Deoxycholic acid enhancement of lymphocyte migration through direct interaction with the intestinal vascular endothelium.

BACKGROUND AND AIM: The small intestine plays a central role in gut immunity, and enhanced lymphocyte migration is involved in the pathophysiology of various enteropathy. Bile acid (BA) is closely related to lipid metabolism and gut microbiota and essential for gut homeostasis. However, the effects of BA on gut immunity have not been studied in detail, especially on the small intestine and lymphocyte migration. Therefore, we aimed to investigate the effect of BA on small intestinal lymphocyte microcirculation. METHODS: The effect of deoxycholic acid (DCA), taurocholic acid (tCA), or cholic acid (CA) on the indomethacin (IND)-induced small intestinal enteropathy in mice was investigated. Lymphocyte movements were evaluated after exposure to BA using intravital microscopy. The effects of BA on surface expression of adhesion molecules on the vascular endothelium and lymphocytes through BA receptors were examined in vitro. RESULTS: IND-induced small intestinal enteropathy was histologically aggravated by DCA treatment alone. The expression of adhesion molecules ICAM-1 and VCAM-1 was significantly enhanced by DCA. Exposure to DCA increased lymphocyte adhesion in the microvessels of the ileum, which was partially blocked by anti-α4ß1 integrin antibody in vivo. The expression of ICAM-1 and VCAM-1 was significantly enhanced by DCA in vitro, which was partially suppressed by the sphingosine-1-phosphate receptor 2 (S1PR2) antagonist. The S1PR2 antagonist significantly ameliorated IND-induced and DCA-exaggerated small intestinal injury. CONCLUSION: DCA exacerbated IND-induced small intestinal enteropathy. DCA directly acts on the vascular endothelium and enhances the expression levels of adhesion molecules partially via S1PR2, leading to enhanced small intestinal lymphocyte migration.

Inflammasome Activation in Ankylosing Spondylitis Is Associated With Gut Dysbiosis.

OBJECTIVE: We undertook this study to evaluate the activation and functional relevance of inflammasome pathways in ankylosing spondylitis (AS) patients and rodent models and their relationship to dysbiosis. METHODS: An inflammasome pathway was evaluated in the gut and peripheral blood from 40 AS patients using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC), flow cytometry, and confocal microscopy, and was compared to that of 20 healthy controls and 10 patients with Crohn's disease. Bacteria was visualized using silver stain in human samples, and antibiotics were administered to HLA-B27-transgenic rats. The NLRP3 inhibitor MCC950 was administered to SKG mice, and ileal and joint tissues were assessed by IHC analysis and real-time qRT-PCR. The role of inflammasome in modulating the interleukin-23 (IL-23)/IL-17 axis was studied ex vivo. RESULTS: Expression levels of Nlrp3, Nlrc4, and Aim2 were increased in the gut of HLA-B27-transgenic rats and reduced by antibiotic treatment (P < 0.05). In curdlan-treated SKG mice, NLRP3 blockade prevented ileitis and delayed arthritis onset (P < 0.05). Compared to healthy controls, AS patients demonstrated overexpression of NLRP3 (fold induction 2.33 versus 22.2; P < 0.001), NLRC4 (fold induction 1.90 versus 6.47; P < 0.001), AIM2 (fold induction 2.40 versus 20.8; P < 0.001), CASP1 (fold induction 2.53 versus 24.8; P < 0.001), IL1B (fold induction 1.07 versus 10.93; P < 0.001), and IL18 (fold induction 2.56 versus 15.67; P < 0.001) in the ileum, and caspase 1 activity was increased (P < 0.01). The score of adherent and invasive mucosa-associated bacteria was higher in AS (P < 0.01) and correlated with the expression of inflammasome components in peripheral blood mononuclear cells (P < 0.001). NLRP3 expression was associated with disease activity (the Ankylosing Spondylitis Disease Activity Score using the C-reactive protein level) (r2 = 0.28, P < 0.01) and with IL23A expression (r2 = 0.34, P < 0.001). In vitro, inflammasome activation in AS monocytes was paralleled by increased serum levels of IL-1ß and IL-18. Induction of IL23A, IL17A, and IL22 was IL-1ß-dependent. CONCLUSION: Inflammasome activation occurs in rodent models of AS and in AS patients, is associated with dysbiosis, and is involved in triggering ileitis in SKG mice. Inflammasomes drive type III cytokine production with an IL-1ß-dependent mechanism in AS patients.

