Gut-associated Lymphoid Tissue Research Articles

Selective IgA Deficiency in Spontaneously Hypertensive Rats With Gut Dysbiosis.

The spontaneously hypertensive rat (SHR) is extensively used to study hypertension. Gut microbiota dysbiosis is a notable feature in SHR for reasons unknown. Immunoglobulin A (IgA) is a major host factor required for gut microbiota homeostasis. We hypothesized that inadequate IgA contributes to gut microbiota dysbiosis in SHR. IgA was measured in feces, cecum, serum, liver, gut-associated lymphoid tissue, and milk from SHR and Wistar Kyoto rats. IgA regulatory factors like IgM, IgG, and pIgR (polymeric immunoglobulin receptor) were analyzed. IgA and IgG antibodies and blood pressure (BP) were measured before and after administrating a bacterial antigen (ie, flagellin). Compared with Wistar Kyoto rats, SHR displayed remarkably near-deficient IgA levels accompanied by compensatory increases in serum IgM and IgG and gut-liver pIgR expression. Inadequate milk IgA in SHR emphasized this immune defect stemmed from the neonatal stage. Reduced IgA+ B cells in circulation and Peyer patches indicated a possible reason for the lower IgA in SHR. Noteworthy, a genetic insufficiency was unlikely because administering flagellin to SHR induced anti-flagellin IgA antibodies. This immune response surprisingly accelerated hypertension development in SHR, suggesting IgA quiescence may help maintain lower BP. This study is the first to reveal IgA deficiency in SHR as one host factor associated with gut microbiota dysbiosis and invigorates future research to determine the pathophysiological role of IgA in hypertension.

The Gut and Kidney Crosstalk in Immunoglobulin A Nephropathy.

Immunoglobulin A nephropathy(IgAN) is the most common primary glomerulonephritis worldwide. The working model for the pathogenesis of IgAN involves a multistep process starting from the production of galactose-deficient and polymeric immunoglobulin A-1 (gd-IgA1) that enters systemic circulation from gut-associated lymphoid tissue (GALT). Galactose-deficient IgA are targeted by endogenous IgG, leading to the formation of circulating immune complexes that deposit in the mesangium and resulting in glomerular inflammation. Disease onset and relapses are often associated with gut infections, supporting the hypothesis that the gut plays an important pathogenic role. In the presence of microbial pathogens or food antigens, activated dendritic cells in the gut mucosa induce T cell dependent and independent B cell differentiation into IgA secreting plasma cells. In IgAN patients, this promotes the systemic release of mucosal gd-IgA1. Not all bacterial strains have the same capacity to elicit IgA production, and little is known about the antigen specificity of the pathogenic gd-IgA1. However, efficacy of treatments targeting gut inflammation support a pathogenic link between the bowel immune system and IgAN. Herein, we review the evidence supporting the role of gut inflammation in IgAN pathogenesis.

Gut Microbiota Interventions for the Management of Obesity: A Literature Review.

The gut microbiota (GM) has been recognized as an important factor in the development of metabolic diseases such as obesity; it has been reported that the composition of the GM differs in obese and lean subjects, suggesting that microbiota dysbiosis can contribute to changes in body weight. Dysbiosis occurs due to an imbalance in the composition of gut bacteria, changes in the metabolic process, or changes in the distribution of microbiota within the gut. Dysbiosis can change the functioning of the intestinal barrier and the gut-associated lymphoid tissues (GALT). Microbial manipulation may help with preventing or treating weight gain and associated comorbidities. Approaches to this may range from dietary manipulation, which is suitable to treat the individual’s microflora, to probiotics, prebiotics, synbiotics, and fecal microbiota transplant (FMT).

Human umbilical cord mesenchymal stem cells regulate immunoglobulin a secretion and remodel the diversification of intestinal microbiota to improve colitis.

