Cells In Intestinal Lamina Propria Research Articles

Colonic mucosal associated invariant T cells in Crohn's disease have a diverse and non-public T cell receptor beta chain repertoire.

Mucosal-Associated Invariant T (MAIT) cells are T cells with a semi-invariant T cell receptor (TCR), recognizing riboflavin precursors presented by a non-polymorphic MR1 molecule. As these precursors are produced by the gut microbiome, we characterized the frequency, phenotype and clonality of MAIT cells in human colons with and without Crohn's disease (CD). The transcriptome of MAIT cells sorted from blood and intestinal lamina propria cells from colectomy recipients were compared with other CD8+ T cells. Colon biopsies from an additional ten CD patients and ten healthy controls (HC) were analyzed by flow cytometry. TCR genes were sequenced from individual MAIT cells from these biopsies and compared with those of MAIT cells from autologous blood. MAIT cells in the blood and colon showed a transcriptome distinct from other CD8 T cells, with more expression of the IL-23 receptor. MAIT cells were enriched in the colons of CD patients, with less NKG2D in inflamed versus uninflamed segments. Regardless of disease, most MAIT cells expressed integrin α4β7 in the colon but not in the blood, where they were enriched for α4β7 expression. TCR sequencing revealed heterogeneity in the colon and blood, with few public sequences associated with cohorts. MAIT cells are enriched in the colons of CD patients and disproportionately express molecules (IL-23R, integrin α4β7) targeted by CD therapeutics, to suggest a pathogenic role for them in CD. Public TCR sequences were neither common nor sufficiently restricted to a cohort to suggest protective or pathogenic antigen-specificities.

Effect of Dietary Chlorella vulgaris or Tetradesmus obliquus on Laying Performance and Intestinal Immune Cell Parameters.

A feeding trial was conducted to investigate the effect of dietary supplementation of Chlorella vulgaris (CV) or Tetradesmus obliquus (TO) on laying performance, egg quality, and gut health indicators of laying hens. A total of 144 Hy-Line Brown laying hens aged 21 weeks were randomly assigned to one of three dietary treatments with eight replicates of six hens. Dietary treatments were as follows: CON, basal diet; CV, basal diet + 5 g C. vulgaris/kg of diet; TO, basal diet + 5 g T. obliquus/kg of diet. The results showed that diets supplemented with CV or TO had insignificant effects on laying performance, egg quality (i.e., Haugh unit and eggshell strength and thickness), jejunal histology, cecal short-chain fatty acids, and antioxidant/immune markers in ileal mucosa samples of laying hens. Compared with the control group, the egg yolk color score was higher (p < 0.05) in laying hens fed on diets containing CV and TO, although the former was a more intense yellow than the latter. Small intestinal lamina propria cells were isolated using flow cytometry to examine the percentages of immune cell subpopulations. Dietary microalgae did not affect B cells or monocytes/macrophages but altered the percentage of CD4+ T cells and CD8- TCR γδ T cells. Collectively, diets supplemented with C. vulgaris or T. obliquus can improve egg yolk color and would modulate host immune development and competence in laying hens.

Maternal antibiotic treatment during pregnancy attenuates the transport and absorption of maternal antibody IgG through TLR4 and TLR2 receptor.

Maternal antibody IgG, the main antibody in colostrum, plays an important role in neonates protection. Commensal microbiota is closely related to host antibody repertoire. However, there are few reports on how maternal gut microbiota affects maternal antibody IgG transfer. In the present study, we investigated the effects of altering the gut microbiota (treated with antibiotics during pregnancy) on maternal IgG transportation and offspring absorption and explored its underlying mechanisms. Results showed that antibiotic treatment during pregnancy significantly decreased maternal cecal microbial richness (Chao1 and Obesrved species) and diversity (Shannon and Simpson). Plasma metabolome enriched significant changes in the process of bile acid secretion pathway, and the concentration of deoxycholic acid, a secondary metabolite of microorganisms was lowered. Flow cytometry analysis indicated that antibiotic treatment promoted the number of B cells and abated the number of T, DC and M1 cells in intestinal lamina propria of dams. Surprisingly, the serum IgG level in antibiotic treated dams was significantly increased, while IgG contents in colostrum was decreased. Moreover, pregnancy antibiotic treatment in dams was reduced the expression of FcRn, TLR4 and TLR2 in breast of dams and in duodenum and jejunum of neonates. Furthermore, TLR4-/- and TLR2-/- knock-out mice showed a lower FcRn expression in breast of dams and in duodenum and jejunum of neonates. These findings suggest that maternal intestine bacteria may affect the maternal IgG transfer through regulating the breast TLR4 and TLR2 of dams.