NOD2 drives early IL-33-dependent expansion of group 2 innate lymphoid cells during Crohn's disease-like ileitis.

Innate lymphoid cells (ILCs) are enriched at barrier surfaces, including the gastrointestinal tract. While most studies have focused on the balance between pathogenic group 1 ILCs (ILC1s) and protective ILC3s in maintaining gut homeostasis and during chronic intestinal inflammation, such as Crohn's disease (CD), less is known regarding ILC2s. Using an established murine model of CD-like ileitis, i.e., the SAMP1/YitFc (SAMP) mouse strain, we showed that ILC2s, compared with ILC1s and ILC3s, were increased within draining mesenteric lymph nodes and ilea of SAMP versus AKR (parental control) mice early, during the onset of disease. Gut-derived ILC2s from CD patients versus healthy controls were also increased and expanded, similarly to ILC1s, in greater proportion compared with ILC3s. Importantly, we report that the intracellular bacteria-sensing protein, nucleotide-binding oligomerization domaining-containing protein 2, encoded by Nod2, the first and strongest susceptibility gene identified for CD, promoted ILC2 expansion, which was dramatically reduced in SAMP mice lacking NOD2 and in SAMP mice raised under germ-free conditions. Furthermore, these effects occurred through a mechanism involving the IL-33/ST2 ligand-receptor pair. Collectively, our results indicate a functional link between NOD2 and ILC2s, regulated by the IL-33/ST2 axis, that mechanistically may contribute to early events leading to CD pathogenesis.

Succinate Produced by Intestinal Microbes Promotes Specification of Tuft Cells to Suppress Ileal Inflammation.

BACKGROUND & AIMS: Countries endemic for parasitic infestations have a lower incidence of Crohn's disease (CD) than nonendemic countries, and there have been anecdotal reports of the beneficial effects of helminths in CD patients. Tuft cells in the small intestine sense and direct the immune response against eukaryotic parasites. We investigated the activities of tuft cells in patients with CD and mouse models of intestinal inflammation. METHODS: We used microscopy to quantify tuft cells in intestinal specimens from patients with ileal CD (n = 19), healthy individuals (n = 14), and TNFΔARE/+ mice, which develop Crohn's-like ileitis. We performed single-cell RNA sequencing, mass spectrometry, and microbiome profiling of intestinal tissues from wild-type and Atoh1-knockout mice, which have expansion of tuft cells, to study interactions between microbes and tuft cell populations. We assessed microbe dependence of tuft cell populations using microbiome depletion, organoids, and microbe transplant experiments. We used multiplex imaging and cytokine assays to assess alterations in inflammatory response following expansion of tuft cells with succinate administration in TNFΔARE/+ and anti-CD3E CD mouse models. RESULTS: Inflamed ileal tissues from patients and mice had reduced numbers of tuft cells, compared with healthy individuals or wild-type mice. Expansion of tuft cells was associated with increased expression of genes that regulate the tricarboxylic acid cycle, which resulted from microbe production of the metabolite succinate. Experiments in which we manipulated the intestinal microbiota of mice revealed the existence of an ATOH1-independent population of tuft cells that was sensitive to metabolites produced by microbes. Administration of succinate to mice expanded tuft cells and reduced intestinal inflammation in TNFΔARE/+ mice and anti-CD3E-treated mice, increased GATA3+ cells and type 2 cytokines (IL22, IL25, IL13), and decreased RORGT+ cells and type 17 cytokines (IL23) in a tuft cell-dependent manner. CONCLUSIONS: We found that tuft cell expansion reduced chronic intestinal inflammation in mice. Strategies to expand tuft cells might be developed for treatment of CD.

Schistosoma mansoni Coinfection Attenuates Murine Toxoplasma gondii-Induced Crohn's-Like Ileitis by Preserving the Epithelial Barrier and Downregulating the Inflammatory Response.

