Immunomodulatory potential of rapamycin-loaded mesoporous silica nanoparticles: pore size-dependent drug loading, release, and in vitro cellular responses.

Rapamycin is a potent immunosuppressive drug that has been recently proposed for a wide range of applications beyond its current clinical use. For some of these proposed applications, encapsulation in nanoparticles is key to ensure therapeutic efficacy and safety. In this work, we evaluate the effect of pore size on mesoporous silica nanoparticles (MSN) as rapamycin nanocarriers. The successful preparation of MSN with 4 different pore sizes was confirmed by dynamic light scattering, zeta potential, transmission electron microscopy and N2 adsorption. In these materials, rapamycin loading was pore size-dependent, with smaller pore MSN exhibiting greater loading capacity. Release studies showed sustained drug release from all MSN types, with larger pore MSN presenting faster release kinetics. In vitro experiments using the murine dendritic cell (DC) line model DC2.4 showed that pore size influenced the biological performance of MSN. MSN with smaller pore sizes presented larger nanoparticle uptake by DC2.4 cells, but were also associated with slightly larger cytotoxicity. Further evaluation of DC2.4 cells incubated with rapamycin-loaded MSN also demonstrated a significant effect of MSN pore size on their immunological response. Notably, the combination of rapamycin-loaded MSN with an inflammatory stimulus (lipopolysaccharide, LPS) led to changes in the expression of DC activation markers (CD40 and CD83) and in the production of the proinflammatory cytokine TNF-α compared to LPS-treated DC without nanoparticles. Smaller-pored MSN induced more substantial reductions in CD40 expression while eliciting increased CD83 expression, indicating potential immunomodulatory effects. These findings highlight the critical role of MSN pore size in modulating rapamycin loading, release kinetics, cellular uptake, and subsequent immunomodulatory responses.

CD23+IgG1+ memory B cells are poised to switch to pathogenic IgE production in food allergy.

Food allergy is caused by allergen-specific immunoglobulin E (IgE) antibodies, but little is known about the B cell memory of persistent IgE responses. Here, we describe, in human pediatric peanut allergy, a population of CD23+IgG1+ memory B cells arising in type 2 immune responses that contain high-affinity peanut-specific clones and generate IgE-producing cells upon activation. The frequency of CD23+IgG1+ memory B cells correlated with circulating concentrations of IgE in children with peanut allergy. A corresponding population of "type 2-marked" IgG1+ memory B cells was identified in single-cell RNA sequencing experiments. These cells differentially expressed interleukin-4 (IL-4)- and IL-13-regulated genes, such as FCER2/CD23+, IL4R, and germline IGHE, and carried highly mutated B cell receptors (BCRs). In children with high concentrations of serum peanut-specific IgE, high-affinity B cells that bind the main peanut allergen Ara h 2 mapped to the population of "type 2-marked" IgG1+ memory B cells and included clones with convergent BCRs across different individuals. Our findings indicate that CD23+IgG1+ memory B cells transcribing germline IGHE are a unique memory population containing precursors of high-affinity pathogenic IgE-producing cells that are likely to be involved in the long-term persistence of peanut allergy.

The memory of pathogenic IgE is contained within CD23 + IgG1 + memory B cells poised to switch to IgE in food allergy.

Food allergy is caused by allergen-specific IgE antibodies but little is known about the B cell memory of persistent IgE responses. Here we describe in human pediatric peanut allergy CD23 + IgG1 + memory B cells arising in type 2 responses that contain peanut specific clones and generate IgE cells on activation. These 'type2-marked' IgG1 + memory B cells differentially express IL-4/IL-13 regulated genes FCER2 / CD23, IL4R , and germline IGHE and carry highly mutated B cell receptors (BCRs). Further, high affinity memory B cells specific for the main peanut allergen Ara h 2 mapped to the population of 'type2-marked' IgG1 + memory B cells and included convergent BCRs across different individuals. Our findings indicate that CD23 + IgG1 + memory B cells transcribing germline IGHE are a unique memory population containing precursors of pathogenic IgE. One-Sentence Summary: We describe a unique population of IgG + memory B cells poised to switch to IgE that contains high affinity allergen-specific clones in peanut allergy.

Allergen recognition by specific effector Th2 cells enables IL-2-dependent activation of regulatory T-cell responses in humans.