Combination Therapy with Indigo and Indirubin for Ulcerative Colitis via Reinforcing Intestinal Barrier Function.

Indigo and indirubin, the active molecules of traditional Chinese medicine indigo naturalis, exert therapeutic activity for ulcerative colitis (UC). Indigo and indirubin are isomers and have distinctive profiles in anti-inflammation, immune regulation, intestinal microbiota regulation, oxidative stress regulation, and intestinal mucosal repair for UC treatment. Thus, exploring its combined administration's integrated advantages for UC is critical. This study is aimed at clarifying the effect and mechanisms of the combined administration of indigo and indirubin on colitis mouse models. The results showed that all the treatment groups could improve the disease symptoms, and the combined administration showed the best effect. Additionally, compared with indigo and indirubin alone, the combination group could significantly reinforce intestinal barrier function by increasing the expression of E-cadherin, occludin, ZO-1, and MUC2 and improving intestinal permeability. The treatment groups significantly improved the expression of cytokines, including TNF-α, IFN-γ, IL-12, IL-23, and IL-17A, and indirubin presented the most potent anti-inflammatory effect. Furthermore, all the treatment groups reduced the infiltration of the immune cells in intestinal lamina propria and the production of ROS/RNS. Notably, indigo exhibited a more substantial capacity to regulate natural killer (NK) cells, ILC3, neutrophils, and dendritic cells, followed by the combination group and indirubin alone. Finally, all the treatment groups modulated intestinal microbiota composition, increased the proportion of beneficial microbiota, and decreased the proportion of microbiota. Our results indicated that indigo and indirubin synergistically reinforced the intestinal barrier function, which may be associated with integrating the indirubin anti-inflammatory and intestinal microbiota regulating strength and indigo immune and ROS/RNS regulation advantage.

초기 육계 사료 내 미세조류(Tetradesmus sp.) 첨가에 따른 장관환경 변화

This study aimed to determine the effects of dietary microalgae <i>(Tetradesmus sp.</i> (TO)) on intestinal immunity and microbiota of pre-starter broilers. One hundred and twenty 1-day-old birds (Ross 308) were allocated to two dietary treatment groups with six blocks in a randomized complete block design. The two experimental diets consisted of a corn-soybean meal-based basal diet and a diet with 0.5% TO powder instead of cornstarch in the basal diet. After feeding the experimental diets for ten days, all birds’ body weight and feed intake were measured, and representative eight birds were selected from each treatment group. Small intestinal lamina propria cells were isolated using flow cytometry to examine the frequency of immune cells. Cecal feces were harvested for 16s rRNA gut microbiota analysis and fecal IgA levels. Here, we found that 0.5% TO supplementation increased CD3⁺CD4⁺ T cells in the small intestine, but decreased CD3⁺CD8⁺ T cells in the small intestine. Gut microbial analysis showed that TO supplementation significantly increased the alpha diversity of the gut microbiome. Taxonomic analysis showed that TO treatment increased the abundance of Firmicutes and decreased that of Bacteroidetes at the phylum level. The distribution of <i>Enterobacteriaceae</i> containing many harmful bacteria at the family level, was lower in the TO group. In the LEfSe analysis, the TO group had a significantly enriched abundance of <i>Agathobaculum</i> at the genus level. Overall, results show that <i>Tetradesmus sp.</i> supplementation influences intestinal T-cell immunity and induces the expansion of beneficial gut microbes in pre-starter broiler chickens.

Differential Modulation of Innate Antiviral Profiles in the Intestinal Lamina Propria Cells of Chickens Infected with Infectious Bursal Disease Viruses of Different Virulence.