Background and aims: Mice orally infected with T. gondii develop Crohn's disease (CD)-like enteritis associated with severe mucosal damage and a systemic inflammatory response, resulting in high morbidity and mortality. Previously, helminthic infections have shown therapeutic potential in experimental colitis. However, the role of S. mansoni in T. gondii-induced CD-like enteritis has not been elucidated. Our study investigated the mechanisms underlying T. gondii-induced ileitis and the potential therapeutic effect of S. mansoni coinfection. Methods: C57BL/6 mice were infected by subcutaneous injection of cercariae of the BH strain of S. mansoni, and 7-9 weeks later, they were orally infected with cysts of the ME49 strain of T. gondii. After euthanasia, the ileum was removed for histopathological analysis; staining for goblet cells; immunohistochemistry characterizing mononuclear cells, lysozyme expression, apoptotic cells, and intracellular pathway activation; and measuring gene expression levels by real-time PCR. Cytokine concentrations were measured in the serial serum samples and culture supernatants of the ileal explants, in addition to myeloperoxidase (MPO) activity. Results:T. gondii-monoinfected mice presented dense inflammatory cell infiltrates and ulcerations in the terminal ileum, with abundant cell extrusion, apoptotic bodies, and necrosis; these effects were absent in S. mansoni-infected or coinfected animals. Coinfection preserved goblet cells and Paneth cells, remarkably depleted in T. gondii-infected mice. Densities of CD4- and CD11b-positive cells were increased in T. gondii- compared to S. mansoni-infected mice and controls. MPO was significantly increased among T. gondii-mice, while attenuated in coinfected animals. In T. gondii-infected mice, the culture supernatants of the explants showed increased concentrations of TNF-alpha, IFN-gamma, and IL-17, and the ileal tissue revealed increased expression of the mRNA transcripts for IL-1 beta, NOS2, HMOX1, MMP3, and MMP9 and activation of NF-kappa B and p38 MAPK signaling, all of which were counterregulated by S. mansoni coinfection. Conclusion:S. mansoni coinfection attenuates T. gondii-induced ileitis by preserving mucosal integrity and downregulating the local inflammatory response based on the activation of NF-kappa B and MAPK. The protective function of prior S. mansoni infection suggests the involvement of innate immune mechanisms and supports a conceptually new approach to the treatment of chronic inflammatory diseases, including CD.

Pituitary Adenylate Cyclase-Activating Polypeptide-A Neuropeptide as Novel Treatment Option for Subacute Ileitis in Mice Harboring a Human Gut Microbiota.

The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) is well-known for its important functions in immunity and inflammation. Data regarding anti-inflammatory properties of PACAP in the intestinal tract are limited, however. In our present preclinical intervention study we addressed whether PACAP treatment could alleviate experimental subacute ileitis mimicking human gut microbiota conditions. Therefore, secondary abioitic mice were subjected to human fecal microbiota transplantation (FMT) and perorally infected with low-dose Toxoplasma gondii to induce subacute ileitis on day 0. From day 3 until day 8 post-infection, mice were either treated with synthetic PACAP38 or placebo. At day 9 post-infection, placebo, but not PACAP treated mice exhibited overt macroscopic sequelae of intestinal immunopathology. PACAP treatment further resulted in less distinct apoptotic responses in ileal and colonic epithelia that were accompanied by lower T cell numbers in the mucosa and lamina propria and less secretion of pro-inflammatory cytokines in intestinal ex vivo biopsies. Notably, ileitis-associated gut microbiota shifts were less distinct in PACAP as compared to placebo treated mice. Inflammation-ameliorating effects of PACAP were not restricted to the intestines, but could also be observed in extra-intestinal including systemic compartments as indicated by lower apoptotic cell counts and less pro-inflammatory cytokine secretion in liver and lungs taken from PACAP treated as compared to placebo control mice, which also held true for markedly lower serum TNF and IL-6 concentrations in the former as compared to the latter. Our preclinical intervention study provides strong evidence that synthetic PACAP alleviates subacute ileitis and extra-intestinal including systemic sequelae of T cell-driven immunopathology. These findings further support PACAP as a novel treatment option for intestinal inflammation including inflammatory bowel diseases (IBD).

Should We Divide Crohn's Disease Into Ileum-Dominant and Isolated Colonic Diseases?

