Intestinal MAdCAM-1 imaging as biomarker for prognostic in murine models of multiple sclerosis.

INTRODUCTION: Multiple sclerosis (MS) is an autoimmune disease of the central nervous system. Recent evidence suggests that lymphocyte trafficking in the intestines could play a key role in its etiology. Nevertheless, it is not clear how intestinal tissue is involved in the disease onset nor its evolution. In the present study, we aimed to evaluate intestinal inflammation dynamic throughout the disease course and its potential impact on disease progression. METHODS: We used tissue immunophenotyping (immunohistofluorescence and flow cytometry) and a recently described molecular magnetic resonance imaging (MRI) method targeting mucosal addressin cell adhesion molecule-1 (MAdCAM-1) to assess intestinal inflammation in vivo in two distinct animal models of MS (Experimental Autoimmune Encephalomyelitis - EAE) at several time points of disease progression. RESULTS: We report a positive correlation between disease severity and MAdCAM-1 MRI signal in two EAE models. Moreover, high MAdCAM-1 MRI signal during the asymptomatic phase is associated with a delayed disease onset in progressive EAE and to a lower risk of conversion to a secondary-progressive form in relapsing-remitting EAE. During disease evolution, in line with a bi-directional immune communication between the gut and the central nervous system, we observed a decrease in T-CD4+ and B lymphocytes in the ileum concomitantly with their increase in the spinal cord. CONCLUSION: Altogether, these data unveil a crosstalk between intestinal and central inflammation in EAE and support the use of molecular MRI of intestinal MAdCAM-1 as a new biomarker for prognostic in MS patients.

Effects of a gut-selective integrin-targeted therapy in male mice exposed to early immune activation, a model for the study of autism spectrum disorder.

To clarify the role of gut mucosal immunity in ASD, we evaluated, in the early-life immune activation (EIA) mouse model, the effects of administration of a monoclonal antibody directed against the integrin alpha4 beta7 (α4ß7 mAb), blocking the leukocyte homing into the gut mucosa. EIA is a double-hit variant of the maternal immune-activation (MIA) model, including both prenatal (Poly I:C) and postnatal (LPS) immune challenges. In C57BL6/J EIA male adult offspring mice, IL-1ß and IL-17A mRNA colonic tissue content increased when compared with controls. Cytofluorimetric analyses of lymphocytes isolated from mesenteric lymph-nodes (MLN) and spleens of EIA mice show increased percentage of total and CD4+α4ß7+, unstimulated and stimulated IL-17A+ and stimulated IFN-γ+ lymphocytes in MLN and CD4+α4ß7+ unstimulated and stimulated IL-17A+ and stimulated IFN-γ+ lymphocytes in the spleen. Treatment with anti-α4ß7 mAb in EIA male mice was associated with colonic tissue IL-1ß, and IL-17A mRNA content and percentage of CD4+ IL-17A+ and IFN-γ+ lymphocytes in MLN and spleens comparable to control mice. The anti-α4ß7 mAb treatment rescue social novelty deficit showed in the three-chamber test by EIA male mice. Increased levels of IL-6 and IL-1ß and decreased CD68 and TGF-ß mRNAs were also observed in hippocampus and prefrontal cortex of EIA male mice together with a reduction of BDNF mRNA levels in all brain regions examined. Anti-α4ß7 mAb treatment restored the expression of BDNF, TGF-ß and CD68 in hippocampus and prefrontal cortex. Improvement of the gut inflammatory status, obtained by a pharmacological agent acting exclusively at gut level, ameliorates some ASD behavioral features and the neuroinflammatory status. Data provide the first preclinical indication for a therapeutic strategy against gut-immune activation in ASD subjects with peripheral increase of gut-derived (α4ß7+) lymphocytes expressing IL-17A.

Advancements in understanding the role of intestinal dysbacteriosis mediated mucosal immunity in IgA nephropathy.

