One of the best characterized mouse models of the inflammatory bowel diseases (IBD; Crohn's disease, ulcerative colitis) is the CD4+CD45RBhigh T cell transfer model of chronic colitis. Following our relocation to Texas Tech University Health Sciences Center (TTUHSC), we observed a dramatic reduction in the incidence of moderate-to-severe colitis from a 16-year historical average of 90% at Louisiana State University Health Sciences Center (LSUHSC) to <30% at TTUHSC. We hypothesized that differences in the commensal microbiota at the 2 institutions may account for the differences in susceptibility to T cell-induced colitis. Using bioinformatic analyses of 16S rRNA amplicon sequence data, we quantified and compared the major microbial populations in feces from healthy and colitic mice housed at the 2 institutions. We found that the bacterial composition differed greatly between mice housed at LSUHSC vs TTUHSC. We identified several genera strongly associated with, and signficantly overrepresented in high responding RAG-/- mice housed at LSUHSC. In addition, we found that colonization of healthy TTUHSC RAG-/- mice with feces obtained from healthy or colitic RAG-/- mice housed at LSUHSC transferred susceptibility to T cell-induced colitis such that the recipients developed chronic colitis with incidence and severity similar to mice generated at LSUHSC. Finally, we found that the treatment of mice with preexisting colitis with antibiotics remarkably attenuated disease. Taken together, our data demonstrate that specific microbial communities determine disease susceptibility and that manipulation of the intestinal microbiota alters the induction and/or perpetuation of chronic colitis.

We demonstrate a novel strategy using affinity extraction (AE) LC-MS to directly measure drug exposure and target engagement, two critical pharmacological questions, with a single assay. The assay measures total drug and target concentration at the site of therapeutic action, as well as the amount of target bound to drug. The case study presented applies the strategy to measure drug engagement of a membrane bound receptor (CD40) that is critical to immune regulation in colon biopsies collected from monkey dosed with an anti-CD40 antibody. Unlike other techniques that measure receptor occupancy, such as flow cytometry, this technique does not rely on viable cells allowing measurement of frozen samples in a remote setting from the clinic.

Background:Inflammation-associated lymphangiogenesis (IAL) is frequently observed in inflammatory bowel diseases. IAL is believed to limit inflammation by enhancing fluid and immune cell clearance. Although monocytes/macrophages (M&PHgr;) are known to contribute to intestinal pathology in inflammatory bowel disease, their role in intestinal IAL has never been studied mechanistically. We investigated contributions of monocytes/M&PHgr; to the development of intestinal inflammation and IAL. Methods:Because inflammatory monocytes express CC chemokine receptor 2 (CCR2), we used CCR2 diphtheria toxin receptor transgenic (CCR2.DTR) mice, in which monocytes can be depleted by diphtheria toxin injection, and CCR2−/− mice, which have reduced circulating monocytes. Acute or chronic colitis was induced by dextran sodium sulfate or adoptive transfer of CD4+CD45RBhigh T cells, respectively. Intestinal inflammation was assessed by flow cytometry, immunofluorescence, disease activity, and histopathology, whereas IAL was assessed by lymphatic vessel morphology and density. Results:We demonstrated that intestinal M&PHgr; expressed vascular endothelial growth factor-C/D. In acute colitis, monocyte-depleted mice were protected from intestinal injury and showed reduced IAL, which was reversed after transfer of wild-type monocytes into CCR2−/− mice. In chronic colitis, CCR2 deficiency did not attenuate inflammation but reduced IAL. Conclusions:We propose a dual role of M&PHgr; in (1) promoting acute inflammation and (2) contributing to IAL. Our data suggest that intestinal inflammation and IAL could occur independently, because IAL was reduced in the absence of monocytes/M&PHgr;, even when inflammation was present. Future inflammatory bowel disease therapies might exploit promotion of IAL and suppression of M&PHgr; independently, to restore lymphatic clearance and reduce inflammation.

