Lamina Propria Of Small Intestine Research Articles

Lactococcus lactis subsp. Cremoris C60 restores T Cell Population in Small Intestinal Lamina Propria in Aged Interleukin-18 Deficient Mice.

Lactic acid bacteria (LAB), a major commensal bacterium in the small intestine, are well known beneficial bacteria which promote establishment of gut-centric immunity, such as anti-inflammation and anti-infection. In this report, we show that a LAB strain Lactococcus lactis subsp. Cremoris C60 possess an ability to activate antigen presenting cells, such as dendritic cells (DCs), and intestinal T cells which possibly support to maintain healthy intestinal immunological environment in aging process. We found that CD4+ T cells in the small intestine are dramatically decreased in aged Interleukin-18 knock out (IL-18KO) mice, associated with the impairment of IFN-γ production in the CD4+ T cells, especially in small intestinal lamina propria (LP). Surprisingly, heat killed-C60 (HK-C60) diet completely recovered the CD4+ T cells population and activity in SI-LP and over activated the population in Peyer’s patches (PPs) of IL-18KO mice. The HK-C60 diet was effective approach not only to restore the number of cells, but also to recover IFN-γ production in the CD4+ T cell population in the small intestine of IL-18-deficient mice. As a possible cause in the age-associated impairment of CD4+ T cells activity in IL-18KO mice, we found that the immunological activity was downregulated in the IL-18-deficient DCs. The cytokines production and cellular activation markers expression were downregulated in the IL-18-deficient bone marrow derived dendritic cells (BMDCs) at the basal level, however, both activities were highly upregulated in HK-C60 stimulation as compared to those of WT cells. Antigen uptake was also attenuated in the IL-18-deficient BMDCs, and it was significantly enhanced in the cells as compared to WT cells in HK-60 stimulation. An in vitro antigen presentation assay showed that IFN-γ production in the CD4+ T cells was significantly enhanced in the culture of IL-18-deficient BMDCs compared with WT cells in the presence of HK-C60. Thus, we conclude that HK-C60 diet possesses an ability to restore T cells impairment in the small intestine of IL-18-deficient environment. In addition, the positive effect is based on the immunological modification of DCs function which directory influences into the promotion of effector CD4+ T cells generation in the small intestine.

Fecal microbiota transplantation from patients with autoimmune encephalitis modulates Th17 response and relevant behaviors in mice

The significance of the microbiota-gut-brain axis has been increasingly recognized as a major modulator of autoimmunity. Here, we aim to characterize the gut microbiota of a large cohort of treatment-naïve anti-N-methyl-d-aspartate receptor (anti-NMDAR) encephalitis patients relative to that of healthy controls (HCs). Relative to HCs, anti-NMDAR encephalitis patients had a decreased microbiome alpha-diversity index, marked disturbances of gut microbial composition and intestinal permeability damage. Disturbed microbiota in anti-NMDAR encephalitis patients might be linked with different clinical characteristics. Imputed KEGG analysis revealed perturbations of functional modules in the gut microbiomes of anti-NMDAR encephalitis. Compared to HCs, microbiota-depleted mice receiving fecal microbiota transplantation (FMT) from anti-NMDAR encephalitis patients had hypersensitivity and cognitive impairment. Furthermore, anti-NMDAR encephalitis FMT mice showed altered T cells in the spleen and small intestine lamina propria with an increased Th17 cells. Overall, this study first suggests that the anti-NMDAR encephalitis microbiome itself can influence neurologic, Th17 response and behavioral function. The gut microbiota is a potential therapeutic target for anti-NMDAR encephalitis.

