Intestinal Mononuclear Cells Research Articles

P0126 Single-cell analysis reveals enrichment of mucosal Th17-polarized T cells in Acute Severe Ulcerative Colitis

Abstract Background Acute severe ulcerative colitis (ASUC), a severe and potentially life-threatening manifestation of ulcerative colitis (UC) affects up to 25% of patients over their lifetime and requires urgent medical intervention. T cell subsets and myeloid cells play a critical role in the pathogenesis of mucosal inflammation. In this study, we aimed to perform an unbiased analysis of mucosal immune cell populations in patients with ASUC and to identify ASUC-specific cell type alterations. Methods Ten adult ASUC patients (defined by the Truelove and Witts criteria), 7 patients with active UC (not fulfilling ASUC criteria), and 5 normal volunteers (NV) were recruited (Fig. 1A). Intestinal mononuclear cells were isolated from endoscopically obtained intestinal biopsies and analyzed by single-cell RNA-sequencing (scRNA-seq). Linear mixed-effects models were applied to compare lineage-normalized cell type fractions across groups, while adjusting for relevant covariates including age, sex, and body mass index. Results Unsupervised clustering of 127,795 cells, expressing 38,224 unique genes, identified 45 distinct cell types (Fig. 1B). Among the salient findings specific to lymphoid cells, re-clustering of the T cell/NK cell compartment identified 9 cell subsets (Fig. 1C-D). Notably, TH17 cells (RORA, CTSH, CXCR6, and CCL20) and cycling TH17 cells (MKI67, PCLAF, SMC4, NUSAP1, and RRM2) were significantly enriched in patients with ASUC compared to those with active UC (adjusted P=0.019 and P=0.027, respectively) and NV (adjusted P<0.001 for both) (Fig. 1E-F). Among myeloid cells, of the 11 delineated subsets, patients with ASUC showed a significant reduction in lineage-normalized fractions of type 2 conventional dendritic cells (cDC2s) (CD1C, FCER1A) relative to both active UC and NV (adjusted P=0.017 for both) (Fig. 1G). Additionally, a cluster of CD14+ monocytes (CD14, FCN1, and LYZ) was significantly elevated in patients with ASUC compared to NV (adjusted P=0.032). Conclusion Elevated TH17 cell frequencies distinguish ASUC from UC. While further investigations, including protein-level validation of transcriptomic data and an assessment of the impact of cellular perturbation on treatment outcome, are ongoing, these findings shed light on a potentially underappreciated role for TH17-polarized T cells in ASUC.

Interferon-Regulated Expression of Cellular Splicing Factors Modulates Multiple Levels of HIV-1 Gene Expression and Replication.

Type I interferons (IFN-Is) are pivotal in innate immunity against human immunodeficiency virus I (HIV-1) by eliciting the expression of IFN-stimulated genes (ISGs), which encompass potent host restriction factors. While ISGs restrict the viral replication within the host cell by targeting various stages of the viral life cycle, the lesser-known IFN-repressed genes (IRepGs), including RNA-binding proteins (RBPs), affect the viral replication by altering the expression of the host dependency factors that are essential for efficient HIV-1 gene expression. Both the host restriction and dependency factors determine the viral replication efficiency; however, the understanding of the IRepGs implicated in HIV-1 infection remains greatly limited at present. This review provides a comprehensive overview of the current understanding regarding the impact of the RNA-binding protein families, specifically the two families of splicing-associated proteins SRSF and hnRNP, on HIV-1 gene expression and viral replication. Since the recent findings show specifically that SRSF1 and hnRNP A0 are regulated by IFN-I in various cell lines and primary cells, including intestinal lamina propria mononuclear cells (LPMCs) and peripheral blood mononuclear cells (PBMCs), we particularly discuss their role in the context of the innate immunity affecting HIV-1 replication.

From In Vitro to In Vivo: A Rational Flowchart for the Selection and Characterization of Candidate Probiotic Strains in Intestinal Disorders.

Experimental and clinical evidence has demonstrated the potential of probiotic strains in the prevention or treatment of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). However, there is little data on what the methodology leading to the identification of such strains should be. In this work, we propose a new flowchart to identify strains with probiotic potential for the management of IBS and IBD, which we tested on a collection of 39 lactic acid bacteria and Bifidobacteria strains. This flowchart included in vitro tests of immunomodulatory properties on intestinal and peripheral blood mononuclear cells (PBMCs), assessment of the barrier-strengthening effect by measuring transepithelial electric resistance (TEER) and quantification of short-chain fatty acids (SCFAs) and aryl hydrocarbon receptor (AhR) agonists produced by the strains. The in vitro results were then combined in a principal component analysis (PCA) to identify strains associated with an anti-inflammatory profile. To validate our flowchart, we tested the two most promising strains identified in the PCA in mouse models of post-infectious IBS or chemically induced colitis to mimic IBD. Our results show that this screening strategy allows the identification of strains with potential beneficial effects on colonic inflammation and colonic hypersensitivity.

