ORMDL3 regulates NLRP3 inflammasome activation by maintaining ER-mitochondria contacts in human macrophages and dictates ulcerative colitis patient outcome.

Genome-wide association studies in inflammatory bowel disease have identified risk loci in the orosomucoid-like protein 3/ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3) gene to confer susceptibility to ulcerative colitis (UC), but the underlying functional relevance remains unexplored. Here, we found that a subpopulation of the UC patients who had higher disease activity shows enhanced expression of ORMDL3 compared to the patients with lower disease activity and the non-UC controls. We also found that the patients showing high ORMDL3 mRNA expression have elevated interleukin-1ß cytokine levels indicating positive correlation. Further, knockdown of ORMDL3 in the human monocyte-derived macrophages resulted in significantly reduced interleukin-1ß release. Mechanistically, we report for the first time that ORMDL3 contributes to a mounting inflammatory response via modulating mitochondrial morphology and activation of the NLRP3 inflammasome. Specifically, we observed an increased fragmentation of mitochondria and enhanced contacts with the endoplasmic reticulum (ER) during ORMDL3 over-expression, enabling efficient NLRP3 inflammasome activation. We show that ORMDL3 that was previously known to be localized in the ER also becomes localized to mitochondria-associated membranes and mitochondria during inflammatory conditions. Additionally, ORMDL3 interacts with mitochondrial dynamic regulating protein Fis-1 present in the mitochondria-associated membrane. Accordingly, knockdown of ORMDL3 in a dextran sodium sulfate -induced colitis mouse model showed reduced colitis severity. Taken together, we have uncovered a functional role for ORMDL3 in mounting inflammation during UC pathogenesis by modulating ER-mitochondrial contact and dynamics.

Cooperative STAT3-NFkB signaling modulates mitochondrial dysfunction and metabolic profiling in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) continues to pose a significant health challenge and is often diagnosed at advanced stages. Metabolic reprogramming is a hallmark of many cancer types, including HCC and it involves alterations in various metabolic or nutrient-sensing pathways within liver cells to facilitate the rapid growth and progression of tumours. However, the role of STAT3-NFκB in metabolic reprogramming is still not clear. APPROACH AND RESULTS: Diethylnitrosamine (DEN) administered animals showed decreased body weight and elevated level of serum enzymes. Also, Transmission electron microscopy (TEM) analysis revealed ultrastructural alterations. Increased phosphorylated signal transducer and activator of transcription-3 (p-STAT3), phosphorylated nuclear factor kappa B (p-NFκß), dynamin related protein 1 (Drp-1) and alpha-fetoprotein (AFP) expression enhance the carcinogenicity as revealed in immunohistochemistry (IHC). The enzyme-linked immunosorbent assay (ELISA) concentration of IL-6 was found to be elevated in time dependent manner both in blood serum and liver tissue. Moreover, immunoblot analysis showed increased level of p-STAT3, p-NFκß and IL-6 stimulated the upregulation of mitophagy proteins such as Drp-1, Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK-1). Meanwhile, downregulation of Poly [ADP-ribose] polymerase 1 (PARP-1) and cleaved caspase 3 suppresses apoptosis and enhanced expression of AFP supports tumorigenesis. The mRNA level of STAT3 and Drp-1 was also found to be significantly increased. Furthermore, we performed high-field 800 MHz Nuclear Magnetic Resonance (NMR) based tissue and serum metabolomics analysis to identify metabolic signatures associated with the progression of liver cancer. The metabolomics findings revealed aberrant metabolic alterations in liver tissue and serum of 75th and 105th days of intervention groups in comparison to control, 15th and 45th days of intervention groups. Tissue metabolomics analysis revealed the accumulation of succinate in the liver tissue samples, whereas, serum metabolomics analysis revealed significantly decreased circulatory levels of ketone bodies (such as 3-hydroxybutyrate, acetate, acetone, etc.) and membrane metabolites suggesting activated ketolysis in advanced stages of liver cancer. CONCLUSION: STAT3-NFκß signaling axis has a significant role in mitochondrial dysfunction and metabolic alterations in the development of HCC.

