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Targeting human CD2 with the monoclonal antibody (mAb) CB.219 reduces intestinal inflammation in a colitis model where T cells carry human CD2. Here, we asked whether this mAb has adverse effects on infection control. Mice expressing human CD2 on T cells (huCD2tg) were orally infected with Toxoplasma (T.) gondii and treated with the human CD2-specific mAb CB.219 in a preventive setting. The intestinal T. gondii loads in CB.219 treated mice did not differ from the control group. Histologically, huCD2tg mice showed moderate ileal inflammation that did not change with CB.219 treatment. In the ileum, CB.219 treatment reduced the protein levels of interferon-γ, transforming growth factor β and interleukin-6, whereas interleukin-18 mRNA was slightly increased. The infiltration of neutrophils, macrophages, and T cells into the ileum was unaffected by CB.219 treatment. However, CB.219 treatment decreased the numbers of forkhead box P3(+) regulatory T cells (Treg) in ileum and liver of huCD2tg mice. This was confirmed in vitro using human peripheral blood mononuclear cells. Taken together, targeting CD2(+) T cells by the human CD2 mAb CB.219 does not prevent beneficial immune reactions necessary for pathogen control. Further experiments will address gut specificity, underlying mechanisms, and general applicability of CB.219 treatment.

Increased levels of the matrix metalloproteinases-2 and -9 (also referred to gelatinase-A and -B, respectively) can be detected in intestinal inflammation. We have recently shown that selective gelatinase blockage by the synthetic compound RO28-2653 ameliorates acute murine ileitis and colitis. We here investigated whether RO28-2653 exerts anti-inflammatory effects in acute Campylobacter jejuni-induced enterocolitis of gnotobiotic IL-10(-/-) mice generated following antibiotic treatment. Mice were perorally infected with C. jejuni (day 0) and either treated with RO28-2653 (75 mg/kg body weight/day) or placebo from day 1 until day 6 post infection (p.i.) by gavage. Irrespective of the treatment, infected mice displayed comparable pathogen loads within the gastrointestinal tract. Following RO28-2653 administration, however, infected mice exhibited less severe symptoms such as bloody diarrhea as compared to placebo controls. Furthermore, less distinct apoptosis but higher numbers of proliferating cells could be detected in the colon of RO28-2653-treated as compared to placebo-treated mice at day 7 p.i. Remarkably, gelatinase blockage resulted in lower numbers of T- and B-lymphocytes as well as macrophages and monocytes in the colonic mucosa of C. jejuni-infected gnotobiotic IL-10(-/-) mice. Taken together, synthetic gelatinase inhibition exerts anti-inflammatory effects in experimental campylobacteriosis.

Tim-3 has opposing roles in innate and adaptive immunities. It not only dampens CD4+ and CD8+ T cells responses but also enhances the ability of macrophages to eliminate intracellular pathogens. After peroral infection with 100 cysts of Toxoplasma gondii genetically susceptible C57BL/6 mice develop an unchecked Th1 response associated with the development of small intestinal immunopathology. Here we report that upon infection with T. gondii, both susceptible C57BL/6 and resistant BALB/c mice exhibit increased frequencies of Tim-3+ cells in spleens and mesenteric lymph nodes. The number of Tim-3+ cells was significantly higher in C57BL/6 than in BALB/c mice. Tim-3 was expressed by macrophages, dendritic, natural killer, as well as CD4+ and CD8+ T cells. Highest frequencies of Tim-3+ cells were observed at the peak of Th1 responses (day 7 post infection) concurrent with the development of ileal immunopathology. Infected Tim-3-deficient BALB/c mice did not develop ileal immunopathology nor did their parasite loads differ from those in wildtype BALB/c mice. Thus, although Tim-3 is markedly upregulated upon infection and differentially regulated in susceptible and resistant mice upon infection with T. gondii, the absence of Tim-3 is not sufficient to overcome the genetic resistance of BALB/c mice to the development of Th1-driven small intestinal immunopathology.