IgA nephropathy, presently recognized as the foremost primary glomerular disorder, emerges as a principal contributor to renal failure globally, with its pathogenesis yet to be fully elucidated. Extensive research has highlighted the critical role of gut microbiome in the onset and progression of IgA nephropathy, underscoring its importance in accurately delineating the disease's etiology. For example, gut microbiome dysbacteriosis can lead to the production of nephritogenic IgA1 antibodies, which form immune complexes that deposit in the kidneys, causing inflammation and damage. The gut microbiome, a source of numerous bioactive compounds, interacts with the host and plays a regulatory role in gut-immune axis modulation, earning it the moniker of the "second brain." Recent investigations have particularly emphasized a significant correlation between IgA nephropathy and gut microbiome dysbacteriosis. This article offers a detailed overview of the pathogenic mechanisms of IgA nephropathy, specifically focusing on elucidating how alterations in the gut microbiome are associated with anomalies in the intestinal mucosal system in IgA nephropathy. Additionally, it describes the possible influence of gut microbiome on recurrent IgA nephropathy following kidney transplantation. Furthermore, it compiles potential therapeutic interventions, offering both theoretical and practical foundations for the management of IgA nephropathy. Lastly, the challenges currently faced in the therapeutic approaches to IgA nephropathy are discussed.

Impact of Early ARV Initiation on Relative Proportions of Effector and Regulatory CD8 T Cell in Mesenteric Lymph Nodes and Peripheral Blood During Acute SIV Infection of Rhesus Macaques.

CD8 T cells are key players in the clearance of human immunodeficiency virus (HIV)-infected cells, such that CD8 T-cell dysfunction contributes to viral persistence despite antiretroviral (ARV) therapy. Mesenteric lymph nodes (MLNs) are major sites of gut mucosal immunity. While different CD8 T cell subsets such as CD8 alpha-alpha (CD8αα), CD8 alpha-beta (CD8αß), CD8 regulatory T cells (Treg), and mucosa-associated invariant T cells (MAIT) are present in the gut and exhibit distinct functions, their dynamics remain poorly understood due to the lack of accessibility to these tissues in humans. We thus assessed CD8 T cells in MLNs versus peripheral blood in simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs) following early ARV therapy initiation. SIV infection was associated with an increase over time of both CD8αß and CD8αα T cells in the blood and MLNs, whereas early ARV initiation significantly decreased the frequencies of CD8αα but not CD8αß T cells in MLNs. A significant decrease in the expression of chemokine receptors CCR6 and CXCR3 by CD8 T cells, which are essential for T-cell trafficking to the inflammatory sites, was observed in chronically SIV-infected RMs. Surprisingly, while MAIT cells are increased in ARV-treated RMs, their frequencies in MLN are extremely low and were not impacted by ARV. The acute infection resulted in an early CD39+FoxP3+ CD8 Tregs increase in both compartments, which was normalized after early ARV. Frequencies of CD8 Treg cells were positively correlated with frequencies of CD4 Tregs and accordingly negatively correlated with the Th17/Treg ratio in the blood but not in MLNs. Overall, our results underscore the difference in CD8 T-cell subset dynamics in the blood and MLNs. IMPORTANCE Changes in CD8 T-cell subsets during acute SIV/HIV infections and following early ARV initiation in gut lymphoid tissues are poorly understood. Using an acute SIV infection model in rhesus macaques, we assessed the impact of early ARV, initiated 4 days postinfection, on relative proportions of CD8 T-cell subsets in MLNs compared to blood. We found that acute SIV infection and early ARV initiation differentially affect the distribution of effector CD8 T cells, CD8 MAIT cells, and CD8 Tregs in MLNs compared to blood. Overall, early ARV initiation maintains the frequency of effector CD8 T cells while reducing immunosuppressive CD39+ CD8 Tregs. Our study provides deeper insight into the dynamics of the CD8 T-cell compartment in gut mucosal immune surveillance during acute SIV infection and following early ARV initiation.