Exposures to uranium and arsenic alter intraepithelial and innate immune cells in the small intestine of male and female mice

Human exposures to environmental metals, including uranium (U) and arsenic (As) are a global public health concern. Chronic exposures to U and As are linked to many adverse health effects including, immune suppression and autoimmunity. The gastrointestinal (GI) tract is home to many immune cells vital in the maintenance of systemic immune health. However, very little is known about the immunotoxicity of U and As at this site. The present study examined the burden of U and As exposure in the GI tract as well as the resultant immunotoxicity to intraepithelial lymphocytes (IELs) and innate immune cells of the small intestine following chronic drinking water exposures of male and female mice to U (in the form of uranyl acetate, UA) and As (in the form of sodium arsenite, As3+). Exposure to U or As3+ resulted in high levels of U or As in the GI tract of male and female mice, respectively. A reduction of small intestinal CD4+ IELs (TCRαβ+, CD8αα+) was found following As3+ exposure, whereas U produced widespread suppression of CD4− IEL subsets (TCRαβ+ and TCRγδ+). Evaluation of innate immune cell subsets in the small intestinal lamina propria revealed a decrease in mature macrophages, along with a corresponding increase in immature/proinflammatory macrophages following As3+ exposures. These data show that exposures to two prevalent environmental contaminants, U and As produce significant immunotoxicity in the GI tract. Collectively, these findings provide a critical framework for understanding the underlying immune health issues reported in human populations chronically exposed to environmental metals.

Nasal vaccination of β7 integrin-deficient mice retains elevated IgA immunity.

Understanding the migration of lymphocytes to nonintestinal mucosal sites is fundamental to developing mucosal vaccination strategies. Studies have shown that nasal and oral immunization with cholera toxin (CT) stimulates, in addition to α4β7+ , the induction of αE (CD103)β7+ B cells. To determine the extent to which αE-associated β7 contributes to antigen (Ag)-specific immunoglobulin (Ig)A responses in the upper respiratory tract, nasal CT vaccination was performed in wild-type (wt) and β7-/- mice. At 16days postprimary immunization, upper respiratory tract IgA responses were greater in β7-/- mice than in wt mice. IgA induction by distal β7-/- Peyer's patches, mesenteric lymph nodes and small intestinal lamina propria was minimal, in contrast to elevated gut IgA responses in wt mice. By 42days postprimary immunization, β7-/- gut IgA responses were restored, and upper respiratory tract Ag-specific IgA responses were equivalent to those of wt mice. Examination of homing receptor expression and cell-sorting experiments revealed that β7-/- mice have increased usage of β1 and αE integrins by upper respiratory tract B cells, suggesting that alternative integrins can facilitate lymphocyte migration to the upper respiratory tract, especially in the absence of β7.

Analysis of tissue specific mechanisms that control the maintenance of Foxp3+ regulatory T cells in the intestine

Abstract Our lab studies the tissue specific pathways implicated in the maintenance of intestinal Foxp3+ regulatory T cells (Tregs). Previously we used K14 transgenic mice lacking peripheral TCR-MHCII to show that the small intestine lamina propria (siLP) is a niche for natural Tregs that can be filled and maintained independently of Ag-specific signals. K14 siLP Tregs display an effector phenotype (CD62loCD44hiCD103+ICOS+) similar to WT. Effector Tregs are known to be largely peripheral tissue-resident cells that require continued TCR and costimulatory signals for their maintenance. In contrast, we find that in K14 mice the blockade of the CTLA4/CD28—but not ICOS—pathway reduces the proliferation of siLP Tregs while preserving their effector phenotype (CD62loCD44hi). Thus, our results suggest that a population of thymically derived effector Tregs that depend on costimulatory signals can be maintained in the siLP independently of Ag-specific signals. B7-1/B7-2 and CD28/CTLA4 are cell surface molecules that interact through cell-cell contact. By microscopic analyses we show that a population of siLP Tregs localize inside of organized lymphoid structures known as isolated lymphoid follicles (ILF). B cells are the largest lymphocyte population within ILFs. Interestingly, our preliminary data suggest that B cells play a role in maintaining siLP Tregs in steady state, as the depletion of B cells with Rituximab reduces Treg numbers in the intestine of huCD20 mice. Additional imaging studies show ILF Tregs in intimate contact with B cells, suggesting that B cells might influence Tregs numbers by a cell-cell contact dependent mechanism. Thus, our data highlight the intricate signaling network that regulates intestinal Treg homeostasis.

