Lamina Propria Macrophages Research Articles

MKL1 expressed in macrophages contributes to the development of murine colitis

Mice deficient in the megakaryoblastic leukaemia 1 (Mkl1) gene experience less severe dextran sulphate sodium (DSS)-induced colitis, implying that Mkl1 plays a pathological role in inflammatory bowel disease (IBD). However, the contribution of Mkl1 to the development of colitis remains to be elucidated. The expression of Mkl1 is higher in the colonic lamina propria macrophages (LPMac) of DSS-treated mice than in those of control mice. Therefore, we established a transgenic mouse line that overexpresses human MKL1 (MKL1-Tg) specifically in cells of the monocyte/macrophage lineage, in order to investigate the potential role of macrophage MKL1 in the pathogenesis of colitis. MKL1-Tg mice displayed spontaneous colon shortening and rectal prolapse. Flow cytometric and quantitative RT-PCR analyses revealed that, in MKL1-Tg mice compared to littermate controls, the population of LPMac was decreased and had an altered inflammatory phenotype indicative of impaired anti-inflammatory properties, whereas bone marrow-derived macrophages from MKL1-Tg mice skewed towards M1 polarisation. In addition, MKL1-Tg mice had higher susceptibility to DSS-induced colitis than their littermate controls. These observations indicated that MKL1 crucially contributes to the development of colitis via the regulation of the function of macrophages, suggesting that it may be a potential therapeutic target for the prevention of IBD.

Gastric and Duodenal Pseudomelanosis in a Patient With Chronic Kidney Disease and Oral Iron Supplementation

A 74 year-old lady has a history of hypertension (HTN), diabetes mellitus (DM), mild chronic kidney disease, coronary artery disease, cerebrovascular accident, osteoarthritis, Iron deficiency anemia and gastroesophageal reflux disease. Her medications include ferrous sulphate and hydralazine. She had an upper GI endoscopy for evaluation of iron deficiency anemia and refractory GERD. Endoscopy showed dark, pigmented, granular appearing mucosa in gastric antrum, lesser curvature of gastric body and duodenal bulb (pseudomelanosis). Pathology from gastric biopsies showed reactive gastropathy and gastric siderosis with deposits of granular, darkly pigmented material showing stromal predominant deposition with presence in macrophages and epithelium. This material stained positive with Prussian blue stain and was negative for Melan-A stain. Pseudomelanosis of stomach and duodenum is associated with various clinical conditions including HTN, DM, chronic kidney disease, gastrointestinal hemorrhage and use of various medications including ferrous sulphate, furosemide, hydralazine, propranolol and hydrochlorothiazide. Differential diagnosis includes hemosiderosis/hemochromatosis, brown bowel syndrome and malignant melanoma, which could be differentiated by using special stains and immunohistochemical markers. Histologically, iron pigments are deposited in lysosomes of macrophages in lamina propria. These pigments are detected by Prussian blue Stain and Fontana-Masson stains of macrophages in the lamina propria. It had been seen mostly in older women similar to our patient. Most cases are asymptomatic, however dyspeptic symptoms have been described occasionally. Long term prognosis and consequences of this condition is unknown. No specific treatment is recommended.Figure: EGD revealed Pseudomelanosis in the stomach.Figure: This material stains positive in areas with a Prussian blue iron immunohistochemical stain and is negative for Melan-A stain.Figure: Deposits of granular, darkly pigmented material displays a nonspecific stromal and macrophage predominant pattern.

The Stimulation of Macrophages with TLR Ligands Supports Increased IL-19 Expression in Inflammatory Bowel Disease Patients and in Colitis Models.

IL-19, a member of the IL-10 cytokine family that signals through the IL-20 receptor type I (IL-20Rα:IL-20Rβ), is a cytokine whose function is not completely known. In this article, we show that the expression of IL19 in biopsies of patients with active ulcerative colitis was increased compared with patients with quiescent ulcerative colitis and that colitis was attenuated in IL-19-deficient mice. The disruption of the epithelial barrier with dextran sodium sulfate leads to increased IL-19 expression. Attenuated colitis in IL-19-deficient animals was associated with reduced numbers of IL-6-producing macrophages in the inflamed colonic lamina propria. Microbial-driven expression of IL-19 by intestinal macrophages may contribute to the pathogenesis of inflammatory bowel disease.

