Integrated omics analysis reveals the immunologic characteristics of cystic Peyer's patches in the cecum of Bactrian camels.

Bactrian camels have specific mucosa-associated lymphoid tissue (MALT) throughout the large intestine, with species-unique cystic Peyer's patches (PPS) as the main type of tissue. However, detailed information about the molecular characteristics of PPS remains unclear. This study applied a transcriptomic analysis, untargeted metabolomics, and 16S rDNA sequencing to compare the significant differences between PPS and the adjacent normal intestine tissues (NPPS) during the healthy stage of three young Bactrian camels. The results showed that samples from PPS could be easily differentiated from the NPPS samples based on gene expression profile, metabolites, and microbial composition, separately indicated using dimension reduction methods. A total of 7,568 up-regulated and 1,266 down-regulated differentially expressed genes (DEGs) were detected, and an enrichment analysis found 994 DEGs that participated in immune-related functions, and a co-occurance network analysis identified nine hub genes (BTK, P2RX7, Pax5, DSG1, PTPN2, DOCK11, TBX21, IL10, and HLA-DOB) during multiple immunologic processes. Further, PPS and NPPS both had a similar pattern of most compounds among all profiles of metabolites, and only 113 differentially expressed metabolites (DEMs) were identified, with 101 of these being down-regulated. Deoxycholic acid (DCA; VIP = 37.96, log2FC = -2.97, P = 0), cholic acid (CA; VIP = 13.10, log2FC = -2.10, P = 0.01), and lithocholic acid (LCA; VIP = 12.94, log2FC = -1.63, P = 0.01) were the highest contributors to the significant dissimilarities between groups. PPS had significantly lower species richness (Chao1), while Firmicutes (35.92% ± 19.39%), Bacteroidetes (31.73% ± 6.24%), and Proteobacteria (13.96% ± 16.21%) were the main phyla across all samples. The LEfSe analysis showed that Lysinibacillus, Rikenellaceae_RC9_gut_group, Candidatus_Stoquefichus, Mailhella, Alistipes, and Ruminococcaceae_UCG_005 were biomarkers of the NPPS group, while Escherichia_Shigella, Synergistes, Pyramidobacter, Odoribacter, Methanobrevibacter, Cloacibacillus, Fusobacterium, and Parabacteroides were significantly higher in the PPS group. In the Procrustes analysis, the transcriptome changes between groups showed no significant correlations with metabolites or microbial communities, whereas the alteration of metabolites significantly correlated with the alteration of the microbial community. In the co-occurrence network, seven DEMs (M403T65-neg, M329T119-neg, M309T38-neg, M277T42-2-neg, M473T27-neg, M747T38-1-pos, and M482t187-pos) and 14 genera (e.g., Akkermansia, Candidatus-Stoquefichus, Caproiciproducens, and Erysipelatoclostridium) clustered much more tightly, suggesting dense interactions. The results of this study provide new insights into the understanding of the immune microenvironment of the cystic PPS in the cecum of Bactrian camels.

Plasmodium chabaudi Infection Alters Intestinal Morphology and Mucosal Innate Immunity in Moderately Malnourished Mice.

Plasmodium falciparum is a protozoan parasite which causes malarial disease in humans. Infections commonly occur in sub-Saharan Africa, a region with high rates of inadequate nutrient consumption resulting in malnutrition. The complex relationship between malaria and malnutrition and their effects on gut immunity and physiology are poorly understood. Here, we investigated the effect of malaria infection in the guts of moderately malnourished mice. We utilized a well-established low protein diet that is deficient in zinc and iron to induce moderate malnutrition and investigated mucosal tissue phenotype, permeability, and innate immune response in the gut. We observed that the infected moderately malnourished mice had lower parasite burden at the peak of infection, but damaged mucosal epithelial cells and high levels of FITC-Dextran concentration in the blood serum, indicating increased intestinal permeability. The small intestine in the moderately malnourished mice were also shorter after infection with malaria. This was accompanied with lower numbers of CD11b+ macrophages, CD11b+CD11c+ myeloid cells, and CD11c+ dendritic cells in large intestine. Despite the lower number of innate immune cells, macrophages in the moderately malnourished mice were highly activated as determined by MHCII expression and increased IFNγ production in the small intestine. Thus, our data suggest that malaria infection may exacerbate some of the abnormalities in the gut induced by moderate malnutrition.