S100A8 and S100A9 Are Important for Postnatal Development of Gut Microbiota and Immune System in Mice and Infants.

BACKGROUND & AIMS: After birth, the immune system matures via interactions with microbes in the gut. The S100 calcium binding proteins S100A8 and S100A9, and their extracellular complex form, S100A8-A9, are found in high amounts in human breast milk. We studied levels of S100A8-A9 in fecal samples (also called fecal calprotectin) from newborns and during infancy, and their effects on development of the intestinal microbiota and mucosal immune system. METHODS: We collected stool samples (n = 517) from full-term (n = 72) and preterm infants (n = 49) at different timepoints over the first year of life (days 1, 3, 10, 30, 90, 180, and 360). We measured levels of S100A8-A9 by enzyme-linked immunosorbent assay and analyzed fecal microbiomes by 16S sRNA gene sequencing. We also obtained small and large intestine biopsies from 8 adults and 10 newborn infants without inflammatory bowel diseases (controls) and 8 infants with necrotizing enterocolitis and measured levels of S100A8 by immunofluorescence microscopy. Children were followed for 2.5 years and anthropometric data and medical information on infections were collected. We performed studies with newborn C57BL/6J wild-type and S100a9-/- mice (which also lack S100A8). Some mice were fed or given intraperitoneal injections of S100A8 or subcutaneous injections of Staphylococcus aureus. Blood and intestine, mesenterial and celiac lymph nodes were collected; cells and cytokines were measured by flow cytometry and studied in cell culture assays. Colon contents from mice were analyzed by culture-based microbiology assays. RESULTS: Loss of S100A8 and S100A9 in mice altered the phenotypes of colonic lamina propria macrophages, compared with wild-type mice. Intestinal tissues from neonatal S100-knockout mice had reduced levels of CX3CR1 protein, and Il10 and Tgfb1 mRNAs, compared with wild-type mice, and fewer T-regulatory cells. S100-knockout mice weighed 21% more than wild-type mice at age 8 weeks and a higher proportion developed fatal sepsis during the neonatal period. S100-knockout mice had alterations in their fecal microbiomes, with higher abundance of Enterobacteriaceae. Feeding mice S100 at birth prevented the expansion of Enterobacteriaceae, increased numbers of T-regulatory cells and levels of CX3CR1 protein and Il10 mRNA in intestine tissues, and reduced body weight and death from neonatal sepsis. Fecal samples from term infants, but not preterm infants, had significantly higher levels of S100A8-A9 during the first 3 months of life than fecal samples from adults; levels decreased to adult levels after weaning. Fecal samples from infants born by cesarean delivery had lower levels of S100A8-A9 than from infants born by vaginal delivery. S100 proteins were expressed by lamina propria macrophages in intestinal tissues from infants, at higher levels than in intestinal tissues from adults. High fecal levels of S100 proteins, from 30 days to 1 year of age, were associated with higher abundance of Actinobacteria and Bifidobacteriaceae, and lower abundance of Gammaproteobacteria-particularly opportunistic Enterobacteriaceae. A low level of S100 proteins in infants' fecal samples associated with development of sepsis and obesity by age 2 years. CONCLUSION: S100A8 and S100A9 regulate development of the intestinal microbiota and immune system in neonates. Nutritional supplementation with these proteins might aide in development of preterm infants and prevent microbiota-associated disorders in later years.

Differential Dynamics of Regulatory T-Cell and Th17 Cell Balance in Mesenteric Lymph Nodes and Blood following Early Antiretroviral Initiation during Acute Simian Immunodeficiency Virus Infection.