Non-pathogenic Escherichia coli (Ec) strains K12 (EcK12) and Nissle 1917 (EcN) are used for gene technology and probiotic treatment of intestinal inflammation, respectively. We investigated intestinal colonization and potential pro-inflammatory properties of EcK12, EcN, and commensal E. coli (EcCo) strains in Toxoplasma (T.) gondii-induced acute ileitis. Whereas gnotobiotic animals generated by quintuple antibiotic treatment were protected from ileitis, mice replenished with conventional microbiota suffered from small intestinal necrosis 7 days post-T. gondii infection (p.i.). Irrespective of the Ec strain, recolonized mice revealed mild to moderate histopathological changes in their ileal mucosa. Upon stable recolonization with EcK12, EcN, or EcCo, development of inflammation was accompanied by pro-inflammatory responses at day 7 p.i., including increased ileal T lymphocyte and apoptotic cell numbers compared to T. gondii-infected gnotobiotic controls. Strikingly, either Ec strain was capable to translocate to extra-intestinal locations, such as MLN, spleen, and liver. Taken together, Ec strains used in gene technology and probiotic treatment are able to exert inflammatory responses in a murine model of small intestinal inflammation. In conclusion, the therapeutic use of Ec strains in patients with broad-spectrum antibiotic treatment and/or intestinal inflammation should be considered with caution.

Enterocolitis caused by Campylobacter jejuni represents an important socioeconomic burden worldwide. The host-specific intestinal microbiota is essential for maintaining colonization resistance (CR) against C. jejuni in conventional mice. Notably, CR is abrogated by shifts of the intestinal microbiota towards overgrowth with commensal E. coli during acute ileitis. Thus, we investigated whether oral transplantation (TX) of ileal microbiota derived from C. jejuni susceptible mice with acute ileitis overcomes CR of healthy conventional animals. Four days following ileitis microbiota TX or ileitis induction and right before C. jejuni infection, mice displayed comparable loads of main intestinal bacterial groups as shown by culture. Eight days following ileitis induction, but not ileal microbiota TX, however, C. jejuni could readily colonize the gastrointestinal tract of conventional mice and also translocate to extra-intestinal tissue sites such as mesenteric lymph nodes, spleen, liver, and blood within 4 days following oral infection. Of note, C. jejuni did not further deteriorate histopathology following ileitis induction. Lack of C. jejuni colonization in TX mice was accompanied by a decrease of commensal E. coli loads in the feces 4 days following C. jejuni infection. In summary, oral ileal microbiota TX from susceptible donors is not sufficient to abrogate murine CR against C. jejuni.

In experimental models of and humans with intestinal inflammation, increased levels of the matrix-degrading gelatinases MMP-2 and -9 in inflamed tissues can be detected. The synthetic collagen analogue (Gly-Pro-Hyp)10, (GPO)10, has been identified as a relevant binding structure for proMMP-2/-9 and promotes enzymatic activity of proMMP-2. Since targeted MMP strategies might offer promising anti-inflammatory treatment options, we for the first time studied in vivo actions exerted by (GPO)10 applying an acute dextrane sulfate sodium (DSS) induced colitis model. Seven-day intraperitoneal (GPO)10 treatment ameliorated clinical symptoms and histopathological colonic changes as compared to placebo controls with severe colitis. (GPO)10-treated mice displayed a diminished influx of neutrophils, and T- and B-lymphocytes into their colonic mucosa whereas numbers of regulatory T-cells and regenerative cells were higher as compared to placebo controls. Furthermore, IL-6 secretion was down-regulated in ex vivo colonic biopsies derived from (GPO)10-treated mice whereas higher concentrations of the anti-inflammatory cytokine IL-10 in extra-intestinal compartments such as MLN and spleen could be detected. Strikingly, influx of inflammatory cells into lungs was abolished following (GPO)10 application. We therefore propose (GPO)10 as a promising effective and safe treatment option of intestinal and extra-intestinal inflammatory conditions in humans.

Peroral infection with Toxoplasma gondii results in a Th1-type immunopathology characterized by small intestinal necrosis and is dependent on IL-18. In the present study, we investigated whether treatment with IL-18 binding protein (IL-18bp) prevents ileal pathology. We observed increased expression of IL-18bp in intestinal biopsies of mice following infection. Whereas small intestines of control mice showed severe necrosis with complete destruction of the small intestinal architecture, mice treated with IL-18bp daily displayed only mild inflammatory changes including flattening of villi and edema in the space between the epithelium and lamina propria. Small intestinal parasite loads and concentrations of pro-inflammatory cytokines did not differ in control and IL-18bp-treated mice. Binding of IL-18 to immobilized IL-18bp revealed a remarkably slow dissociation rate, indicating high affinity. Using chimeric mice we observed that bone marrow-derived rather than stromal cells were the primary source of IL-18 that resulted in small intestinal pathology following peroral infection with T. gondii. In conclusion, the results presented here suggest that IL-18bp may be an effective and safe treatment for small intestinal inflammation. Antigen-presenting rather than epithelial cells appear to be the main source of IL-18 in T. gondii-induced small intestinal inflammation.