Physiologic expression of AhR regulates CX3CR1+ resident intestinal macrophages and promotes diabetes in NOD mice

Abstract Susceptibility to type 1 diabetes (T1D) is influenced by both genetic and environmental factors. The environmental sensor, the aryl hydrocarbon receptor (AhR), is expressed in mucosal immune cells and can be activated by metabolites derived from the diet and microbiome. We hypothesized that these metabolites may influence the autoimmune response and subsequent susceptibility to T1D. To test this hypothesis, we generated NOD AhR knockout mice and examined the intestinal immune response and development of T1D. Female AhR knockout mice had reduced insulitis at 12 weeks of age, and a decreased incidence and onset of hyperglycemia in comparison to their wild type littermates. To determine whether physiologic expression of AhR was altering the immune response, we analyzed CD4+ T cell subsets and identified an increase in IL-10R+Foxp3+ Tregs in the lamina propria (LP) of AhR knockout mice. We also discovered a population of resident-like macrophages (CD11b+CX3CR1+) in the small intestine LP that were negatively regulated by AhR. These cells have a very strong positive correlation to LP Tregs, and correlate with protection against insulitis in NOD mice. To identify the potential source of AhR ligands that regulate these resident-like macrophages, mice were fed an AhR ligand-deficient diet. These cells were not further regulated by dietary ligands, suggesting that AhR activation originates from the intestinal microbiome. Current studies are aimed at determining how AhR regulates the expression of LP resident-like macrophages and how these cells impose their immunoregulatory functions during T1D pathogenesis.

Toll-like receptor 7 protects against intestinal inflammation and restricts the development of tissue-resident memory CD8+ T cells

Abstract The maintenance of intestinal homeostasis depends on a complex interaction between microbiota, intestinal epithelial barrier, and the immune system. Alteration in of one of these components could lead to the development of inflammatory bowel diseases (IBD). Norovirus infection of mice with a mutation in the Crohn’s disease susceptibility gene Atg16L1 induces intestinal inflammation. Moreover, persistent norovirus infection leads to intestinal virus-specific CD8+ T cells responses. However, the role of the enteric virome in IBD is still poorly understood. Toll-like receptor 7 (TLR7) recognizes single-stranded RNA viruses. Here, we investigate the role of TLR7 in intestinal homeostasis and inflammation. At steady state, Tlr7−/− mice have an approx. 10-fold increase in small and large intestinal lamina propria (LP) granzyme B+ tissue-resident memory (Trm) CD8+ T cells compared to WT mice (WT: 5.5, Tlr7−/−: 59.5 %, p < 0.005), reminiscent of persistent norovirus infection. Furthermore, Tlr7−/− mice were more susceptible to dextran sulfate sodium (DSS) colitis with more severe inflammation (Histoscore: WT: 7.6, Tlr7−/−: 12.7, p < 0.005), increased disease activity index (WT: 5.5, Tlr7−/−: 7.4, p < 0.05), and increased secretion of IFNg (WT: 5.2, Tlr7−/−: 24.2 ng/ml, p < 0.005) and TNFα (WT: 108.6, Tlr7−/−: 191.8 pg/ml, p < 0.05). Increased colonic inflammation was associated with increased LP Trm CD8+ T cells (WT: 3.9, Tlr7−/−: 42.0 %, p < 0.005). Our data shows that TLR7-deficiency promotes the development of LP Trm CD8+ T cells and increases susceptibility to DSS colitis. In conclusion, TLR7 plays an important role in maintaining immune response to intestinal viruses and protects against development of colitis.