Increased frequencies of immunosuppressive regulatory T cells (Tregs) are associated with gut lymphoid tissue fibrosis and dysfunction which, in turn, contribute to disease progression in chronic simian immunodeficiency virus/human immunodeficiency virus (SIV/HIV) infection. Mesenteric lymph nodes (MLNs), which drain the large and small intestine, are critical sites for the induction and maintenance of gut mucosal immunity. However, the dynamics of Tregs in MLNs are not well understood due to the lack of accessibility to these tissues in HIV-infected individuals. Here, the dynamics of Tregs in blood and MLNs were assessed in SIV-infected rhesus macaques (RMs) following early antiretroviral drug (ARV) initiation. Early ARV initiation reduced T-cell immune activation, as assessed by HLA-DR/CD39 expression, and prevented the depletion of memory CCR6+ Th17 cells in both blood and MLNs. Untreated animals showed higher frequencies of Tregs, CD39+ Tregs, thymic Tregs, and new memory CD4 populations sharing similarity with Tregs as CTLA4+ PD1- and CTLA4+ PD1- FoxP3+ T cells. Despite early ARV treatment, the frequencies of these Treg subsets remained unchanged within the MLNs and, in contrast to blood normalization, the Th17/Treg ratio remained distorted in MLNs. Furthermore, our results highlighted that the expressions of IDO-1, TGFß1 and collagen-1 mRNA remained unchanged in MLN of ARV-treated RMs. ARV interruption did not affect T-cell immune activation and Th17/Treg ratios in MLN. Altogether, our data demonstrated that early ARV initiation within the first few days of SIV infection is unable to reduce the frequencies and homing of various subsets of Tregs within the MLNs which, in turn, may result in tissue fibrosis, impairment in MLN function, and HIV persistence.IMPORTANCE Tregs contribute to SIV/HIV disease progression by inhibition of antiviral specific responses and effector T-cell proliferation. Tregs also cause tissue fibrosis via transforming growth factor ß1 production and collagen deposition, which are associated with microbial translocation and generalized immune activation. Early ARV initiation upon viral exposure is recommended globally and results in improved immune function recovery and reduced viral persistence. Here, using an acute SIV infection model of rhesus macaques, we demonstrated for the first time that despite clear improvements in mucosal CD4 T cells, in contrast to blood, Treg frequencies in MLNs remained elevated following early ARV initiation. The particular Th17/Treg balance observed in MLNs can contribute, in part, to the maintenance of mucosal fibrosis during suppressive ARV treatment. Our results provide a better understanding of gut mucosal immune dynamics following early ARV initiation. These findings suggest that Treg-based treatments could serve as a novel immunotherapeutic approach to decrease gut mucosal damage during SIV/HIV infections.

Relative defects in mucosal immunity predict acute graft-versus-host disease.

Impairment of gut mucosal immunity by the transplant process could facilitate translocation of commensal bacteria and thereby augment the graft-versus-host response. To begin to assess the influence of gut mucosal immunity on the development of acute graft-versus-host disease (GVHD), we conducted a prospective study in 24 pediatric allogeneic hematopoietic cell transplant recipients, assessing 4 fecal markers of mucosal immunity: calprotectin, soluble CD8 (sCD8), soluble intracellular adhesion molecule 1, and ß-defensin-2. Stool samples were collected prospectively on transplant days 0, +5, +10, and +15 and analyzed by ELISA. Lower levels on day +5 (calprotectin and ß-defensin-2) and day +10 (calprotectin, ß-defensin-2, and sCD8) were associated with subsequent acute GVHD. The most striking difference was with calprotectin on day +10. Patients with levels below 424 mg/kg had an incidence of 77.8%, whereas those with levels above this threshold had a cumulative incidence of 0% (P = .002). Relative defects in gut mucosal immunity may be important in the pathogenesis of acute GVHD.

Glycyrrhiza polysaccharides may have an antitumor effect in γδT cells through gut microbiota and TLRs/NF-κB pathway in mice.