Campylobacter jejuni is one of the predominant causes for foodborne bacterial infections worldwide. We investigated whether signaling of C. jejuni-lipoproteins and -lipooligosaccharide via Toll-like-receptor (TLR) -2 and -4, respectively, is inducing intestinal and extra-intestinal immune responses following infection of conventional IL-10(-/-) mice with chronic colitis. At day 3 following oral infection, IL-10(-/-) mice lacking TLR-2 or TLR-4 harbored comparable C. jejuni strain ATCC 43431 loads in their colon. Interestingly, infected TLR-4(-/-) IL-10(-/-) mice displayed less compromized epithelial barrier function as indicated by lower translocation rates of live gut commensals into mesenteric lymphnodes (MLNs), and exhibited less distinct B lymphocyte responses in their colonic mucosa as compared to naїve IL-10(-/-) controls. Furthermore, in extra-intestinal compartments such as MLNs and spleens, abundance of myeloid cells was less distinct whereas relative percentages of activated T helper cells and cytotoxic T cells were higher in spleens and dendritic cells more abundant in MLNs of infected IL-10(-/-) animals lacking TLR-4 as compared to IL-10(-/-) controls. Taken together, in conventionally colonized IL-10(-/-) mice, TLR-4, but not TLR-2, is involved in mediating extra-intestinal pro-inflammatory immune responses following C. jejuni infection. Thus, conventional IL-10(-/-) mice are well suited to further dissect mechanisms underlying Campylobacter infections in vivo.

Diseases such as liver fibrosis and intestinal inflammation are characterized by accumulated components of the extracellular matrix (ECM). Given that fibrillar collagen structures were shown to serve as storage site for inactive proforms of matrixmetalloproteinases (MMPs), modulating this MMP-collagen interaction might offer a rational interventional (therapeutic) approach to enhance degradation of accumulated ECM. The synthetic triple helical collagen analogue (Gly-Pro-Hyp)10 - (GPO)10 - was shown to trigger release and enzymatic activation of collagen sequestered proMMP-2. In the presented study, we, for the first time, investigated how MMP-(GPO)10 interaction impacts cellular responses in vitro. We found that recombinant proMMP-2 induced proliferation of hepatic stellate cells (HSC), which was enhanced after addition of (GPO)10 reaching comparable levels following incubation with fully activated MMP-2. In addition, (GPO)10 induced HSC migration similar to the platelet-derived growth factor subunit-B. Further, the MMP-2-dependent invasion of HT1080 fibrosarcoma cells through an ECM membrane was enhanced after addition of (GPO)10. Since cellular proliferation and migration concomitant with matrix degradation is stimulated, we conclude that the MMP-(GPO)10 interaction also functions in a physiological environment. Thus, a potential therapeutic effect of (GPO)10 should be further tested in animal models for MMP-associated diseases such as colitis or fibrosis.