Abstract Background Following relocation to our current institution we observed a 40% reduction in the incidence of T cell-induced colitis in our well-established mouse model of IBD. The objectives of this study were to: a) quantify and compare the colonic microbiota in healthy and colitic mice obtained from our current (TTUHSC) and former insitution (LSUHSC), and b) determine whether colonization of healthy mice housed at our current institution with feces from healthy or colitic mice from LSUHSC alters the incidence and/or severity of colitis. Methods DNA from frozen feces was isolated using standard protocols and 16S rRNA sequencing was performed using the Illumina MiSeq platform. For some studies, RAG1−/−mice were colonized (via gastric gavage) with 40 mg donor feces 1 week prior to adoptive transfer of naïve (CD4+CD45RBhigh) T-cells. Results Intestinal microbial populations are markedly different between the two institutions in healthy and colitic animals. Transplant of fecal microbiota from LSUHSC mice with chronic colitis into healthy RAG−/− recipients accelerates the onset, increases the incidence (>95%) and enhances the severity of chronic colitis following T cell transfer. However, colonization of healthy WT or RAG−/− mice (in the absence of T cell transfer) with colitic feces did not induce disease over the 8 week observation period. Conclusion We conclude that colonization of healthy RAG−/− mice with dysbiotic but nonpathogenic microbiota obtained from colitic mice markedly increases the incidence and severity of chronic colitis in the T cell transfer model.

The intestinal mucosal surface in all vertebrates is exposed to enormous numbers of microorganisms that include bacteria, archaea, fungi and viruses. Coexistence of the host with the gut microbiota represents an active and mutually beneficial relationship that helps to shape the mucosal and systemic immune systems of both mammals and teleosts (ray-finned fish). Due to the potential for enteric microorganisms to invade intestinal tissue and induce local and/or systemic inflammation, the mucosal immune system has developed a number of protective mechanisms that allow the host to mount an appropriate immune response to invading bacteria, while limiting bystander tissue injury associated with these rohn’s disease lcerative colitis athobiont ysbiosis cells immune responses. Failure to properly regulate mucosal immunity is thought to be responsible for the development of chronic intestinal inflammation. The objective of this review is to present our current understanding of the role that intestinal bacteria play in vertebrate health and disease. While our primary focus will be humans and mice, we also present the new and exciting comparative studies being performed in zebrafish to model host–microbe interactions. © 2016 Elsevier B.V. All rights reserved.

Background and PurposeThe lymphatic system maintains tissue homeostasis by unidirectional lymph flow, maintained by tonic and phasic contractions within subunits, ‘lymphangions’. Here we have studied the effects of the inflammatory cytokine IL‐1β on tonic contraction of rat mesenteric lymphatic muscle cells (RMLMC).Experimental ApproachWe measured IL‐1β in colon‐conditioned media (CM) from acute (AC‐CM, dextran sodium sulfate) and chronic (CC‐CM, T‐cell transfer) colitis‐induced mice and corresponding controls (Con‐AC/CC‐CM). We examined tonic contractility of RMLMC in response to CM, the cytokines h‐IL‐1β or h‐TNF‐α (5, 10, 20 ng·mL−1), with or without COX inhibitors [TFAP (10−5 M), diclofenac (0.2 × 10−5 M)], PGE2 (10−5 M)], IL‐1‐receptor antagonist, Anakinra (5 μg·mL−1), or a selective prostanoid EP4 receptor antagonist, GW627368X (10−6 and 10−7 M).Key ResultsTonic contractility of RMLMC was reduced by AC‐ and CC‐CM compared with corresponding control culture media, Con‐AC/CC‐CM. IL‐1β or TNF‐α was not found in Con‐AC/CC‐CM, but detected in AC‐ and CC‐CM. h‐IL‐1β concentration‐dependently decreased RMLMC contractility, whereas h‐TNF‐α showed no effect. Anakinra blocked h‐IL‐1β‐induced RMLMC relaxation, and with AC‐CM, restored contractility to RMLMC. IL‐1β increased COX‐2 protein and PGE2 production in RMLMC.. PGE2 induced relaxations in RMLMC, comparable to h‐IL‐1β. Conversely, COX‐2 and EP4 receptor inhibition reversed relaxation induced by IL‐1β.Conclusions and ImplicationsThe IL‐1β‐induced decrease in RMLMC tonic contraction was COX‐2 dependent, and mediated by PGE2. In experimental colitis, IL‐1β and tonic lymphatic contractility were causally related, as this cytokine was critical for the relaxation induced by AC‐CM and pharmacological blockade of IL‐1β restored tonic contraction.