Role for B-cell lymphoma 6 in intestinal innate lymphoid cell homeostasis and inflammatory disease

Abstract Group 3 innate lymphoid cells (ILC3) are critical for intestinal homeostasis. The functional activity of ILC3 must be tightly regulated as cytokines (e.g. IL-17 and IL-22) produced by these cells are critical for antimicrobial immune defense but can also contribute to intestinal disease when produced in excess. This regulation is achieved via incompletely defined transcriptional networks that include B-cell lymphoma 6 (BCL6), which we recently demonstrated is critical for maintaining ILC1- and ILC3-specific gene expression programs during homeostasis (Pokrovskii et al 2019). We now show that Bcl6-deficient small intestine lamina propria ILC3 exhibit elevated expression of the transcription factors retinoic acid receptor-related orphan nuclear receptor alpha (RORα) and RORγt. These factors are known to support ILC3 cytokine expression, potentially explaining elevated Il17a and Il17f expression by Bcl6-deficient ILC3. Of note, expression of Il22ra2 (IL-22 binding) was also markedly increased in these ILC3, likely limiting bioavailability of IL-22. Accordingly, these mice exhibit marked reduction in IL-22-dependent expression of epithelium-associated anti-microbial peptides in the terminal ileum. Of note, Bcl6ΔILC mice exhibited augmented susceptibility to dextran sodium sulfate-induced inflammatory bowel disease. Thus, BCL6 expression in ILC3-intrinsic BCL6 expression is important for mucosal immune defense and limiting intestinal inflammation.

Specific microbiota enhances intestinal IgA levels by inducing TGF-β in T follicular helper cells of Peyer's patches in mice.

In humans and mice, mucosal immune responses are dominated by IgA antibodies and the cytokine TGF-β, suppressing unwanted immune reactions but also targeting Ig class switching to IgA. It had been suggested that eosinophils promote the generation and maintenance of mucosal IgA-expressing plasma cells. Here, we demonstrate that not eosinophils, but specific bacteria determine mucosal IgA production. Co-housing of eosinophil-deficient mice with mice having high intestinal IgA levels, as well as the intentional microbiota transfer induces TGF-β expression in intestinal T follicular helper cells, thereby promoting IgA class switching in Peyer's patches, enhancing IgA+ plasma cell numbers in the small intestinal lamina propria and levels of mucosal IgA. We show that bacteria highly enriched for the genus Anaeroplasma are sufficient to induce these changes and enhance IgA levels when adoptively transferred. Thus, specific members of the intestinal microbiota and not the microbiota as such regulate gut homeostasis, by promoting the expression of immune-regulatory TGF-β and of mucosal IgA.

A34 EOSINOPHILS ALTER STEADY-STATE SMALL INTESTINAL IL-1α AND IL-1β LEVELS BUT ARE NOT REQUIRED FOR THE MAINTENANCE OF IGA-SECRETING LAMINA PROPRIA-RESIDENT PLASMA CELLS, SECRETORY IGA OR SERUM IGA LEVELS

Abstract Background During homeostasis eosinophils are highly abundant in the lamina propria of the small intestine. Eosinophils have been reported to play a role in promoting barrier function in the steady state intestinal tract, and in the control of intestinal colonization by harmless or symbiotic members of the bacterial microbiota. However, the role eosinophils play during enteric infection with a bacterial pathogen is unknown. Aims Our study aims to confirm the previously reported role of eosinophils in supporting intestinal barrier function and to investigate how the absence of eosinophils affects intestinal colonization by an enteric bacterial pathogen. Methods We used wildtype BALB/c and eosinophil-deficient (dblGATA knockout) BALB/c mice that had been cohoused, or bred as littermate controls, to normalize bacterial microbiota populations between mice used for experiments. To investigate steady state barrier function in these mice we used naïve mice to quantify levels of small intestinal IL-1α and IL-1β by cytometric bead arrays, and we used ELISAs to measure levels of immunoglobulin A (sIgA) and serum IgA. Levels of lamina propria-resident B220-IgA+ plasma cells were quantified using flow cytometry. To investigate the contributions of eosinophils to enteric bacterial infection, mice were orally infected with Salmonella enterica serovar Typhimurium. Salmonella burdens along the intestinal tract as well as in the liver and spleen were quantified. Results We found that levels of IL-1α and IL-1β were significantly decreased in the small intestine of naive eosinophil-deficient mice, compared to wildtype mice. In naïve wildtype and eosinophil-deficient littermate control mice, we did not detect any differences in sIgA or serum IgA levels. Additionally, levels of IgA producing plasma cells were similar in the small intestinal lamina propria between wildtype and eosinophil-deficient mice. Following oral Salmonella infection, Salmonella burdens were similar between wildtype and eosinophil-deficient mice both 24 hours and three days post-infection. Conclusions Our data supports a role for eosinophils in modifying steady-state cytokine levels in the intestinal tract. For the first time we report data on IgA levels from littermate controls of wildtype and eosinophil-deficient mice, and contrary to previously published reports, we found that eosinophils are not critical for the maintenance of intestinal sIgA, serum IgA or lamina propria resident IgA producing plasma cells. Our data further concluded that an absence of eosinophils did not impact control of Salmonella infection. Funding Agencies CIHR