Rare Finding of Concomitant Pseudomelanosis of Stomach and Duodenum

Introduction: Pseudomelanosis (PM) of the stomach and duodenum is a rare entity and a striking finding diagnosed by upper endoscopy. It is characterized by dark pigmented stripes that, while benign in nature, are histologically composed of pigment-laden macrophages. We describe the case of an 83-year-old woman, with a complicated medical history, who was was found to have both pseudomelanosis gastri and duodeni. Although both are rare, gastric pseudomelanosis appears to be even more unusual than duodenal pseudomelanosis, with few reported cases documented in the literature thus far. Case Presentation: An 83-year-old woman past medical history of atrial fibrillation, chronic obstructive pulmonary disease, pulmonary hypertension, heart failure, non-Hodgkin Lymphoma, monoclonal gammopathy of undetermined significance (MGUS) who was referred to gastroenterology by cardiology for a determination of her bleeding risk in the context of an episode of self resolved gastrointestinal bleeding (melena) while on anticoagulation (Rivaroxaban). Patient underwent a diagnostic upper endoscopy which revealed mucosal changes in the stomach and duodenum consistent with pseudomelanosis (Figure 1&2). Pathology report revealed of pigment-laden macrophages in the lamina propria.Figure: Pseudomelanosis in the gastric antrum.Figure: Pseudomelanosis in the duodenum.Discussion: Although a benign finding, gastric and duodenal pseudomelanosis is such an infrequent and manifest condition that it can lead to an extensive, expensive, and ultimately unnecessary work-up. It is very rare to see both pseudomelanosis of stomach an duodenum in the same patient. It involves the collection of dark pigmented granules inside the macrophages of the duodenal villi's lamina propria, usually within the apical tips. Electron probe microanalysis has shown that these macrophages often contain lipomelanin, ceroid, iron sulfide, and hemosiderin. However, gastric and duodenal pseudomelanosis represents a diagnostic challenge for physicians because the precise etiology and clinical significance of this condition remain to be determined. It is typically seen in older women and has been associated with several clinical conditions, some of which include: hypertension, hemochromatosis, diabetes, chronic renal disease, and gastrointestinal bleeding. Gastric and duodenal pseudomelanosis has also been seen in connection with medications containing iron sulfate as well as certain antihypertensive medications, like furosemide, hydrochlorothiazide, propranolol, and hydralazine.

Heme-oxygenase-1 Production by Intestinal CX3CR1+ Macrophages Helps to Resolve Inflammation and Prevents Carcinogenesis.

CX3CR1+ macrophages in the intestinal lamina propria contribute to gut homeostasis through the immunomodulatory interleukin IL10, but there is little knowledge on how these cells or the CX3CR1 receptor may affect colorectal carcinogenesis. In this study, we show that CX3CR1-deficient mice fail to resolve gut inflammation despite high production of IL10 and have increased colitis and adenomatous polyps in chemical and genetic models of colon carcinogenesis. Mechanistically, CX3CL1-mediated engagement of the CX3CR1 receptor induced upregulation of heme-oxygenase-1 (HMOX-1), an antioxidant and anti-inflammatory enzyme. CX3CR1-deficient mice exhibited significantly lower expression of HMOX-1 in their adenomatous colon tissues. Combining LPS and CX3CL1 displayed a strong synergistic effect in vitro, but HMOX-1 levels were significantly lower in KO macrophages. Cohousing of wild-type and CX3CR1-/- mice during the AOM/DSS treatment attenuated disease severity in CX3CR1-/- mice, indicating the importance of the microbiome, but did not fully reinstate HMOX-1 levels and did not abolish polyp formation. In contrast, pharmacologic induction of HMOX-1 in vivo by cobalt protoporphyrin-IX treatment eradicated intestinal inflammation and fully protected KO mice from carcinogenesis. Taken together, our results establish an essential role for the receptor CX3CR1 in gut macrophages in resolving inflammation in the intestine, where it helps protects against colitis-associated cancer by regulating HMOX-1 expression. Cancer Res; 77(16); 4472-85. ©2017 AACR.