Crohn's disease (CD) is an inflammatory bowel disease that can involve any region of the gastrointestinal tract. First described in 1932 as terminal ileitis or regional enteritis, it predominately involves the ileum with or without colonic involvement. Isolated colonic CD was first described in 1960 and since then the phenotypic classification of CD has evolved to stratify patients into isolated ileal, ileocolonic, or isolated colonic involvement. In the current review we evaluate the published literature regarding differences in epidemiology, natural history, pathogenesis, response to therapy, and disease monitoring, when stratified by disease location. Based on the available evidence consideration could be given to a new classification for CD, which splits it into ileum dominant (isolated ileal and ileocolonic) and isolated colonic disease. This may allow for a more optimized approach to clinical care and scientific research for CD.

Mesenteric Lymphatic Alterations Observed During DSS Induced Intestinal Inflammation Are Driven in a TLR4-PAMP/DAMP Discriminative Manner.

Background: Inflammatory bowel disease (IBD) is characterized by both acute and chronic phase inflammation of the gastro-intestinal (GI) tract that affect a large and growing number of people worldwide with little to no effective treatments. This is in part due to the lack of understanding of the disease pathogenesis and also the currently poorly described involvement of other systems such as the lymphatics. During DSS induced colitis, mice also develop a severe inflammation of terminal ileum with many features similar to IBD. As well as inflammation within the ileum we have previously demonstrated lymphatic remodeling within the mesentery and mesenteric lymph nodes of DSS-treated mice. The lymphatic remodeling includes lymphangiogenesis, lymphatic vessel dilation and leakiness, as well as cellular infiltration into the surrounding tissue and peripheral draining lymph nodes. Methods: Intestinal inflammation was induced in C57BL/6 mice by administration of 2.5% DSS in drinking water for 7 days. Mice were treated with TLR4 blocker C34 or Polymyxin-B (PMXB) daily from days 3 to 7 of DSS treatment via I.P. injection, and their therapeutic effects on disease activity and lymphatic function were examined. TLR activity and subsequent effect on lymphangiogenesis, lymphadenopathy, and mesenteric lymph node cellular composition were assessed. Results: DSS Mice treated with TLR4 inhibitor, C34, had a significantly improved disease phenotype characterized by reduced ileal and colonic insult. The change correlated with significant reduction in colonic and mesenteric inflammation, resolved mesenteric lymphangiectasia, and CD103+ DC migration similar to that of healthy control. PMXB treatment however did not resolve inflammation within the colon or associated mesenteric lymphatic dysfunction but did however prevent lymphadenopathy within the MLN through alteration of CCL21 gradients and CD103+ DC migration. Conclusions: TLR4 appears to mediate several changes within the mesenteric lymphatics, more specifically it is shown to have different outcomes whether stimulation occurs through pathogen derived factors such as LPS or tissue derived DAMPs, a novel phenomenon.

TL1A (TNFSF15) and DR3 (TNFRSF25): A Co-stimulatory System of Cytokines With Diverse Functions in Gut Mucosal Immunity.

TL1A and its functional receptor DR3 are members of the TNF/TNFR superfamilies of proteins. Binding of APC-derived TL1A to lymphocytic DR3 provides co-stimulatory signals for activated lymphocytes. DR3 signaling affects the proliferative activity of and cytokine production by effector lymphocytes, but also critically influences the development and suppressive function of regulatory T-cells. DR3 was also found to be highly expressed by innate lymphoid cells (ILCS), which respond to stimulation by TL1A. Several recent studies with transgenic and knockout mice as well as neutralizing or agonistic antibodies for these two proteins, have clearly shown that TL1A/DR3 are important mediators of several chronic immunological disorders, including Inflammatory Bowel Disease (IBD). TL1A and DR3 are abundantly localized at inflamed intestinal areas of patients with IBD and mice with experimental ileitis or colitis and actively participate in the immunological pathways that underlie mucosal homeostasis and intestinal inflammation. DR3 signaling has demonstrated a dichotomous role in mucosal immunity. On the one hand, during acute mucosal injury it exerts protective functions by ameliorating the severity of acute inflammatory responses and facilitating tissue repair. On the other hand, it critically participates in the pro-inflammatory pathways that underlie chronic inflammatory responses, such as those that take place in IBD. These effects are mediated through modulation of the relative mucosal abundance and function of Th1, Th2, Th17, Th9, and Treg lymphocytes, but also of all types of ILCs. Recently, an important role was demonstrated for TL1A/DR3 as potential mediators of intestinal fibrosis that is associated with the presence of gut inflammation. These accumulating data have raised the possibility that TL1A/DR3 pathways may represent a valid therapeutic target for chronic immunological diseases. Nevertheless, applicability of such a therapeutic approach will greatly rely on the net result of TL1A/DR3 manipulation on the various cell populations that will be affected by this approach.