Hepatocyte growth factor ameliorates dextran sodium sulfate‑induced colitis in a mouse model by altering the phenotype of intestinal macrophages.

Hepatocyte growth factor (HGF) serves key roles in cell motility, proliferation and immunoregulatory functions. However, the effect of HGF on macrophages is unclear. The present study aimed to elucidate the effect of HGF on the phenotypic alterations of intestinal lamina propria mononuclear cells (LPMCs). Colitis was induced in a mouse model using dextran sodium sulfate (DSS). Subsequently, LPMCs were isolated from the mice with chronic colitis and the expression levels of cytokine‑encoding genes in the LPMCs were determined. CD11b‑positive macrophages isolated from LPMCs were cultured with HGF, and alterations in the levels of M1 or M2 markers were evaluated by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and flow cytometry. In addition, the cytokine levels were assessed using RT‑qPCR and ELISA. HGF shifted the phenotype of macrophages from M1 to M2‑like, as determined by increased mRNA expression levels of arginase‑1, CD206 and IL‑10, and reduced mRNA expression levels of CD86 and IL‑6 in mice with DSS‑induced colitis. Moreover, HGF could ameliorate DSS‑induced colitis owing to its immunosuppressive effect on immune cells. These findings indicated that HGF treatment may not only promote the regeneration of epithelial cells but also lead to tissue repair by phenotypic alteration of M1 macrophages to M2‑like macrophages.

SRSF1 acts as an IFN-I-regulated cellular dependency factor decisively affecting HIV-1 post-integration steps.

Efficient HIV-1 replication depends on balanced levels of host cell components including cellular splicing factors as the family of serine/arginine-rich splicing factors (SRSF, 1-10). Type I interferons (IFN-I) play a crucial role in the innate immunity against HIV-1 by inducing the expression of IFN-stimulated genes (ISGs) including potent host restriction factors. The less well known IFN-repressed genes (IRepGs) might additionally affect viral replication by downregulating host dependency factors that are essential for the viral life cycle; however, so far, the knowledge about IRepGs involved in HIV-1 infection is very limited. In this work, we could demonstrate that HIV-1 infection and the associated ISG induction correlated with low SRSF1 levels in intestinal lamina propria mononuclear cells (LPMCs) and peripheral blood mononuclear cells (PBMCs) during acute and chronic HIV-1 infection. In HIV-1-susceptible cell lines as well as primary monocyte-derived macrophages (MDMs), expression levels of SRSF1 were transiently repressed upon treatment with specific IFNα subtypes in vitro. Mechanically, 4sU labeling of newly transcribed mRNAs revealed that IFN-mediated SRSF1 repression is regulated on early RNA level. SRSF1 knockdown led to an increase in total viral RNA levels, but the relative proportion of the HIV-1 viral infectivity factor (Vif) coding transcripts, which is essential to counteract APOBEC3G-mediated host restriction, was significantly reduced. In the presence of high APOBEC3G levels, however, increased LTR activity upon SRSF1 knockdown facilitated the overall replication, despite decreased vif mRNA levels. In contrast, SRSF1 overexpression significantly impaired HIV-1 post-integration steps including LTR transcription, alternative splice site usage, and virus particle production. Since balanced SRSF1 levels are crucial for efficient viral replication, our data highlight the so far undescribed role of SRSF1 acting as an IFN-modulated cellular dependency factor decisively regulating HIV-1 post-integration steps.

Simvastatin Reduces Protection and Intestinal T Cell Responses Induced by a Norovirus P Particle Vaccine in Gnotobiotic Pigs.

Noroviruses (NoVs) are a leading cause of acute gastroenteritis worldwide. P particles are a potential vaccine candidate against NoV. Simvastatin is a cholesterol-reducing drug that is known to increase NoV infectivity. In this study, we examined simvastatin’s effects on P particle-induced protective efficacy and T-cell immunogenicity using the gnotobiotic pig model of human NoV infection and diarrhea. Pigs were intranasally inoculated with three doses (100 µg/dose) of GII.4/VA387-derived P particles together with monophosphoryl lipid A and chitosan adjuvants. Simvastatin-fed pigs received 8 mg/day orally for 11 days prior to challenge. A subset of pigs was orally challenged with 10 ID50 of a NoV GII.4/2006b variant at post-inoculation day (PID) 28 and monitored for 7 days post-challenge. Intestinal and systemic T cell responses were determined pre- and postchallenge. Simvastatin abolished the P particle’s protection and significantly increased diarrhea severity after NoV infection. Simvastatin decreased proliferation of virus-specific and non-specific CD8 T cells in duodenum and virus-specific CD4 and CD8 T cells in spleen and significantly reduced numbers of intestinal mononuclear cells in vaccinated pigs. Furthermore, simvastatin significantly decreased numbers of duodenal CD4+IFN-γ+, CD8+IFN-γ+ and regulatory T cells and total duodenal activated CD4+ and CD8+ T cells in vaccinated pigs pre-challenge at PID 28. Following challenge, simvastatin prevented the IFN-γ+ T cell response in spleen of vaccinated pigs. These results indicate that simvastatin abolished P particle vaccine-induced partial protection through, at least in part, impairing T cell immunity. The findings have specific implications for the development of preventive and therapeutic strategies against NoV gastroenteritis, especially for the elderly population who takes statin-type drugs.