The intestinal mucosal surface in all vertebrates is exposed to enormous numbers of microorganisms that include bacteria, archaea, fungi and viruses. Coexistence of the host with the gut microbiota represents an active and mutually beneficial relationship that helps to shape the mucosal and systemic immune systems of both mammals and teleosts (ray-finned fish). Due to the potential for enteric microorganisms to invade intestinal tissue and induce local and/or systemic inflammation, the mucosal immune system has developed a number of protective mechanisms that allow the host to mount an appropriate immune response to invading bacteria, while limiting bystander tissue injury associated with these rohn’s disease lcerative colitis athobiont ysbiosis cells immune responses. Failure to properly regulate mucosal immunity is thought to be responsible for the development of chronic intestinal inflammation. The objective of this review is to present our current understanding of the role that intestinal bacteria play in vertebrate health and disease. While our primary focus will be humans and mice, we also present the new and exciting comparative studies being performed in zebrafish to model host–microbe interactions. © 2016 Elsevier B.V. All rights reserved.

Chronic intestinal inflammation in patients with inflammatory bowel diseases (IBD) is fueled by complex and poorly understood interactions between T cells and myeloid cells that are constantly recruited from blood into the affected regions of the gut. We undertook this study to characterize the interplay between colitis-induced myeloid cells and CD4 T cells in CD45RBhigh T cell-induced colitis model. Within massively expanded CD11b+Gr-1+ splenocytes in colitic mice, only Ly6Chigh inflammatory monocytes suppressed proliferation and production of cytokines by CD4 T cells. Suppression was mediated by cell-contact, nitric oxide (NO), and partially by IFN-γ and prostaglandins. Ly6Chigh cells isolated from colitic colons showed upregulation of inducible NO synthase (iNOS) and arginase-1 and were more potent suppressors than those from spleen. Morphological and phenotypical analysis revealed that colon Ly6Chigh cells were macrophage-like (CD115-CD64+) but did not express MHC-II, CD11c, or F4/80. They secreted IL-10 and IL-1β/IL-6 and, upon co-culture with naïve T cells, promoted their differentiation into foxp3+ T cells and Th17 cells, respectively. Taken together, recruitment of inflammatory monocytes into inflamed intestine enhances their suppressive properties and may also arrest their further differentiation. Thus, monocyte recruitment to areas of chronic inflammation may represent an important homeostatic mechanism aimed at restraining and/or reshaping T cell responses.