Porcine lactoferrin-derived peptide LFP-20 modulates immune homoeostasis to defend lipopolysaccharide-triggered intestinal inflammation in mice.

The performance of immune system is vital for defending the body from pathogens, and it plays a crucial role in health homoeostasis. In a previous study, we have shown that LFP-20, a twenty-amino acid antimicrobial peptide in the N terminus of porcine lactoferrin, modulated inflammatory response in colitis. Here, we further investigated the effects of LFP-20 on immune homoeostasis to elucidate the mechanism of its anti-inflammation action. A lipopolysaccharide (LPS)-triggered systemic inflammatory response mice model was established. On the basis of observed mucosal lesions and apoptosis in small intestine, we found increased macrophage and neutrophil infiltration in ileum after LPS stimulation. Expectedly, LFP-20 pre-treatment attenuated the LPS-mediated immune disorders in ileum. Moreover, the flow cytometry results indicated pre-treatment with LFP-20 sustained the balance of CD3+CD8+ T cells, B cells and natural killer cells in LPS-triggered immune disturbance. Simultaneously, we demonstrated LFP-20 modulated the secretion of both activated Th1-related IL-12p70, interferon-γ, TNF-α and Th2-related IL-4, IL-5 and IL-6. Furthermore, we found LFP-20 facilitated a balanced Th1 and Th2 response, which triggered cellular defence mechanisms and induced B cells to produce opsonising antibodies belonging to certain IgG subclasses to defend against LPS stimulation. Collectively, our study indicated pre-treatment with LFP-20 could defend against LPS-triggered systemic inflammatory response in mice via modulating immune homoeostasis.

Death Receptor 3 Signaling Controls the Balance between Regulatory and Effector Lymphocytes in SAMP1/YitFc Mice with Crohn's Disease-Like Ileitis.

Death receptor 3 (DR3), a member of the tumor necrosis factor receptor (TNFR) superfamily, has been implicated in regulating T-helper type-1 (TH1), type-2 (TH2), and type-17 (TH17) responses as well as regulatory T cell (Treg) and innate lymphoid cell (ILC) functions during immune-mediated diseases. However, the role of DR3 in controlling lymphocyte functions in inflammatory bowel disease (IBD) is not fully understood. Recent studies have shown that activation of DR3 signaling modulates Treg expansion suggesting that stimulation of DR3 represents a potential therapeutic target in human inflammatory diseases, including Crohn's disease (CD). In this study, we tested a specific DR3 agonistic antibody (4C12) in SAMP1/YitFc (SAMP) mice with CD-like ileitis. Interestingly, treatment with 4C12 prior to disease manifestation markedly worsened the severity of ileitis in SAMP mice despite an increase in FoxP3+ lymphocytes in mesenteric lymph node (MLN) and small-intestinal lamina propria (LP) cells. Disease exacerbation was dominated by overproduction of both TH1 and TH2 cytokines and associated with expansion of dysfunctional CD25-FoxP3+ and ILC group 1 (ILC1) cells. These effects were accompanied by a reduction in CD25+FoxP3+ and ILC group 3 (ILC3) cells. By comparison, genetic deletion of DR3 effectively reversed the inflammatory phenotype in SAMP mice by promoting the expansion of CD25+FoxP3+ over CD25-FoxP3+ cells and the production of IL-10 protein. Collectively, our data demonstrate that DR3 signaling modulates a multicellular network, encompassing Tregs, T effectors, and ILCs, governing disease development and progression in SAMP mice with CD-like ileitis. Manipulating DR3 signaling toward the restoration of the balance between protective and inflammatory lymphocytes may represent a novel and targeted therapeutic modality for patients with CD.

T-bet-independent Th1 response induces intestinal immunopathology during Toxoplasma gondii infection.