Type 2 allergen-specific T cells are essential for the induction and maintenance of allergies to foods, and Tregs specific for these allergens are assumed to be involved in their resolution. However, it has not been convincingly demonstrated whether allergen-specific Treg responses are responsible for the generation of oral tolerance in humans. We observed that sustained food allergen exposure in the form of oral immunotherapy resulted in increased frequency of Tregs only in individuals with lasting clinical tolerance. We sought to identify regulatory components of the CD4+ T-cell response to food allergens by studying their functional activation over time in vitro and in vivo. Two subsets of Tregs expressing CD137 or CD25/OX40 were identified with a delayed kinetics of activation compared with clonally enriched pathogenic effector Th2 cells. Treg activation was dependent on IL-2 derived from effector T cells. In vivo exposure to peanut in the form of an oral food challenge of allergic subjects induced a delayed and persistent activation of Tregs after initiation of the allergen-specific Th2 response. The novel finding of our work is that a sustained wave of Treg activation is induced by the release of IL-2 from Th2 effector cells, with the implication that therapeutic administration of IL-2 could improve current OIT approaches.

IgG memory B cells expressing IL4R and FCER2 are associated with atopic diseases.

BACKGROUND: Atopic diseases are characterized by IgE antibody responses that are dependent on cognate CD4 T cell help and T cell-produced IL-4 and IL-13. Current models of IgE cell differentiation point to the role of IgG memory B cells as precursors of pathogenic IgE plasma cells. The goal of this work was to identify intrinsic features of memory B cells that are associated with IgE production in atopic diseases. METHODS: Peripheral blood B lymphocytes were collected from individuals with physician diagnosed asthma or atopic dermatitis (AD) and from non-atopic individuals. These samples were analyzed by spectral flow cytometry, single cell RNA sequencing (scRNAseq), and in vitro activation assays. RESULTS: We identified a novel population of IgG memory B cells characterized by the expression of IL-4/IL-13 regulated genes FCER2/CD23, IL4R, IL13RA1, and IGHE, denoting a history of differentiation during type 2 immune responses. CD23+ IL4R+ IgG+ memory B cells had increased occurrence in individuals with atopic disease. Importantly, the frequency of CD23+ IL4R+ IgG+ memory B cells correlated with levels of circulating IgE. Consistently, in vitro stimulated B cells from atopic individuals generated more IgE+ cells than B cells from non-atopic subjects. CONCLUSIONS: These findings suggest that CD23+ IL4R+ IgG+ memory B cells transcribing IGHE are potential precursors of IgE plasma cells and are linked to pathogenic IgE production.

Modulating the systemic and local adaptive immune response after fracture improves bone regeneration during aging.

Tissue injury leads to the well-orchestrated mobilization of systemic and local innate and adaptive immune cells. During aging, immune cell recruitment is dysregulated, resulting in an aberrant inflammatory response that is detrimental for successful healing. Here, we precisely define the systemic and local immune cell response after femur fracture in young and aging mice and identify increased toll-like receptor signaling as a potential culprit for the abnormal immune cell recruitment observed in aging animals. Myd88, an upstream regulator of TLR-signaling lies at the core of this aging phenotype, and local treatment of femur fractures with a Myd88 antagonist in middle-aged mice reverses the aging phenotype of impaired fracture healing, thus offering a promising therapeutic target that could overcome the negative impact of aging on bone regeneration.

Mice carrying an epithelial deletion of the glucocorticoid receptor NR3C1 develop a higher tumor load in experimental colitis-associated cancer.