It is well-known that intestinal microbiota are required for the induction of chronic gut inflammation in the CD4+CD45RBhigh→ RAG-1-/- mouse model of chronic colitis. Following our relocation from LSU Health Sciences Center (LSUSHC) to Texas Tech University Health Sciences Center (TTUHSC), we observed a significant reduction in the incidence and severity of disease (30-40%) compared to our previous 15 year history of ∼85% at LSUHSC. The objectives of this study were to: a) ascertain whether differences in the gut microbiota may account for the differences in disease incidence and b) determine whether colonization of RAG-1-/- (RAG ko) mice with feces obtained from colitic mice increases the incidence and severity of colitis in the T cell transfer model. DNA from freshly frozen feces was isolated using standard protocols and 16S rRNA sequencing was performed using the Roche 454 platform. For some studies, mice were colonized (via gastric gavage) with 40 mg donor feces 1 week prior to adoptive transfer of naïve (CD4+CD45RBhigh) T-cells. Microbiome analysis revealed marked shifts in the relative abundance of several major bacterial communities present in feces obtained from LSUHSC versus TTUHSC mice. For example, the relative abundance of Firmicutes and Bacteroidetes was ∼70 and 20%, respectively in healthy TTUHSC-RAG ko mice whereas these same 2 phyla represented 48 and 50%, respectively in healthy LSUHSC-RAG ko animals. Interestingly, the fecal microbiota from TTUHSC-RAG ko mice revealed significantly higher abundances of Proteobacteria, Verrucomicrobia and Tenericutes when compared to the microbiota obtained from healthy LSUHSC-RAG ko mice. Not surprisingly, induction of chronic colitis in LSUHSC-RAG ko mice via adoptive transfer of naïve T cells, produced a marked dysbiosis most notably characterized by an increased abundance of Verrucomicrobia together with corresponding and marked decrease in Bacteroidetes. Adoptive transfer of naive T cells into healthy TTUHSC-RAG ko mice that were first colonized with healthy LSUHSC-RAG ko feces induced mild-to-moderate colitis in ∼80% of the mice at 8 weeks post T cell transfer. In addition, we found that T cell transfer into TTUHSC-RAG ko mice that were first colonized with feces from colitic LSUHSC-RAG ko mice induced robust colitis in >90% of these recipients (called LSUHSCc→TTUHSC mice). Furthermore, T cell transfer into TTUHSC-RAG ko mice that had been colonized with feces from colitic LSUHCc→TTUHSC mice induced moderate-to-severe colitis in virtually all (>98%) of these recipients (called TTUHSCc→TTUHSC mice). Importantly, colonization of healthy wild type mice or TTUHSC-RAG ko mice (in the absence of T cell transfer) with colitic feces from TTUHSCc→TTUHSC mice did not induce disease over the 8 week observation period. We conclude that colonization of healthy RAG ko mice with dysbiotic microbiota obtained from colitic mice markedly increases the severity and incidence of chronic colitis in the T cell transfer model. Our data also suggest that the increase in severity and incidence of disease is not due to the presence of intestinal pathogens and can only be produced in mice with a dysregulated immune system (supported by grants from the DOD-W81XWH-11-1-0666 and NIH- RO1 DK 091269).

Chronic colitis is accompanied by extensive myelopoiesis and accumulation of CD11b+Gr-1+ cells in spleens and secondary lymphoid tissues. Although cells with similar phenotype have been described in cancer, chronic infection, or autoimmunity, where they were associated with suppression of T cell responses, little is known regarding how these cells affect CD4 T cell responses in the context of chronic intestinal inflammation. Therefore, we undertook this study to characterize the interplay between colitis-induced myeloid cells and CD4 T cell. Within the CD11b+Gr-1+ population, only monocytes (Ly6GnegLy6Chigh) but not other myeloid cell subsets suppressed proliferation and production of cytokines by CD4 T cells. Suppression was mediated by cell-contact, NO and partially by IFN-γ and PGs. Interestingly, Ly6Chigh MDCs, isolated from colitic colons, showed up-regulation of iNOS and arginase-1 and were more potent suppressors than those isolated from spleen. On a single-cell level, MDCs inhibited Th1 responses but enhanced generation of foxp3+ T cells. MDCs, cocultured with activated/Teffs, isolated from inflamed colons under hypoxic (1% O2) conditions typical for the inflamed intestine, suppressed proliferation but not their production of proinflammatory cytokines and chemokines. Taken together, expansion of monocytes and MDCs and activation of their suppressive properties may represent a homeostatic mechanism aimed at restraining excessive T cell activation during chronic inflammatory settings. The contribution of immunosuppressive monocytes/MDCs to chronic colitis and their role in shaping T cell responses in vivo require further investigation.