Establishment of an In Vitro Co-Culture Model of the Piglet Gut to Study Inflammatory Response and Barrier Integrity.

In intensive farming, piglets are exposed to various challenges that activate intestinal inflammatory processes, negatively affecting animal health and leading to economic losses. To study the role of the inflammatory response on epithelial barrier integrity, co-culture systems that mimic in vivo complexity are more and more preferred over cell monocultures. In this study, an in vitro gut co-culture model consisting of intestinal porcine epithelial cells and porcine peripheral blood mononuclear cells was established. The model provides an appropriate tool to study the role of the inflammatory response on epithelial barrier integrity and to screen for feed and food components, exerting beneficial effects on gut health. In the established model, inflammation-like reactions and damage of the epithelial barrier, indicated by a decrease of transepithelial electrical resistance, were elicited by activation of peripheral blood mononuclear cells via one of 3 stimuli: lipopolysaccharide, lipoteichoic acid, or concanavalin A. Two phytogenic substances that are commonly used as feed additives, licorice extract and oregano oil, have been shown to counteract the drop in transepithelial electrical resistance values in the gut co-culture model. The established co-culture model provides a powerful in vitro tool to study the role of intestinal inflammation on epithelial barrier integrity. As it consists of porcine epithelial and porcine blood cells it perfectly mimics in vivo conditions and imitates the inter-organ communication of the piglet gut. The developed model is useful to screen for nutritional components or drugs, having the potential to balance intestinal inflammation and strengthen the epithelial barrier integrity in piglets.

Reduction in the resident intestinal myelomonocytic cell population occurs during ApcMin/+ mouse intestinal tumorigenesis.

With its significant contribution to cancer mortality globally, advanced colorectal cancer (CRC) requires new treatment strategies. However, despite recent good results for mismatch repair (MMR)-deficient CRC and other malignancies, such as melanoma, the vast majority of MMR-proficient CRCs are resistant to checkpoint inhibitor (CKI) therapy. MMR-proficient CRCs commonly develop from precursor adenomas with enhanced Wnt-signalling due to adenomatous polyposis coli (APC) mutations. In melanomas with enhanced Wnt signalling due to stabilized β-catenin, immune anergy and resistance to CKI therapy has been observed, which is dependent on micro-environmental myelomonocytic (MM) cell depletion in melanoma models. However, MM populations of colorectal adenomas or CRC have not been studied. To characterize resident intestinal MM cell populations during the early stages of tumorigenesis, the present study utilized the ApcMin/+ mouse as a model of MMR-proficient CRC, using enhanced green fluorescent protein (EGFP) expression in the mouse lysozyme (M-lys)lys-EGFP/+ mouse as a pan-myelomonocytic cell marker and a panel of murine macrophage surface markers. Total intestinal lamina propria mononuclear cell (LPMNC) numbers significantly decreased with age (2.32±1.39×107 [n=4] at 33 days of age vs. 1.06±0.24×107 [n=8] at 109 days of age) during intestinal adenoma development in ApcMin/+ mice (P=0.05; unpaired Student's t-test), but not in wild-type littermates (P=0.35). Decreased total LPMNC numbers were associated with atrophy of intestinal lymphoid follicles and the absence of MM/lymphoid cell aggregates in ApcMin/+ mouse intestine, but not spleen, compared with wild-type mice. Furthermore, during the early stage of intestinal adenoma development, there was a two-fold reduction of M-lys expressing cells (P=0.05) and four-fold reduction of ER-HR3 (macrophage sub-set) expressing cells (P=0.05; two tailed Mann-Whitney U test) in mice with reduced total intestinal LPMNCs (n=3). Further studies are necessary to determine the relevance of these findings to immune-surveillance of colorectal adenomas or MMR-proficient CRC CKI therapy resistance.

