Gut microbiota-derived short-chain fatty acids regulate IL-17 production by mouse and human intestinal γδ T cells.

Gut interleukin-17A (IL-17)-producing γδ T cells are tissue-resident cells that are involved in both host defense and regulation of intestinal inflammation. However, factors that regulate their functions are poorly understood. In this study, we find that the gut microbiota represses IL-17 production by cecal γδ T cells. Treatment with vancomycin, a Gram-positive bacterium-targeting antibiotic, leads to decreased production of short-chain fatty acids (SCFAs) by the gut microbiota. Our data reveal that these microbiota-derived metabolites, particularly propionate, reduce IL-17 and IL-22 production by intestinal γδ T cells. Propionate acts directly on γδ T cells to inhibit their production of IL-17 in a histone deacetylase-dependent manner. Moreover, the production of IL-17 by human IL-17-producing γδ T cells from patients with inflammatory bowel disease (IBD) is regulated by propionate. These data contribute to a better understanding of the mechanisms regulating gut γδ T cell functions and offer therapeutic perspectives of these cells.

Anatomical Variation in Mesenteric Macrophage Phenotypes in Crohn's Disease.

INTRODUCTION: Clinical trials are currently investigating whether an extended mesenteric resection for ileocecal resections could reduce postoperative recurrence in Crohn's disease. Resection of the mesorectum, which contains proinflammatory macrophages, during proct(ocol)ectomy, is associated with reduced recurrent inflammation and improved wound healing. We aimed to characterize the macrophages in the ileocecal mesentery, which were compared with those in the mesorectum, to provide a biological rationale for the ongoing trials. METHODS: In 13 patients with Crohn's disease and 4 control patients undergoing a proctectomy, tissue specimens were sampled at 3 locations from the mesorectum: distal (rectum), middle, and proximal (sigmoid). In 38 patients with Crohn's disease and 7 control patients undergoing ileocecal resections, tissue specimens also obtained from 3 locations: adjacent to the inflamed terminal ileum, adjacent to the noninflamed ileal resection margin, and centrally along the ileocolic artery. Immune cells from these tissue specimens were analyzed by flow cytometry for expression of CD206 to determine their inflammatory status. RESULTS: In the mesorectum, a gradient from proinflammatory to regulatory macrophages from distal to proximal was observed, corresponding to the adjacent inflammation of the intestine. By contrast, the ileocecal mesentery did not contain high amounts of proinflammatory macrophages adjacent to the inflamed tissue, and a gradient toward a more proinflammatory phenotype was seen in the central mesenteric area. DISCUSSION: Although the mesentery is a continuous structure, the mesorectum and the ileocecal mesentery show different immunological characteristics. Therefore, currently, there is no basis to perform an extended ileocecal resection in patients with Crohn's disease.

Sex-related differences in markers of immune activation in virologically suppressed HIV-infected patients.

OBJECTIVES: Gender-specific studies remain a neglected area of biomedical research. Recent reports have emphasized that sex-related biological factors may affect disease progression during HIV-1 infection. The aim of this study was to investigate the influence of sex on the levels of immune activation in the gut and in peripheral blood of individuals with HIV treated with fully suppressive antiretroviral therapy (ART). METHODS: Thirty individuals with HIV undergoing long-term fully suppressive ART were enrolled in this study. Lamina propria lymphocytes (LPL) and peripheral blood mononuclear cells (PBMCs) were isolated from gut biopsies collected by pancolonoscopy and peripheral blood samples. The expression of markers of immune activation was evaluated by multi-parametric flow cytometry. This is a sub analysis of ClinicalTrials.gov Identifier: NCT02276326 RESULTS: We observed differences in the levels of immune activation in the gut and in PBMCs, with values higher in the gut compartment compared to PBMCs. In addition, we found that the mean value of the levels of immune activation was higher in the women than in the men. Finally, we measured the markers of immune activation by mean relative difference (MRD) and confirmed the higher value in the women. CONCLUSION: A significant sex-related difference in the level of immune activation was observed in a population of individuals with HIV on long-term ART. A more complete characterization of these differences may support the introduction of sex-specific approaches in the clinical management of individuals with HIV.

Accelerating effects of blebbistatin on relaxation process of cell membrane permeabilized trachea and taenia cecum from guinea pig.