BackgroundMesenchymal stem cell (MSC) therapy has emerged as a promising novel therapeutic strategy for managing inflammatory bowel disease (IBD) mainly via dampening inflammation, regulating immune disorders, and promoting mucosal tissue repair. However, in the process, the associated changes in the gut microbiota and the underlying mechanism are not yet clear.MethodsIn the present study, dextran sulfate sodium (DSS) was used to induce colitis in mice. Mice with colitis were treated with intraperitoneal infusions of MSCs from human umbilical cord mesenchymal stem cells (HUMSCs) and evaluated for severity of inflammation including weight reduction, diarrhea, bloody stools, histopathology, and mortality. The proportion of regulatory T cells (Tregs) and immunoglobulin A-positive (IgA+) plasmacytes in gut-associated lymphoid tissue were determined. The intestinal and fecal levels of IgA were tested, and the proportion of IgA-coated bacteria was also determined. Fecal microbiome was analyzed using 16S rRNA gene sequencing analyses.ResultsTreatment with HUMSCs ameliorated the clinical abnormalities and histopathologic severity of acute colitis in mice. Furthermore, the proportion of Tregs in both Peyer’s patches and lamina propria of the small intestine was significantly increased. Meanwhile, the proportion of IgA+ plasmacytes was also substantially higher in the MSCs group than that of the DSS group, resulting in elevated intestinal and fecal levels of IgA. The proportion of IgA-coated bacteria was also upregulated in the MSCs group. In addition, the microbiome alterations in mice with colitis were partially restored to resemble those of healthy mice following treatment with HUMSCs.ConclusionsTherapeutically administered HUMSCs ameliorate DSS-induced colitis partially via regulating the Tregs–IgA response, promoting the secretion of IgA, and facilitating further the restoration of intestinal microbiota, which provides a potential therapeutic mechanism for HUMSCs in the treatment of IBD.

Peculiarities of Endoplasmic Reticulum Stress Regulator XBP1 Expression in the Gut-Associated Lymphoid Tissue of Wistar Rats under Chronic Stress

Peculiarities of Endoplasmic Reticulum Stress Regulator XBP1 Expression in the Gut-Associated Lymphoid Tissue of Wistar Rats under Chronic Stress

Lipid Compositions of Total Parenteral Nutrition Affect Gut Peyer's Patches and Morphology in Mice

IntroductionAlthough parenteral nutrition (PN) is the only option for providing adequate nutrition to patients who cannot tolerate oral ingestion, it severely impairs intestinal barrier function in terms of morphology and immunity. While addition of either soybean oil (SO) or fish oil (FO) to PN partially reverses these defects, the effects of the oil composition (FO/SO ratio) on morphology and gut-associated lymphoid tissues (GALT) have yet to be elucidated. We focused on the effects of the FO/SO ratio in PN on the number of lymphocytes in Peyer's patches, immunoglobulin A levels, and intestinal structures. MethodsMale ICR mice (n = 61) were randomized into five groups; oral nutrition (Chow, n = 14) and four groups receiving PN without oral nutrition. PN solutions contained fat emulsions with the following FO:SO ratios: 0:1 (SO, n = 12), 1:11.5 (11.5FSO, n = 17),1:2 (1:2FSO, n = 13) and 1:0 (FO, n = 5). All mice underwent jugular vein catheter insertion. The PN groups were given isocaloric and isonitrogenous nutritional support with 20% of total calories from fat emulsions with equivalent fat delivery in 11.9 g/kg/d. After 5 d of each feeding, Peyer's patches lymphocytes were isolated from the small intestine, counted and analyzed with flowcytometry for determination of their phenotypes (αβTCR+, γδTCR+, CD4+, CD8+ and B cells). Villus height and crypt depth of the jejunum and ileum were evaluated with hematoxylin-eosin staining. Immunoglobulin A levels in the intestinal washings were also determined. ResultsNumbers of total lymphocytes and B lymphocytes in PP were increased in the 1:2 FSO-PN but neither in the 1:11.5 FSO nor the FO group, as compared to the SO group. There were no marked differences among the groups in numbers neither of total T cells nor in any of T cell phenotypes determined. The 1:2 FSO group showed significantly greater villus height and crypt depth than the SO group. IgA levels did not differ significantly among the four PN groups. ConclusionsThe PN with 1:2 FSO (FO:SO = 1:2) maintained lymphocyte numbers in PP and intestinal villus morphology at levels nearly the same as those obtained with chow feeding. An appropriate ratio of FO to SO in PN is expected to prevent immunological impairment and morphological atrophy of the gut associated with lack of oral nutrition.