The Bile Acid Receptor GPBAR1 Regulates the M1/M2 Phenotype of Intestinal Macrophages and Activation of GPBAR1 Rescues Mice from Murine Colitis

GPBAR1 (TGR5 or M-BAR) is a G protein-coupled receptor for secondary bile acids that is highly expressed in monocytes/macrophages. In this study, we aimed to determine the role of GPBAR1 in mediating leukocyte trafficking in chemically induced models of colitis and investigate the therapeutic potential of BAR501, a small molecule agonist for GPBAR1. These studies demonstrated that GPBAR1 gene ablation enhanced the recruitment of classically activated macrophages in the colonic lamina propria and worsened the severity of inflammation. In contrast, GPBAR1 activation by BAR501 reversed intestinal inflammation in the trinitrobenzenesulfonic acid and oxazolone models by reducing the trafficking of Ly6C+ monocytes from blood to intestinal mucosa. Exposure to BAR501 shifted intestinal macrophages from a classically activated (CD11b+, CCR7+, F4/80-) to an alternatively activated (CD11b+, CCR7-, F4/80+) phenotype, reduced the expression of inflammatory genes (TNF-α, IFN-γ, IL-1β, IL-6, and CCL2 mRNAs), and attenuated the wasting syndrome and severity of colitis (≈70% reduction in the Colitis Disease Activity Index). The protective effect was lost in Gpbar1-/- mice. Exposure to BAR501 increased the colonic expression of IL-10 and TGF-β mRNAs and the percentage of CD4+/Foxp3+ cells. The beneficial effects of BAR501 were lost in Il-10-/- mice. In a macrophage cell line, regulation of IL-10 by BAR501 was GPBAR1 dependent and was mediated by the recruitment of CREB to its responsive element in the IL-10 promoter. In conclusion, GPBAR1 is expressed in circulating monocytes and colonic macrophages, and its activation promotes a IL-10-dependent shift toward an alternatively activated phenotype. The targeting of GPBAR1 may offer therapeutic options in inflammatory bowel diseases.

Macrophage dysfunction initiates colitis during weaning of infant mice lacking the interleukin-10 receptor.

Infants with defects in the interleukin 10 receptor (IL10R) develop very early onset inflammatory bowel disease. Whether IL10R regulates lamina propria macrophage function during infant development in mice and whether macrophage-intrinsic IL10R signaling is required to prevent colitis in infancy is unknown. Here we show that although signs of colitis are absent in IL10R-deficient mice during the first two weeks of life, intestinal inflammation and macrophage dysfunction begin during the third week of life, concomitant with weaning and accompanying diversification of the intestinal microbiota. However, IL10R did not directly regulate the microbial ecology during infant development. Interestingly, macrophage depletion with clodronate inhibited the development of colitis, while the absence of IL10R specifically on macrophages sensitized infant mice to the development of colitis. These results indicate that IL10R-mediated regulation of macrophage function during the early postnatal period is indispensable for preventing the development of murine colitis.

Cooperation of Gastric Mononuclear Phagocytes with Helicobacter pylori during Colonization

Helicobacter pylori, the dominant member of the human gastric microbiota, elicits immunoregulatory responses implicated in protective versus pathological outcomes. To evaluate the role of macrophages during infection, we employed a system with a shifted proinflammatory macrophage phenotype by deleting PPARγ in myeloid cells and found a 5- to 10-fold decrease in gastric bacterial loads. Higher levels of colonization in wild-type mice were associated with increased presence of mononuclear phagocytes and in particular with the accumulation of CD11b+F4/80hiCD64+CX3CR1+ macrophages in the gastric lamina propria. Depletion of phagocytic cells by clodronate liposomes in wild-type mice resulted in a reduction of gastric H. pylori colonization compared with nontreated mice. PPARγ-deficient and macrophage-depleted mice presented decreased IL-10-mediated myeloid and T cell regulatory responses soon after infection. IL-10 neutralization during H. pylori infection led to increased IL-17-mediated responses and increased neutrophil accumulation at the gastric mucosa. In conclusion, we report the induction of IL-10-driven regulatory responses mediated by CD11b+F4/80hiCD64+CX3CR1+ mononuclear phagocytes that contribute to maintaining high levels of H. pylori loads in the stomach by modulating effector T cell responses at the gastric mucosa.

Activation-Induced TIM-4 Expression Identifies Differential Responsiveness of Intestinal CD103+ CD11b+ Dendritic Cells to a Mucosal Adjuvant.