Antibiotic-associated dysbiosis affects the ability of the gut microbiota to control intestinal inflammation upon fecal microbiota transplantation in experimental colitis models

BackgroundThe gut microbiota plays a central role in host physiology and in several pathological mechanisms in humans. Antibiotics compromise the composition and functions of the gut microbiota inducing long-lasting detrimental effects on the host. Recent studies suggest that the efficacy of different clinical therapies depends on the action of the gut microbiota. Here, we investigated how different antibiotic treatments affect the ability of the gut microbiota to control intestinal inflammation upon fecal microbiota transplantation in an experimental colitis model and in ex vivo experiments with human intestinal biopsies.ResultsMurine fecal donors were pre-treated with different antibiotics, i.e., vancomycin, streptomycin, and metronidazole before FMT administration to colitic animals. The analysis of the gut microbiome, fecal metabolome, and the immunophenotyping of colonic lamina propria immune cells revealed that antibiotic pre-treatment significantly influences the capability of the microbiota to control intestinal inflammation. Streptomycin and vancomycin-treated microbiota failed to control intestinal inflammation and were characterized by the blooming of pathobionts previously associated with IBD as well as with metabolites related to the presence of oxidative stress and metabolism of simple sugars. On the contrary, the metronidazole-treated microbiota retained its ability to control inflammation co-occurring with the enrichment of Lactobacillus and of innate immune responses involving iNKT cells. Furthermore, ex vivo cultures of human intestinal lamina propria mononuclear cells and iNKT cell clones from IBD patients with vancomycin pre-treated sterile fecal water showed a Th1/Th17 skewing in CD4+ T-cell populations; metronidazole, on the other hand, induced the polarization of iNKT cells toward the production of IL10.ConclusionsDiverse antibiotic regimens affect the ability of the gut microbiota to control intestinal inflammation in experimental colitis by altering the microbial community structure and microbiota-derived metabolites.6E6mfZzU_A62fWsXVW3UYFVideo

Repertoire analysis of memory T-cell receptors in Japanese patients with inflammatory bowel disease.

Background and AimThe T‐cell receptor (TCR) repertoire was assessed in response to various antigens and was considered to be associated with the pathogenesis of inflammatory bowel disease (IBD). Thus, we performed TCR repertoire analysis to examine the pathology of IBD from changes in the TCR repertoire of memory T cells in the intestinal lamina propria mononuclear cells (LPMCs) and peripheral blood mononuclear cells (PBMCs) of patients with IBD.MethodsLPMCs in the surgical specimens and PBMCs were isolated from 12 patients with IBD (5 patients with ulcerative colitis [UC] and 7 patients with Crohn's disease [CD]). PBMCs were collected from 10 healthy individuals as controls. Comprehensive TCR sequence analyses of adaptor‐ligation polymerase chain reaction (PCR) products were performed using MiSeq.ResultsThe diversity of TCR‐α and TCR‐β in PBMCs was significantly lower in patients with IBD than that in controls (P = 0.00084 and 0.0013, respectively). Comparisons of TCR diversity in LPMCs and PBMCs between CD and UC showed that the diversity in LPMC was not affected by diseases, whereas that in PBMCs was significantly lower in CD than in UC (P = 0.045 and 0.049, respectively). Some TCR clones may have shown a specific increase or decrease in CD and UC, and many clones were common to both LPMCs and PBMCs in the same patients.ConclusionThe diversity of TCR clones in LPMCs and PBMCs in patients with IBD was significantly lower than that of PBMCs in controls. TCR diversity in PBMCs was particularly low in patients with CD.

P027 Repertoire analysis of memory T-cell receptors in Japanese patients with inflammatory bowel disease

Abstract Background The T-cell receptor (TCR) repertoire was assessed in response to various antigens and was considered to be associated with the pathogenesis of inflammatory bowel disease (IBD). Thus, we performed TCR repertoire analysis to examine the pathology of IBD from changes in the TCR repertoire of memory T cells in the intestinal lamina propria mononuclear cells (LPMCs) and peripheral blood mononuclear cells (PBMCs) of patients with IBD. Methods LPMCs in the surgical specimens and PBMCs were isolated from 12 patients with IBD [five patients with ulcerative colitis (UC) and seven patients with Crohn’s disease (CD)]. PBMCs were collected from 10 healthy individuals as controls. Comprehensive TCR sequence analyses of adaptor-ligation PCR products were performed using MiSeq. Results The diversity of TCR-α and -β in PBMCs was significantly lower in patients with IBD than that in controls (p = 0.00084 and 0.0013, respectively). Comparisons of TCR diversity in LPMCs and PBMCs between CD and UC showed that the diversity in LPMC was not affected by diseases, whereas that in PBMCs was significantly lower in CD than in UC (p = 0.045 and 0.049, respectively). Some TCR clones may have shown a specific increase or decrease in CD and UC and many clones were common to both LPMCs and PBMCs in the same patients. Conclusion The diversity of TCR clones in LPMCs and PBMCs in patients with IBD was significantly lower than that of PBMCs in controls. TCR diversity in PBMCs was particularly low in patients with CD.