In vitro-induced Foxp3+ CD8+ regulatory T cells suppress allergic IgE response in the gut.

Several subsets of CD8+ T cells are known to have a suppressive function in different tissues and diseases in mice and humans. Due to the lack of a consensus on the phenotype of regulatory CD8+ T cells and very low frequency in the body, its clinical use as adoptive cellular therapy has not advanced much. In the present work, using DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (Aza), we efficiently and stably differentiated naïve CD8+ T cells (CD8+ CD25- CD44- cells) into the CD8+ Foxp3+ regulatory CD8+ T cells (CD8 Tregs). We also generated OVA peptide257-264 -specific CD8+ Foxp3+ Tregs. Compared with activated CD8 T cells, Aza plus TGF-β-induced CD8+ Foxp3+ Tregs showed significantly increased surface expression of CD39, CD73, CD122, CD62L, and CD103, and secreted TGF-βand suppressed the proliferation of effector CD4+ T cells. Interestingly, CD8+ Foxp3+ Tregs exhibited low expression of perforin and granzyme required for cytotoxic function. Analysis of chemokine receptors showed that TGF-β+Aza induced CD8+ Foxp3+ Tregs expressed gut-tropic chemokine receptors CCR6 and CCR9, and chemokine receptors CCR7 and CXCR3 required for mobilization into the spleen, lymph nodes, and gut-associated lymphoid tissues. Adoptive transfer of induced CD8+ Foxp3+ Tregs restored cholera toxin-induced breakdown of oral tolerance to OVA by regulating OVA-specific IgE and IgG1. Altogether, we showed an efficient method to generate antigen-specific CD8+ Foxp3+ Tregs, and the adoptive transfer of these cells induces oral tolerance by suppressing allergic response and maintaining intestinal homeostasis.

Gut microbiota and COVID-19: An intriguing pediatric perspective.

Gastrointestinal (GI) involvement has been reported in approximately 50% of patients with coronavirus disease 2019 (COVID-19), which is due to the pathogenic role of inflammation and the intestinal function of the angiotensin-converting enzyme 2 and its receptor. Accumulating adult data has pointed out that gut dysbiosis might occur in these patients with a potential impact on the severity of the disease, however the role of gut microbiota in susceptibility and severity of COVID-19 disease in children is still poorly known. During the last decades, the crosstalk between gut and lung has been largely recognized resulting in the concept of “gut-lung axis” as a central player in modulating the development of several diseases. Both organs are involved in the common mucosal immune system (including bronchus-associated and gut-associated lymphoid tissues) and their homeostasis is crucial for human health. In this framework, it has been found that the role of GI dysbiosis is affecting the homeostasis of the gut-liver axis. Of note, a gut microbiome imbalance has been linked to COVID-19 severity in adult subjects, but it remains to be clarified. Based on the increased risk of inflammatory diseases in children with COVID-19, the potential correlation between gut microbiota dysfunction and COVID-19 needs to be studied in this population. We aimed to summarize the most recent evidence on this striking aspect of COVID-19 in childhood.

Relationship between intestinal hyperpermeability and obesity

Obesity is a combination of genetic, environmental factors, and systemic inflammation of adipose tissue. In the last decade, more and more evidence suggests that intestinal microbiota is an environmental factor that plays a crucial role in obesity and associated metabolic disorders. Here, we review the association between intestinal microbiota and obesity based on the literature data available to us. The intestinal flora, in the equilibrium state of conventional bacteria, protects the health of the host and helps the development of the immune system. The genome, diet, lifestyle, and epigenetic changes of the host can pathologically alter the composition of the microbiota. In dysbiosis, the development of the gut-associated lymphoid tissue (GALT) associated with the intestinal tract is impaired and the integrity of the intestinal barrier is impaired. Due to the consequent intestinal hyperpermeability, components of pathogenic pathogens such as lipopolysaccharides enter the bloodstream. These components bind to receptors on adipose tissue immune cells as ligands for molecular samples with pathogenic properties and induce adipose tissue dysfunction. The secretion of inflammatory cytokines in adipose tissue is increased. This induces persistent low chronic inflammation, which is responsible for the development of obesity. The damage to health caused by the hyperpermeability of the intestinal barrier can be reduced by interventions, or restored early in the process. Knowing the relationships will help prevent and treat obesity.