CLUH functions as a negative regulator of inflammation in human macrophages and determines ulcerative colitis pathogenesis.

Altered mitochondrial function without a well-defined cause has been documented in patients with ulcerative colitis (UC). In our efforts to understand UC pathogenesis, we observed reduced expression of clustered mitochondrial homolog (CLUH) only in the active UC tissues compared with the unaffected areas from the same patient and healthy controls. Stimulation with bacterial Toll-like receptor (TLR) ligands similarly reduced CLUH expression in human primary macrophages. Further, CLUH negatively regulated secretion of proinflammatory cytokines IL-6 and TNF-α and rendered a proinflammatory niche in TLR ligand-stimulated macrophages. CLUH was further found to bind to mitochondrial fission protein dynamin related protein 1 (DRP1) and regulated DRP1 transcription in human macrophages. In the TLR ligand-stimulated macrophages, absence of CLUH led to enhanced DRP1 availability for mitochondrial fission, and a smaller dysfunctional mitochondrial pool was observed. Mechanistically, this fissioned mitochondrial pool in turn enhanced mitochondrial ROS production and reduced mitophagy and lysosomal function in CLUH-knockout macrophages. Remarkably, our studies in the mouse model of colitis with CLUH knockdown displayed exacerbated disease pathology. Taken together, this is the first report to our knowledge explaining the role of CLUH in UC pathogenesis, by means of regulating inflammation via maintaining mitochondrial-lysosomal functions in the human macrophages and intestinal mucosa.

Role of mitochondria in regulating immune response during bacterial infection.

Mitochondria are dynamic organelles of eukaryotes involved in energy production and fatty acid oxidation. Besides maintaining ATP production, calcium signaling, cellular apoptosis, and fatty acid synthesis, mitochondria are also known as the central hub of the immune system as it regulates the innate immune pathway during infection. Mitochondria mediated immune functions mainly involve regulation of reactive oxygen species production, inflammasome activation, cytokine secretion, and apoptosis of infected cells. Recent findings indicate that cellular mitochondria undergo constant biogenesis, fission, fusion and degradation, and these dynamics regulate cellular immuno-metabolism. Several intracellular pathogens target and modulate these normal functions of mitochondria to facilitate their own survival and growth. De-regulation of mitochondrial functions and dynamics favors bacterial infection and pathogens are able to protect themselves from mitochondria mediated immune responses. Here, we will discuss how mitochondria mediated anti-bacterial immune pathways help the host to evade pathogenic insult. In addition, examples of bacterial pathogens modulating mitochondrial metabolism and dynamics will also be elaborated. Study of these interactions between the mitochondria and bacterial pathogens during infection will lead to a better understanding of the mitochondrial metabolism pathways and dynamics important for the establishment of bacterial diseases. In conclusion, detailed studies on how mitochondria regulate the immune response during bacterial infection can open up new avenues to develop mitochondria centric anti-bacterial therapeutics.

Role of mitochondrial dysfunction in the pathogenesis of rheumatoid arthritis: Looking closely at fibroblast- like synoviocytes.

Rheumatoid arthritis (RA) is a chronic, autoimmune, and inflammatory disease that primarily targets the joints, leading to cartilage and bone destruction.Fibroblast-like synoviocytes (FLS) are specialized cells of the synovial lining in the joint that plays a fundamental role in the development of RA. Particularly, FLS of RA patients (RA-FLS) in the joint exhibit specific characteristics like higher invading and immunogenic properties, hyperproliferation, and reduced apoptotic capacity, suggesting a dysfunctional mitochondrial pool in these cells. Mitochondria are emerging as a potential organelle that can decide cellular immunometabolism, invasion properties, and cell death. Accordingly, multiplestudies established that mitochondria are crucial in establishing RA. However, the underlying mechanism of impaired mitochondrial function in RA remains poorly understood. This review will provide an overview of the mitochondrial role in the progression of RA, specifically in the context of FLS biology. We will also outline how mitochondria-centric therapeutics can be achieved that would yield novel avenues of research in pathological mediation and prevention.