Replacement of fishmeal with processed canola meal in diets for juvenile Nile tilapia (Oreochromis niloticus): Growth performance, mucosal innate immunity, hepatic oxidative status, liver and intestine histology

The present study was carried out to evaluate the effect of processed canola meal (PCM) as fishmeal (FM) substitute in juvenile Nile tilapia diet. Firstly, canola meal (CM) was dephytinized using 2000 IU/kg phytase and subjected to methanolic ammonia solution (MAS). Finally, five isonitrogenous and isoenergetic diets were formulated by inclusion of 0 (control), 12.5, 25, 37.5 and 50% PCM at the expense of dietary FM. Each experimental diet was fed to three randomly allotted groups of 17 juvenile Nile tilapia with an average body weight of 3.5 ± 0.1 g for 36 days. Fish were feed three times a day until apparent satiation. Results showed that there were no statistically significant differences regarding SGR, final body weight and WG between control group and PCM2 (i.e., diet with 25% PCM inclusion level) (p > .05); however receiving diets with higher PCM content (>25%) resulted in significantly decreased growth indices (p < .05). Meanwhile, FCR, FI and apparent digestibility coefficient of protein (ADCprotein) did not differ among experimental groups (p > .05). Digestive enzyme activities, mucosal innate immunity, liver antioxidant enzyme activities and liver tissue malondialdehyde content (MDA) were not significantly affected by dietary inclusion of PCM (p > .05). However, relative gene expression of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) were significantly increased by dietary PCM content increment (p < .05). In line with growth performance, histological observations revealed that those fish received higher levels of dietary PCM content (i.e. 37.5 and 50%) showed intensive intestinal and hepatic mononuclear immune cell infiltration, lamina propria expansion and intestinal villus detachment and shortening (p < .05). In conclusion, despite containing lower glucosinolate, phytic acid, phenolic compounds and tannins well within the recommended thresholds for aquatic animals nutrition, diets containing PCM beyond 25% negatively affected the growth performance and intestine and liver tissue histoarchitecture of juvenile Nile tilapia. However, further complementary studies are needed regarding the effects observed on gut histology and expression of antioxidant enzyme genes.

Mucosal expression of S100A12 (calgranulin C) and S100A8/A9 (calprotectin) and correlation with serum and fecal concentrations in dogs with chronic inflammatory enteropathy

S100A12 and S100A8/A9 (calprotectin) are released from activated mononuclear cells and belong to the group of damage associated molecular patterns. Fecal S100A12 and S100A8/A9 concentrations have been suggested as biomarkers of intestinal inflammation in dogs with chronic inflammatory enteropathies (CIE). However, the mucosal cellular infiltrate in dogs with CIE is primarily lymphocytic-plasmacytic. Whether fecal S100A12 and S100A8/A9 levels reflect the number and/or activity of intestinal mucosal mononuclear cells, or whether these proteins are also produced by other cells has not been investigated. Thus, the aim of this study was to evaluate intestinal mucosal S100A12 and S100A8/A9 positivity and a potential relationship with the respective protein concentrations in serum and fecal samples in dogs with CIE.Serum (single sample), fecal samples (from 3 consecutive days), and gastrointestinal tissue biopsies (i.e., stomach, duodenum, ileum, and colon) were evaluated from 21 dogs with CIE. Serum and fecal S100A12 and S100A8/A9 concentrations were measured by analytically validated in-house ELISAs. Tissue biopsies underwent routine histopathology and immunohistochemical evaluation for S100A12 and S100A8/A9 positivity (S100A12+ and S100A8/A9+, each recorded as positive cells/mm2).S100A12+ and S100A8/A9+ cells were identified in all segments of the gastrointestinal tract, but were predominantly localized in the lamina propria (LP). Duodenal LP S100A12 positivity correlated statistically significantly with that in the stomach and ileum (ρ = 0.66 and 0.69, both p < 0.01), but was inversely correlated with the severity of macrophage infiltration in the duodenum (ρ=-0.47, p = 0.042). Ileal LP S100A8/A9 positivity correlated positively with the extent of ileal neutrophil and macrophage infiltration (ρ=0.61, p = 0.047). Fecal S100A12 concentrations strongly correlated with the number of S100A12+ cells along the entire gastrointestinal tract (ρ = 0.76, p = 0.028), whereas serum S100A12 concentrations were inversely correlated to colonic S100A12+ cell counts (ρ=-0.50, p = 0.043). Mucosal S100A8/A9+ cell counts were not associated with the corresponding fecal or serum S100A8/A9 concentrations.These results suggest that the intestinal mucosa in dogs with CIE contains an increased number of activated (pro-inflammatory) phagocytes expressing and secreting the S100A12 protein, but the macrophage population seen on routine histopathology is predominantly mature (anti-inflammatory) with a reduced or absent expression of S100A12 and a normal or increased expression of S100A8/A9. However, the distribution of intestinal S100A8/A9 expression requires further study.