Local assessment of the immunohistochemical expression of Foxp3+ regulatory T lymphocytes in the different pathological forms associated with bovine paratuberculosis

BackgroundMycobacterium avium subsp. paratuberculosis infected animals show a variety of granulomatous lesions, from focal forms with well-demarcated granulomas restricted to the gut-associated lymphoid tissue (GALT), that are seen in the initial phases or latency stages, to a diffuse granulomatous enteritis, with abundant (multibacillary) or scant (paucibacillary) bacteria, seen in clinical stages. Factors that determine the response to the infection, responsible for the occurrence of the different types of lesion, are still not fully determined. It has been seen that regulatory T cells (Treg) play an important role in various diseases where they act on the limitation of the immunopathology associated with the immune response. In the case of paratuberculosis (PTB) the role of Treg lymphocytes in the immunity against Map is far away to be completely understood; therefore, several studies addressing this subject have appeared recently. The aim of this work was to assess, by immunohistochemical methods, the presence of Foxp3+ T lymphocytes in intestinal samples with different types of lesions seen in cows with PTB.MethodsIntestinal samples of twenty cows showing the different pathological forms of PTB were evaluated: uninfected controls (n = 5), focal lesions (n = 5), diffuse paucibacillary (n = 5) and diffuse multibacillary (n = 5) forms. Foxp3+ lymphocyte distribution was assessed by differential cell count in intestinal lamina propria (LP), gut-associated lymphoid tissue (GALT) and mesenteric lymph node (MLN).ResultsA significant increase in the number of Foxp3+ T cells was observed in infected animals with respect to control group, regardless of the type of lesion. However, when the different categories of lesion were analyzed independently, all individuals with PTB lesions showed an increase in the amount of Foxp3+ T lymphocytes compared to the control group but this increase was only significant in cows with focal lesions and, to a lesser extent, in animals with diffuse paucibacillary forms. The former showed the highest numbers, significantly different from those found in cows with diffuse lesions, where no differences were noted between the two forms. No specific distribution pattern was observed within the granulomatous lesions in any of the groups.ConclusionsThe increase of Foxp3+ T cells in focal forms, that have been associated with latency or resistance to infection, suggest an anti-inflammatory action of these cells at these stages, helping to prevent exacerbation of the inflammatory response, as occurs in diffuse forms, responsible for the appearance of clinical signs.

SMAD4 Suppresses Colitis-associated Carcinoma Through Inhibition of CCL20/CCR6-mediated Inflammation

Background & AimsWe previously reported that colon epithelial cell silencing of Smad4 increased epithelial expression of inflammatory genes, including the chemokine c-c motif chemokine ligand 20 (CCL20), and increased susceptibility to colitis-associated cancer. Here, we examine the role of the chemokine/receptor pair CCL20/c-c motif chemokine receptor 6 (CCR6) in mediating colitis-associated colon carcinogenesis induced by SMAD4 loss. MethodsIn silico analysis of SMAD4, CCL20, and CCR6 messenger RNA expression was performed on published transcriptomic data from human ulcerative colitis (UC), and colon and rectal cancer samples. Immunohistochemistry for CCL20 and CCR6 was performed on human tissue microarrays comprising human UC-associated cancer specimens, Mice with conditional, epithelial-specific Smad4 loss with and without germline deletion of the Ccr6 gene were subjected to colitis and followed for up to 3 months. Tumors were quantified histologically, and immune cell populations were analyzed by flow cytometry and immunostaining. ResultsIn human UC-associated cancers, loss of epithelial SMAD4 was associated with increased CCL20 expression and CCR6+ cells. SMAD4 loss in mouse colon epithelium led to enlarged gut-associated lymphoid tissues and recruitment of immune cells to the mouse colon epithelium and stroma, particularly T regulatory, Th17, and dendritic cells. Loss of CCR6 abrogated these immune responses and significantly reduced the incidence of colitis-associated tumors observed with loss of SMAD4 alone. ConclusionsRegulation of mucosal inflammation is central to SMAD4 tumor suppressor function in the colon. A key downstream node in this regulation is suppression of epithelial CCL20 signaling to CCR6 in immune cells. Loss of SMAD4 in the colon epithelium increases CCL20 expression and chemoattraction of CCR6+ immune cells, contributing to greater susceptibility to colon cancer.