Blebbistatin, a potent inhibitor of myosin II, is known to suppress smooth muscle contraction without affecting myosin light chain phosphorylation level. In order to clarify the regulatory mechanisms of blebbistatin on phasic and tonic smooth muscles in detail, we examined the effects of blebbistatin on relaxation process by Ca2+ removal after Ca2+-induced contraction of ß-escin skinned (cell membrane permeabilized) trachea and taenia cecum preparations from guinea pigs. Blebbistatin significantly suppressed the force during relaxation both in skinned trachea and taenia cecum. The data fitting analysis of the relaxation processes indicates that blebbistatin accelerates slow (latch-like) bridge dissociation.

Intravital imaging of eosinophils: Unwrapping the enigma.

Eosinophils are traditionally associated with allergic and parasitic inflammation. More recently, eosinophils have also been shown to have roles in diverse processes including development, intestinal health, thymic selection, and B-cell survival with the majority of these insights being derived from murine models and in vitro assays. Despite this, tools to measure the dynamic activity of eosinophils in situ have been lacking. Intravital microscopy is a powerful tool that enables direct visualization of leukocytes and their dynamic behavior in real-time in a wide range of processes in both health and disease. Until recently eosinophil researchers have not been able to take full advantage of this technology due to a lack of tools such as genetically encoded reporter mice. This mini-review examines the history of intravital microscopy with a focus on eosinophils. The development and use of eosinophil-specific Cre (EoCre) mice to create GFP and tdTomato fluorescent reporter animals is also described. Genetically encoded eosinophil reporter mice combined with intravital microscopy provide a powerful tool to add to the toolbox of technologies that will help us unravel the mysteries still surrounding this cell.

Changes in cecal morphology, cell proliferation, antioxidant enzyme, volatile fatty acids, lipopolysaccharide, and cytokines in piglets during the postweaning period.

Weaning stresses often induce markedly structural and functional changes in the small intestine. However, little data are available on the changes of the morphology, function, and health in the cecum of piglets post-weaning. This study was conducted to measure the effects of weaning on the cecum in piglets. Forty piglets were weaned at 21 d and samples were collected at different time points (days 0, 1, 3, 7, and 14) post-weaning. The crypt depth, crypt width, and total epithelial cell numbers increased on days 7 and 14, compared with days 0, 1, and 3 (P < 0.001). The value of Ki67/total epithelial cells was highest on day 0 compared with all other days (P < 0.05). Besides, a higher concentration of malondialdehyde was observed on day 3 (P < 0.001). The activity of CuZn-superoxide dismutase (P < 0.05) enhanced from day 0 to 1 and the activity of catalase increased from day 1 to 3 (P < 0.001). In addition, days 3, 7, and 14 had greater acetic, propanoic, and butyric acid contents than on day 1 (P < 0.001). The pH and monocarboxylate transporter 1 (MCT1) expression increased from day 0 to 1 and from day 3 to 7 and decreased between days 1 and 3 (P < 0.001). The mRNA expression of solute carrier family 9 member A3 (SLC9A3) decreased on day 1 compared to all other postweaning days (P < 0.05). The abundance of toll-like receptor 4 (TLR4) and interferon-γ (IFN-γ) mRNA expression increased (P < 0.05) during the first 24 h after weaning. The concentration of lipopolysaccharide increased from day 3 to 7, then decreased on day 14 (P < 0.05). The mRNA expression of tumor necrosis factor-α (TNF-α) enhanced from day 7 to 14 (P < 0.05). The abundance of phosphorylated mammalian target of rapamycin protein was lower on day 14 than day 0 (P < 0.05). Taken together, these results show that weaning in piglets influences intestinal morphology, function, and health in the cecum.

Osteoprotegerin-dependent M cell self-regulation balances gut infection and immunity.