Myeloid-Derived Suppressor Cell-Derived Arginase-1 Oppositely Modulates IL-17A and IL-17F Through the ESR/STAT3 Pathway During Colitis in Mice.

Myeloid-derived suppressor cells (MDSC) play a crucial role in regulating the intestinal immune response during colitis. We previously revealed an essential role of MDSC in promoting TH17 cell polarization, which was found to be arginase-1 (Arg-1)-dependent; however, the underlying mechanism remains obscure. Here we report that percentage of MDSC decreased in Arg myeKO mice during DSS-induced colitis. IL-17A levels reduced but IL-17F levels increased significantly in the colorectum of Arg myeKO mice, leading to severe tissue damage and high risk of mortality rate. Activation of estrogen receptor (ESR) increased pSTAT3 level in MDSC and consequently led to elevated percentage of MDSC and more Arg-1 and inducible nitric oxide synthase expression in MDSC. Increased level of IL-17A and reduced level of IL-17F alleviated colitis in mice consequently. Together, these findings demonstrate a protective role of MDSC-derived Arg-1 during colitis after activates ESR/STAT3 signaling in MDSC. High level of Arg-1 favors accumulation of IL-17A, but reduced IL-17F expression in the colorectum of mice and ultimately leading to relief of colitis, indicating a potential clinical impact of MDSC-derived Arg-1 for controlling inflammatory bowel disease.

Effect of Combined Antimicrobial Therapy on Ischemia/Reperfusion Myocardial Injury in Rats with Acute Inflammation of the Large Intestine against the Background of Alimentary Obesity.

We studied the effect of combined antimicrobial therapy with amoxicillin, metronidazole, and clarithromycin on the severity of ischemia/reperfusion myocardial injury in Wistar rats with alimentary obesity and acute inflammation of the large intestine. General ischemia/reperfusion was reproduced on Langendorff-perfused isolated hearts and infarct size was estimated. Acute inflammation of the large intestine was accompanied by an increase in the blood levels of proinflammatory cytokines. The presence of obesity and acute inflammation of the large intestine did not significantly affect the infarct size in comparison with the control. Administration of antimicrobial drugs to animals with obesity and acute inflammation of the large intestine led to a significant increase in the infarct size, which should be considered when prescribing antimicrobial therapy to patients with comorbidity.

The Functions and Therapeutic Potential of Heat Shock Proteins in Inflammatory Bowel Disease-An Update.

Inflammatory bowel disease (IBD) is a multifactorial human intestinal disease that arises from numerous, yet incompletely defined, factors. Two main forms, Crohn's disease (CD) and ulcerative colitis (UC), lead to a chronic pathological form. Heat shock proteins (HSPs) are stress-responsive molecules involved in various pathophysiological processes. Several lines of evidence link the expression of HSPs to the development and prognosis of IBD. HSP90, HSP70 and HSP60 have been reported to contribute to IBD in different aspects. Moreover, induction and/or targeted inhibition of specific HSPs have been suggested to ameliorate the disease consequences. In the present review, we shed the light on the role of HSPs in IBD and their targeting to prevent further disease progression.

Bacterial immunogenic α-galactosylceramide identified in the murine large intestine: dependency on diet and inflammation.

The glycosphingolipid, α-galactosylceramide (αGalCer), when presented by CD1d on antigen-presenting cells, efficiently activates invariant natural killer T (iNKT) cells. Thereby, it modulates immune responses against tumors, microbial and viral infections, and autoimmune diseases. Recently, the production of αGalCer by Bacteroidetes from the human gut microbiome was elucidated. Using hydrophilic interaction chromatography coupled to MS2, we screened murine intestinal tracts to identify and quantify αGalCers, and we investigated the αGalCer response to different dietary and physiologic conditions. In both the cecum and the colon of mice, we found 1-15 pmol of αGalCer per milligram of protein; in contrast, mice lacking microbiota (germ-free mice) and fed identical diet did not harbor αGalCer. The identified αGalCer contained a ß(R)-hydroxylated hexadecanoyl chain N-linked to C18-sphinganine, which differed from what has been reported with Bacteroides fragilis Unlike ß-anomeric structures, but similar to αGalCers from B. fragilis, the synthetic form of the murine αGalCer induced iNKT cell activation in vitro. Last, we observed a decrease in αGalCer production in mice exposed to conditions that alter the composition of the gut microbiota, including Western type diet, colitis, and influenza A virus infection. Collectively, this study suggests that αGalCer is produced by commensals in the mouse intestine and reveals that stressful conditions causing dysbiosis alter its synthesis. The consequences of this altered production on iNKT cell-mediated local and systemic immune responses are worthy of future studies.