NK cell spatial dynamics and IgA responses in gut-associated lymphoid tissues during SIV infections

HIV infection induces tissue damage including lymph node (LN) fibrosis and intestinal epithelial barrier disruption leading to bacterial translocation and systemic inflammation. Natural hosts of SIV, such as African Green Monkeys (AGM), do not display tissue damage despite high viral load in blood and intestinal mucosa. AGM mount a NK cell-mediated control of SIVagm replication in peripheral LN. We analyzed if NK cells also control SIVagm in mesenteric (mes) LN and if this has an impact on gut humoral responses and the production of IgA known for their anti-inflammatory role in the gut. We show that CXCR5 + NK cell frequencies increase in mesLN upon SIVagm infection and that NK cells migrate into and control viral replication in B cell follicles (BCF) of mesLN. The proportion of IgA+ memory B cells were increased in mesLN during SIVagm infection in contrast to SIVmac infection. Total IgA levels in gut remained normal during SIVagm infection, while strongly decreased in intestine of chronically SIVmac-infected macaques. Our data suggest an indirect impact of NK cell-mediated viral control in mesLN during SIVagm infection on preserved BCF function and IgA production in intestinal tissues.

Ingested (Oral) Adrenocorticotropic Hormone Inhibits IL-17 in the Central Nervous System in the Mouse Model of Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory autoimmune disease of the CNS that resembles multiple sclerosis and provides a useful animal model for the evaluation of mechanisms of action for potential immunomodulatory therapies. We have previously shown that oral adrenocorticotropic hormone (ACTH) decreased IL-17 in the gut lamina propria and the spleen and increased CD4+ Foxp3+ T regulatory cells and IL-10 in the spleen during EAE in the C57BL/6 mouse. However, we did not investigate the specific cellular alterations of proinflammatory and anti-inflammatory factors in the CNS. The aim was to determine if oral ACTH would have a similar clinical effect on inflammatory cytokines in the gut and define specific cellular effects in the CNS in an alternative strain of mice. SJL/J mice were immunized with proteolipid protein peptide 138-151 and gavaged with scrambled ACTH (scrambled α-melanocyte-stimulating hormone) or ACTH 1-39 during ongoing disease. Ingested (oral) ACTH attenuated ongoing clinical EAE disease, decreased IL-6 production, and increased T regulatory cells in the lamina propria and decreased CD4+ and γδ IL-17 production in the CNS. Ingested ACTH attenuated EAE clinical disease by decreasing IL-6 in the gut-associated lymphoid tissue and decreasing IL-17 in the CNS.

Potential Health Benefits of Functional Foods in Boosting Immunity and Relationship between Immunosuppressant Diseases and Severity of Viral Infections-COVID-19 – A Review Article

COVID-19 pandemic has been a global challenge to all the nations and has really affected the sense of normalcy in the lives of People. It has been a sense of awakening that has made people realize the role of immunotherapy in fighting against such situations. It has been observed that the strong gut-associated lymphoid tissue (GALT) which accounts as a dominant role in the immunity has a very important role to play in strengthening the immune system. Dysbiosis could be a major cause of many health disorders and immune suppressants diseases. It was strongly observed that patients with compromised immune system badly affected in COVID-19. In this review, we are trying to find out the association between functional foods such as Probiotics, Prebiotics, and various other Nutraceuticals in strengthening the immune system and its potential benefit to act as preventive medicine in curbing down incidence or management of COVID-19.

The injuries of spleen and intestinal immune system induced by 2-Gy 60Co γ-ray whole-body irradiation