Microfold cells (M cells) are responsible for antigen uptake to initiate immune responses in the gut-associated lymphoid tissue (GALT). Receptor activator of nuclear factor-κB ligand (RANKL) is essential for M cell differentiation. Follicle-associated epithelium (FAE) covers the GALT and is continuously exposed to RANKL from stromal cells underneath the FAE, yet only a subset of FAE cells undergoes differentiation into M cells. Here, we show that M cells express osteoprotegerin (OPG), a soluble inhibitor of RANKL, which suppresses the differentiation of adjacent FAE cells into M cells. Notably, OPG deficiency increases M cell number in the GALT and enhances commensal bacterium-specific immunoglobulin production, resulting in the amelioration of disease symptoms in mice with experimental colitis. By contrast, OPG-deficient mice are highly susceptible to Salmonella infection. Thus, OPG-dependent self-regulation of M cell differentiation is essential for the balance between the infectious risk and the ability to perform immunosurveillance at the mucosal surface.

Cellular elements in the developing caecum of Japanese quail (Coturnix coturnix japonica): morphological, morphometrical, immunohistochemical and electron-microscopic studies.

The present study aims to investigate the histological, histochemical and electron microscopic changes of the caecal proximal part of Japanese quail during both pre- and post-hatching periods starting from the 2nd embryonic day (ED) until four weeks post-hatching. On the 2nd and 3rd ED, the primordia of caeca appeared as bilateral swelling on the wall of the hindgut. On the 7th ED, the lamina propria/submucosa contained the primordia of glands. On the 8th ED, rodlet cells could be observed amongst the epithelial cells. On the 9th ED, the caeca began to divide into three parts with more developed layers. With age, the height and number of villi increased. On the 13th ED, immature microfold cells (M-cells) could be identified between the surface epithelium of the villi. The caecal tonsils (CTs) appeared in the form of aggregations of lymphocytes, macrophages, dendritic cells and different types of leukocytes. Telocytes and crypts of Lieberkuhn were observed at this age. On hatching day, the crypts of Lieberkuhn were well-defined and formed of low columnar epithelium, goblet cells, and enteroendocrine cells. Post-hatching, the lumen was filled with villi that exhibited two forms: (1) tongue-shaped villi with tonsils and (2) finger-shaped ones without tonsils. The villi lining epithelium contained simple columnar cells with microvilli that were dispersed with many goblet cells, in addition to the presence of a high number of intra-epithelial lymphocytes and basophils. Moreover, the submucosa was infiltrated by numerous immune cells. CD3 immunomarker was expressed in intraepithelial lymphocytes, while CD20 immunomarker showed focal positivity in CTs. In conclusion, the caecal immune structures of quails at post-hatching were more developed than those in pre-hatching life. The high frequency of immune cells suggests that this proximal part may be a site for immunological surveillance in the quail caecum. The cellular organisation of the caecum and its relation to the immunity was discussed.

Interleukin 1 beta and Matrix Metallopeptidase 3 Contribute to Development of Epidermal Growth Factor Receptor-Dependent Serrated Polyps in Mouse Cecum.

BACKGROUND & AIMS: Transgenic mice (HBUS) that express the epidermal growth factor receptor (EGFR) ligand HBEGF (heparin-binding epidermal growth factor-like growth factor) and a constitutively active G protein-coupled receptor (US28) in intestinal epithelial cells develop serrated polyps in the cecum. Development of serrated polyps depends on the composition of the gut microbiota and is associated with bacterial invasion of the lamina propria, accompanied by induction of inflammation and up-regulation of interleukin 1 beta (IL1B) and matrix metalloproteinase (MMP) 3 in the cecum. We investigated the mechanisms by which these changes contribute to development of serrated polyps. METHODS: We performed studies with C57BL/6 (control) and HBUS mice. To accelerate polyp development, we increased the exposure of the bacteria to the lamina propria by injecting HBUS mice with diphtheria toxin, which binds transgenic HBEGF expressed by the epithelial cells and causes apoptosis. Mice were given injections of IL1B-neutralizing antibody and the MMP inhibitor N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid. Intestinal tissues were collected from mice and analyzed by histology, reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence, and flow cytometry. We examined fibroblast subsets in polyps using single-cell RNA sequencing. RESULTS: Administration of diphtheria toxin to HBUS mice accelerated development of serrated polyps (95% of treated mice developed polyps before 100 days of age, compared with 53% given vehicle). IL1B stimulated subsets of platelet-derived growth factor receptor alpha+ (PDGRFA+) fibroblasts isolated from cecum, resulting in increased expression of MMP3. Neutralizing antibodies against IL1B or administration of the MMP inhibitor reduced the number of serrated polyps that formed in the HBUS mice. Single-cell RNA sequencing analysis showed subsets of fibroblasts in serrated polyps that express genes that regulate matrix fibroblasts and inflammation. CONCLUSIONS: In studies of mice, we found that barrier breakdown and expression of inflammatory factors contribute to development of serrated polyps. Subsets of cecal PDGFRA+ fibroblasts are activated by release of IL1B from myeloid cells during the early stages of serrated polyp development. MMP3 produced by PDGFRA+ fibroblasts is important for serrated polyp development. Our findings confirm the functions of previously identified serrated polyp-associated molecules and indicate roles for immune and stromal cells in serrated polyp development.