Macrophage and dendritic cell (DC) populations residing in the intestinal lamina propria (LP) are highly heterogeneous and have disparate yet collaborative roles in the promotion of adaptive immune responses towards intestinal antigen. Under steady-state conditions, macrophages are efficient at acquiring antigen but are non-migratory. In comparison, intestinal DC are inefficient at antigen uptake but migrate to the mesenteric lymph nodes (mLN) where they present antigen to T cells. Whether such distinction in the roles of DC and macrophages in the uptake and transport of antigen is maintained under immunostimulatory conditions is less clear. Here we show that the scavenger and phosphatidylserine receptor T cell Immunoglobulin and Mucin (TIM)-4 is expressed by the majority of LP macrophages at steady-state, whereas DC are TIM-4 negative. Oral treatment with the mucosal adjuvant cholera toxin (CT) induces expression of TIM-4 on a proportion of CD103+ CD11b+ DC in the LP. TIM-4+ DC selectively express high levels of co-stimulatory molecules after CT treatment and are detected in the mLN a short time after appearing in the LP. Importantly, intestinal macrophages and DC expressing TIM-4 are more efficient than their TIM-4 negative counterparts at taking up apoptotic cells and soluble antigen ex vivo. Taken together, our results show that CT induces phenotypic changes to migratory intestinal DC that may impact their ability to take up local antigens and in turn promote the priming of mucosal immunity.

Diet-dependent, microbiota-independent regulation of IL-10-producing lamina propria macrophages in the small intestine.

Intestinal resident macrophages (Mϕs) regulate gastrointestinal homeostasis via production of an anti-inflammatory cytokine interleukin (IL)-10. Although a constant replenishment by circulating monocytes is required to maintain the pool of resident Mϕs in the colonic mucosa, the homeostatic regulation of Mϕ in the small intestine (SI) remains unclear. Here, we demonstrate that direct stimulation by dietary amino acids regulates the homeostasis of intestinal Mϕs in the SI. Mice that received total parenteral nutrition (TPN), which deprives the animals of enteral nutrients, displayed a significant decrease of IL-10-producing Mϕs in the SI, whereas the IL-10-producing CD4+ T cells remained intact. Likewise, enteral nutrient deprivation selectively affected the monocyte-derived F4/80+ Mϕ population, but not non-monocytic precursor-derived CD103+ dendritic cells. Notably, in contrast to colonic Mϕs, the replenishment of SI Mϕs and their IL-10 production were not regulated by the gut microbiota. Rather, SI Mϕs were directly regulated by dietary amino acids. Collectively, our study highlights the diet-dependent, microbiota-independent regulation of IL-10-producing resident Mϕs in the SI.

Intrinsic Gastrointestinal Macrophages: Their Phenotype and Role in Gastrointestinal Motility.

There is an increasing awareness of the role of macrophages in the regulation and maintenance of gastrointestinal function in health and disease. This work has proceeded in the context of an increased understanding of the complex phenotypic variation in macrophages throughout the body and has shown previously unidentified roles for macrophages in diseases such as gastroparesis, postoperative ileus, and inflammatory bowel disease. Opportunities for exploiting the phenotypic modulation of tissue resident macrophages have been identified as possible therapies for some of these diseases. In addition, macrophages are an established component of the innate immune system and can respond to variations and changes in the intestinal microbiome and potentially mediate part of the impact of the microbiota on intestinal health. We reviewed the latest work on novel concepts in defining macrophage phenotype, discuss possible mechanisms of action for tissue-resident macrophages in the gut, address the significance of microbiome effects on macrophage phenotype, and review the known and possible roles of macrophages in motility disorders of the gastrointestinal tract.

Neuro-immune Interactions Drive Tissue Programming in Intestinal Macrophages

Proper adaptation to environmental perturbations is essential for tissue homeostasis. In the intestine, diverse environmental cues can be sensed by immune cells, which must balance resistance to microorganisms with tolerance, avoiding excess tissue damage. By applying imaging and transcriptional profiling tools, we interrogated how distinct microenvironments in the gut regulate resident macrophages. We discovered that macrophages exhibit a high degree of gene-expression specialization dependent on their proximity to the gut lumen. Lamina propria macrophages (LpMs) preferentially expressed a pro-inflammatory phenotype when compared to muscularis macrophages (MMs), which displayed a tissue-protective phenotype. Upon luminal bacterial infection, MMs further enhanced tissue-protective programs, and this was attributed to swift activation of extrinsic sympathetic neurons innervating the gut muscularis and norepinephrine signaling to β2 adrenergic receptors on MMs. Our results reveal unique intra-tissue macrophage specialization and identify neuro-immune communication between enteric neurons and macrophages that induces rapid tissue-protective responses to distal perturbations.