Infectious bursal disease virus (IBDV) is one of the most important infectious diseases of poultry around the world. Gut-associated lymphoid tissues (GALT) are the first line of defense of the host against the infection. The purpose of this study was to investigate the role of innate immune antiviral signaling triggered by Toll-like receptor 3 (TLR3), as well as macrophage activation and cytokine response in the intestinal lamina propria (ILP) cells after the oral challenge of IBDV in relation to IBDV virulence and disease pathogenesis. The results showed that the expression levels of TLR3, IRF7, IFN-α/β and the corresponding downstream antiviral factors OAS, PKR and Mx were all upregulated in the SPF chicken ILP cells at 8 h post-infection (hpi) and 12 hpi. Similarly, macrophages were activated, with the initial macrophage M1 activation observed at 8 hpi, but then it rapidly shifted to a non-protective M2-type. Both Th1 (IFN-γ, TNF-α, IL-12) and Th2 (IL-4 and IL-10) types of cytokines were differentially upregulated during the early stage of infection; however, the Th1 cytokines exhibited stronger activation before 8 hpi compared to those of the Th2 cytokines. Interestingly, differential regulations of gene expression induced by different IBDV strains with different virulence were detected. The HLJ0504-like very virulent (vv) IBDV strain NN1172 induced stronger activation of TLR3-IFN-α/β pathway, macrophages and the Th1/2 cytokines’ expression, compared to those induced by the attenuated strain B87 at 8 hpi and 12 hpi in the ILP cells. In conclusion, the innate antiviral response mediated by the TLR3-IRF7 pathway, macrophage activation and cytokine expression in the GALT cells at the early stage of IBDV infection was differentially modulated, and the HLJ0504-like vvIBDV strain triggered stronger activation than the attenuated vaccine strain, and that may play an important role in the progression of disease.

Ontogeny of ROR\u03b3t+ cells in the intestine of newborns and its role in the development of experimental necrotizing enterocolitis

BackgroundNeonates possess an immature and plastic immune system, which is a major cause of some diseases in newborns. Necrotizing enterocolitis (NEC) is a severe and devastating intestinal disease that typically affects premature infants. However, the development of intestinal immune cells in neonates and their roles in the pathological process of NEC have not been elucidated.ResultsWe examined the ontogeny of intestinal lamina propria lymphocytes in the early life of mice and found a high percentage of RORγt+ cells (containing inflammatory Th17 and ILC3 populations) during the first week of life. Importantly, the proportion of RORγt+ cells of intestinal lamina propria further increased in both NEC mice and patients tissue than the control. Furthermore, the application of GSK805, a specific antagonist of RORγt, inhibited IL-17A release and ameliorated NEC severity.ConclusionsOur data reveal the high proportion of RORγt+ cells in newborn mice may directly contribute to the development of NEC.

CD127+ CD94+ innate lymphoid cells expressing granulysin and perforin are expanded in patients with Crohn\u2019s disease

Phenotypic definition of helper ILC1 and NK cells is problematic due to overlapping markers. Recently we showed the identification of cytotoxic ILC3s characterized by expression of CD94. Here we analyse CD127+ ILCs and NK cells in intestinal lamina propria from healthy donors and Crohn’s disease patients and identify two populations of CD127+CD94+ ILCs, designated population A and B, that can be distinguished on the expression of CD117, CD18 and cytotoxic molecules. Population B expresses granulysin, a cytotoxic molecule linked to bacterial lysis and/or chemotaxis of monocytes. Granulysin protein is secreted by population B cells upon stimulation with IL-15. Activation of population B in the presence of TGF-β strongly reduces the expression of cytotoxic effector molecules of population B. Strikingly, samples from individuals that suffer from active Crohn’s disease display enhanced frequencies of granulysin-expressing effector CD127+CD94+ ILCs in comparison to controls. Thus this study identifies group 1 ILC populations which accumulate in inflamed intestinal tissue of Crohn’s disease patients and may play a role in the pathology of the disease.

Ingestion of organic acids and cinnamaldehyde improves tissue homeostasis of piglets exposed to enterotoxic Escherichia coli (ETEC).