Histological and immunohistochemical studies of the proximal caecum and caecal tonsils of quail (Coturnix coturnix japonica).

The proximal caecum in quails consists of lymphoid and non-lymphoid structures. The caecal tonsils in the proximal part of the caecum are units of gut-associated lymphoid tissue in poultry. This study aimed to examine the histological characteristics of the proximal caecum, as well as compositions of dendritic cells (DCs) and antigen-presenting cells (APCs) in the caecal tonsil of quails. Tissue sections were stained with Crossman's triple, periodic acid-Schiff, Gordon and Sweet's silver, Congo red and methyl green-pyronin dyes, as well as immunohistochemically by the streptavidin-biotin-peroxidase complex method. Caecal lymphoid tissue was located in the lamina propria and submucosa. Germinative centres were observed within the lymphoid tissue. Reticular fibres were mainly distributed in the border area of the germinal centre with only a few fibres scattered in the centre. Plasma cells were observed in the subepithelial region and germinal centres. Eosinophil granulocytes were prevalent in the lymphoid tissue. Additionally, CD83-immunoreactive DCs and MHC class II immunoreactive APCs were present in the subepithelial area and diffuse lymphoid tissue. While DCs were seen in the germinal centres of tonsillar units, APCs were rarely present in the germinal centres, but they were noticed around the germinal centres. In conclusion, the histological structure of the proximal caecum in quails and the distributions of some immunological cells in the caecal tonsils were revealed. Therefore, the defensive role of the caecal tonsils in the digestive system may be better understood, and comparative studies may be carried out.

The Adenomatous Polyposis Coli (APC) mutation spectra in different anatomical regions of the large intestine in colorectal cancer.

The enhanced incidence of colorectal cancer (CRC) in the U.S.A. has been linked to promutagens, such as heterocyclic aromatic amines, in the western diet that are produced by high temperature cooking of meat. However, a prior analysis of driver nonsense mutations in the Adenomatous Polyposis Coli (APC) gene, which is mutated in 75% of human CRC, indicated that the C·G → A·T transversions produced by this class of mutagens were not enriched but actually lower than what would be statistically anticipated. Moreover, the APC mutation patterns in the U.S.A. vs. China were indistinguishable despite differences in diet. In the present study, we have dissected the APC mutation pattern in tumors that arise in the different anatomical regions of the large intestine. The results show that the nonsense mutation pattern in APC differ in the different regions and that there is a statistically significant increase in C·G → A·T transversions in the rectum vs. the other regions, albeit, the percent of C·G → A·T mutations still remains lower than predicted based on random mutagenesis.

Pathway of oxfendazole from the host into the worm: Trichuris suis in pigs.

It is well known that the efficacy of a single oral dose of benzimidazoles against Trichuris spp. infections in humans and animals is poor, but is currently still used in control programmes against human trichuriasis. However, the route of the benzimidazoles from the treated host to Trichuris remains unknown. As parts of adult Trichuris are situated intracellularly in the caecum, they might be exposed to anthelmintic drugs in the intestinal content as well as the mucosa. In this study, the pathway of oxfendazole and its metabolites was explored using a T. suis-pig infection model, by simultaneously measuring drug concentrations within the worms and the caecal mucosa, caecal tissue, caecal content and plasma of pigs over time after a single oral dose of 5 mg/kg oxfendazole. Additionally, for comparison to the in vivo study, drug uptake and metabolism of oxfendazole by T. suis was examined after in vitro incubation. Oxfendazole and metabolites were quantified by High Performance Liquid Chromatography. Multivariate linear regression analysis showed a strong and highly significant association between OFZ concentrations within T. suis and in plasma, along with a weaker association between OFZ concentrations in caecal tissue/mucosa and T. suis, suggesting that oxfendazole reaches T. suis after absorption from the gastrointestinal tract and enters the worms by the blood-enterocyte pathway. The fenbendazole sulfone level in T. suis was highly affected by the concentrations in plasma. In addition, correlations between drug concentrations in the host compartments, were generally highest for this metabolite. In comparison to oxfendazole, the correlation between plasma and content was particularly high for this metabolite, suggesting a high level of drug movement between these compartments and the possible involvement of the enterohepatic circulation.