Macrophage accumulation in gut mucosa differentiates AIDS from chronic SIV infection in rhesus macaques.

The relationship between recruitment of mononuclear phagocytes to lymphoid and gut tissues and disease in HIV and SIV infection remains unclear. To address this question, we conducted cross-sectional analyses of dendritic cell (DC) subsets and CD163(+) macrophages in lymph nodes (LNs) and ileum of rhesus macaques with acute and chronic SIV infection and AIDS. In LNs significant differences were only evident when comparing uninfected and AIDS groups, with loss of myeloid DCs and CD103(+) DCs from peripheral and mesenteric LNs, respectively, and accumulation of plasmacytoid DCs and macrophages in mesenteric LNs. In contrast, there were fourfold more macrophages in ileum lamina propria in macaques with AIDS compared with chronic infection, and this increased to 40-fold in Peyer's patches. Gut macrophages exceeded plasmacytoid DCs and CD103(+) DCs by ten- to 17-fold in monkeys with AIDS but were at similar low frequencies as DCs in chronic infection. Gut macrophages in macaques with AIDS expressed IFN-α and TNF-α consistent with cell activation. CD163(+) macrophages also accumulated in gut mucosa in acute infection but lacked expression of IFN-α and TNF-α. These data reveal a relationship between inflammatory macrophage accumulation in gut mucosa and disease and suggest a role for macrophages in AIDS pathogenesis.

Glycol methacrylate embedding for the histochemical study of the gastrointestinal tract of dogs naturally infected with Leishmania infantum.

In canine visceral leishmaniasis a diffuse chronic inflammatory exudate and an intense parasite load throughout the gastrointestinal tract (GIT) has been previously reported. However, these studies did not allow a properly description of canine cellular morphology details. The aim of our study was to better characterize these cells in carrying out a qualitative and quantitative histological study in the gastrointestinal tract of dogs naturally infected with Leishmania infantum by examining gut tissues embedded in glycol methacrylate. Twelve infected adult dogs were classified in asymptomatic and symptomatic. Five uninfected dogs were used as controls. After necropsy, three samples of each gut segment, including oesophagus, stomach, duodenum, jejunum, ileum, cecum, colon, and rectum were collected and fixed in Carnoy’s solution for glycol methacrylate protocols. Sections were stained with hematoxylin-eosin, toluidine blue borate, and periodic acid-Schiff stain. Leishmania amastigotes were detected by immunohistochemistry employed in both glycol methacrylate and paraffin embedded tissues. The quantitative histological analysis showed higher numbers of plasma cells, lymphocytes and macrophages in lamina propria of all segments of GIT of infected dogs compared with controls. The parasite load was more intense and cecum and colon, independently of the clinical status of these dogs. Importantly, glycol methacrylate embedded tissue stained with toluidine blue borate clearly revealed mast cell morphology, even after mast cell degranulation. Infected dogs showed lower numbers of mast cells in all gut segments than controls. Despite the glycol methacrylate (GMA) protocol requires more attention and care than the conventional paraffin processing, this embedding procedure proved to be especially suitable for the present histological study, where it allowed to preserve and observe cell morphology in fine detail.

Defective macrophage handling of Escherichia coli in Crohn's disease.

Escherichia coli can be isolated from lamina propria macrophages in Crohn's disease (CD), and their intramacrophage persistence may provide a stimulus for inflammation. To further determine the contributions of macrophage dysfunction and E. coli pathogenicity to this, we aimed to compare in vitro functioning of macrophages from patients with CD and healthy controls (HC) in response to infection with CD-derived adherent-invasive E. coli (AIEC) and less pathogenic E. coli strains. Monocyte-derived macrophages were cultured from patients with CD and HC. Intramacrophage survival of E. coli strains (CD-derived adherent-invasive [AI] and non-AI strains and laboratory strain K-12) was compared. Macrophage cytokine release (tumor necrosis factor alpha [TNFα], interleukin [IL]-23, IL-8 and IL-10) and monocyte phagoctyosis and respiratory burst function were measured after E. coli infection. For CD patients, laboratory data were correlated with clinical phenotype, use of immunomodulation, and CD risk alleles (NOD2, IL-23R, ATG16L1 and IRGM). Attenuated TNFα and IL-23 release from CD macrophages was found after infection with all E. coli strains. There was prolonged survival of CD-derived AIEC, CD-derived non-AIEC and E. coli K-12 in macrophages from CD patients compared to within those from HC. No abnormality of monocyte phagocytosis or respiratory burst function was detected in CD. Macrophage dysfunction in CD was not influenced by phenotype, use of immunomodulation or genotype. CD macrophage responses to infection with E. coli are deficient, regardless of clinical phenotype, CD genotype or E. coli pathogenicity. This suggests host immunodeficiency is an important contributor to intramacrophage E. coli persistence in CD.