Organic acids (OA) and phytogenic compounds have been used in pig feeding as alternatives to antibiotic growth promoters. However, few studies have evaluated the systemic effect of the combination of these additives. The aim of this study was to assess the impact of an organic acid-based feed additive (OAFA), containing a blend of OA and cinnamaldehyde, on the tissue integrity of bacterially challenged piglets. Thirty weaned piglets 21 d old were used in a 19-d trial. Pigs received a standard diet during the first 7 d and afterward were allotted to five treatments. Dietary treatments were: Control (basal diet), Escherichia coli (basal diet and challenge with E. coli), colistin (basal diet + 200 mg colistin/kg feed + challenge with E. coli), OAFA1 (basal diet + 1 kg OAFA/ton feed + challenge with E. coli), and OAFA2 (basal diet + 2 kg OAFA/ton feed + challenge with E. coli). Seven days after the beginning of the treatment, the animals were challenged with an enterotoxic strain of E. coli (K88) for pigs. Five days after the challenge, all animals were euthanized for tissue sampling for histological and oxidative stress (intestine and liver) analysis. The reduced glutathione (GSH), ferric-reducing ability potential (FRAP), and free-radical scavenging ability (ABTS) assays were used to evaluate the intestinal antioxidant defense. Lipid peroxidation and superoxide anion production were evaluated through the levels of thiobarbituric acid-reactive substances (TBARS) and nitroblue tetrazolium (NBT) reduction assay, respectively. Animals fed the OAFA (1 and 2) diets had a decrease (P < 0.05) on histological changes in the intestine, liver, mesenteric lymph nodes, and spleen. Greater villus height (VH) and a higher ratio of VH to crypt depth (CD) were observed in animals of the OAFA2 group compared with the control and E. coli groups. The colistin and OAFA groups decreased (P < 0.05) the number of inflammatory cells in intestinal lamina propria. OAFA2 group increased (P < 0.05) intestinal cell proliferation. Colistin and OAFA2 supplementation induced a decrease (P < 0.05) in the levels of TBARS in both the intestine and liver compared with the E. coli group. In addition, an increase (P < 0.05) in GSH and FRAP ileal levels was observed in the OAFA2 group compared with E. coli group. These results show that the supplementation with OAFA in the diet of weaned piglets, especially at a dose of 2 kg/ton (OAFA2) protected tissues against enterotoxigenic Escherichia coli (ETEC) damage.

Beneficial effects of dual TORC1/2 inhibition on chronic experimental colitis

Background and aimAZD8055, a new immunosuppressive reagent, a dual TORC1/2 inhibitor, had been used successfully in animal models for heart transplantation. The aim of this study was to evaluate the effects and mechanisms of AZD8055 on chronic intestinal inflammation. MethodsDextran sulfate sodium (DSS) - induced chronic colitis was used to investigate the effects of AZD8055 on the development of colitis. Colitis activity was monitored by body weight assessment, colon length, histology and cytokine profile analysis. ResultsAZD8055 treatment significantly alleviated the severity of colitis, as assessed by colonic length and colonic damage. In addition, AZD8055 treatment decreased the colonic CD4+ T cell numbers and reduced both Th1 and Th17 cell activation and cytokine production. The percentages of Treg cells in the colon were also expanded by AZD8055 treatment. Furthermore, AZD8055 effectively inhibited mTOR downstream proteins and signal transducer and activator of transcription related proteins in CD4+ T cells of intestinal lamina propria. ConclusionsThese findings increased our understanding of DSS-induced colitis and shed new lights on mechanisms of digestive tract chronic inflammation. Dual TORC1/2 inhibition showed potent anti-inflammatory and immune regulation effects by targeting critical signaling pathways. The results supported the strategy of using dual mTOR inhibitor to treat inflammatory bowel disease.

The NLRP3 inflammasome mediates DSS-induced intestinal inflammation in Nod2 knockout mice.