Dynamic scaling and stochastic fractal in nucleation and growth processes.

A class of nucleation and growth models of a stable phase is investigated for various different growth velocities. It is shown that for growth velocities v ≈ s ( t ) / t and v ≈ x / τ ( x ), where s ( t ) and τ are the mean domain size of the metastable phase (M-phase) and the mean nucleation time, respectively, the M-phase decays following a power law. Furthermore, snapshots at different time t that are taken to collect data for the distribution function c ( x , t ) of the domain size x of the M-phase are found to obey dynamic scaling. Using the idea of data-collapse, we show that each snapshot is a self-similar fractal. However, for v = const ., such as in the classical Kolmogorov-Johnson-Mehl-Avrami model, and for v ≈ 1 / t, the decays of the M-phase are exponential and they are not accompanied by dynamic scaling. We find a perfect agreement between numerical simulation and analytical results.

A machine learning-based approach to determine infection status in recipients of BBV152 (Covaxin) whole-virion inactivated SARS-CoV-2 vaccine for serological surveys.

Data science has been an invaluable part of the COVID-19 pandemic response with multiple applications, ranging from tracking viral evolution to understanding the vaccine effectiveness. Asymptomatic breakthrough infections have been a major problem in assessing vaccine effectiveness in populations globally. Serological discrimination of vaccine response from infection has so far been limited to Spike protein vaccines since whole virion vaccines generate antibodies against all the viral proteins. Here, we show how a statistical and machine learning (ML) based approach can be used to discriminate between SARS-CoV-2 infection and immune response to an inactivated whole virion vaccine (BBV152, Covaxin). For this, we assessed serial data on antibodies against Spike and Nucleocapsid antigens, along with age, sex, number of doses taken, and days since last dose, for 1823 Covaxin recipients. An ensemble ML model, incorporating a consensus clustering approach alongside the support vector machine model, was built on 1063 samples where reliable qualifying data existed, and then applied to the entire dataset. Of 1448 self-reported negative subjects, our ensemble ML model classified 724 to be infected. For method validation, we determined the relative ability of a random subset of samples to neutralize Delta versus wild-type strain using a surrogate neutralization assay. We worked on the premise that antibodies generated by a whole virion vaccine would neutralize wild type more efficiently than delta strain. In 100 of 156 samples, where ML prediction differed from self-reported uninfected status, neutralization against Delta strain was more effective, indicating infection. We found 71.8% subjects predicted to be infected during the surge, which is concordant with the percentage of sequences classified as Delta (75.6%-80.2%) over the same period. Our approach will help in real-world vaccine effectiveness assessments where whole virion vaccines are commonly used.

FOXO3a acetylation regulates PINK1, mitophagy, inflammasome activation in murine palmitate-conditioned and diabetic macrophages.

Nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing 3 inflammasome and mitophagy play an important role in cytokine release and diabetes progression; however, the role of saturated fatty acid that is induced under such conditions remains little explored. Therefore, the present study evaluates mechanisms regulating mitophagy and inflammasome activation in primary murine diabetic and palmitate-conditioned wild-type (WT) peritoneal macrophages. Peritoneal macrophage, from the diabetic mice and WT mice, challenged with LPS/ATP and palmitate/LPS/ATP, respectively, showed dysfunctional mitochondria as assessed by their membrane potential, mitochondrial reactive oxygen species (mtROS) production, and mitochondrial DNA (mtDNA) release. A defective mitophagy was observed in the diabetic and palmitate-conditioned macrophages stimulated with LPS/ATP as assessed by translocation of PTEN-induced kinase 1 (PINK1)/Parkin or p62 in the mitochondrial fraction. Consequently, increased apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) oligomerization, caspase-1 activation, and IL1ß secretion were observed in LPS/ATP stimulated diabetic and palmitate-conditioned macrophages. LPS/ATP induced Forkhead box O3a (FOXO3a) binding to PINK1 promoter and increased PINK1 mRNA expression in WT macrophages. However, PINK1 mRNA and protein expression were significantly decreased in diabetic and palmitate-conditioned macrophages in response to LPS/ATP. Palmitate-induced acetyl CoA promoted FOXO3a acetylation, which prevented LPS/ATP-induced FOXO3a binding to the PINK1 promoter. C646 (P300 inhibitor) and SRT1720 (SIRT1 activator) prevented FOXO3a acetylation and restored FOXO3a binding to the PINK1 promoter, PINK1 mRNA expression, and mitophagy in palmitate-conditioned macrophages treated with LPS/ATP. Also, a significant decrease in the LPS/ATP-induced mtROS production, mtDNA release, ASC oligomerization, caspase-1 activation, and IL-1ß release was observed in the palmitate-conditioned macrophages. Similarly, modulation of FOXO3a acetylation also prevented LPS/ATP-induced mtDNA release and inflammasome activation in diabetic macrophages. Therefore, FOXO3a acetylation regulates PINK1-dependent mitophagy and inflammasome activation in the palmitate-conditioned and diabetic macrophages.