Coordinated production of IFN-γ by innate and adaptive immune cells is central for host defense, but can also trigger immunopathology. The investigation of the lymphoid cell-specific contribution to the IFN-γ-mediated intestinal pathology during Toxoplasma gondii infection identified CD4+ T cells as a key cell population responsible for IFN-γ-dependent intestinal inflammation and Paneth cell loss, where T-bet-dependent group 1 innate lymphoid cells have a minor role in driving the parasite-induced immunopathology. This was evident from the analysis of T-bet deficiency that did not prevent the intestinal inflammation and instead revealed that T-bet-deficient CD4+ Th1 cells are sufficient for T. gondii-triggered acute ileitis and Paneth cell loss. These results revealed that T-bet-independent Th1 effector cells are major functional mediators of the type I immunopathological response during acute gastrointestinal infection.

The Xenobiotic Transporter Mdr1 Enforces T Cell Homeostasis in the Presence of Intestinal Bile Acids.

CD4+ T cells are tightly regulated by microbiota in the intestine, but whether intestinal T cells interface with host-derived metabolites is less clear. Here, we show that CD4+ T effector (Teff) cells upregulated the xenobiotic transporter, Mdr1, in the ileum to maintain homeostasis in the presence of bile acids. Whereas wild-type Teff cells upregulated Mdr1 in the ileum, those lacking Mdr1 displayed mucosal dysfunction and induced Crohn's disease-like ileitis following transfer into Rag1-/- hosts. Mdr1 mitigated oxidative stress and enforced homeostasis in Teff cells exposed to conjugated bile acids (CBAs), a class of liver-derived emulsifying agents that actively circulate through the ileal mucosa. Blocking ileal CBA reabsorption in transferred Rag1-/- mice restored Mdr1-deficient Teff cell homeostasis and attenuated ileitis. Further, a subset of ileal Crohn's disease patients displayed MDR1 loss of function. Together, these results suggest that coordinated interaction between mucosal Teff cells and CBAs in the ileum regulate intestinal immune homeostasis.

Epicutaneous Tolerance Induction to a Bystander Antigen Abrogates Colitis and Ileitis in Mice.

BACKGROUND: Although inflammatory bowel disease (IBD) is a failure in maintaining tolerance to the intestinal microbiota, few studies have investigated the use of immunologic tolerance as a treatment approach for IBD. We hypothesized that induction of immune tolerance at a distal site could suppress intestinal inflammation through a process of bystander regulation. METHODS: Epicutaneous tolerance was induced by topical application of ovalbumin (OVA) using a Viaskin patch for 48 hours. In some experiments, a single feed of ovalbumin was used to drive epicutaneous tolerance-induced regulatory T cells (Tregs) to the intestine. The mechanism of tolerance induction was tested using neutralizing antibodies against TGF-ß, IL-10, and Treg depletion using Foxp3-DTR mice. The capacity of skin-draining Tregs, or epicutaneous tolerance, to prevent or treat experimental IBD was tested using T-cell transfer colitis, dextran sodium sulfate (DSS) colitis, and ileitis in SAMP-YITFc mice. Weight loss, colonic inflammatory cytokines and histology were assessed. RESULTS: Epicutaneous exposure to ovalbumin induced systemic immune tolerance by a TGF-ß-dependent, but IL-10 and iFoxp3 Treg-independent mechanism. Skin draining Tregs suppressed the development of colitis. Epicutaneous tolerance to a model antigen prevented intestinal inflammation in the dextran sodium sulfate and SAMP-YITFc models and importantly could halt disease in mice already experiencing weight loss in the T-cell transfer model of colitis. This was accompanied by a significant accumulation of LAP and Foxp3 Tregs in the colon. CONCLUSIONS: This is the first demonstration that epicutaneous tolerance to a model antigen can lead to bystander suppression of inflammation and prevention of disease progression in preclinical models of IBD.

Role of Gut Inflammation in Altering the Monocyte Compartment and Its Osteoclastogenic Potential in HLA-B27-Transgenic Rats.