Crohn’s disease (CD) is a chronic disorder of the gastrointestinal tract characterized by inflammation and intestinal epithelial injury. Loss of function mutations in the intracellular bacterial sensor NOD2 are major risk factors for the development of CD. In the absence of robust bacterial recognition by NOD2 an inflammatory cascade is initiated through alternative PRRs leading to CD. In the present study, MCC950, a specific small molecule inhibitor of NLR pyrin domain-containing protein 3 (NLRP3), abrogated dextran sodium sulfate (DSS)-induced intestinal inflammation in Nod2−/− mice. NLRP3 inflammasome formation was observed at a higher rate in NOD2-deficient small intestinal lamina propria cells after insult by DSS. NLRP3 complex formation led to an increase in IL-1β secretion in both the small intestine and colon of Nod2ko mice. This increase in IL-1β secretion in the intestine was attenuated by MCC950 leading to decreased disease severity in Nod2ko mice. Our work suggests that NLRP3 inflammasome activation may be a key driver of intestinal inflammation in the absence of functional NOD2. NLRP3 pathway inhibition can prevent intestinal inflammation in the absence of robust NOD2 signaling.

Myeloid Cells in the Immune Response to Giardia

Abstract Infection with the protozoan parasite, Giardia, produces symptoms associated with intestinal distress, but does not induce intestinal inflammation. Long-term sequellae of this infection include malnutrition, Irritable Bowel Syndrome and Chronic Fatigue Syndrome. Previous work indicates that T-cells are critical for clearance of this parasite; however, it is unclear which cells are responsible for the activation of T-cells. As such, our goal was to examine the contribution of myeloid cells to Giardia immunity as possible T-cell activators, effector cells and/or immune regulators. Small intestinal lamina propria cells were isolated from Giardia-infected wild-type or CCR2 deficient mice. Flow cytometry was used to determine cell phenotypes and to monitor cytokine response. EdU labeling was used to examine in situ proliferation of resident macrophages. Lastly, wild-type mice were treated with an anti-CSF1R monoclonal antibody to deplete macrophage populations. Flow cytometry demonstrated that CD11b+, F4/80+, CD11cint, MHCII+, Ly6Cint, CX3CR1int cells accumulate in the small intestine and express ARG1 and NOS2 during infection. Intracellular cytokine staining revealed an increase in IL-10 with a concurrent decrease in TNF-α. Macrophage accumulation was the same in wild-type and CCR2-deficient mice, and EdU labeling suggested in situ proliferation of resident macrophages rather than recruitment of inflammatory monocytes. Mice treated with anti-CSF1R eliminated the parasites as well as control mice. This study demonstrates that macrophages with a regulatory phenotype accumulate in the small intestine during Giardia infections and may explain why intestinal inflammation is not present during Giardia infections.

Regulatory role of NKG2D+ NK cells in intestinal lamina propria by secreting double-edged Th1 cytokines in ulcerative colitis.

The role of intestinal lamina propria (LP) NKG2D+ NK cells is unclear in regulating Th1/Th2 balance in ulcerative colitis (UC). In this study, we investigated the frequency of LP NKG2D+ NK cells in DSS-induced colitis model and intestinal mucosal samples of UC patients, as well as the secretion of Th1/Th2/Th17 cytokines in NK cell lines after MICA stimulation. The role of Th1 cytokines in UC was validated by bioinformatics analysis. We found that DSS-induced colitis in mice was characterized by a Th2-mediated process. In acute phrase, the frequency of LP NKG2D+ lymphocytes increased significantly and decreased in remission, while the frequency of LP NKG2D+ NK cells decreased significantly in acute phase and increased in remission. No obvious change was found in the frequency of total LP NK cells. Similarly, severe UC patients had a higher expression of mucosal NKG2D and a lower number of NKG2D+ NK cells than mild to moderate UC. In NK cell lines, the MICA stimulation could induce a predominant secretion of Th1 cytokines (TNF, IFN-γ). Furthermore, in bioinformatics analysis, mucosal Th1 cytokine of TNF, showed a double-edged role in UC when compared to the Th1-mediated disease of Crohn's colitis. In conclusion, LP NKG2D+ NK cells partially played a regulatory role in UC through secreting Th1 cytokines to regulate the Th2-predominant Th1/Th2 imbalance, despite of the concomitant pro-inflammatory effects of Th1 cytokines.

CD103+ Dendritic Cell Function Is Altered in the Colons of Patients with Ulcerative Colitis.