Lamina propria macrophage phenotypes in relation to Escherichia coli in Crohn's disease.

BackgroundAbnormal handling of E. coli by lamina propria (LP) macrophages may contribute to Crohn’s disease (CD) pathogenesis. We aimed to determine LP macrophage phenotypes in CD, ulcerative colitis (UC) and healthy controls (HC), and in CD, to compare macrophage phenotypes according to E. coli carriage.MethodsMucosal biopsies were taken from 35 patients with CD, 9 with UC and 18 HCs. Laser capture microdissection was used to isolate E. coli-laden and unladen LP macrophages from ileal or colonic biopsies. From these macrophages, mRNA was extracted and cytokine and activation marker expression measured using RT-qPCR.ResultsE. coli-laden LP macrophages were identified commonly in mucosal biopsies from CD patients (25/35, 71 %), rarely in UC (1/9, 11 %) and not at all in healthy controls (0/18). LP macrophage cytokine mRNA expression was greater in CD and UC than healthy controls. In CD, E. coli-laden macrophages expressed high IL-10 & CD163 and lower TNFα, IL-23 & iNOS irrespective of macroscopic inflammation. In inflamed tissue, E. coli-unladen macrophages expressed high TNFα, IL-23 & iNOS and lower IL-10 & CD163. In uninflamed tissue, unladen macrophages had low cytokine mRNA expression, closer to that of healthy controls.ConclusionIn CD, intra-macrophage E. coli are commonly found and LP macrophages express characteristic cytokine mRNA profiles according to E. coli carriage. Persistence of E. coli within LP macrophages may provide a stimulus for chronic inflammation.

Regulation of the gut microbiota and intestinal homeostasis by phagocyte NADPH oxdiase (INC1P.351)

Abstract The phagocyte NADPH oxidase generates reactive oxygen species (ROS) for microbial killing, and its impairment gives rise to Chronic Granulomatous Disease (CGD), a primary immunodeficiency characterized by recurrent infections and granulomatous lesions. Mutations in Nox2 (Cybb), a critical subunit of the oxidase, contribute 66% of CGD. In our study, we found spontaneous inflammation in the intestine of naïve Cybb-/- mice, indicated by higher levels of IL-1b, IL-6 and TNFα in lamina propria macrophages, as well as increased levels of IFNγ and IL-17A in CD4+ T cells. In addition, Klebsiella pneumoniae, a bacterium frequently found in granulomatous lesions in CGD patients, were detected in the spleens and mesenteric lymph nodes (MLNs) of naïve Cybb-/- mice, and IP administration of this pathobiont strain of K. pneumonia led to rapid mortality of wild-type mice. Oral administration of this K. pneumoniae induced intestinal inflammation in wild-type and Cybb-/- mice, but translocation of K. pneumoniae to MLNs was drastically enhanced in Cybb-/- mice and bone marrow chimeras with a neutrophil-specific deficiency in NOX2. Thus, our results demonstrate the importance of neutrophil-derived ROS in restricting dissemination of pathobionts from the intestine under steady-state conditions. Therefore, our study underscores a critical role for the NADPH oxidase in regulating intestinal homeostasis and provides insights into intestinal complications in CGD patients.