Estradiol overcomes adiponectin-resistance in diabetic mice by regulating skeletal muscle adiponectin receptor 1 expression.

Adiponectin and insulin resistance creates a vicious cycle that exacerbates type 2 diabetes. Earlier, we observed that female leptin receptor-deficient BLKS mice (BKS-db/db) were more sensitive to an adiponectin mimetic GTDF than males, which led us to explore if E2 plays a crucial role in modulation of adiponectin-sensitivity. Male but not female BKS-db/db mice were resistant to metabolic effects of globular adiponectin treatment. Male BKS-db/db displayed reduced skeletal muscle AdipoR1 protein expression, which was consequent to elevated polypyrimidine tract binding protein 1 (PTB) and miR-221. E2 treatment in male BKS-db/db, and ovariectomized BALB/c mice rescued AdipoR1 protein expression via downregulation of PTB and miR-221, and also directly increased AdipoR1 mRNA by its classical nuclear receptors. Estrogen receptor regulation via dietary or pharmacological interventions may improve adiponectin resistance and consequently ameliorate insulin resistance in type 2 diabetes.

Insights from a Pan India Sero-Epidemiological survey (Phenome-India Cohort) for SARS-CoV2.

To understand the spread of SARS-CoV2, in August and September 2020, the Council of Scientific and Industrial Research (India) conducted a serosurvey across its constituent laboratories and centers across India. Of 10,427 volunteers, 1058 (10.14%) tested positive for SARS-CoV2 anti-nucleocapsid (anti-NC) antibodies, 95% of which had surrogate neutralization activity. Three-fourth of these recalled no symptoms. Repeat serology tests at 3 (n = 607) and 6 (n = 175) months showed stable anti-NC antibodies but declining neutralization activity. Local seropositivity was higher in densely populated cities and was inversely correlated with a 30-day change in regional test positivity rates (TPRs). Regional seropositivity above 10% was associated with declining TPR. Personal factors associated with higher odds of seropositivity were high-exposure work (odds ratio, 95% confidence interval, p value: 2.23, 1.92-2.59, <0.0001), use of public transport (1.79, 1.43-2.24, <0.0001), not smoking (1.52, 1.16-1.99, 0.0257), non-vegetarian diet (1.67, 1.41-1.99, <0.0001), and B blood group (1.36, 1.15-1.61, 0.001).

6,7-Dihydroxycoumarin ameliorates crystal-induced necroptosis during crystal nephropathies by inhibiting MLKL phosphorylation.