Tumor immunotherapy can be a suitable cancer treatment option in certain instances. Here we investigated the potential immunomodulatory effect of oral glycyrrhiza polysaccharides (GCP) on the antitumor function of γδT cells in intestinal epithelial cells in mice. We found that GCP can inhibit tumor growth and was involved in the regulation of systemic immunosuppression. GCP administration also promoted the differentiation of gut epithelia γδT cells into IFN-γ-producing subtype through regulation of local cytokines in gut mucosa. GCP administration increased local cytokine levels through gut microbiota and the gut mucosa Toll-like receptors / nuclear factor kappa-B pathway. Taken together, our results suggest that GCP might be a suitable candidate for tumor immunotherapy, although further clinical research, including clinical trials, are required to validate these results.

Cytokine-Mediated Regulation of Innate Lymphoid Cell Plasticity in Gut Mucosal Immunity.

Mucosal barriers are active sites that encounter a bombardment of antigenic stimuli derived from both the commensal flora and a variety of pathogens, as well as from environmental insults. As such, the ability to mount appropriate innate immune responses is an important first line of defense that confers protection to the host. Central to innate immunity are innate lymphoid cells (ILCs), which were first described a decade ago, and represent a family of heterogeneous cells driven by specific transcription factors and exhibit distinct cytokine profiles that are shared with their CD4+ T-helper cell counterparts. ILCs are particularly enriched at mucosal surfaces, and the tissue microenvironment and cytokine milieu in which ILCs reside are critical factors that drive the behavior and overall function of these cells. In fact, ILCs situated at mucosal barriers must be able to temper their response to a constant exposure of environmental antigens, but also promptly react to pathogens or signals that are potentially harmful to the host. In this context, the ability of ILCs to readily transdifferentiate in response to their dynamic surroundings has become a vigorous area of research, and defining specific mechanism(s) of ILC plasticity is at the advent of discovery. This review will summarize what is currently known regarding the network of cytokines and regulatory elements that enable ILCs to readily transform, based on the range of diverse signals and signal gradients they encounter that lead to either protective or pathogenic function(s), with focus on the gut mucosal immune system.

Effects of Probiotics on the Expression of Cathelicidins in Response to Stimulation by Salmonella Minnesota Lipopolysaccharides in the Proventriculus and Cecum of Broiler Chicks.

The aim of this study was to determine whether probiotic-feeding affected the expression of cathelicidins (CATHs), a major family of antimicrobial peptides, in response to lipopolysaccharides (LPS) challenge in the proventriculus and cecum of broiler chicks. One-day-old male Chunky broiler chicks were fed with or without 0.4% probiotics for 7 days (P-group and non-P-group, respectively). Then, they were orally challenged with no LPS (0-LPS), 1 µg LPS (1-LPS), or 100 µg LPS (100-LPS) (n=5 in all groups) in Experiment 1, and with no LPS or 1 µg LPS (n=6 in all groups) in Experiment 2. Five hours after LPS challenge, the proventriculi and ceca were collected to analyze CATHs expression. Expression of CATHs was examined at first by reverse transcription-polymerase chain reaction (RT-PCR) using the 0-LPS chicks of non-P-group. The differences in CATHs expression upon probiotics-feeding and LPS were analyzed by real time-PCR. All four CATHs (CATH1, 2, 3 and 4) were expressed in the proventriculus and cecum of chicks. In the proventriculus, the expression of CATHs after LPS challenge did not show significant differences between non-P and P-groups in Experiment 1 and 2. In the cecum, the interactions of the effects of probiotics and LPS on the expression of CATH2 in Experiment 1 and CATH1 and 2 in Experiment 2 were significant, and their expression in 1-LPS chicks was higher in P-group than in non-P-group. However, CATH3 and 4 did not show any significant differences between non-P- and P-groups challenged with LPS. These results suggest that probiotics-feeding may stimulate the immunodefense system mediated by CATH2 and possibly CATH1 against infection by Gram-negative bacteria in the cecum.