Identification of Murine Innate Lymphoid Cell Subsets in Barrier Tissues and Their Draining Lymph Nodes Using Flow Cytometry.

Innate lymphoid cells (ILCs) are present in most tissues within the body but have been most extensively studied within mucosal barriers such as the lung and intestine. Isolation of immune cells from such tissues requires enzymatic digestion, and the number and composition of the cells released are dependent upon robust protocols tailored to the tissue of study. Here, detailed methodologies to isolate ILCs from various barrier sites and their draining lymph nodes (LNs) are described. Flow cytometry staining and gating strategies for identification and quantification of ILCs are then provided. Combined, these provide an efficient means to study ILCs within the small intestine lamina propria, lung, ear skin, and LNs.

Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131 Induce the Expression of the REG3 Family in the Small Intestine of Mice via the Stimulation of Dendritic Cells and Type 3 Innate Lymphoid Cells.

Accumulating evidence clarifies that intestinal barrier function, for example, by the mucus layer, antimicrobial peptides, immune systems, and epithelial tight junctions, plays crucial roles in maintaining our health. We reported previously that yogurt fermented with Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131 induced the gene expression of the regenerating family member 3 (REG3) family, which encodes antimicrobial peptides in the small intestine, although it was unclear how the yogurt activated the intestinal cells related to it. Here, we evaluated the cytokine production from the intestinal immune cells stimulated by these strains in vitro and in vivo to elucidate the mechanism for the induction of the REG3 family by the yogurt. The results showed that stimulation by both strains induced interleukin (IL)-23 production from bone marrow-derived dendritic cells (DCs) and IL-22 production from small intestinal lamina propria (LP) cells. In addition, oral administration of these strains to mice increased IL-23p19+ LPDCs and IL-22+ type 3 innate lymphoid cells and induced the expression of Reg3g in small intestinal tissue. Moreover, we showed that the activities for the induction of IL-23 by DCs were strain dependent on L. bulgaricus and S. thermophilus and that S. thermophilus 1131, which is the predominant species in the yogurt, exhibited relatively higher activity compared to the other strains of S. thermophilus. Our findings suggested that these yogurt starter strains, L. bulgaricus 2038 and S. thermophilus 1131, have the potential to maintain and improve intestinal barrier function by stimulating immune cells in the LP.

High-fat diet-derived free fatty acids impair the intestinal immune system and increase sensitivity to intestinal epithelial damage