Protective Effects of Aryl Hydrocarbon Receptor Signaling in Celiac Disease Mucosa and in Poly I:C-Induced Small Intestinal Atrophy Mouse Model

Aryl hydrocarbon receptor (AhR), a transcription factor activated by a large number of natural and synthetic agents, modulates the activity of immune cells in the gut and represents an important link between the environment and immune-mediated pathologies. In this study, we investigated the role of AhR in celiac disease (CD), a gluten-driven enteropathy. AhR expression was evaluated in intestinal biopsies taken from patients with CD and controls by real-time polymerase chain reaction (PCR), immunohistochemistry and flow cytometry. AhR was also analyzed in ex vivo organ cultures of duodenal biopsies taken from inactive CD patients incubated in presence or absence of peptic-tryptic digest of gliadin. IFN-γ, TNF-α, granzyme B, and perforin expression was evaluated in anti-CD3/CD28-activated intestinal lamina propria mononuclear cells (LPMC) and intestinal intra-epithelial cells (IEL) of active CD patients cultured in the presence or absence of the AhR agonist 6-formylindolo(3, 2-b)carbazole (Ficz). Finally, the protective role of AhR was evaluated in a mouse model of poly I:C-driven small intestine damage. AhR RNA transcripts were reduced in active CD samples as compared to inactive CD and normal controls. Flow cytometry confirmed such results and showed a reduction of AhR in both IEL and LPMC of active CD patients. The addition of a peptic-tryptic digest of gliadin to ex vivo organ cultures of duodenal biopsies taken from inactive CD patients reduced AhR expression. Treatment of CD IEL and LPMC with Ficz reduced the levels of inflammatory cytokines, granzyme B and perforin. Mice injected with Ficz were protected against poly I:C-induced intestinal lesions. Our findings suggest that defective AhR-driven signals could contribute to amplify pathogenic responses in the gut of CD patients.

Hydrolyzed fumonisin B1 induces less inflammatory responses than fumonisin B1 in the co-culture model of porcine intestinal epithelial and immune cells

Fumonisin B1 (FB1), mainly produced by Fusarium verticillioides and Fusarium proliferatum, can be converted to the less toxic metabolite hydrolyzed FB1 (HFB1) by enzymatic degradation. The application of an FB1degrading enzyme as a feed additive is a strategy to reduce fumonisin exposure of animals. However, the difference between the effect of FB1 and HFB1 on porcine intestinal immunity is poorly documented. We investigated the toxic effects of FB1 and HFB1 exposure on porcine gut barrier function and intestinal immunity by using a co-culture model of intestinal porcine epithelial cells (IPEC-J2) and porcine peripheral blood mononuclear cells (PBMCs). First, we confirmed that Fusarium mycotoxin (deoxynivalenol; DON), in the presence of an endotoxin (lipopolysaccharide: LPS), disrupted gut permeability of IPEC-J2 and induced inflammatory response in the co-culture system. FB1 induced additional damage to gut barrier function and promoted pro-inflammatory responses in the presence of LPS and DON compared to only LPS/DON treatment. In the co-culture system, FB1/LPS/DON induced increased cell death of PBMCs and pro-inflammatory cytokines than LPS/DON treatment. In contrast, the application of HFB1 resulted in reduced levels of chemokines and pro-inflammatory cytokines together with marginal immune cell death compared to FB1/LPS/DON in the IPEC-J2/PBMC co-culture system. These findings suggest that FB1 aggravates LPS/DON-induced intestinal inflammation, and HFB1 showed less toxicity to immune response. Therefore, enzymatic degradation of FB1 to HFB1 could be an effective strategy to reduce intestinal inflammation in pigs.

GPR31-dependent dendrite protrusion of intestinal CX3CR1+ cells by bacterial metabolites.