Quantification of FITC-labelled probiotic Lactobacillus salivarius DSPV 001P during gastrointestinal transit in broilers.

The knowledge related to the fate of probiotics in the complex environment of the intestinal microbiota in broilers is just beginning to be elucidated; however, it is not yet well understood. A good method to investigate the mechanisms by which probiotics mediate their effects is to mark probiotic bacteria and trace them. The aim of this research was to develop a new method to estimate in vivo fluorescein isothiocyanate (FITC)-labelled Lactobacillus salivarius DSPV 001P counts during passage through the gastrointestinal tract (GIT) of broilers. Forty-five, 1 d old Cobb broilers were used in this trial. Programmed necropsies were performed 30 min, 6 h, and 12 h after the administration of the probiotic bacterium, and samples of liver, crop, duodenum, caecum, and bursa of fabricius were collected. To determine the spatial and temporal transit of L. salivarius DSPV 001P in broilers, the number of bacteria as well as its respective fluorescent signal produced by FITC were measured. In order to observe the relationship between the variables, a logistic regression analysis was applied. The amount of fluorescence could be used as an indicator of fluorescent probiotic bacteria in the crop and duodenum 30 min after probiotic bacterium supplementation. In addition, the fluorescent signal could be used to estimate bacterial counts in caecum 6 and 12 h after L. salivarius DSPV 001P administration. To the best of our knowledge, this research is the first in vivo trial to employ the bacterial FITC-labelling technique in order to enumerate probiotic bacteria during gastrointestinal transit in broilers.

Metabolism of sn-1(3)-Monoacylglycerol and sn-2-Monoacylglycerol in Caecal Enterocytes and Hepatocytes of Brown Trout (Salmo trutta).

sn-2-Monoacylglycerol (2-MAG) and sn-1(3)-monoacylglycerol [1(3)-MAG] are important but yet little studied intermediates in lipid metabolism. The current study compared the metabolic fate of 2-MAG and 1(3)-MAG in isolated caecal enterocytes and hepatocytes of brown trout (Salmo trutta). 1(3)-Oleoyl [9,10-3H(N)]-glycerol and 2-Oleoyl [9,10-3H(N)]-glycerol were prepared by pancreatic lipase digestion of triolein [9,10-3H(N)]. The 1(3)-MAG and 2-MAG were efficiently absorbed by enterocytes and hepatocytes at similar rates. The 2-MAG was quickly resynthesized into TAG through the monoacylglycerol acyltransferase (EC: 2.3.1.22, MGAT) pathway in both tissues, whereas 1(3)-MAG was processed into TAG and phospholipids at a much slower rate, suggesting 2-MAG was the preferred substrates for MGAT. Further analysis showed that 1(3)-MAG was synthesized into 1,3-DAG, but there were no accumulation of 1,3-DAG in either enterocytes or hepatocytes, which contrasts that of mammalian studies. Some of the 1(3)-MAG may be acylated to 1,2(2,3)-DAG and then utilized for TAG synthesis. Alternatively, 1(3)-MAG can be hydrolyzed to free fatty acid and glycerol, and re-synthesized into TAG through the glycerol-3-phosphate (Gro-3-P) pathway. The overall data suggested that the limiting step of the intracellular 1(3)-MAG metabolism is the conversion of 1(3)-MAG itself.

Mitochondrial pathways are involved in Eimeria tenella-induced apoptosis of chick embryo cecal epithelial cells.

