity , Cell Line , Enterotoxins/chemistry , Female , Hemolysin Proteins/toxicity , Host Microbial Interactions , Host Specificity , Humans , Inflammasomes/drug effects , Inflammasomes/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Pyroptosis/drug effects , Virulence Factors/toxicityABSTRACT
Calotropis procera, a latex producing plant is known to possess medicinal properties including its beneficial effect in gastrointestinal disorders. The anti-inflammatory effect of its latex in various experimental models is noteworthy and in light of this the present study was carried out with an objective to evaluate its efficacy in ulcerative colitis, an inflammatory condition of the colon. Colitis was induced in rats by acetic acid and the rats were divided into four groups where one group served as experimental control and the other groups were treated with two doses of methanol extract of dried latex of C. procera (MeDL; 50 and 150 mg/kg) and mesalazine (MSZ; 300 mg/kg). The study also included normal control (NC) group for comparison of various parameters related to colon like macroscopic changes, ulcer score, adherent mucus content, weight/length ratio, small intestinal transit, oxidative stress and inflammatory markers, tissue histology and immunoreactivity of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and nuclear factor kappa beta (NFκB) subunit p65. Treatment of colitic rats with MeDL produced a significant reduction in colonic mucosal damage as revealed by macroscopic and microscopic evaluation and normalization of tissue levels of oxidative stress markers and pro-inflammatory mediators. The protection afforded by MeDL was also evident from its restorative effect on tissue histology and expression of COX-2, iNOS and NFκB(p65). This study shows that by targeting oxidative stress and NFκB(p65) mediated pro-inflammatory signaling, the latex of C. procera affords protection in colitis and its effect was comparable to that of mesalazine. This study suggests that latex of C. procera could serve as a promising therapeutic option for treating inflammatory conditions of the colon.
Subject(s)
Calotropis , Colitis/metabolism , Colitis/prevention & control , Inflammation Mediators/metabolism , Latex/therapeutic use , Oxidative Stress/drug effects , Acetic Acid/toxicity , Animals , Colitis/chemically induced , Disease Models, Animal , Female , Inflammation/chemically induced , Inflammation/drug therapy , Inflammation/metabolism , Inflammation Mediators/antagonists & inhibitors , Latex/isolation & purification , Latex/pharmacology , Male , Methanol/pharmacology , Methanol/therapeutic use , Oxidative Stress/physiology , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , Plant Extracts/therapeutic use , Rats , Rats, Wistar , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
Metformin, an antidiabetic drug, is well known for its multifarious properties and its ability to modulate inflammatory cascade. Ulcerative colitis (UC) is an inflammatory condition of the colon where drugs exhibiting anti-inflammatory property have been shown to induce and maintain remission. The objective of the present study was to evaluate the efficacy of metformin against acetic-acid induced colitis in rat. The study included five groups of rats namely normal control, experimental control, drug treated groups (50 and 500mg/kg of metformin, MET50, MET500 and 300mg/kg of mesalazine, MSZ300). Parameters like small intestinal transit and colonic macroscopic changes, ulcer score, weight/length (W/L) ratio, levels of oxidative stress and inflammatory markers, tissue histology and expression of COX-2, iNOS, NFκB(p65) were evaluated. The results of this study show that treatment with metformin significantly decreased colonic mucosal damage, maintained oxidative homeostasis and normalized intestinal transit and W/L ratio in a dose-dependent manner. The restorative effect of metformin on colonic mucosa was accompanied by a marked reduction in the tissue levels of pro-inflammatory mediators and immunoreactivity of COX-2, iNOS and NFκB(p65). Further, its protective effect was found to be comparable to that of mesalazine. This study shows that metformin targets oxidative stress and down regulates transcription factor NFκB(p65) mediated pro-inflammatory signaling and has a therapeutic potential in treating inflammatory conditions of the colon.