Small intestinal mononuclear cells that express CX3CR1 (CX3CR1+ cells) regulate immune responses1-5. CX3CR1+ cells take up luminal antigens by protruding their dendrites into the lumen1-4,6. However, it remains unclear how dendrite protrusion by CX3CR1+ cells is induced in the intestine. Here we show in mice that the bacterial metabolites pyruvic acid and lactic acid induce dendrite protrusion via GPR31 in CX3CR1+ cells. Mice that lack GPR31, which was highly and selectively expressed in intestinal CX3CR1+ cells, showed defective dendrite protrusions of CX3CR1+ cells in the small intestine. A methanol-soluble fraction of the small intestinal contents of specific-pathogen-free mice, but not germ-free mice, induced dendrite extension of intestinal CX3CR1+ cells in vitro. We purified a GPR31-activating fraction, and identified lactic acid. Both lactic acid and pyruvic acid induced dendrite extension of CX3CR1+ cells of wild-type mice, but not of Gpr31b-/- mice. Oral administration of lactate and pyruvate enhanced dendrite protrusion of CX3CR1+ cells in the small intestine of wild-type mice, but not in that of Gpr31b-/- mice. Furthermore, wild-type mice treated with lactate or pyruvate showed an enhanced immune response and high resistance to intestinal Salmonella infection. These findings demonstrate that lactate and pyruvate, which are produced in the intestinal lumen in a bacteria-dependent manner, contribute to enhanced immune responses by inducing GPR31-mediated dendrite protrusion of intestinal CX3CR1+ cells.

Thymopentin ameliorates dextran sulfate sodium-induced colitis by triggering the production of IL-22 in both innate and adaptive lymphocytes.

Background: Ulcerative colitis (UC) is a chronic inflammatory gastrointestinal disease, notoriously challenging to treat. Previous studies have found a positive correlation between thymic atrophy and colitis severity. It was, therefore, worthwhile to investigate the effect of thymopentin (TP5), a synthetic pentapeptide corresponding to the active domain of the thymopoietin, on colitis.Methods: Dextran sulfate sodium (DSS)-induced colitis mice were treated with TP5 by subcutaneous injection. Body weight, colon length, colon weight, immune organ index, disease activity index (DAI) score, and the peripheral blood profile were examined. The immune cells of the spleen and colon were analyzed by flow cytometry. Histology was performed on isolated colon tissues for cytokine analysis. Bacterial DNA was extracted from mouse colonic feces to assess the intestinal microbiota. Intestinal lamina propria mononuclear cells (LPMCs), HCT116, CT26, and splenocytes were cultured and treated with TP5.Results: TP5 treatment increased the body weight and colon length, decreased the DAI score, and restored colon architecture of colitic mice. TP5 also decreased the infiltration of immune cells and expression levels of pro-inflammatory cytokines such as IL-6. Importantly, the damaged thymus and compromised lymphocytes in peripheral blood were significantly restored by TP5. Also, the production of IL-22, both in innate and adaptive lymphoid cells, was triggered by TP5. Given the critical role of IL-22 in mucosal host defense, we tested the effect of TP5 on mucus barrier and gut microbiota and found that the number of goblet cells and the level of Mucin-2 expression were restored, and the composition of the gut microbiome was normalized after TP5 treatment. The critical role of IL-22 in the protective effect of TP5 on colitis was further confirmed by administering the anti-IL-22 antibody (αIL-22), which completely abolished the effect of TP5. Furthermore, TP5 significantly increased the expression level of retinoic acid receptor-related orphan receptor γ (RORγt), a transcription factor for IL-22. Consistent with this, RORγt inhibitor abrogated the upregulation of IL-22 induced by TP5.Conclusion: TP5 exerts a protective effect on DSS-induced colitis by triggering the production of IL-22 in both innate and adaptive lymphocytes. This study delineates TP5 as an immunomodulator that may be a potential drug for the treatment of UC.

Immunological responsiveness of intestinal tissue explants and mucosal mononuclear cells to ex vivo stimulation

BackgroundThere are limited data on the immunological responsiveness of healthy intestinal tissue when it is cultured and stimulated ex vivo. Such an ex vivo model has the potential to be a valuable tool in understanding disease pathogenesis and as a preclinical tool for the assessment of candidate therapeutic agents used to treat inflammatory bowel disease (IBD). AimWe undertook a comprehensive study to evaluate ex vivo immunological responses of intestinal tissue and isolated mucosal mononuclear cells (MMC) to a broad range of stimuli. MethodsColorectal biopsies (explants) were obtained from healthy participants by flexible sigmoidoscopy and were placed either directly into culture or digested to isolate MMC prior to placement in culture. Explants or MMC were treated with polyinosinic:polycytidylic acid (Poly IC), phytohemagglutinin (PHA), lipopolysacccharides from E Coli (LPS), anti-CD3/CD28 antibodies, or IL-1β/TNF-α for 24 h. Supernatants were assayed for 40 inflammatory biomarkers using multiplexed enzyme-linked immunosorbent assay (ELISA). The isolated MMCs were further characterized using twelve color flow cytometry. ResultsExplants have greater weight adjusted constitutive expression of inflammatory biomarkers than MMCs. Biomarker responses varied as a function of immunogen and use of intact tissue or isolated cells. PHA applied to intact explants was the most effective agent in inducing biomarker changes. Stimulation induced activated and memory cellular phenotypes in both explants and MMCs. ConclusionsThe breadth and magnitude of responses from intact and enzymatically digested intestinal tissue explants stimulated with exogenous immunogens are complex and vary by tissue form and treatment. Overall, PHA stimulation of intact explants produced the most robust responses in normal human colorectal tissue. This system could potentially serve as a preliminary model of the disease state, suitable for small scale screening of new therapeutic agents prior to using IBD patient derived tissue.