Accumulating evidence suggests that Eimeria tenella severely damages the intestinal mucosa in infected poultry, resulting in deadly haemorrhagic typhlocolitis and major economic losses. Damage to host tissue is believed to arise mainly from apoptosis, which is, in general, intimately related to mitochondrial function. However, it is unclear whether mitochondria-dependent apoptotic pathways are specifically involved in parasite-induced apoptosis of chick embryo cecal epithelial cells. Because the mitochondrial permeability transition pore (MPTP) and caspase-9 are important elements in these pathways, we studied the effects of their respective inhibitors (i.e., cyclosporine A [CsA] and Z-LEHD-FMK, respectively) in primary cultures of chicken embryonic cecum epithelial cells using histopathological techniques, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assays, flow cytometry (FCM) and ELISA. Results indicated that the inhibitors significantly decreased (p < 0.01) DNA injury, apoptosis and caspase-9 and caspase-3 activity of chick embryo cecal epithelial cells at 24, 48, 72, 96 and 120 h after E. tenella infection. Thus, our data supported that mitochondria-dependent apoptotic pathways were involved in apoptosis of parasitised chick embryo cecal epithelial cells.

Probiotic feeding affects T cell populations in blood and lymphoid organs in chickens.

This study was performed to evaluate the effects of Lactobacillus acidophilus bacteria as a probiotic on chicken T cell subset populations in peripheral blood and lymphoid tissues. Thirty chickens were divided into three groups and fed sterilised cow milk, a mixture of milk and L. acidophilus (probiotic), or neither, as the control group. Chickens were euthanised after 14 and 21 days, and whole blood and ileal, bursal, and caecal tonsillar tissues were collected. The populations of T cell subsets, including CD4+, CD8+, and TCR1+ cells, were evaluated by immunohistochemistry and flow cytometry. After 21 days of treatment the percentage of blood CD4+, CD8+, and TCR1+ cells was significantly higher in the probiotic-fed group than in the control group. After 14 days of treatment, a significantly greater number of CD4+ T cells were found in the ileum of probiotic-fed chickens than in chickens from the other two groups. This difference was even greater after 21 days. In addition, after 21 days, a significantly greater number of TCR1+ cells were found in the caecal tonsils of milk-fed chickens than in chickens from the control group. The findings indicate that probiotics may alter the distribution of T cells in the blood and lymphoid tissues in young chickens; however, transient changes in lymphoid tissues indicate that probiotics likely do not permanently affect mucosal immunity.

Characterization of host responses induced by Toll-like receptor ligands in chicken cecal tonsil cells.

The innate responses of cecal tonsils against invading microorganisms are mediated by conserved pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs). TLRs expressed by mammalian and avian immune system cells have the capability to recognize pathogen-associated molecular patterns (PAMPs). Although, the role of TLR ligands in innate and adaptive responses in chickens has been characterized in spleen and bursa of Fabricius, considerably less is known about responses in cecal tonsils. The aim of the current study was to assess responses of mononuclear cells from cecal tonsils to treatment with the TLR2, TLR4 and TLR21 ligands, Pam3CSK4, lipopolysaccharide (LPS), and CpG oligodeoxynucleotide (ODN), respectively. All three ligands induced significant up-regulation of interferon (IFN)-γ, interleukin (IL)-1ß, IL-6 and CxCLi2/IL-8, whereas no significant changes were observed in expression of IL-13 or the antimicrobial peptides, avian ß-defensin (AvBD) 1, AvBD2 and cathelicidin 3 (CATHL-3). In general, CpG ODN elicited the highest cytokine responses by cecal tonsil mononuclear cells, inducing significantly higher expression compared to LPS and Pam3CSK4, for IFNγ, IL-1ß, IL-6 and CxCLi2 at various time points. These findings suggest the potential use of TLR21 ligands as mucosal vaccine adjuvants, especially in the context of pathogens of the intestinal tract.

Ppm1b negatively regulates necroptosis through dephosphorylating Rip3.