Human intestinal innate myeloid cells can be divided into 3 subsets: HLA-DRCD14 cells, HLA-DRCD103 dendritic cells (DCs), and HLA-DRCD14CD103 cells. CD103 DCs generate Treg cells and Th17 cells in the ileum, but their function in the colon remains largely unknown. This study characterized CD103 DCs in the colon and investigated whether these cells are implicated in the pathogenesis of ulcerative colitis (UC). Normal intestinal mucosa was obtained from intact sites of patients with colorectal cancer (n = 24). Noninflamed and inflamed colonic tissues were obtained from surgically resected specimens of patients with UC (n = 13). Among LinCD45HLA-DR intestinal lamina propria cells, CD14 cells and CD103 DCs were sorted and analyzed for microRNA expression of cytokines and toll-like receptors by quantitative real-time polymerase chain reaction. In addition, IL-4/IL-5/IL-13/IL-17/IFN-γ production and Foxp3 expression by naive T cells cultured with CD14 cells and CD103 DCs were analyzed. CD103 DCs in the normal colon showed lower expression of toll-like receptors and proinflammatory cytokines than CD14 cells. Coculture with naive T cells revealed that CD103 DCs generated Treg cells. CD103 DCs from patients with UC did not generate Treg cells, but they induced IFN-γ-, IL-13-, and IL-17-producing CD4 T cells and showed higher expression of IL6 (P < 0.0001), IL23A (P < 0.05), IL12p35 (P < 0.05), and TNF (P < 0.05). In patients with UC, CD103 DCs show the impaired ability to generate Treg cells, but exhibit a colitogenic function inducing Th1/Th2/Th17 responses. These findings show how human CD103 DCs could contribute to the pathogenesis of UC.

Identification of a human intestinal myeloid cell subset that regulates gut homeostasis.

Inappropriate activation of T helper (Th) cells, such as Th1 and Th17 cells, is implicated in the pathogenesis of chronic inflammatory disorders including ulcerative colitis (UC). CX3CR1high macrophages contribute to intestinal homeostasis through various mechanisms in mice. However, whether mononuclear phagocytes with regulatory functions are present in the human colon is not clearly defined. We investigated whether innate myeloid cells that suppress activation of effector T cells exist in the human intestinal mucosa. Among intestinal lamina propria cells, Lin- HLA-DRhigh CD14+ CD163high cells were subdivided into CD160low and CD160high cells. Both subsets produced high levels of IL-10. CD163high CD160high cells suppressed effector T cell proliferation, whereas CD163high CD160low cells induced Th17 differentiation. Patients with UC exhibited increased numbers of CD163high CD160low cells, while showing profoundly decreased numbers of CD163high CD160high cells. In this context, CD163high CD160high cells had higher CD80/CD86 expression and lower IL10RB expression, and these cells did not suppress effector T cell proliferation. The CD163high CD160high subset in normal intestinal mucosa inhibits inappropriate Th1/Th17 responses through suppression of their proliferation, and its number and suppressive activity are impaired in patients with UC. These findings indicate how human innate immune cells might prevent UC development.