Human Gut-Associated Natural Killer Cells in Health and Disease.

It is well established that natural killer (NK) cells are involved in both innate and adaptive immunity. Indeed, they can recognize molecules induced at the cell surface by stress signals and virus infections. The functions of NK cells in the gut are much more complex. Gut NK cells are not precisely organized in lymphoid aggregates but rather scattered in the epithelium or in the stroma, where they come in contact with a multitude of antigens derived from commensal or pathogenic microorganisms in addition to components of microbiota. Furthermore, NK cells in the bowel interact with several cell types, including epithelial cells, fibroblasts, macrophages, dendritic cells, and T lymphocytes, and contribute to the maintenance of immune homeostasis and development of efficient immune responses. NK cells have a key role in the response to intestinal bacterial infections, primarily through production of IFNγ, which can stimulate recruitment of additional NK cells from peripheral blood leading to amplification of the anti-bacterial immune response. Additionally, NK cells can have a role in the pathogenesis of gut autoimmune inflammatory bowel diseases (IBDs), such as Crohn's Disease and Ulcerative Colitis. These diseases are considered relevant to the generation of gastrointestinal malignancies. Indeed, the role of gut-associated NK cells in the immune response to bowel cancers is known. Thus, in the gut immune system, NK cells play a dual role, participating in both physiological and pathogenic processes. In this review, we will analyze the known functions of NK cells in the gut mucosa both in health and disease, focusing on the cross-talk among bowel microenvironment, epithelial barrier integrity, microbiota, and NK cells.

Comparative Assessment of Small and Large Intestine Biopsies for Ex Vivo HIV-1 Pathogenesis Studies.

Ex vivo mucosal explants have become a mainstay of HIV-1 studies using human tissue. In this study, we examine the baseline phenotypic and virologic differences between biopsies derived from the small intestine (SI) and large intestine (LI) for use in ex vivo explant studies. To do this, we collected endoscopic mucosal biopsies from both SI and LI from the same healthy, HIV-seronegative participants. Mucosal mononuclear cell phenotypes and quantity were compared using flow cytometry. Comparative HIV-1 infectibility of the explants was assessed using an ex vivo explant HIV-1 infection assay. We found that all biopsies had similar numbers of T cells per biopsy. While the percentage of CD4+ T cells from SI biopsies expressed significantly more activation markers (CD38, HLA-DR) and HIV coreceptors (CXCR4, CCR5), the absolute numbers of activated CD4+ T cells were similar between both sites. LI explants, however, supported more efficient HIV-1 infection, as evidenced by earlier rise in p24 accumulation and greater percent of infected explants at limiting infectious doses. These results suggest that explants from LI biopsies support more efficient HIV-1 infection than SI biopsies, despite similar numbers of available, activated HIV-1 target cells. These findings highlight important differences in LI and SI explants, which must be considered in designing and interpreting ex vivo HIV-1 infection studies, and suggest that factors within the tissue other than target cell number and activation state may play a role in regulating HIV-1 infection.

Clinical importance of IL-22 cascade in IBD.

IL-22 is a relatively new cytokine that is characterized by several unique biological properties. In the intestines, the effect of IL-22 is restricted mainly to non-lymphoid cells such as epithelial cells. Interestingly, the expression pattern and major cellular source of IL-22 have distinct difference between large and small intestines. IL-22 possesses an ability to constitutively activate STAT3 for promoting epithelial cell regeneration and reinforcing mucosal barrier integrity through stimulating the expression of anti-bacterial peptide and mucins. Of note, IL-22 is characterized as a two-faced cytokine that can play not only protective but also deleterious roles in the intestinal inflammation depending on the cytokine environment such as the expression levels of IL-23, T-bet, and IL-22 binding protein. Most importantly, clinical relevance of IL-22 to inflammatory bowel disease has been well highlighted. Mucosal healing, which represents the current therapeutic goal for IBD, can be induced by IL-22. Indeed, indigo naturalis, which can activate IL-22 pathway through Ahr, has been shown in a clinical trial to exhibit a strong therapeutic effect on ulcerative colitis. Despite the beneficial effect of IL-22, continuous activation of the IL-22 pathway increases the risk of colitis-associated cancer, particularly in patients with an extended history of IBD. This review article discusses how IL-22 regulates colitis, how beneficial versus deleterious effects of IL-22 is determined, and why IL-22 represents a promising target for IBD therapy.