OBJECTIVE: To investigate the relationship between intestinal inflammation and the central and peripheral innate immune system in the pathogenesis of HLA-B27-associated spondyloarthritis using an HLA-B27-transgenic (B27-Tg) rat model. METHODS: The myeloid compartment of the blood and bone marrow (BM) of B27-Tg rats, as well as HLA-B7-Tg and non-Tg rats as controls, was evaluated by flow cytometry. Plasma from rats was assessed by enzyme-linked immunosorbent assay for levels of CCL2 and interleukin-1α (IL-1α). Rats were treated with antibiotics for 4 weeks, and the myeloid compartment of the blood and BM was evaluated by flow cytometry. The osteoclastogenic potential of BM-derived cells from antibiotic-treated rats, in the presence or absence of tumor necrosis factor (TNF), was evaluated in vitro. RESULTS: B27-Tg rats had substantially higher numbers of circulating Lin-CD172a+CD43low monocytes as compared to control animals, and this was significantly correlated with higher levels of plasma CCL2. Antibiotic treatment of B27-Tg rats markedly reduced the severity of ileitis, plasma levels of CCL2 and IL-1α, and number of BM and blood Lin-CD172a+CD43low monocytes, a cell subset shown in the present study to have the greatest in vitro osteoclastogenic potential. Antibiotic treatment also prevented the TNF-dependent enhancement of osteoclastogenesis in B27-Tg rats. CONCLUSION: Microbiota-dependent intestinal inflammation in B27-Tg rats directly drives the systemic inflammatory and bone-erosive potential of the monocyte compartment.

VEN-120, a Recombinant Human Lactoferrin, Promotes a Regulatory T Cell [Treg] Phenotype and Drives Resolution of Inflammation in Distinct Murine Models of Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Inflammatory bowel disease [IBD] is characterised by a disruption of immune homeostasis, which is tightly regulated to protect against harmful pathogens yet not react to commensal antigens. Animal studies indicate that regulatory T cells [Treg] modulate the immune response to prevent IBD development. Lactoferrin [LF] is an endogenous anti-inflammatory pleiotropic protein secreted at high concentrations in colostrum and at mucosal sites. However, the effect of LF on specific T lymphocyte populations has not been studied. Here, we identify a novel mechanism by which a recombinant human LF, VEN-120, regulates T cell populations in health and disease. METHODS: Two murine models of intestinal inflammation, the dextran sodium sulphate colitis model and the TNFΔARE/+ model of ileitis, were used to study the anti-inflammatory and T cell modulating ability of VEN-120. Flow cytometry was used to evaluate T cell populations within the lamina propria and mesenteric lymph nodes, and to evaluate the effect of VEN-120 on CD4+ T cells in vitro. RESULTS: VEN-120 reduced inflammation in both models of IBD, accompanied by increased Tregs in the intestinal lamina propria. Treatment of CD4+ T cells in vitro resulted in an upregulation of Treg genes and skewing towards a Treg population. This in vitro T cell skewing translated to an increase of Treg homing to the intestinal lamina propria and associated lymph tissue in healthy mice. CONCLUSIONS: These data provide a novel immunological mechanism by which VEN-120 modulates T cells to restrict inflammatory T cell-driven disease.

Intestinal, extra-intestinal and systemic sequelae of Toxoplasma gondii induced acute ileitis in mice harboring a human gut microbiota.

BACKGROUND: Within seven days following peroral high dose infection with Toxoplasma gondii susceptible conventionally colonized mice develop acute ileitis due to an underlying T helper cell (Th) -1 type immunopathology. We here addressed whether mice harboring a human intestinal microbiota developed intestinal, extra-intestinal and systemic sequelae upon ileitis induction. METHODOLOGY/PRINCIPAL FINDINGS: Secondary abiotic mice were generated by broad-spectrum antibiotic treatment and associated with a complex human intestinal microbiota following peroral fecal microbiota transplantation. Within three weeks the human microbiota had stably established in the murine intestinal tract as assessed by quantitative cultural and culture-independent (i.e. molecular 16S rRNA based) methods. At day 7 post infection (p.i.) with 50 cysts of T. gondii strain ME49 by gavage human microbiota associated (hma) mice displayed severe clinical, macroscopic and microscopic sequelae indicating acute ileitis. In diseased hma mice increased numbers of innate and adaptive immune cells within the ileal mucosa and lamina propria and elevated intestinal secretion of pro-inflammatory mediators including IFN-γ, IL-12 and nitric oxide could be observed at day 7 p.i. Ileitis development was accompanied by substantial shifts in intestinal microbiota composition of hma mice characterized by elevated total bacterial loads and increased numbers of intestinal Gram-negative commensals such as enterobacteria and Bacteroides / Prevotella species overgrowing the small and large intestinal lumen. Furthermore, viable bacteria translocated from the inflamed ileum to extra-intestinal including systemic compartments. Notably, pro-inflammatory immune responses were not restricted to the intestinal tract as indicated by increased pro-inflammatory cytokine secretion in extra-intestinal (i.e. liver and kidney) and systemic compartments including spleen and serum. CONCLUSION/SIGNIFICANCE: With respect to the intestinal microbiota composition "humanized" mice display acute ileitis following peroral high dose T. gondii infection. Thus, hma mice constitute a suitable model to further dissect the interactions between pathogens, human microbiota and vertebrate host immunity during acute intestinal inflammation.