A role of SIRPα/CD172a on eosinophil stability in secondary lymphoid tissues

Event Abstract Back to Event A role of SIRPα/CD172a on eosinophil stability in secondary lymphoid tissues Noel Verjan Garcia1, 2, 3* and Masayuki Miyasaka2, 3, 4* 1 University of Tolima, Animal Health, Colombia 2 Osaka University, Department of Microbiology and Immunology Osaka University Graduate School of Medicine, Japan 3 Osaka University, Japan 4 Osaka University, Japan Eosinophils are pleiotropic and multifunctional leukocytes involved in a variety of cellular processes. Previously, we reported that intestinal eosinophils constitutively express high levels of an ITIM-containing receptor, SIRPα/CD172a, that appears to regulate eosinophil homeostasis through the interaction with CD47, with substantial effects on its degranulation and survival (Verjan Garcia N et al, J. Immunol. 187:2268, 2011). SIRPα Cyto−/− eosinophils that express a mutated SIRPα/CD172a protein lacking most of its cytoplasmic domain showed increased degranulation and reduced viability, which were accompanied by intestinal lymphoid tissues (P.P and MLN) and splenic red pulp hyperplasia. Here, we analyzed the effect of disruption of SIRPα/CD172a signaling on various cell populations including peritoneal cells, small intestinal lamina propria cells, and those from the mesenteric lymph nodes (MLNs), Peyer’s patches (PPs) and peripheral blood under both steady state and inflammatory conditions. SIRPα Cyto−/− mice showed increased number of leukocytes in the peritoneal cavity and MLNs, whereas cells in the small intestinal lamina propria were reduced. The numbers of cells in the peripheral blood and PPs were also slightly increased in SIRPα Cyto−/− mice. In addition, B220+MHCII+ B-cells were substantially increased in the lamina propria of the small intestine, peritoneal cavity and MLN of SIRPα Cyto−/− mice, whereas these cells were proportionally reduced in PP and PBL. The B cells were often located close to degranulating SIRPα Cyto−/− eosinophils in In the small intestinal lamina propria, whereas in the spleen, degranulating eosinophils and MBP+CD63+ amorphous structures appeared to be phagocytosed in the marginal zone and red pulp of CD47−/− mice. In MLNs eosinophils were abundant and often found within the high endothelial venules (HEVs) under steady state conditions, however, when a Th2-biased inflammation was induced by intraperitoneal injection of IL-25, a substantial number of eosinophils accumulated in the T-cell area of MLNs and red pulp of the spleen in SIRPα Cyto−/− mice compared to WT mice. These data are consistent with the hypothesis that SIRPα signaling is required for eosinophil stability in the steady state and that, in the absence of this signaling, dysregulated eosinophil degranulation/survival was associated with MHCII+B220+ B-cell increase in intestinal lamina propria, peritoneal cavity and MLNs. Under inflammatory conditions, degranulating eosinophils seem to leave these tissues and accumulate in the spleen and MLNs, where phagocytes remove damaged cells, which might have resulted in no obvious tissue damage. Acknowledgements We thank Dr. Per-Arne Oldenborg for providing the CD47 -/- mice.

Activation of Pattern Recognition Receptors Up-Regulates Metallothioneins, Thereby Increasing Intracellular Accumulation of Zinc, Autophagy, and Bacterial Clearance by Macrophages

Continuous stimulation of pattern recognition receptors (PRRs), including nucleotide-binding oligomerization domain-2 (NOD2) (variants in NOD2 have been associated with Crohn's disease), alters the phenotype of myeloid-derived cells, reducing production of inflammatory cytokines and increasing microbe clearance. We investigated the mechanisms by which microbial clearance increases in macrophages under these conditions. Monocytes were purified from human peripheral blood mononuclear cells and differentiated to monocyte-derived macrophages (MDMs). We also isolated human intestinal macrophages. Bacterial clearance by MDMs was assessed in gentamicin protection assays. Effects of intracellular zinc and autophagy were measured by flow cytometry, immunoblot, reverse-transcription polymerase chain reaction, and microscopy experiments. Small interfering RNAs were used to knock down specific proteins in MDMs. NOD2-/- and C57BL/6J mice, maintained in a specific pathogen-free facility, were given antibiotics, muramyl dipeptide (to stimulate NOD2), or dextran sodium sulfate; intestinal lamina propria cells were collected and analyzed. Chronic stimulation of human MDMs through NOD2 up-regulated the expression of multiple genes encoding metallothioneins, which bind and regulate levels of intracellular zinc. Intestinal myeloid-derived cells are stimulated continually through PRRs; metallothionein expression was up-regulated in human and mouse intestinal myeloid-derived cells. Continuous stimulation of NOD2 increased the levels of intracellular zinc, thereby increasing autophagy and bacterial clearance. The metal-regulatory transcription factor-1 (MTF-1) was required for regulation of metallothionein genes in human MDMs. Knockdown of MTF-1 did not affect baseline clearance of bacteria by MDMs. However, the increase in intracellular zinc, autophagy, and bacterial clearance observed with continuous NOD2 stimulation was impaired in MDMs upon MTF-1 knockdown. The addition of zinc or induction of autophagy restored bacterial clearance to MDMs after metallothionein knockdown. NOD2 synergized with the PRRs Toll-like receptors 5 and 9 increase the effects of metallothioneins in MDMs. In mice, the intestinal microbiota contributed to the regulation in expression of metallothioneins, levels of zinc, autophagy, and bacterial clearance by intestinal macrophages. In studies of human MDMs and in mice, continuous stimulation of PRRs induces expression of metallothioneins. This leads to increased levels of intracellular zinc and enhanced clearance of bacteria via autophagy in macrophages.