Study of the effects of spray drying in whey-starch on the probiotic capacity of Lactobacillus rhamnosus 64 in the gut of mice.

AIMS: To evaluate the effects of spray drying of Lactobacillus rhamnosus 64 on its capacity to modulate the gut immune response and on the attenuation of TNBS-induced colitis in mice. METHODS AND RESULTS: Lactobacillus rhamnosus 64 was spray dried in cheese whey-starch solution and administered to mice for 3, 6 or 10 consecutive days. Peritoneal macrophage phagocytic activity, secretory IgA levels in the small intestinal fluid and TNFα, IFNγ, IL-10, IL-6 and IL-2 levels in homogenates of the small and large intestine were determined. The effects of spray drying were also evaluated in an acute model of Trinitrobenzenesulfonic acid (TNBS)-induced colitis. A shift in the regulation of immune parameters, particularly the cytokine profile, was observed for mice treated with the spray-dried culture, compared to the profile observed in animals that received the strain as fresh culture (FC). The spray-dried culture of L. rhamnosus 64 showed anti-inflammatory properties in murine model of TNBS-induced colitis. CONCLUSIONS: The spray-drying process of L. rhamnosus 64 in whey-starch modified its immunomodulating capacity in healthy animals and conferred enhanced protection in an in vivo model of inflammation. SIGNIFICANCE AND IMPACT OF THE STUDY: Probiotic capacity can be affected by spray drying in relation to the properties observed for the strain as an overnight FC. This fact should be taken into account when producing the culture for its application in the industry.

Immunoregulation effects of different γδT cells and toll-like receptor signaling pathways in neonatal necrotizing enterocolitis.

The aim of the study was to observe cytokine and T-cell-related toll-like-receptor (TLR) changes in intestinal samples of neonatal necrotizing enterocolitis patients.Four necrotic bowels were collected from neonatal NEC patients with gestational ages of 28 to 29 weeks in our hospital, whereas 4 neonatal patients who underwent intestinal atresia surgery served as the controls. Intestinal flora was examined and IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, IFN-γ, and IL-17 expressions in resected intestine samples, as well as in isolated gamma delta T (γδT) cells, were analyzed immunohistochemically and via quantitative RT-PCR. γδT cells were isolated from the intestinal intraepithelial lymphocytes (IELs) and their TLR4/TLR9 distribution in the intestinal tissues was determined by flow cytometry.The bacterial flora of the neonatal NEC patients' contained significantly higher amounts of Gram-negative Enterobacteriaceae, Klebsiella, and Bacteroides but anaerobic Gram-positive Bifidobacteria occurred significantly less in the NEC than the control group. IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, IFN-γ, and IL-17 expressions in the resected intestine samples and in isolated γδT cells were enhanced in NEC samples compared to the controls. γδT cells were less prevalent in NEC-derived intestinal tissues, but their TLR4/TLR9 expressions were significantly enhanced.The changed bacterial flora in preterm neonatal NEC patients led to an obvious inflammation of the intestines, which was accompanied by reductions of γδT cell localizations to the intestine and a shift of their surface expressions to TLR4 and TLR9.

Expression of Ifnlr1 on Intestinal Epithelial Cells Is Critical to the Antiviral Effects of Interferon Lambda against Norovirus and Reovirus.