In Japan and other Asian countries, increased fat uptake induced by a westernized diet is thought to be associated with an increased incidence of inflammatory bowel disease, colorectal cancer and food allergies; however, the mechanism for this remains unclear. High-fat diet (HFD)-fed mice are common animal models used to examine the effect of fat intake in vivo. HFDs are reported to exacerbate DSS-induced colitis and intestinal tumorigenesis, but the effect of HFDs on the intestines before disease induction is often overlooked. We found that the intestinal and gut-associated lymphoid tissue (GALT) morphology of HFD-fed mice differed from that of standard diet (SD)-fed mice. To clarify the mechanism by which fat intake increases intestinal diseases, we analyzed the morphological and immunological aspects of the intestines of HFD-fed mice as well as the molecular mechanisms and physiology.Feeding an HFD for 3 weeks induced atrophy of the small intestine, colon and GALT and reduced the number of small intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). Feeding an HFD for only one day reduced the number of small intestinal (SI)-IELs and SI-LPLs. The effect of feeding a 3-week HFD continued for 2 weeks after returning to the SD. The effect of the HFD on the intestinal immune system was independent of the gut microbes. We hypothesized that the cytotoxicity of the abundant HFD-derived free fatty acids in the intestinal lumen impairs the intestinal immune system. Both saturated and unsaturated free fatty acids were toxic to intestinal T-cells in vitro. Orally administering free fatty acids reduced the number of SI-IELs and LPLs. Using a lipase inhibitor to reduce the luminal free fatty acids attenuated the HFD-induced changes in the intestinal immune system, while using a statin to reduce the serum free fatty acids did not. Thus, HFD-induced free fatty acids damaged the intestines; this effect was termed “intestinal lipotoxicity”. Because sustained reduction of SI-LPLs after HFD feeding exacerbated indomethacin-induced small intestinal damage, lipotoxicity to the human intestines incurred by consuming a westernized diet in Japan may increase intestinal diseases such as IBD, colorectal cancer or food allergies.

Induction of Intestinal Th17 Cells by Flagellins From Segmented Filamentous Bacteria.

T-helper-17 (Th17) cells are a subset of CD4+ T cells that can produce the cytokine interleukin (IL)-17 and play vital roles in protecting the host from bacterial and fungal infections, especially at the mucosal surface. These are abundant in the small intestinal lamina propria (SILP) and their differentiation are associated with the colonization of the intestinal flora. Segmented filamentous bacteria (SFB) drew the attention of researchers due to their unique ability to drive the accumulation of Th17 cells in the SI LP of mice. Recent work has highlighted that SFB used microbial adhesion-triggered endocytosis (MATE) to transfer SFB antigenic proteins into small intestinal epithelial cells (SI ECs) and modulate host immune homeostasis. However, which components of SFB are involved in this immune response process remains unclear. Here, we examined the roles of SFB flagellins in Th17 cells induction using various techniques, including ELISA, ELISPOT, and RNA-seq in vitro and in vivo. The results show that the immune function of SFB flagellins is similar to SFB, i.e., induces the appearance of CD4+ T helper cells that produce IL-17 and IL-22 (Th17 cells) in the SI LP. Furthermore, treatment of mice with SFB flagellins lead to a significant increase in the expression of genes associated with the IL-17 signaling pathway, such as IL-6, IL-1β, TNF-α, IL-17A, IL-17F, and IL-22. In addition, SFB flagellins have an intimate relationship with intestinal epithelial cells, influencing the expression of epithelial cell-specific genes such as Nos2, Duox2, Duoxa2, SAA3, Tat, and Lcn2. Thus, we propose that SFB flagellins play a significant role in the involvement of SFB in the induction of intestinal Th17 cells.

γδ T Cell-Secreted XCL1 Mediates Anti-CD3-Induced Oral Tolerance.

Oral tolerance is defined as the specific suppression of cellular and/or humoral immune responses to an Ag by prior administration of the Ag through the oral route. Although the investigation of oral tolerance has classically involved Ag feeding, we have found that oral administration of anti-CD3 mAb induced tolerance through regulatory T (Treg) cell generation. However, the mechanisms underlying this effect remain unknown. In this study, we show that conventional but not plasmacytoid dendritic cells (DCs) are required for anti-CD3-induced oral tolerance. Moreover, oral anti-CD3 promotes XCL1 secretion by small intestine lamina propria γδ T cells that, in turn, induces tolerogenic XCR1+ DC migration to the mesenteric lymph node, where Treg cells are induced and oral tolerance is established. Consistent with this, TCRδ-/- mice did not develop oral tolerance upon oral administration of anti-CD3. However, XCL1 was not required for oral tolerance induced by fed Ags, indicating that a different mechanism underlies this effect. Accordingly, oral administration of anti-CD3 enhanced oral tolerance induced by fed MOG35-55 peptide, resulting in less severe experimental autoimmune encephalomyelitis, which was associated with decreased inflammatory immune cell infiltration in the CNS and increased Treg cells in the spleen. Thus, Treg cell induction by oral anti-CD3 is a consequence of the cross-talk between γδ T cells and tolerogenic DCs in the gut. Furthermore, anti-CD3 may serve as an adjuvant to enhance oral tolerance to fed Ags.