The auto-phosphorylation of murine receptor-interacting protein 3 (Rip3) on Thr 231 and Ser 232 in the necrosome is required to trigger necroptosis. However, how Rip3 phosphorylation is regulated is still largely unknown. Here we identified protein phosphatase 1B (Ppm1b) as a Rip3 phosphatase and found that Ppm1b restricts necroptosis in two settings: spontaneous necroptosis caused by Rip3 auto-phosphorylation in resting cells, and tumour necrosis factor-α (TNF)-induced necroptosis in cultured cells. We revealed that Ppm1b selectively suppresses necroptosis through the dephosphorylation of Rip3, which then prevents the recruitment of mixed lineage kinase domain-like protein (Mlkl) to the necrosome. We further showed that Ppm1b deficiency (Ppm1b(d/d)) in mice enhanced TNF-induced death in a Rip3-dependent manner, and the role of Ppm1b in inhibiting necroptosis was evidenced by elevated Rip3 phosphorylation and tissue damage in the caecum of TNF-treated Ppm1b(d/d) mice. These data indicate that Ppm1b negatively regulates necroptosis through dephosphorylating Rip3 in vitro and in vivo.

Visualization of the entire differentiation process of murine M cells: suppression of their maturation in cecal patches.

The microfold (M) cell residing in the follicle-associated epithelium is a specialized epithelial cell that initiates mucosal immune responses by sampling luminal antigens. The differentiation process of M cells remains unclear due to limitations of analytical methods. Here we found that M cells were classified into two functionally different subtypes based on the expression of Glycoprotein 2 (GP2) by newly developed image cytometric analysis. GP2-high M cells actively took up luminal microbeads, whereas GP2-negative or low cells scarcely ingested them, even though both subsets equally expressed the other M-cell signature genes, suggesting that GP2-high M cells represent functionally mature M cells. Further, the GP2-high mature M cells were abundant in Peyer's patch but sparse in the cecal patch: this was most likely due to a decrease in the nuclear translocation of RelB, a downstream transcription factor for the receptor activator of nuclear factor-κB signaling. Given that murine cecum contains a protrusion of beneficial commensals, the restriction of M-cell activity might contribute to preventing the onset of any excessive immune response to the commensals through decelerating the M-cell-dependent uptake of microorganisms.

Effect of whole wheat feeding on selected immune parameters in growing male turkeys.

The objective of this study was to determine the effect of whole wheat feeding on selected parameters of humoral and cell-mediated immunity in growing turkeys. A total of 210 one-day-old heavy-type Hybrid Converter male turkeys were randomly divided into three different dietary treatment groups, each consisting of 7 replicate pens of 10 birds per pen. Until 4 wk of age, all birds were fed a commercial diet formulated to meet nutrient requirements. From 5 to 12 wk of age, diets were composed of wheat (ground-pelleted or whole grain) and protein-fat-mineral-vitamin concentrate. The total wheat concentration in diets was 500 or 600 g/kg in the feeding periods of 5-8 and 9-12 wk of birds' age, respectively. Whole grain wheat had a 0, 50 or 100% share of the total wheat amount in the daily ration in treatment groups W0, W50 or W100, respectively. Commercial vaccines against ND (Newcastle disease) and TRT (Turkey rhinotracheitis) were administered to turkeys via the drinking water on days 20 and 30, respectively. Over the entire experiment, a significant linear decrease was observed in body weight gains (BWG) with increasing dietary levels of whole grain wheat. As a result the BWG of control turkeys (W0) were significantly higher than the BWG of group W100 birds (P = 0.002). A significant linear increase in feed conversion ratio (FCR) was observed with increasing dietary levels of whole grain wheat (P < 0.001). The levels of antibodies against TRT and ND viruses after immunization were significantly higher in both the W50 and W100 group, in comparison to group W0 (P = 0.006 and P = 0.001, respectively). Turkeys from group W50, in comparison to those from groups W0 and W100, had a significantly higher percentage of CD4+ T cell subpopulation within the lymphocytes isolated from blood and ileal mucosa, as well as CD4+ CD8+ and CD8+ T cell subpopulations within the blood immunocompetent cells (P = 0.022, P = 0.029, P = 0.009 and P = 0.011, respectively). In the cecal tonsils, the percentage of CD8+ T cell subpopulation was significantly lower in group W50 than in groups W0 and W100 (P = 0.014). The results of our study indicate that diluting diets with whole grain wheat stimulates the non-specific cell-mediated defense mechanisms of the gastrointestinal immune system in turkeys, thus positively affecting humoral response after vaccination.