AIMS: Mineralization of crystalline particles and the formation of renal calculi contribute to the pathogenesis of crystal nephropathies. Several recent studies on the biology of crystal handling implicated intrarenal crystal deposition-induced necroinflammation in their pathogenesis. We hypothesized that 6,7-dihydroxycoumarin (DHC) inhibit intrarenal crystal cytotoxicity and necroinflammation, and ameliorate crystal-induced chronic kidney disease (CKD). MAIN METHODS: An unbiased high content screening coupled with fluorescence microscopy was used to identify compounds that inhibit CaOx crystal cytotoxicity. The ligand-protein interactions were identified using computational models e.g. molecular docking and molecular dynamics simulations. Furthermore, mice and rat models of oxalate-induced CKD were used for in-vivo studies. Renal injury, crystal deposition, and fibrosis were assessed by histology analysis. Western blots were used to quantify the protein expression. Data were expressed as boxplots and analyzed using one way ANOVA. KEY FINDINGS: An unbiased high-content screening in-vitro identified 6,7-DHC as a promising candidate. Further, 6,7-DHC protected human and mouse cells from calcium oxalate (CaOx) crystal-induced necroptosis in-vitro as well as mice and rats from oxalate-induced CKD in either preventive or therapeutic manner. Computational modeling demonstrated that 6,7-DHC interact with MLKL, the key protein in the necroptosis machinery, and inhibit its phosphorylation by ATP, which was evident in both in-vitro and in-vivo analyses. SIGNIFICANCE: Together, our results indicate that 6,7-DHC possesses a novel pharmacological property as a MLKL inhibitor and could serve as a lead molecule for further development of coumarin-based novel MLKL inhibitors. Furthermore, our data identify 6,7-DHC as a novel therapeutic strategy to combat crystal nephropathies.

Modulation of host mitochondrial dynamics during bacterial infection.

Mitochondria is a dynamic organelle of the cell that can regulate and maintain cellular ATP level, ROS production, calcium signaling and immune response. In order to retain their shape and distribution, mitochondria go through coordinated cycles of fission and fusion. Further, dysfunctional mitochondria are selectively eliminated from the cell via mitophagy to synchronize mitochondrial quality control and cellular homeostasis. In addition, mitochondria when in close proximity with the endoplasmic reticulum can alter the signaling pathways and some recent findings also reveal a direct correlation between the mitochondrial localization in the cell to the immune response elicited against the invading pathogen. These modulations in the mitochondrial network are collectively termed as 'mitochondrial dynamics'. Diverse bacteria, virus and parasitic pathogens upon infecting a cell can alter the host mitochondrial dynamics in favor of their multiplication and this in turn can be a major determinant of the disease outcome. Pharmacological perturbations in these pathways thus could lead to generation of additional therapeutic opportunities. This review will focus on the pathogenic modulation of the host mitochondrial dynamics, specifically during the bacterial infections and describes how dysregulated mitochondrial dynamics facilitates the pathogen's ability to establish efficient infection.

Standardized Xylocarpus moluccensis fruit fraction mitigates collagen-induced arthritis in mice by regulating immune response.

OBJECTIVE: This study was undertaken to evaluate the effect of Xylocarpus moluccensis fruit fraction (F018) on the pathogenesis of collagen-induced arthritis in mice. METHODS: Arthritis was induced by intradermal injection of collagen (2 mg/ml) with complete Freund's adjuvant in DBA/1J mice. F018 was administered orally at 1, 3 and 10 mg/kg for 20 days. Disease progression and mechanism were assessed by micro-CT analysis, RT-PCR, flow cytometry assay, myeloperoxidase (MPO) and MTT assay. RESULTS: F018 at 3 and 10 mg/kg significantly reduced paw thickness, clinical score, mononuclear cell infiltration and collagen layer depletion in the knee section of collagen-induced arthritis (CIA) mice when compared with collagen-induced arthritis mice alone. Furthermore, F018 treatment in collagen-induced arthritis mice significantly recovered bone volume and trabecular number and decreased the trabecular space by modulating RANKL and OPG mRNA expression in the synovial tissue. F018 treatment in collagen-induced arthritis mice significantly attenuated spleen index, lymphocyte proliferation and paw myeloperoxidase (MPO) activity, pro-inflammatory cytokine TNFα, IL1ß, and IL6 mRNA expression and enhanced IL10 mRNA expression in paw tissue. Furthermore, F018 treatment in collagen-induced arthritis mice significantly reduced splenic dendritic cell maturation and Th17 cells. In culture, F018 significantly decreased collagen-induced arthritis-FLS proliferation and promoted apoptosis. CONCLUSION: F018 may serve as a potential curative agent for arthritis.

The gut-liver-kidney axis: Novel regulator of fatty liver associated chronic kidney disease.