Lambda interferon (IFN-λ) has potent antiviral effects against multiple enteric viral pathogens, including norovirus and rotavirus, in both preventing and curing infection. Because the intestine includes a diverse array of cell types, however, the cell(s) upon which IFN-λ acts to exert its antiviral effects is unclear. Here, we sought to identify IFN-λ-responsive cells by generation of mice with lineage-specific deletion of the receptor for IFN-λ, Ifnlr1 We found that expression of IFNLR1 on intestinal epithelial cells (IECs) in the small intestine and colon is required for enteric IFN-λ antiviral activity. IEC Ifnlr1 expression also determines the efficacy of IFN-λ in resolving persistent murine norovirus (MNoV) infection and regulates fecal shedding and viral titers in tissue. Thus, the expression of Ifnlr1 by IECs is necessary for the response to both endogenous and exogenous IFN-λ. We further demonstrate that IEC Ifnlr1 expression is required for the sterilizing innate immune effects of IFN-λ by extending these findings in Rag1-deficient mice. Finally, we assessed whether our findings pertained to multiple viral pathogens by infecting mice specifically lacking IEC Ifnlr1 expression with reovirus. These mice phenocopied Ifnlr1-null animals, exhibiting increased intestinal tissue titers and enhanced reovirus fecal shedding. Thus, IECs are the critical cell type responding to IFN-λ to control multiple enteric viruses. This is the first genetic evidence that supports an essential role for IECs in IFN-λ-mediated control of enteric viral infection, and these findings provide insight into the mechanism of IFN-λ-mediated antiviral activity.IMPORTANCE Human noroviruses (HNoVs) are the leading cause of epidemic gastroenteritis worldwide. Type III interferons (IFN-λ) control enteric viral infections in the gut and have been shown to cure mouse norovirus, a small-animal model for HNoVs. Using a genetic approach with conditional knockout mice, we identified IECs as the dominant IFN-λ-responsive cells in control of enteric virus infection in vivo Upon murine norovirus or reovirus infection, Ifnlr1 depletion in IECs largely recapitulated the phenotype seen in Ifnlr1-/- mice of higher intestinal tissue viral titers and increased viral shedding in the stool. Moreover, IFN-λ-mediated sterilizing immunity against murine norovirus requires the capacity of IECs to respond to IFN-λ. These findings clarify the mechanism of action of this cytokine and emphasize the therapeutic potential of IFN-λ for treating mucosal viral infections.

Oxazolone-Induced Colitis as a Model of Th2 Immune Responses in the Intestinal Mucosa.

Murine models of intestinal inflammation have been widely used in biomedical research. Similarities in anatomy and physiology between such murine models and patients with inflammatory bowel diseases may allow a better understanding of the pathogenesis of Crohn's disease and ulcerative colitis. Additionally, models of intestinal inflammation may be used for the analysis of potentially new therapeutic agents. One key class of models consists of chemically induced inflammation models. Within this group, colitis induced by the haptenizing agent oxazolone is an important model that results in induction of acute or chronic inflammation of the large bowel. Here, we describe the induction and the analysis of this experimental colitis model.

Donor interleukin-22 and host type I interferon signaling pathway participate in intestinal graft-versus-host disease via STAT1 activation and CXCL10.

Acute graft-versus-host disease (aGVHD) remains a major complication following allogeneic hematopoietic cell transplantation, limiting the success of this therapy. We previously reported that interleukin-22 (IL-22) participates to aGVHD development, but the underlying mechanisms of its contribution remain poorly understood. In this study, we analyzed the mechanism of the pathological function of IL-22 in intestinal aGVHD. Ex-vivo colon culture experiments indicated that IL-22 was able to induce Th1-like inflammation via signal transducer and activator of transcription factor-1 (STAT1) and CXCL10 induction in the presence of type I interferon (IFN). To evaluate a potential synergy between IL-22 and type I IFN in aGVHD, we transplanted recipient mice, either wild-type (WT) or type I IFN receptor deficient (IFNAR(-/-)), with bone marrow cells and WT or IL-22 deficient (IL-22(-/-)) T cells. We observed a decreased GVHD severity in IFNAR(-/-) recipient of IL-22(-/-) T cells, which was associated with a lower level of STAT1 activation and reduced CXCL10 expression in the large intestine. Finally, immunohistochemistry staining of STAT1 performed on gastrointestinal biopsies of 20 transplanted patients showed exacerbated STAT1 activation in gastrointestinal tissues of patients with aGVHD as compared with those without aGVHD. Thus, interfering with both IL-22 and type I IFN signaling may provide a novel approach to limit aGVHD.

Evaluation of CD40 and CD80 receptors in the colonic mucosal membrane of children with inflammatory bowel disease.