Accumulating evidence shows that myeloid-derived suppressor cells (MDSC) expand in chronic inflammatory settings, including inflammatory bowel diseases (IBD), and have potent suppressive effects on the T cells. MDSC are pathologically activated heterogeneous population of myeloid cells consisting of neutrophils, monocytes, and their precursors. Expansion of MDSC is thought to represent a homeostatic mechanism aimed at dampening chronic inflammation and attenuating collateral tissue damage. One of the mechanisms of T cell suppression used by MDSC is arginase-1-mediated depletion of nonessential amino acid L-arginine. Because T cells with enhanced reactivity towards bacterial antigens in the gut play an important contributing role in IBD, we decided to test the effectiveness of pegylated arginase-1 (Peg-Arg1) in mediating the suppression of mouse and human CD4 T cells. Naive CD4 T cells were isolated from spleens of wild-type (WT) mice. Some of these cells were polarized in vitro into Th1 and Th17 subsets according to the standard protocols. Mononuclear cells were isolated from mice with chronic colitis, which was induced by the adoptive transfer of CD45RBhigh T cells into the immunodeficient mice. Human CD4 T cells were isolated from peripheral blood of healthy volunteers. All cells were cultured in vitro in the presence of Peg-Arg1 or control Peg-BSA. We found that addition of Peg-Arg1 dramatically suppressed proliferation and production of IL-2, IFN-g, and IL-17 from mouse naïve CD4 T cells, Th1, and Th17 cells, respectively. We also found that suppression was due to the induction of apoptosis, which was rescued by addition of excess L-arg. Naïve T cells were more sensitive to Peg-Arg1-induced apoptosis than in vitro polarized T-cells. Moreover, Peg-Arg1 treatment of colon LP mononuclear cells isolated from colitic mice was effective at suppressing their proliferation and IFN-g production. Finally, treatment of human anti-CD3/CD28-activated CD4 T cells with Peg-Arg1 was accompanied by dose-dependent suppression of their proliferation and cytokine production. However, Peg-Arg1 treatment did not trigger human T cell apoptosis but arrested their proliferation in the G0-G1 phase of the cell cycle. Taken together, we demonstrated that Peg-Arg1-mediated depletion of L-arginine is very effective at suppressing mouse or human CD4 T cells through either induction of apoptosis or triggering cell cycle arrest and dysfunction, respectfully. Our data suggest that catabolism of amino acid mediated by Peg-Arg or related enzymes could be an effective approach for the treatment of IBD in the future. Supported in part by the Career Development Award #2923 to DVO.

Background: Inflammatory arthritis is a chronic disease, resulting in synovitis and subchondral and bone area destruction, which can severely affect a patient's quality of life. The most common form of inflammatory arthritis is rheumatoid arthritis (RA) in which many of the disease mechanisms are not well understood. The collagen-induced arthritis (CIA) mouse model is similar to RA as it exhibits joint space narrowing and bone erosion as well as involves inflammatory factors and cellular players that have been implicated in RA pathogenesis. Quantitative data for disease progression in RA models is difficult to obtain as serum blood markers may not always reflect disease state and physical disease indexes are subjective. Thus, it is important to develop tools to objectively assess disease progression in CIA.Results: Micro-CT (Computed Tomography) is a relatively mature technology that has been used to track a variety of anatomical changes in small animals. In this study, micro-CT scans of several joints of control and CIA mice were acquired at 0, 4, 7, and 9 weeks after the immunization with collagen type II. Each micro-CT scan was analyzed by applying a segmentation algorithm to individual slices in each image set to provide 3-dimensional representations of specific bones including the humerus, femur, and tibia. From these representations, the volume and mean density of these bones were measured and compared. This analysis showed that both the volume and the density of each measured bone of the CIA mice were significantly smaller than those of the controls at week 7.Conclusions: This study demonstrates that micro-CT can be used to quantify bone changes in the CIA mouse model as an alternative to disease index assessments. In conclusion, micro-CT could be useful as a non-invasive method to monitor the efficacy of new treatments for RA tested in small animals.

BACKGROUND:L-selectin (CD62L) and β(7) integrins are important for trafficking of naive T cells under steady-state conditions. The objectives of this study were to dissect the requirements for T cell-associated CD62L and β(7) integrins during initiation, progression, and regulation of chronic colitis. METHODS:Using the T-cell transfer model, we compared colitogenic potential between T cells lacking one or both of these molecules with wild-type T cells. To assess trafficking of cells to the secondary lymphoid tissue and the gut, we performed co-homing experiments. RESULTS:Adoptive transfer of wild-type, CD62L(-/-) or β(7)(-/-) single-deficient T cells induced moderate to severe disease with slightly different kinetics. However, transfer of CD62L(-/-) β(7)(-/-) double-deficient (DKO) T cells produced significantly attenuated gut inflammation, which correlated with fewer T cells and reduced levels of proinflammatory cytokines in the colon lamina propria. Our subsequent experiments established that lack of colitogenic potential of these cells was due to inability of DKO T cells to home to the secondary lymphoid tissue. Furthermore, homing of in vitro-generated effector DKO T cells to the inflamed intestine was significantly impaired. Lastly, DKO regulatory T cells were ineffective at suppressing colitis induced by wild-type T cells. CONCLUSIONS:We established that T cells can use either CD62L(-/-) or β(7)(-/-) integrins to induce chronic colitis, but lack of both abrogates their colitogenic potential. Effector T cells critically rely on β(7) integrin during their recruitment to the inflamed intestinal mucosa. Finally, regulation of intestinal inflammation by regulatory T cells requires one or both of these adhesion molecules.