Increased interest in understanding the liver-kidney axis in health and disease during the last decade unveiled multiple recent evidence that suggested a strong association of fatty liver diseases with chronic kidney disease (CKD). Low-grade systemic inflammation is thought to be the major contributing factor to the pathogenesis of CKD associated with fatty liver. However, other contributing factors largely remained unclear, for example, gut microbiota and intestinal barrier integrity. Homeostasis of the gut microbiome is very crucial for the health of an individual. Imbalance in the gut microbiota leads to various diseases like fatty liver disease and CKD. On the contrary, disease conditions can also distinctly change gut microbiota. In this review, we propose the pathogenic role of the gut-liver-kidney axis in the development and progression of CKD associated with chronic fatty liver diseases, either non-alcoholic fatty liver disease or non-alcoholic steatohepatitis in experimental models and humans. Further, we discuss the therapeutic potential and highlight the future research directions for therapeutic targeting of the gut-liver-kidney axis.

Huntingtin-interacting protein 1 (HIP1) regulates arthritis severity and synovial fibroblast invasiveness by altering PDGFR and Rac1 signalling.

OBJECTIVES: While new treatments for rheumatoid arthritis (RA) have markedly improved disease control by targeting immune/inflammatory pathways, current treatments rarely induce remission, underscoring the need for therapies that target other aspects of the disease. Little is known about the regulation of disease severity and joint damage, which are major predictors of disease outcome, and might be better or complementary targets for therapy. In this study, we aimed to discover and characterise a new arthritis severity gene. METHODS: An unbiased and phenotype-driven strategy including studies of unique congenic rat strains was used to identify new arthritis severity and joint damage genes. Fibroblast-like synoviocytes (FLS) from rats and patients with RA expressing or not Huntingtin-interacting protein 1 (HIP1) were studied for invasiveness, morphology and cell signalling. HIP1 knockout mice were used in in vivo confirmatory studies. Paired t-test was used. RESULTS: DNA sequencing and subcongenic strains studied in pristane-induced arthritis identified a new amino acid changing functional variant in HIP1. HIP1 was required for the increased invasiveness of FLS from arthritic rats and from patients with RA. Knocking down HIP1 expression reduced receptor tyrosine kinase-mediated responses in RA FLS, including RAC1 activation, affecting actin cytoskeleton and cell morphology and interfering with the formation of lamellipodia, consistent with reduced invasiveness. HIP1 knockout mice were protected in KRN serum-induced arthritis and developed milder disease. CONCLUSION: HIP1 is a new arthritis severity gene and a potential novel prognostic biomarker and target for therapy in RA.

Human LACC1 increases innate receptor-induced responses and a LACC1 disease-risk variant modulates these outcomes.

Functional consequences for most inflammatory disease-associated loci are incompletely defined, including in the LACC1 (C13orf31) region. Here we show that human peripheral and intestinal myeloid-derived cells express laccase domain-containing 1 (LACC1); LACC1 is expressed in both the cytoplasm and mitochondria. Upon NOD2 stimulation of human macrophages, LACC1 associates with the NOD2-signalling complex, and is critical for optimal NOD2-induced signalling, mitochondrial ROS (mtROS) production, cytokine secretion and bacterial clearance. LACC1 constitutively associates with succinate dehydrogenase (SDH) subunit A, and amplifies pattern recognition receptor (PRR)-induced SDH activity, an important contributor to mtROS production. Relative to LACC1 Ile254, cells transfected with Crohn's disease-risk LACC1 Val254 or LACC1 with mutations of the nearby histidines (249,250) have reduced PRR-induced outcomes. Relative to LACC1 Ile254 carriers, Val254 disease-risk carrier macrophages demonstrate decreased PRR-induced mtROS, signalling, cytokine secretion and bacterial clearance. Therefore, LACC1 is critical for amplifying PRR-induced outcomes, an effect that is attenuated by the LACC1 disease-risk variant.

Peptide utilizing carbon starvation gene yjiY is required for flagella mediated infection caused by Salmonella.