2546 Rare Case of Duodenal Melanosis in a 72-Year-Old Male

INTRODUCTION: Pseudomelanosis (PM) is a rare condition of unknown etiology. Prevalence of this condition is not well documented however, there have been several case reports. It is described as speckled black pigmentation of intestinal mucosa. Underlying pathogenesis and significance of PM is not well understood. Reports have associated it with medications, including anti-hypertensives and iron supplements and systemic diseases such as chronic renal disease, iron deficiency and diabetes mellitus. It is most commonly discovered as an incidental finding during endoscopy and frequently seen in the duodenum, however, few cases have also reported it in the stomach and jejunum. CASE DESCRIPTION/METHODS: A 72-year-old male with multiple co-morbidities including diabetes, hypertension, peripheral arterial disease and chronic kidney disease presented with epigastric pain, nausea and hematemesis. There was no melena or hematochezia. Last colonoscopy 10-years ago was significant for diverticulosis and hemorrhoids. His home medications included aspirin, clopidogrel, amlodipine, hydralazine, hydrochlorothiazide, atorvastatin, levothyroxine, furosemide and baclofen. On evaluation, digital rectal exam was significant for dark stools and hemoglobin decrease from 11.4 to 9.2 over 8 months. Upper endoscopy showed normal appearing esophagus and gastric mucosa. Dark pigmentation of the duodenal mucosa was noted and biopsied. Pathology findings were consistent with intestinal pseudomelanosis. Patient's symptoms resolved without any additional intervention. He was discharged on pantoprazole and continued on aspirin, but his clopidogrel was discontinued. DISCUSSION: Prognosis of PM is not well understood, but it is considered a benign condition. Unlike melanosis coli, which is a similar finding of black/brown speckles in the colon that has been associated with colon cancer. Histochemical studies have shown the pigments found in PM are likely secondary to accumulation of iron sulfide, ferrous sulfate or lipomenin within the lysosomes of macrophages in lamina propria of small intestine. Current theories suggest PM develops secondary to impaired iron transport coupled with sulfur from anti-hypertensive medications. In our patient, his medical conditions and anti-hypertensive medications likely contributed to this benign finding. Although, considered a benign condition, it is imperative to identify this condition during endoscopy to exclude other serious conditions.

Proliferation of Resident Macrophages Is Dispensable for Protection during Giardia duodenalis Infections.

Infection with the intestinal parasite Giardia duodenalis is one of the most common causes of diarrheal disease in the world. Previous work has demonstrated that the cells and mechanisms of the adaptive immune system are critical for clearance of this parasite. However, the innate system has not been as well studied in the context of Giardia infection. We have previously demonstrated that Giardia infection leads to the accumulation of a population of CD11b+, F4/80+, ARG1+, and NOS2+ macrophages in the small intestinal lamina propria. In this report, we sought to identify the accumulation mechanism of duodenal macrophages during Giardia infection and to determine if these cells were essential to the induction of protective Giardia immunity. We show that F4/80+, CD11b+, CD11cint, CX3CR1+, MHC class II+, Ly6C−, ARG1+, and NOS2+ macrophages accumulate in the small intestine during infections in mice. Consistent with this resident macrophage phenotype, macrophage accumulation does not require CCR2, and the macrophages incorporate EdU, indicating in situ proliferation rather than the recruitment of monocytes. Depletion of macrophages using anti-CSF1R did not impact parasite clearance nor development of regulatory T cell or Th17 cellular responses, suggesting that these macrophages are dispensable for protective Giardia immunity.

Increased Innate Lymphoid Cell 3 and IL-17 Production in Mouse Lamina Propria Stimulated with Giardia lamblia.