Infecção natural pelo vírus da diarreia viral bovina (BVDV) em bovinos de corte no Estado do Pará

Background: Bovine viral diarrhea virus (BVDV) is a pathogen that causes great economic losses in dairy and beef herds.The main disturbances caused by this virus are related to abortion, fetal malformation and mummification, and the birth ofweak calves. In addition, this disease may result in persistently infected animals, as well as individual immune tolerance tothe virus. However, the symptoms are nonspecific and variable, and dependent on the age of affected animal. The objectiveof the present study was to report three cases with clinical signs of BVDV infection, with subsequent confirmation of thediagnosis by reverse transcription - polymerase chain reaction technique (RT-PCR).Cases: In a beef production farm located at the state of Para, Brazil, three animals presented severe diarrhea; the feces wereliquefied and greenish-yellow in color; crusted lesions were visible and scattered over various parts of the animals’ bodies,including the muzzle, nostrils, periauricular region, and hooves; the tongue mucosa was undergoing an intense desquamationprocess. One of the affected animals, a 6-month-old calf, was euthanized following the recommendations of the “Brazilianguide to good practices for animal euthanasia” of the Ethics, Bioethics and Animal Welfare Committee / Federal Council ofVeterinary Medicine. After euthanasia, this calf was necropsied and some of its organs, including the tongue and skin, weresent to the Biological Institute of Sao Paulo State for histopathology examination (HE) and identification and isolation of thevirus. Positive samples for the presence of BVDV were diagnosed by RT-PCR, confirming the suspicion and explaining thesigns observed in that animal and reported in other dead animals of the farm. Because RT-PCR was performed in ulcerativelesions present in the tongue, negative reactions were detected for bovine herpesvirus (BoHV) and positive reactions forBVDV. The cutaneous tissue was marked with lymphocytic infiltration, observed in both epidermis and dermis, which refersto crusted lesions distributed in several regions of the animal’s body. In the lungs, we observed rare areas of emphysema andmarked lymphocytic interstitial pneumonia with hemorrhagic spots, correlated with symptoms of respiratory disease. Also,marked renal damage and spleen lesions were identified, indicating glomerulonephritis and lymphocytic splenitis.Discussion: This study evidenced the occurrence of BVDV in two calves and one adult animal, which is in agreementwith the described higher incidence of symptomatic infection in young animals. Clinical signs of severe greenish-yellowdiarrhea and crusted lesions distributed throughout several regions of the animals’ bodies, including the muzzle, nostrils,periauricular region, and hooves were described, with intense tongue epithelium desquamation, which is a particular andcharacteristic symptom of Mucosal Disease - the classical form of the physiopathogeny caused by bovine viral diarrheavirus. The clinical findings of severe diarrhea are possibly related to the histopathological results, which showed hyaliniza-tion of the intestinal mucosa and rare mononuclear cells in the liver nodules. Pulmonary histopathological findings of rareareas of lymphocytic interstitial pneumonia, severe emphysema, and hemorrhagic foci refer to respiratory diseases. Basedon the clinical signs and laboratory findings, we conclude that the affected animals showed classic symptoms of bovineviral diarrhea. This diagnosis highlights the presence of the disease in cattle raised in the state of Para, Brazil.

Intestinal mononuclear cells primed by systemic interleukin-12 display long-term ability to aggravate colitis in mice.

To address whether the burst of systemic interleukin-12 (IL-12) influences intestinal inflammation elicited by luminal stimuli, we induced IL-12 release by cDNA injection in C57BL/6 mice and simultaneously started dextran sulphate sodium administration. The sequence of the inflammatory response triggered by IL-12 release was characterized by assessing myeloperoxidase activity and histological damage in colon samples on days 1, 3, 5 and 7 after colitis induction. To evaluate the persistence of IL-12 priming, colitis was induced in mice 7 or 60 days after cDNA injection. Under IL-12 influence, the development of acute colitis presented a faster and selective infiltration of inflammatory mononuclear cells in the lamina propria. Recruitment was driven by systemic cytokines rather than luminal antigens. Interestingly, when colitis was triggered 7 or 60 days after the cytokine storm, cells maintained the ability to worsen clinical signs of intestinal inflammation. Together, a systemic IL-12 burst effectively primed intestinal cells that became more prone to develop inflammatory responses. Activation was long-lasting because intestinal cells maintained their inflammatory potential and their ability to aggravate colitis.