The glucocorticoid receptor NR3C1 is expressed in multiple cell types in the gut and elsewhere. Intestinal epithelial cells both produce and respond to glucocorticoids in different physiological and pathological contexts. In experimental colitis, glucocorticoids have been shown to exert a dual role, dampening inflammation while producing a deterioration in animal status, including death. Mice with tamoxifen-inducible, intestinal epithelial-specific deletion of NR3C1 (NR3C1ΔIEC mice) are protected against experimental colitis, suggesting glucocorticoid epithelial actions are deleterious. Since glucocorticoids modulate epithelial proliferation, it follows that they may affect the development of colon cancer. In this study, we set out to test this hypothesis using the dextran sulfate sodium-azoxymethane model of colitis-associated cancer. Knockout (KO) mice were found to exhibit a twofold higher tumor load but similar incidence and tumor size. Tumors had a higher trend to extend close to the submucosal layer (36% vs. 0%) in NR3C1ΔIEC mice, and overexpressed Lgr5, Egfr, and Myc, consistent with distinct expression of proliferative/stemness markers. Snai1 and Snai2 were upregulated specifically in tumors of NR3C1ΔIEC mice, suggesting enhanced epithelial to mesenchymal transition in the absence of the intestinal epithelial glucocorticoid (GC) receptor. We conclude that endogenous GC epithelial signaling is involved in colitis-associated cancer.NEW & NOTEWORTHY Mice carrying a tamoxifen-inducible deletion of the glucocorticoid receptor in intestinal epithelial cells (NR3C1ΔIEC mice) and their corresponding controls were subjected to the azoxymethane-dextran sulfate sodium model of colitis-associated cancer. KO mice exhibit a twofold higher tumor load, with a higher trend to extend close to the submucosal layer (36% vs. 0%), but with similar incidence and tumor size. Colonic tumors in NR3C1ΔIEC mice showed signs of increased neoplastic transformation and tumor-associated inflammation.

Immunoregulatory Effects of Porcine Plasma Protein Concentrates on Rat Intestinal Epithelial Cells and Splenocytes.

Serum protein concentrates have been shown to exert in vivo anti-inflammatory effects. Specific effects on different cell types and their mechanism of action remain unraveled. We aimed to characterize the immunomodulatory effect of two porcine plasma protein concentrates, spray dried serum (SDS) and an immunoglobulin concentrate (IC), currently used as animal nutritional supplements with established in vivo immunomodulatory properties. Cytokine production by the intestinal epithelial cell line IEC18 and by primary cultures of rat splenocytes was studied. The molecular pathways involved were explored with specific inhibitors and gene knockdown. Our results indicate that both products induced GROα and MCP-1 production in IEC18 cells by a MyD88/NF-κB-dependent mechanism. Inhibition of TNF production was observed in rat primary splenocyte cultures. The immunoglobulin concentrate induced IL-10 expression in primary splenocytes and lymphocytes. The effect on TNF was independent of IL-10 production or the stimulation of NF-kB, MAPKs, AKT, or RAGE. In conclusion, SDS and IC directly regulate intestinal and systemic immune response in murine intestinal epithelial cells and in T lymphocytes and monocytes.

Epithelial deletion of the glucocorticoid receptor (Nr3c1) protects the mouse intestine against experimental inflammation.

BACKGROUND AND PURPOSE: Glucocorticoids are the first line treatment for the flare-ups of inflammatory bowel disease, but they have significant limitations. The objective of this study is to investigate whether glucocorticoid epithelial actions contribute to such limitations. EXPERIMENTAL APPROACH: Intestinal epithelium glucocorticoid receptor knockout mice (Nr3c1ΔIEC ) received dextran sulfate sodium (DSS) to induce colitis. Inflammatory status was assessed by morphological and biochemical methods, and corticoid production was measured in colonic explants. Some mice were administered budesonide. KEY RESULTS: After 7 days of DSS Nr3c1ΔIEC , mice exhibited 23.1% lower disease activity index (DAI) and 37% lower diarrheal score than WT mice, with decreased weight loss in days 5-7 of colitis, attenuated tissue damage, reduced colonic expression of S100A9 and STAT3 phosphorylation, and a better overall state. Ki67 immunoreactivity was increased at the crypt base, indicating enhanced epithelial proliferation. Mice administered budesonide (6 µg·day-1 PO) showed modest antiinflammatory effects but increased weight loss, which was prevented in knockout mice. Epithelial deletion of the glucocorticoid receptor also protected mice in a protracted colitis protocol. Conversely, knockout mice presented a worse status compared to the control group at 1 day post DSS. In a separate experiment, colonic corticosterone production was shown to be significantly increased in knockout mice at 7 days of colitis but not at earlier stages. CONCLUSIONS AND IMPLICATIONS: The intestinal epithelial glucocorticoid receptor has deleterious effects in experimental colitis induced by DSS, probably related to inhibition of epithelial proliferative responses leading to impaired wound healing and reduced endogenous corticosterone production.

Intestinal epithelial deletion of the glucocorticoid receptor NR3C1 alters expression of inflammatory mediators and barrier function.