INTRODUCTION: The most prevalent inflammatory bowel diseases (IBD) include ulcerative colitis (UC) and Crohn's disease (CD). Immune processes play a vital role in the etiopathogenesis of these conditions, involving both cellular and humoral response mechanisms. The aim of this study was to quantify CD40- and CD80-positive cells in the biopsy specimens of large intestinal mucosa from children with IBD. MATERIALS AND METHOD: The study comprised 38 children aged between 3-17 years (mean 11.5±3.7 years) - 20 boys (52.6 %) and 18 girls (47.4%). Eighteen patients were diagnosed with UC on the basis of clinical manifestation, endoscopic and histopathological findings. Mean age of this subgroup was 11.55±4.07 years. A group of 10 children (mean age 12.30±2.83) diagnosed with CD was also included. The control group comprised 10 IBD-free children (mean age 10.28±4.07 years). The surface expressions of CD40 and CD80 were analyzed in large intestine mucosa biopsy specimens, fixed in formaldehyde, embedded in paraffin, and cut with a microtome into 4 µm slices. RESULTS: The number of CD40- and CD80-positive cells in the large intestinal mucosa of children with Crohn's disease and ulcerative colitis was significantly higher than in the controls. The highest number of CD40+ and CD80+ cells was observed in the caecal mucosal membrane of Crohn's disease patients and in the rectal mucosa of individuals with ulcerative colitis. CONCLUSION: IBD is characterized by elevated, segment-specific, expression of CD40 and CD80.

The Gut-Associated Lymphoid Tissues in the Small Intestine, Not the Large Intestine, Play a Major Role in Oral Prion Disease Pathogenesis.

UNLABELLED: Prion diseases are infectious neurodegenerative disorders characterized by accumulations of abnormally folded cellular prion protein in affected tissues. Many natural prion diseases are acquired orally, and following exposure, the early replication of some prion isolates upon follicular dendritic cells (FDC) within gut-associated lymphoid tissues (GALT) is important for the efficient spread of disease to the brain (neuroinvasion). Prion detection within large intestinal GALT biopsy specimens has been used to estimate human and animal disease prevalence. However, the relative contributions of the small and large intestinal GALT to oral prion pathogenesis were unknown. To address this issue, we created mice that specifically lacked FDC-containing GALT only in the small intestine. Our data show that oral prion disease susceptibility was dramatically reduced in mice lacking small intestinal GALT. Although these mice had FDC-containing GALT throughout their large intestines, these tissues were not early sites of prion accumulation or neuroinvasion. We also determined whether pathology specifically within the large intestine might influence prion pathogenesis. Congruent infection with the nematode parasite Trichuris muris in the large intestine around the time of oral prion exposure did not affect disease pathogenesis. Together, these data demonstrate that the small intestinal GALT are the major early sites of prion accumulation and neuroinvasion after oral exposure. This has important implications for our understanding of the factors that influence the risk of infection and the preclinical diagnosis of disease. IMPORTANCE: Many natural prion diseases are acquired orally. After exposure, the accumulation of some prion diseases in the gut-associated lymphoid tissues (GALT) is important for efficient spread of disease to the brain. However, the relative contributions of GALT in the small and large intestines to oral prion pathogenesis were unknown. We show that the small intestinal GALT are the essential early sites of prion accumulation. Furthermore, congruent infection with a large intestinal helminth (worm) around the time of oral prion exposure did not affect disease pathogenesis. This is important for our understanding of the factors that influence the risk of prion infection and the preclinical diagnosis of disease. The detection of prions within large intestinal GALT biopsy specimens has been used to estimate human and animal disease prevalence. However, our data suggest that using these biopsy specimens may miss individuals in the early stages of oral prion infection and significantly underestimate the disease prevalence.

Phenotypic and functional profiling of mouse intestinal antigen presenting cells.

The microbiota that populates the mammalian intestine consists of hundreds of trillions of bacteria that are separated from underlying immune cells by a single layer of epithelial cells. The intestinal immune system effectively tolerates components of the microbiota that provide benefit to the host while remaining poised to eliminate those that are harmful. Antigen presenting cells, especially macrophages and dendritic cells, play important roles in maintaining intestinal homeostasis via their ability to orchestrate appropriate responses to the microbiota. Paramount to elucidating intestinal macrophage- and dendritic cell-mediated functions is the ability to effectively isolate and identify these cells from a complex cellular environment. In this review, we summarize methodology for the isolation and phenotypic characterization of macrophages and DCs from the mouse intestine and discuss how this may be useful for gaining insight into the mechanisms by which mucosal immune tolerance is maintained.

Reprogramming of monocytes by GM-CSF contributes to regulatory immune functions during intestinal inflammation.