Background:Myeloid cells are the most abundant and heterogeneous population of leukocytes. They are rapidly recruited from the blood to areas of inflammation and perform a number of important biological functions. Chronic inflammatory conditions contribute to generation of myeloid-derived suppressor cells (MDSCs). These pathologically activated cells are increasingly recognized as important players in cancer, transplantation, and autoimmunity for their abilities to modulate innate and adaptive immune responses. Methods:Since clinical data on MDSC accumulation in human patients affected with inflammatory bowel diseases (IBD) are relatively scarce, most of the information described in this review came from studies using experimental mouse models of IBD. Results:In this review, we discuss possible roles of these cells in chronic immune-mediated disorders focusing on studies conducted in IBD. We will review the available evidence on how MDSCs are involved in modulating T cell responses and look into the complex relationship between Th1, Th17 cells, and myeloid cells. Finally, we will review some recent successes and failures resulted from therapies aimed at manipulating myeloid cell numbers and/or their function. Conclusions:Although MDSCs have been described in animal models of experimental colitis and in patients with IBD, their exact role in IBD pathogenesis is unclear and needs to be studied further. Information obtained from these studies will be useful to better understand the cross talk between myeloid cells in T cells during chronic inflammation and may identify novel pathways to be targeted therapeutically.

INTRODUCTION:We have previously demonstrated that adoptive transfer of naïve CD4(+) T cells devoid of lymphocyte function-associated antigen-1-deficient (LFA-1; CD11a/CD18) into recombination activating gene-1 (RAG-1) deficient (RAG(-/-) ) mice fails to induce chronic colitis whereas transfer of wild type (WT) T-cells induces unrelenting and chronic disease.METHODS:The objectives of this study were to assess the role of lymphocyte function-associated antigen-1 (LFA-1) in enteric antigen (EAg)-induced activation of T cells in vitro and in vivo and to define the importance of this integrin in promoting trafficking of T cells to the mesenteric lymph nodes (MLNs) and colon.RESULTS:We found that EAg-pulsed dendritic cells (DCs) induced proliferation of LFA-1-deficient (CD11a(-/-) ) CD4(+) T cells that was very similar to that induced using WT T cells, suggesting that LFA-1 is not required for activation/proliferation of T cells in vitro. Coculture of WT or CD11a(-/-) T cells with EAg-pulsed DCs induced the generation of similar amounts of interferon-gamma, interleukin (IL)-4, and IL-10, whereas IL-17A production was reduced ≈ 2-fold in cocultures with CD11a(-/-) T cells. Short-term (20-22 hours) trafficking studies demonstrated that while both WT and CD11a(-/-) T cells migrated equally well into the spleen, liver, lungs, small intestine, cecum, and colon, trafficking of CD11a(-/-) T cells to the MLNs was reduced by 50% when compared to WT T cells. When the observation period was extended to 3-7 days posttransfer, we observed ≈ 2-3-fold more WT T cells within the MLNs and colon than CD11a(-/-) T cells, whereas T-cell proliferation (as measured by CFSE dilution) was comparable in both populations.CONCLUSIONS:Taken together, our data suggest that LFA-1 is not required for EAg-induced activation of CD4(+) T cells in vitro or in vivo but is required for trafficking of T cells to the MLNs and homing of colitogenic effector cells to the colon where they initiate chronic gut inflammation.