Intestine lamina propria CD4+T cells promote the bactericidal activity of macrophages via galectin-9 and Tim-3 interaction during Salmonella typhimurium infection (MUC5P.760)

Abstract The intestinal immune system is crucial for protection the host from pathogenic infection and maintenance of mucosal homeostasis. The intestine lamina propria macrophages are main effector cells in innate resistance to intracellular microbial pathogens. In the present study, we found that Salmonella typhimurium infection augmented the Tim-3 expression on intestinal lamina propria CD4+T cells and enhanced galectin-9 expression on F4/80+CD11c+macrophages. Moreover, CD4+T cells promote the activation and bactericidal activity of F4/80+CD11c+macrophages via galectin-9 and Tim-3 interaction during Salmonella typhimurium infection. Blockade the interaction of Tim-3 and galectin-9 with a-Lactose significantly attenuated the killing of intracellular bacteria by macrophages. Furthermore, the interaction of Tim-3 and galectin-9 also promoted the formation and activation of inflammasome, which leads to the cleavage of caspase-1 and IL-1β. The secretion of active IL-1β further improved bactericidal activity of macrophages and expression of galectin-9 on macrophages. These results demonstrated the critical role of the cross-talk between CD4+T cell and macrophage, in particularly the interaction of Tim-3 and galectin-9, in antimicrobial immunity and control intestinal pathogen infection.

Subversion of Human Intestinal Mucosa Innate Immunity by a Crohn's Disease‐associated E. coli

Adherent‐invasive Escherichia coli (AIEC), associated with Crohn's disease, are likely candidate contributory factors in the disease. However, signaling pathways involved in human intestinal mucosa innate host response to AIEC remain unknown. Here we use a 3‐D model of human intestinal mucosa explant culture to explore the effects of the AIEC strain LF82, on two innate immunity platforms, i.e. the inflammasome through evaluation of caspase‐1 status, and NFkB signaling. We showed that LF82 bacteria enter and survive within a few intestinal epithelial cells and lamina propria macrophages, without altering the mucosa overall architecture. While 4h infection with a Salmonella strain caused crypt disorganization, caspase‐1 activation and mature IL‐18 production, LF82 bacteria were unable to activate caspase‐1 and induce IL‐18 production. In parallel, LF82 bacteria activated NFkB signaling in epithelial cells through IkBα phosphorylation, NFkBp65 nuclear translocation and TNFα secretion. In addition, pharmacological blockade of NFkB activation using the SN50 peptide showed that NFkB activation was crucial for the maintenance of epithelial homeostasis upon LF82 infection. In conclusion, here we decipher at the whole mucosa level the mechanisms of the LF82‐induced subversion of innate immunity that, by maintaining host cell integrity, ensure intracellular bacteria survival.

Mo1828 Reduced Intestinal Leukocyte Exit Heightens the Severity of DSS Colitis in a Murine Model of Lymphovascular Deficiency

process of intestinal inflammation is less understood. While we could demonstrate that HDAC inhibitors induce amelioration of colitis in mice, we are further interested in how far specific HDAC are responsible for inflammatory responses and what are the mechanism behind these effects. Methods: The macrophage cell line RAW 264.7 and the monocyte cell line U937 were analysed for HDAC5-expression using quantitative PCR and Western-blot. The cells were transfected with a HDAC5-encoding plasmid for overexpression or small interfering (si)RNA for knock-down studies. HDAC5 knock out mice were used in dextran sulphate sodium (DSS) induced colitis model. Colon tissue was analysed by histology and lamina propria mononuclear cells (LPMCs) were isolated. All cells were analysed by flow cytometry. Supernatant of colon and cell cultures were analysed by cytometric bead assay. Results: In RAW 264.7 and U937 expression of HDAC5 mRNA down regulated for up to 7 h but presented baseline levels after 24 h. HDAC5-overexpression in RAW 264.7 resulted in an increased LPS-dependent secretion of the pro-inflammatory cytokines tumor necrosis factor alpha, the monocyte chemoattractant protein-1 and interleukin 6. Subsequently, knock-down of HDAC5-mRNA expression by specific siRNA significantly reduced the secretion of these cytokines. These effects were accompanied by increased nuclear factor kappa B activity. Further experimental colitis experiments demonstrate a differential disease pattern for HDAC5 knockout mice compared to wild type mice. This was shown by an increased histological disease score, higher weight loss and severe intestinal bleeding. LPMC isolation followed by extensive FACS analysis of the LPMC confirmed these results by demonstrating changes in macrophage polarization. When the colonic barrier function was analysed in DSS induced colitis experiments, we detected a decreased trans-epithelial resistance and modulated flux of mannitol, HRP and FITC-Dextran. Conclusion: Our data demonstrate a critical function of HDAC5 in the pro-inflammatory immune response of macrophages in the lamina propria as well as a role within the physiological barrier of the gut epithelium, proposing a potential new target for the treatment of inflammatory bowel disease