Human and murine studies showed that GM-CSF exerts beneficial effects in intestinal inflammation. To explore whether GM-CSF mediates its effects via monocytes, we analyzed effects of GM-CSF on monocytes in vitro and assessed the immunomodulatory potential of GM-CSF-activated monocytes (GMaMs) in vivo. We used microarray technology and functional assays to characterize GMaMs in vitro and used a mouse model of colitis to study GMaM functions in vivo. GM-CSF activates monocytes to increase adherence, migration, chemotaxis, and oxidative burst in vitro, and primes monocyte response to secondary microbial stimuli. In addition, GMaMs accelerate epithelial healing in vitro. Most important, in a mouse model of experimental T cell-induced colitis, GMaMs show therapeutic activity and protect mice from colitis. This is accompanied by increased production of IL-4, IL-10, and IL-13, and decreased production of IFN-γ in lamina propria mononuclear cells in vivo. Confirming this finding, GMaMs attract T cells and shape their differentiation toward Th2 by upregulating IL-4, IL-10, and IL-13 in T cells in vitro. Beneficial effects of GM-CSF in Crohn's disease may possibly be mediated through reprogramming of monocytes to simultaneously improved bacterial clearance and induction of wound healing, as well as regulation of adaptive immunity to limit excessive inflammation.

Chagas disease reactivation in HIV-coinfected patients: histopathological aspects.

INTRODUCTION: Chagas disease reactivation has been described in severely immunocompromised patients by various etiologies, including in HIV-coinfected patients. OBJECTIVE: This study aimed to perform histopathological and immunohistochemical evaluation of the brain, myocardium, esophagus and large bowel of autopsied patients with CHD and/or acquired immunodeficiency syndrome in comparison with control patients. MATERIAL AND METHODS: Autopsy reports were reviewed from 1998 to 2012 and eight adult subjects were selected and divided into four groups: RE, CH, AI and CO. Sections of brain, myocardium, esophagus and large bowel were collected from each subject and processed for histological and immunohistochemical analysis. The histological sections stained with HE, Giemsa and picrosirius were used to quantify the density of inflammatory cells, the density of mast cells, and the percentage of collagen, respectively. Immunohistochemical analysis of IL17 and CD31 was performed. RESULTS: The density of mast cells in the myocardium was significantly higher in the CH group than in the other groups. The density of mast cells in the esophagus and in the large bowel was significantly higher when compared to the other groups. The percentage of collagen in the esophagus, myocardium and large bowel was significantly lower in the RE group than in the CO group. The CH group had a higher percentage of collagen in the myocardium and in the large bowel in relation to the other groups. The density of cells immunostained with anti-IL17 was significantly higher in the large bowel and in the myocardium in the CH group than in the CO group. There was higher density of vessels immunostained with anti-CD31 in the myocardium and esophagus of the AI group than in the other groups. There were no significant correlations between the density of mast cells and percentage of collagen in the RE, CO, CH and AI groups. CONCLUSION: Brain lesions observed in patients with CDR, as well as the higher density of cells immunostained with anti-IL17 at these sites, suggest that this cytokine was increasing local inflammation with subsequent tissue damage due to inflammation. Furthermore, the higher density of mast cells in the esophagus and large bowel of these subjects suggests that these cells might play a major role in esophageal and intestinal inflammation.

Understanding how commensal obligate anaerobic bacteria regulate immune functions in the large intestine.

The human gastrointestinal tract is colonised by trillions of commensal bacteria, most of which are obligate anaerobes residing in the large intestine. Appropriate bacterial colonisation is generally known to be critical for human health. In particular, the development and function of the immune system depends on microbial colonisation, and a regulated cross-talk between commensal bacteria, intestinal epithelial cells and immune cells is required to maintain mucosal immune homeostasis. This homeostasis is disturbed in various inflammatory disorders, such as inflammatory bowel diseases. Several in vitro and in vivo studies indicate a role for Faecalibacterium prausnitzii, Bacteroides thetaiotaomicron, Bacteroides fragilis, Akkermansia muciniphila and segmented filamentous bacteria in maintaining intestinal immune homeostasis. These obligate anaerobes are abundant in the healthy intestine but reduced in several inflammatory diseases, suggesting an association with protective effects on human health. However, knowledge of the mechanisms underlying the effects of obligate anaerobic intestinal bacteria remains limited, in part due to the difficulty of co-culturing obligate anaerobes together with oxygen-requiring human epithelial cells. By using novel dual-environment co-culture models, it will be possible to investigate the effects of the unstudied majority of intestinal microorganisms on the human epithelia. This knowledge will provide opportunities for improving human health and reducing the risk of inflammatory diseases.