Purpose The aim of the present study was to investigate the injuries of spleen and intestinal immune system induced by 2 Gy 60Co γ ray in mice. Materials and methods A total of 120 Balb/c mice were randomly divided into two groups: blank control (Ctrl) and model (IR). The IR mice were exposed to a single dose of total body irradiation (2 Gy, dose rate: 1 Gy/min) and sacrificed on 1st, 3rd, 7th, 14th and 21st day after irradiation. The indicators including general observations and body weight, the changes in peripheral hemogram, spleen index, histopathology examination and lymphocyte subsets of spleen. As well as the count and subsets of lymphocyte in gut-associated lymphoid tissue. Results Compared with the Ctrl group, the body weight, spleen index, peripheral blood cell and splenocyte amounts, intraepithelial lymphocytes number decreased significantly after exposure, accompanied by a notable decreased count of lymphocytes in Peyer’s patch and mesenteric lymph nodes. Moreover, ionizing radiation also broke the balance of CD4+/CD8+ and increased the Treg proportion in spleen, which then triggered immune imbalance and immunosuppression. In general, the spleen injuries occurred on 1st day after exposure, worse on 3rd day, and were relieved on 7th day. The intestinal immune injuries were observed on 1st day, and attenuated on 3rd day. On 21st day after exposure, the spleen volume and index have returned to normal, except for the distribution of lymphocyte subpopulations. Furthermore, all indicators of gut-associated lymphoid tissue, except for mesenteric lymph nodes lymphocyte count, had returned to normal levels on 21st day. Conclusion In conclusion, our data showed the injuries of spleen and intestinal immune system induced by 2 Gy 60Co γ ray whole-body irradiation. These findings may provide the bases for further radiation protection in the immunity.

No evidence of uptake or propagation of reindeer CWD prions in environmentally exposed sheep

BackgroundChronic wasting disease (CWD) is a prion disease of cervids first reported in North America in the 1960s. In Europe, CWD was first diagnosed in 2016 in a wild reindeer in Norway. Detection of two more cases in the same mountain area led to the complete culling of this partially confined reindeer population of about 2400 animals. A total of 19 CWD positive animals were identified. The affected area is extensively used for the grazing of sheep during summers. There are many mineral licks intended for sheep in the area, but these have also been used by reindeer. This overlap in area use raised concerns for cross-species prion transmission between reindeer and sheep. In this study, we have used global positioning system (GPS) data from sheep and reindeer, including tracking one of the CWD positive reindeer, to investigate spatial and time-relevant overlaps between these two species. Since prions can accumulate in lymphoid follicles following oral uptake, samples of gut-associated lymphoid tissue (GALT) from 425 lambs and 78 adult sheep, which had grazed in the region during the relevant timeframe, were analyzed for the presence of prions. The recto-anal mucosa associated lymphoid tissue (RAMALT) from all the animals were examined by histology, immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA), and the ileal Peyer's patch (IPP) from a subsample of 37 lambs were examined by histology and IHC, for the detection of prions.ResultsGPS data showed an overlap in area use between the infected reindeer herd and the sheep. In addition, the GPS positions of an infected reindeer and some of the sampled sheep showed temporospatial overlap. No prions were detected in the GALT of the investigated sheep even though the mean lymphoid follicle number in RAMALT and IPP samples were high.ConclusionThe absence of prions in the GALT of sheep that have shared pasture with CWD-infected reindeer, may suggest that transmission of this novel CWD strain to sheep does not easily occur under the conditions found in these mountains. We document that the lymphoid follicle rich RAMALT could be a useful tool to screen for prions in sheep.

Nano-strategies as Oral Drug Delivery Platforms for Treatment of Cancer: Challenges and Future Perspectives.

Oral drug administration is the oldest and widely used method for drug administration. The objectives behind developing an oral drug delivery for the treatment of cancer are to achieve low cost treatment by utilizing novel techniques to target cancer through gut-associated lymphoid tissue (GALT) and to enhance patient comfort and compliance through a hospital-free treatment leading to "Chemotherapy at Home." Unfortunately, due to the physiological environment of the GIT and physicochemical properties of drug candidate, the efficacy of oral drug delivery methods is limited in the treatment of cancer. Due to their low hydrophilicity, high P-gp efflux and restricted intestinal permeability most of the anti-cancer drugs fail to achieve oral bioavailability. The review focuses on the efforts, challenges, opportunities and studies conducted by scientists worldwide on the oral administration of anticancer medications via nanocarriers such as liposomes, SLNs and dendrimers, because of their potential to overcome the epithelial barrier associated with GALT, as well as the applications of different polymers in targeting the cancer. The oral delivery can set newer horizons in cancer therapy to make it more patient friendly.

Cecal Patches Generate Abundant IgG2b-Bearing B Cells That Are Reactive to Commensal Microbiota.