Glucocorticoids (GCs) are important hormones involved in the regulation of multiple physiologic functions. GCs are also widely used in anti-inflammatory/immunosuppressant drugs. GCs are synthesized by the adrenal cortex as part of the hypothalamus-pituitary-adrenal axis and also by intestinal epithelial cells, among other peripheral sites. GCs are one of the main therapy choices for the exacerbations of inflammatory bowel disease, but they are not useful to prolong remission, and development of tolerance with secondary treatment failure is frequent. Thus, GC actions at the intestinal epithelial level are of great importance, both physiologically and pharmacologically. We generated a tamoxifen-inducible nuclear receptor subfamily 3 group C member 1 (NR3C1)ΔIEC mouse model to study the effects of GCs on epithelial cells in vivo. Nr3c1 deletion in epithelial cells of the small intestine and colon was associated with limited colonic inflammation at 1 wk postdeletion, involving augmented epithelial proliferation and mucus production, plus local and systemic immune/inflammatory changes. This phenotype regressed substantially, but not completely, after 2 wk. The mechanism may involve augmented inflammatory signaling by epithelial cells or defective barrier function. We conclude that the epithelial GC receptor plays a significant role in colonic homeostasis in basal conditions, but its deficiency can be compensated in the short term. Future studies are required to assess the impact of Nr3c1 deletion in other conditions such as experimental colitis.-Aranda, C. J., Arredondo-Amador, M., Ocón, B., Lavín, J. L., Aransay, A. M., Martínez-Augustin, O., Sánchez de Medina, F. Intestinal epithelial deletion of the glucocorticoid receptor NR3C1 alters expression of inflammatory mediators and barrier function.

Exogenous leptin reinforces intestinal barrier function and protects from colitis.

Besides its function controlling energy expenditure and food intake, leptin is an important modulator of inflammatory responses. The role of leptin in intestinal inflammation remains controversial, since both pro-inflammatory and anti-inflammatory effects have been reported. This study was carried out to further understand leptin contribution in the inflamed intestinal mucosa. Exogenous PEG-leptin or saline solution was given to C57BL/6 mice for two weeks. After 1 week, acute colitis was induced to C57BL/6 mice using dextran sulfate sodium (DSS) in drinking water. The severity of colitis, inflammatory parameters and mucosal barrier function were evaluated. Overall our results indicate that colitis was less severe in mice receiving leptin, as shown by a decrease in rectal bleeding, epithelial damage and colon inflammatory markers, and improved diarrhea. Leptin-treated mice displayed an increase in the expression of tight junction proteins and proliferative expression markers in colon, indicating a reinforcement in the mucosal barrier function induced by leptin administration. PEG-leptin treatment conferred protection to mice in the DSS model of colitis by reinforcing mucosal barrier function.

Non-classical B Cell Memory of Allergic IgE Responses.

The long-term effectiveness of antibody responses relies on the development of humoral immune memory. Humoral immunity is maintained by long-lived plasma cells that secrete antigen-specific antibodies, and memory B cells that rapidly respond to antigen re-exposure by generating new plasma cells and memory B cells. Developing effective immunological memory is essential for protection against pathogens, and is the basis of successful vaccinations. IgE responses have evolved for protection against helminth parasites infections and against toxins, but IgE is also a potent mediator of allergic diseases. There has been a dramatic increase in the incidence of allergic diseases in recent decades and this has provided the impetus to study the nature of IgE antibody responses. As will be discussed in depth in this review, the IgE memory response has unique features that distinguish it from classical B cell memory.

The Secret Life of IgE-Producing Cells.

IgE antibodies are essential mediators of allergies. In a recent study in Science, Croote et al. (2018) characterize IgE cells isolated from individuals allergic to peanuts. Their findings provide insight into the differentiation of IgE cells in humans and have implications for our understanding of allergic disease.

miR-146a regulates the crosstalk between intestinal epithelial cells, microbial components and inflammatory stimuli.