Peptide metabolism forms an important part of the metabolic network of Salmonella and to acquire these peptides the pathogen possesses a number of peptide transporters. While various peptide transporters known in Salmonella are well studied, very little is known about the carbon starvation (cst) genes, cstA and yjiY, which are also predicted to be involved in peptide metabolism. We investigated the role of these genes in the metabolism and pathogenesis of Salmonella and demonstrated for the first time that cst genes actually participate in transport of specific peptides in Salmonella. Further, we established that the carbon starvation gene yjiY affects the expression of flagella leading to poor adhesion of the bacterium to host cells. In contrast with the previously reported role of the gene cstA in virulence of Salmonella in C. elegans, we showed that yjiY is required for successful colonization of Salmonella in the mouse gut. Thus, cst genes not only contribute to the metabolism of Salmonella but also influence its virulence.

MTMR3 risk allele enhances innate receptor-induced signaling and cytokines by decreasing autophagy and increasing caspase-1 activation.

Inflammatory bowel disease (IBD) is characterized by dysregulated host:microbial interactions and cytokine production. Host pattern recognition receptors (PRRs) are critical in regulating these interactions. Multiple genetic loci are associated with IBD, but altered functions for most, including in the rs713875 MTMR3/HORMAD2/LIF/OSM region, are unknown. We identified a previously undefined role for myotubularin-related protein 3 (MTMR3) in amplifying PRR-induced cytokine secretion in human macrophages and defined MTMR3-initiated mechanisms contributing to this amplification. MTMR3 decreased PRR-induced phosphatidylinositol 3-phosphate (PtdIns3P) and autophagy levels, thereby increasing PRR-induced caspase-1 activation, autocrine IL-1ß secretion, NFκB signaling, and, ultimately, overall cytokine secretion. This MTMR3-mediated regulation required the N-terminal pleckstrin homology-GRAM domain and Cys413 within the phosphatase domain of MTMR3. In MTMR3-deficient macrophages, reducing the enhanced autophagy or restoring NFκB signaling rescued PRR-induced cytokines. Macrophages from rs713875 CC IBD risk carriers demonstrated increased MTMR3 expression and, in turn, decreased PRR-induced PtdIns3P and autophagy and increased PRR-induced caspase-1 activation, signaling, and cytokine secretion. Thus, the rs713875 IBD risk polymorphism increases MTMR3 expression, which modulates PRR-induced outcomes, ultimately leading to enhanced PRR-induced cytokines.

T cell-extrinsic CD18 attenuates antigen-dependent CD4+ T cell activation in vivo.

The ß2 integrins (CD11/CD18) are heterodimeric leukocyte adhesion molecules expressed on hematopoietic cells. The role of T cell-intrinsic CD18 in trafficking of naive T cells to secondary lymphoid organs and in Ag-dependent T cell activation in vitro and in vivo has been well defined. However, the T cell-extrinsic role for CD18, including on APC, in contributing to T cell activation in vivo is less well understood. We examined the role for T cell-extrinsic CD18 in the activation of wild-type CD4(+) T cells in vivo through the adoptive transfer of DO11.10 Ag-specific CD4(+) T cells into CD18(-/-) mice. We found that T cell-extrinsic CD18 was required for attenuating OVA-induced T cell proliferation in peripheral lymph nodes (PLN). The increased proliferation of wild-type DO11.10 CD4(+) T cells in CD18(-/-) PLN was associated with a higher percentage of APC, and these APC demonstrated an increased activation profile and increased Ag uptake, in particular in F4/80(+) APC. Depletion of F4/80(+) cells both reduced and equalized Ag-dependent T cell proliferation in CD18(-/-) relative to littermate control PLN, demonstrating that these cells play a critical role in the enhanced T cell proliferation in CD18(-/-) mice. Consistently, CD11b blockade, which is expressed on F4/80(+) macrophages, enhanced the proliferation of DO11.10 CD4(+) T cells in CD18(+/-) PLN. Thus, in contrast to the T cell-intrinsic essential role for CD18 in T cell activation, T cell-extrinsic expression of CD18 attenuates Ag-dependent CD4(+) T cell activation in PLN in vivo.