Gut-associated lymphoid tissue (GALT), such as Peyer's patches (PPs), are key inductive sites that generate IgA+ B cells, mainly through germinal center (GC) responses. The generation of IgA+ B cells is promoted by the presence of gut microbiota and dietary antigens. However, the function of GALT in the large intestine, such as cecal patches (CePs) and colonic patches (CoPs), and their regulatory mechanisms remain largely unknown. In this study, we demonstrate that the CePs possess more IgG2b+ B cells and have fewer IgA+ B cells than those in PPs from BALB/c mice with normal gut microbiota. Gene expression analysis of postswitched transcripts supported the differential expression of dominant antibody isotypes in B cells in GALT. Germ-free (GF) mice showed diminished GC B cells and had few IgA+ or IgG2b+ switched B cells in both the small and large intestinal GALT. In contrast, myeloid differentiation factor 88- (MyD88-) deficient mice exhibited decreased GC B cells and presented with reduced numbers of IgG2b+ B cells in CePs but not in PPs. Using ex vivo cell culture, we showed that CePs have a greater capacity to produce total and microbiota-reactive IgG2b, in addition to microbiota-reactive IgA, than the PPs. In line with the frequency of GC B cells and IgG2b+ B cells in CePs, there was a decrease in the levels of microbiota-reactive IgG2b and IgA in the serum of GF and MyD88-deficient mice. These data suggest that CePs have a different antibody production profile compared to PPs. Furthermore, the innate immune signals derived from gut microbiota are crucial for generating the IgG2b antibodies in CePs.

FC050: Nefecon® Selectively Modifies the Composition of Circulating Immune Complexes in IGA Nephropathy

Abstract BACKGROUND AND AIMS IgA nephropathy (IgAN) is characterized by the mesangial deposition of IgA-containing immune complexes (IgA-IC). There is, however, no clear association between extent of mesangial IgA-IC deposition and disease severity in IgAN, suggesting that the composition of the IgA-IC, rather than the actual amount, may be critical in determining pathogenic consequences. The NEFIGAN trial (NCT01738035) assessed the safety and efficacy of a novel targeted-release formulation of budesonide (Nefecon®), designed to deliver the drug to the gut-associated lymphoid tissue (GALT)-rich distal ileum in patients with IgAN. The trial comprised a 6-month run-in, a 9-month treatment and a 3-month follow-up phase. A total of 48 patients received Nefecon® 16 mg/day, 51 patients received Nefecon® 8 mg/day and 50 patients received placebo. The key result of the study was that Nefecon® 16 mg/day, added to optimized renin–angiotensin system blockade, reduced proteinuria and stabilized estimated glomerular filtration rate in patients with IgAN. These findings have now been replicated in the NefIgArd study, which released data in 2021. Here, we report the composition of IgA-IC in the serum of patients treated with Nefecon® 16 mg/day or placebo in the NEFIGAN trial, assessed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). This study was supported by a grant from Calliditas Therapeutics AB. METHOD Using Jacalin Agarose and elution with 0.1 M d-galactose, IgA-ICs were affinity-purified from serum of patients who received placebo (n=18) or Nefecon® 16 mg/day (n=18) at the start of treatment (SOT) and end of treatment (EOT). All protein samples were reduced and alkylated prior to digestion with trypsin, dried using a Savant SpeedVac (Thermo Fisher Scientific, UK) and dissolved in 40 µL of 0.1% formic acid. Each sample, spiked with iRT standard peptides (Biognosys, Switzerland), was loaded onto an Evotip (Evosep Biosystems, Denmark) column. Mass spectrometry analyses were performed on the Evosep One (Evosep) platform, coupled to the TIMS TOF Pro (Bruker, US). For protein identification and quantification, raw data were processed with FragPipe (V16), based on at least two peptides per protein. The protein intensities thus generated were Log_2-transformed and internally normalized using IGHA1 protein as standard. For each patient, the normalized protein amount at SOT was subtracted from the amount at EOT. A probabilistic dropout model was applied to the data to estimate fold changes between treatment and placebo groups at EOT. The proteins were ranked using t-statistic, and a gene set enrichment analysis was performed to determine the gene sets significantly affected by Nefecon® 16 mg/day versus placebo. RESULTS Gene ontology analysis revealed that treatment with Nefecon® 16 mg/day led to a significant protein enrichment in IgA-IC associated with pathways driving biological processes involved in the negative regulation of gene expression and transcription, gene silencing and epigenetics. In parallel, treatment with Nefecon® was also associated with an enrichment of proteins associated with extracellular vesicle and exosome formation. CONCLUSION For the first time, we show that it is possible to pharmacologically modify the composition of circulating IgA-IC in IgAN. Treatment with Nefecon® 16 mg/day for 9 months selectively altered the protein composition of IgA-IC in a way that is likely to have significant consequences on the downstream mesangial response to IgA-IC accumulation; this may, in part, explain the antiproteinuric effect of this approach and the longer-term kidney function protection that has been reported in both the NEFIGAN and NefIgArd clinical trials. Studies are ongoing to precisely delineate the impact of these compositional changes on the pathogenicity of IgA-IC in IgAN.