Regulation of miR-146a abundance and its role in intestinal inflammation and particularly in intestinal epithelial cells (IECs) has been poorly studied. Here we study the relationship between bacterial antigens and inflammatory stimuli, and miR-146a expression using IEC lines and models of colitis (trinitrobenzenesulfonic acid (TNBS), dextran sulfate sodium (DSS) and the CD4 + CD62L + T cell transfer model). Specific bacterial antigens and cytokines (LPS, flagelin and IL-1ß/TNF) stimulate miR-146a expression, while peptidoglycan, muramyldipeptide and CpG DNA have no effect. Overexpression of miR-146a by LPS depends on the activation of the TLR4/MyD88/NF-kB and Akt pathways. Accordingly, the induction of miR-146a is lower in TLR4, but not in TLR2 knock out mice in both basal and colitic conditions. miR-146a overexpression in IECs induces immune tolerance, inhibiting cytokine production (MCP-1 and GROα/IL-8) in response to LPS (IEC18) or IL-1ß (Caco-2). Intestinal inflammation induced by chemical damage to the epithelium (DSS and TNBS models) induces miR-146a, but no effect is observed in the lymphocyte transfer model. Finally, we found that miR-146a expression is upregulated in purified IECs from villi vs. crypts. Our results indicate that miR-146a is a key molecule in the interaction among IECs, inflammatory stimuli and the microbiota.

Calprotectin protects against experimental colonic inflammation in mice.

BACKGROUND AND PURPOSE: Calprotectin is a heterodimer composed of two myeloid-related proteins, S100A8 and S100A9, that is abundant in neutrophils and monocytes/macrophages. Faecal levels of calprotectin are used routinely to monitor inflammatory bowel disease activity. EXPERIMENTAL APPROACH: We aimed to assess the role of calprotectin in intestinal inflammation, using the dextran sulfate sodium model of colitis in mice. Calprotectin was administered (50 or 100 µg·day-1 ) by the intrarectal or by i.p. injection (50 µg·day-1 only). The condition of the mice was characterized by morphological and biochemical methods. KEY RESULTS: Intrarectal calprotectin protected significantly against colitis, as shown by lower levels of macroscopic and microscopic damage, colonic myeloperoxidase activity and decreased expression of TNFα and toll-like receptor 4. IL-17 production by spleen and mesenteric lymph node cells was reduced. Calprotectin had no effect on body weight loss or colonic thickening. There were no effects of calprotectin after i.p. injection. Calprotectin had virtually no effects in control, non-colitic mice. Calprotectin had almost no effect on the colonic microbiota but enhanced barrier function. Treatment of rat IEC18 intestinal epithelial cells in vitro with calprotectin induced output of the chemokines CXL1 and CCL2, involving the receptor for advanced glycation end products- and NFκB. CONCLUSION AND IMPLICATIONS: Calprotectin exerted protective effects in experimental colitis when given by the intrarectal route, by actions that appear to involve effects on the epithelium.

Interaction of glucocorticoids with FXR/FGF19/FGF21-mediated ileum-liver crosstalk.

At high doses, glucocorticoids (GC) have been associated with enhanced serum bile acids and liver injury. We have evaluated the effect of GC, in the absence of hepatotoxicity, on FXR/FGF91(Fgf15)/FGF21-mediated ileum-liver crosstalk. Rats and mice (wild type and Fxr-/-, Fgf15-/- and int-Gr-/- strains; the latter with GC receptor (Gr) knockout selective for intestinal epithelial cells), were treated (i.p.) with dexamethasone, prednisolone or budesonide. In both species, high doses of GC caused hepatotoxicity. At a non-hepatotoxic dose, GC induced ileal Fgf15 down-regulation and liver Fgf21 up-regulation, without affecting Fxr expression. Fgf21 mRNA levels correlated with those of several genes involved in glucose and bile acid metabolism. Surprisingly, liver Cyp7a1 was not up-regulated. The expression of factors involved in transcriptional modulation by Fxr and Gr (p300, Drip205, CBP and Smrt) was not affected. Pxr target genes Cyp3a11 and Mrp2 were not up-regulated in liver or intestine. In contrast, the expression of some Pparα target genes in liver (Fgf21, Cyp4a14 and Vanin-1) and intestine (Vanin-1 and Cyp3a11) was altered. In mice with experimental colitis, liver Fgf21 was up-regulated (4.4-fold). HepG2 cells transfection with FGF21 inhibited CYP7A1 promoter (prCYP7A1-Luc2). This was mimicked by pure human FGF21 protein or culture in medium previously conditioned by cells over-expressing FGF21. This response was not abolished by deletion of a putative response element for phosphorylated FGF21 effectors present in prCYP7A1. In conclusion, GC interfere with FXR/FGF19-mediated intestinal control of CYP7A1 expression by the liver and stimulate hepatic secretion of FGF21, which inhibits CYP7A1 promoter through an autocrine mechanism.

Biosimilars: Concepts and controversies.