Innate lymphoid cells (ILCs) are key players during an immune response at the mucosal surfaces, such as lung, skin, and gastrointestinal tract. Giardia lamblia is an extracellular protozoan pathogen that inhabits the human small intestine. In this study, ILCs prepared from the lamina propria of mouse small intestine were incubated with G. lamblia trophozoites. Transcriptional changes in G. lamblia-exposed ILCs resulted in identification of activation of several immune pathways. Secretion of interleukin (IL)-17A, IL-17F, IL-1β, and interferon-γ was increased, whereas levels of IL-13, IL-5, and IL-22, was maintained or reduced upon exposure to G. lamblia. Goup 3 ILC (ILC3) was found to be dominant amongst the ILCs, and increased significantly upon co-cultivation with G. lamblia trophozoites. Oral inoculation of G. lamblia trophozoites into mice resulted in their presence in the small intestine, of which, the highest number of parasites was detected at the 5 days-post infection. Increased ILC3 was observed amongst the ILC population at the 5 days-post infection. These findings indicate that ILC3 from the lamina propria secretes IL-17 in response to G. lamblia, leading to the intestinal pathology observed in giardiasis.

BLT1 mediates commensal bacteria-dependent innate immune signals to enhance antigen-specific intestinal IgA responses

Leukotriene B4 receptor 1 (BLT1) triggers the migration of granulocytes and activated T cells; however, its role in B-cell function remains unclear. Here we report that BLT1 is required to induce the production of antigen-specific IgA against oral vaccine through mediating innate immune signals from commensal bacteria. B cells acquire BLT1 expression during their differentiation to IgA+ B cells and plasma cells in Peyer’s patches and the small intestinal lamina propria, respectively. BLT1 KO mice exhibited impaired production of antigen-specific fecal IgA to oral vaccine despite normal IgG responses to systemically immunized antigen. Expression of MyD88 was decreased in BLT1 KO gut B cells and consequently led to diminished proliferation of commensal bacteria-dependent plasma cells. These results indicate that BLT1 enhances the proliferation of commensal bacteria-dependent IgA+ plasma cells through the induction of MyD88 and thereby plays a key role in the production of antigen-specific intestinal IgA.

Neuroprotection of Resveratrol Against Focal Cerebral Ischemia/Reperfusion Injury in Mice Through a Mechanism Targeting Gut-Brain Axis.

Increasing evidences have shown that resveratrol could protect the brain from ischemic injury; the mechanisms underlying its neuroprotective effects are multifactorial and not fully understood. It remains unclear whether resveratrol could exert neuroprotection through modulating gut-brain axis, which plays important rolesin stroke pathology. In this study, C57BL/6 mice underwent middle cerebral artery occlusion (60min) followed by reperfusion for 3 days. Resveratrol, when applied immediately after MCAO onset for 3 days, promoted Th1/Th2 balance towards Th2 polarization and skewed Treg/Th17 balance towards Treg in the small intestinal lamina propria (SI-LP), and decreased small intestinal pro-inflammatory cytokines expression through modulating intestinal flora at 3days post-ischemia (dpi). Resveratrol attenuated cerebral ischemia-induced increase in the epithelial and vascular permeability of small intestine as evidenced by reduced evans blue extravasasion and decreased protein leakage by feces/plasma albumin ratio at 3 dpi. The blood levels of pro-inflammatory cytokines at 3 dpi were also attenuated by resveratrol due to inhibiting intestinal pro-inflammatory immunity and decreasing epithelial and vascular permeability. Resveratrol robustly protected against post-stroke inflammation-induced blood-brain barrier disruption not only in the cortex but also in the striatum at 3 dpi. Furthermore, resveratrol mediated smaller cerebral infarcts and less neurological deficits via decreasing the levels of pro-inflammatory cytokines in the peri-infarct area at 3 dpi. Our results for the first time demonstrated that resveratrol may inhibit systemic post-stroke inflammation and neuroinflammation via modulating intestinal flora-mediated Th17/Tregs and Th1/Th2 polarity shift in SI-LP, which may be one of the mechanisms underlying the neuroprotective effects of resveratrol.