Increased Th17-Inducing Activity of CD14+ CD163low Myeloid Cells in Intestinal Lamina Propria of Patients With Crohn’s Disease

Abnormal activity of innate immune cells and T-helper (Th) 17 cells has been implicated in the pathogenesis of autoimmune and inflammatory diseases, including Crohn's disease (CD). Intestinal innate immune (myeloid) cells have been found to induce development of Th17 cells in mice, but it is not clear if this occurs in humans or in patients with CD. We investigated whether human intestinal lamina propria cells (LPCs) induce development of Th17 cells and whether these have a role in the pathogenesis of CD. Normal intestinal mucosa samples were collected from patients with colorectal cancer and noninflamed and inflamed regions of mucosa were collected from patients with CD. LPCs were isolated by enzymatic digestion and analyzed for expression of HLA-DR, lineage markers CD14 and CD163 using flow cytometry. Among HLA-DR(high) Lin(-) cells, we identified a subset of CD14(+) CD163(low) cells in intestinal LPCs; this subset expressed Toll-like receptor (TLR) 2, TLR4, and TLR5 mRNAs and produced interleukin (IL)-6, IL-1β, and tumor necrosis factor in response to lipopolysaccharide. In vitro co-culture with naïve T cells revealed that CD14(+) CD163(low) cells induced development of Th17 cells. CD14(+) CD163(low) cells from inflamed regions of mucosa of patients with CD expressed high levels of IL-6, IL-23p19, and tumor necrosis factor mRNAs, and strongly induced Th17 cells. CD14(+) CD163(low) cells from the noninflamed mucosa of patients with CD also had increased abilities to induce Th17 cells compared with those from normal intestinal mucosa. CD14(+) CD163(low) cells in intestinal LPCs from normal intestinal mucosa induce differentiation of naive T cells into Th17 cells; this activity is increased in mucosal samples from patients with CD. These findings show how intestinal myeloid cell types could contribute to pathogenesis of CD and possibly other Th17-associated diseases.

Lactobacillus probiotic protects intestinal epithelium from radiation injury in a TLR-2/cyclo-oxygenase-2-dependent manner

BackgroundThe small intestinal epithelium is highly sensitive to radiation and is a major site of injury during radiation therapy and environmental overexposure.ObjectiveTo examine probiotic bacteria as potential radioprotective agents in...

Oral Immunization with Recombinant Salmonella Induces Mucosal IgA Antibodies in CD4-Deficient Mice

It is well established that CD4+ T cells are major contributors to the induction and regulation of antigen-specific antibody responses in both mucosal and systemic compartments. However, our previous studies have demonstrated that oral immunization of CD4-/- mice with soluble protein plus cholera toxin as adjuvant elicited antigen-specific IgA antibody responses in the gastrointestinal tract. In this study, we investigated whether oral delivery of live bacteria could elicit mucosal immunity in CD4-/- mice. Oral immunization with recombinant Salmonella expressing fragment C of tetanus toxin resulted in serum IgG and IgA anti-tetanus toxoid antibody responses. Furthermore, CD4-/- mice are capable of eliciting secretory IgA antibodies in the intestinal tract. Antibody-forming cell analysis confirmed the antibody titers by revealing significant numbers of tetanus toxoid-specific IgG- and IgA-forming cells in the spleen and IgA-forming cells in intestinal lamina propria. In addition, significant delayed-type hypersensitivity responses were detected in CD4-/- mice given rSalmonella-Tox C. These results suggest that antigen-specific mucosal as well as systemic antibody responses can be induced in the absence of CD4+ T cells after oral immunization with Salmonella.