Coronin 1A is Uniquely Expressed in the Human Follicle Associated Epithelium and is Required For Human M Cell Maturation and Function

IntroductionMicrofold (M) cells are specialized epithelial cells that play an integral role in immune surveillance of the gut luminal environment. They functionally act as gatekeepers within the follicle‐associated epithelium (FAE) of Peyer’s patches to regulate antigen delivery to immune cells of gut‐associated lymphoid tissue. Studies have associated damage in the FAE with chronic inflammatory conditions, such as IBD; however, studies designed to understand how human M cells contribute to disease onset and/or pathogenesis under pathogenic conditions remain incomplete. OBJECTIVE: Since M cells are differentiated from Lgr5+ intestinal stem cells, we proposed to utilize human enteroids to determine the factors that regulate human M cell differentiation and function. Quantitative proteomics analysis of human enteroids differentiated to express M cells revealed enriched expression of coronin 1A, an actin‐remodeling protein involved in phagocytosis and expressed immune cells. Since M cells engulf luminal gut antigens via phagocytosis, we hypothesized that coronin 1A is uniquely expressed in the human FAE and plays a role in gut antigen uptake and transcytosis in human M cells.MethodsHuman ileal enteroids derived from biopsies obtained from healthy donors were grown as monolayers and differentiated (no Wnt3a, R‐spondin‐1) for at least 5 days. To express M cells, cultures were exposed to RANKL and TNF during differentiation. Human expression of coronin 1A was validated by immunoblot and confocal microscopy of ileal biopsies obtained from healthy donors. Stable knockdown (KD) of coronin 1A in human enteroids was achieved by shRNA lentivirus and puromycin selection. M cell monolayers, expressing or lacking coronin 1A, were exposed apically to commensal (HS) and pathogenic strains of E. coli (EPEC, AIEC) to determine the contribution of gut microbes to stimulate M cell transcytosis, as measured by uptake of fluorescent polystyrene latex beads and immunoglobulins.ResultsCoronin 1A was expressed in human enteroid monolayers differentiated to express M cells, but not in monolayers differentiated to mimic the villus epithelium. Similar results were obtained in biopsy sections of the human ileum with coronin 1A detected in the FAE, but not in crypt or villus epithelia. This staining pattern was unique for human, as coronin 1A expression in the mouse ileum was confined to lamina propria and lymphoid follicle immune cells. Coronin 1A KD resulted in decreased expression of glycoprotein 2, a marker of mature M cells, while other markers of the FAE and M cells, including CD14, ICAM1, Spi‐B, SOX8, and FcRN remained unchanged. Coronin 1A KD also resulted in impaired uptake/transcytosis of IgG (via FcRN) as well as pathogen‐stimulated uptake/transcytosis of polystyrene beads by human M cells.ConclusionsThe studies show: (1) human enteroids can be differentiated to model coronin 1A expression in the intact human FAE; (2) coronin 1A expression is necessary for terminal differentiation and function of human M cells; and (3) coronin 1A is required for pathogen‐stimulated uptake and transcytosis of various types of luminal gut cargo. We conclude that M cell‐expressing enteroid monolayers can be used as a relevant human model to further interrogate tropism for host‐pathogen interactions, mechanisms of luminal surveillance, and response to oral vaccine therapies.