Biosimilars are copies of reference biological drugs, developed as the patents for original biologicals expire. They are thus developed to replicate an original biological medicine just a generics are intended to replicate a chemically-synthesized medicine; however, there are important technical and regulatory differences between the two. Unlike chemical drugs, molecular identity cannot generally be established for any two biological drugs. Accordingly, their pharmacological properties cannot be assumed to be the same. This is due to the complexity of the production of biologicals and to the presence of minor natural variations in the molecular structure (collectively known as microheterogeneity). Further, biological production yields slightly different versions of the drug over time, particularly when changes are introduced in the production process. In this case the prechange and postchange versions of the biological are analyzed in what is called a comparability exercise. The comparable versions thus validated are considered not to have any significant differences at the clinical level. Likewise, biosimilars are not identical copies but comparable versions of the original biological drug, also validated through a comparability exercise, although of a much broader scope. Although current knowledge about biosimilars has increased significantly, they still arise a number of controversies and misconceptions, particularly regarding issues like extrapolation of indications, immunogenicity and substitution. This review deals with concepts and controversies in the biosimilar field.

The glucocorticoid budesonide has protective and deleterious effects in experimental colitis in mice.

Glucocorticoids are widely used for the management of inflammatory bowel disease, albeit with known limitations for long-term use and relevant adverse effects. In turn, they have harmful effects in experimental colitis. We aimed to explore the mechanism and possible implications of this phenomenon. Regular and microbiota depleted C57BL/6 mice were exposed to dextran sulfate sodium (DSS) to induce colitis and treated with budesonide. Colonic inflammation and animal status were compared. In vitro epithelial models of wound healing were used to confirm the effects of glucocorticoids. Budesonide was also tested in lymphocyte transfer colitis. Budesonide (1-60µg/day) exerted substantial colonic antiinflammatory effects in DSS colitis. At the same time, it aggravated body weight loss, increased rectal bleeding, and induced general deterioration of animal status, bacterial translocation and endotoxemia. As a result, there was an associated increase in parameters of sepsis, such as plasma NOx, IL-1ß, IL-6, lung myeloperoxidase and iNOS, as well as significant hypothermia. Budesonide also enhanced DSS induced colonic damage in microbiota depleted mice. These effects were correlated with antiproliferative effects at the epithelial level, which are expected to impair wound healing. In contrast, budesonide had significant but greatly diminished deleterious effects in noncolitic mice or in mice with lymphocyte transfer colitis. We conclude that budesonide weakens mucosal barrier function by interfering with epithelial dynamics and dampening the immune response in the context of significant mucosal injury, causing sepsis. This may be a contributing factor, at least in part, limiting clinical usefulness of corticoids in inflammatory bowel disease.

Germ-free and Antibiotic-treated Mice are Highly Susceptible to Epithelial Injury in DSS Colitis.

BACKGROUND AND AIMS: Intestinal microbiota is required to maintain immune homeostasis and intestinal barrier function. At the same time, intraluminal bacteria are considered to be involved in inflammatory bowel disease and are required for colitis induction in animal models, with the possible exception of dextran sulphate sodium [DSS] colitis. This study was carried out to ascertain the mechanism underlying the induction of colitis by DSS in the absence of bacteria. METHODS: Conventional and germ-free [GF] Naval Medical Research Institute [NMRI] mice were used, plus conventional mice treated with an antibiotic cocktail to deplete the intestinal microbiota ['pseudo-GF' or PGF mice]. The differential response to DSS was assessed. RESULTS: Conventional mice developed DSS-induced colitis normally, whereas GF mice showed only minimal inflammation [no colonic thickening, lower myeloperoxidase activity, IL-6, IL-17, TNF-α, and IFN-γ secretion by splenocytes and mesenteric cell cultures, etc.]. However, these mice suffered enhanced haemorrhage, epithelial injury and mortality as a consequence of a weakened intestinal barrier, as shown by lower occludin, claudin 4, TFF3, MUC3, and IL-22. In contrast, PGF mice had a relatively normal, albeit attenuated, inflammatory response, but were less prone to haemorrhage and epithelial injury than GF mice. This was correlated with an increased expression of IL-10 and Foxp3 and preservation barrier-related markers. CONCLUSIONS: We conclude that enteric bacteria are essential for the development of normal DSS-induced colitis. The absence of microbiota reduces DSS colonic inflammation dramatically but it also impairs barrier function, whereas subtotal microbiota depletion has intermediate effects at both levels.