Epithelial-specific Toll-like Receptor (TLR)5 Activation Mediates Barrier Dysfunction in Experimental Ileitis.

BACKGROUND: A large body of evidence supports a central role of TLR5 and its natural ligand, flagellin, in Crohn's disease (CD), with the precise mechanism(s) still unresolved. METHODS: We investigated the role of flagellin/TLR5 in SAMP1/YitFc (SAMP) mice, a spontaneous model of Crohn's disease-like ileitis. RESULTS: Ileal Tlr5 and serum antiflagellin IgG antibodies were increased in SAMP before the onset of inflammation and during established disease; these trends were abrogated in the absence of colonizing commensal bacteria. Irradiated SAMP receiving either wild-type (AKR) or SAMP bone marrow (BM) developed severe ileitis and displayed increased ileal Tlr5 compared with AKR recipients of either SAMP or AKR bone marrow, neither of which conferred ileitis, suggesting that elevated TLR5 in native SAMP is derived primarily from a nonhematopoietic (e.g., epithelial) source. Indeed, ileal epithelial TLR5 in preinflamed SAMP was increased compared with age-matched AKR and germ-free SAMP. TLR5-specific ex vivo activation of SAMP ileal tissues decreased epithelial barrier resistance, indicative of increased permeability, and was accompanied by altered expression of the tight junction proteins, claudin-3, occludin, and zonula occludens-1. CONCLUSIONS: Our results provide evidence that aberrant, elevated TLR5 expression is present in the ileal epithelium of SAMP mice, is augmented in the presence of the gut microbiome, and that TLR5 activation in response to bacterial flagellin results in a deficiency to maintain appropriate epithelial barrier integrity. Together, these findings represent a potential mechanistic pathway leading to the exacerbation and perpetuation of chronic gut inflammation in experimental ileitis and possibly, in patients with Crohn's disease.

Dysbiosis and zonulin upregulation alter gut epithelial and vascular barriers in patients with ankylosing spondylitis.

BACKGROUND: Dysbiosis has been recently demonstrated in patients with ankylosing spondylitis (AS) but its implications in the modulation of intestinal immune responses have never been studied. The aim of this study was to investigate the role of ileal bacteria in modulating local and systemic immune responses in AS. METHODS: Ileal biopsies were obtained from 50 HLA-B27+ patients with AS and 20 normal subjects. Silver stain was used to visualise bacteria. Ileal expression of tight and adherens junction proteins was investigated by TaqMan real-time (RT)-PCR and immunohistochemistry. Serum levels of lipopolysaccharide (LPS), LPS-binding protein (LPS-BP), intestinal fatty acid-BP (iFABP) and zonulin were assayed by ELISA. Monocyte immunological functions were studied in in vitro experiments. In addition the effects of antibiotics on tight junctions in human leukocyte antigen (HLA)-B27 transgenic (TG) rats were assessed. RESULTS: Adherent and invasive bacteria were observed in the gut of patients with AS with the bacterial scores significantly correlated with gut inflammation. Impairment of the gut vascular barrier (GVB) was also present in AS, accompanied by significant upregulation of zonulin, and associated with high serum levels of LPS, LPS-BP, iFABP and zonulin. In in vitro studies zonulin altered endothelial tight junctions while its epithelial release was modulated by isolated AS ileal bacteria. AS circulating monocytes displayed an anergic phenotype partially restored by ex vivo stimulation with LPS+sCD14 and their stimulation with recombinant zonulin induced a clear M2 phenotype. Antibiotics restored tight junction function in HLA-B27 TG rats. CONCLUSIONS: Bacterial ileitis, increased zonulin expression and damaged intestinal mucosal barrier and GVB, characterises the gut of patients with AS and are associated with increased blood levels of zonulin, and bacterial products. Bacterial products and zonulin influence monocyte behaviour.