Campylobacter (C.) jejuni is among the leading bacterial agents causing enterocolitis worldwide. Despite the high prevalence of C. jejuni infections and its significant medical and economical consequences, intestinal pathogenesis is poorly understood. This is mainly due to the lack of appropriate animal models. In the age of 3 months, adult mice display strong colonization resistance (CR) against C. jejuni. Previous studies underlined the substantial role of the murine intestinal microbiota in maintaining CR. Due to the fact that the host-specific gut flora establishes after weaning, we investigated CR against C. jejuni in 3-week-old mice and studied intestinal and extra-intestinal immunopathogenesis as well as age dependent differences of the murine colon microbiota. In infant animals infected orally immediately after weaning C. jejuni strain B2 could stably colonize the gastrointestinal tract for more than 100 days. Within six days following infection, infant mice developed acute enterocolitis as indicated by bloody diarrhea, colonic shortening, and increased apoptotic cell numbers in the colon mucosa. Similar to human campylobacteriosis clinical disease manifestations were self-limited and disappeared within two weeks. Interestingly, long-term C. jejuni infection was accompanied by distinct intestinal immune and inflammatory responses as indicated by increased numbers of T- and B-lymphocytes, regulatory T-cells, neutrophils, as well as apoptotic cells in the colon mucosa. Strikingly, C. jejuni infection also induced a pronounced influx of immune cells into extra-intestinal sites such as liver, lung, and kidney. Furthermore, C. jejuni susceptible weaned mice harbored a different microbiota as compared to resistant adult animals. These results support the essential role of the microflora composition in CR against C. jejuni and demonstrate that infant mouse models resemble C. jejuni mediated immunopathogenesis including the characteristic self-limited enterocolitis in human campylobacteriosis. Furthermore, potential clinical and immunological sequelae of chronic C. jejuni carriers in humans can be further elucidated by investigation of long-term infected infant mice. The observed extraintestinal disease manifestations might help to unravel the mechanisms causing complications such as reactive arthritis or Guillain-Barré syndrome.

Expression of gelatinases A and B, also referred to matrixmetalloproteinases (MMP)-2 and -9, respectively, is increased in inflamed tissues of experimental intestinal inflammation and humans with inflammatory bowel disease (IBDs). Given that we recently reported that treatment with the selective gelatinase inhibitor RO28-2653 ameliorates acute dextrane sulfate sodium (DSS) colitis, we asked whether gelatinase A or B expression is pivotal in mediating large intestinal inflammation. Results from our study reveal that symptoms of acute DSS colitis as well as histopathological colonic changes were ameliorated in MMP-2-, but not MMP-9-deficient mice, and were paralleled by a diminished influx of immune cells. In MMP-2-deficient mice, we observed lower expression of pro-inflammatory cytokines including interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and IL-6 in colonic biopsies and less overgrowth of the colonic lumen by potentially pro-inflammatory enterobacteria from the commensal gut microbiota. We conclude that rather MMP-2 than MMP-9 is causative for the establishment of DSS colitis in mice. The discrepancy of these data to prior reports might be due to substantial differences in the intestinal microbiota composition of the mice bred at different animal facilities impacting susceptibility to inflammatory stimuli. Consequently, a detailed survey of the gut microbiota should be implemented in immunological/inflammatory studies in the future in order to allow comparison of data from different facilities.

In the experimental models of intestinal inflammation and humans with inflammatory bowel diseases (IBD), increased levels of the matrix metalloproteinases (MMPs), MMP-2 and -9 (also referred to as gelatinase A and B, respectively), in inflamed tissue sites can be detected. In the presented study, we investigated potential beneficial effects exerted by doxycycline nonselectively blocking MMPs and the selective gelatinase inhibitor RO28-2653 in acute DSS colitis. Treatment with either compound for 8 days ameliorated clinical colitis pathology with a superior outcome in RO28-2653-treated animals. As compared to placebo controls, histopathological changes in the colon were less distinct following MMP blockage and IL-6 secretion in ex vivo biopsies was downregulated, paralleled by a diminished influx of pro-inflammatory immune cells and lack of overgrowth of the colonic lumen by potentially pro-inflammatory Escherichia coli of the commensal colon flora. We conclude that selective gelatinase inhibition not only exerts beneficial effects by disrupting the vicious cycle of positive feedback between immune cell stimulation and MMP induction but also prevents overgrowth of the colonic lumen by pro-inflammatory E. coli despite a lack of direct anti-bacterial properties, thus unaffecting the commensal gut microbiota. These findings put RO28-2653 into a center stage for development of intervention strategies in human IBD.

Enterocolitis caused by Campylobacter jejuni-infections represents an important socioeconomic burden worldwide. Recent results from novel murine infection models reveal that the intestinal microbiota is essential for maintaining colonization resistance against C. jejuni. We extended these studies to investigate the role of nutrition and obesity in susceptibility to C. jejuni-infection. Gnotobiotic (GB) mice generated by antibiotic treatment, which were fed with a human cafeteria diet (CAF), as well as obese (ob/ob) mice with a conventional microbiota harbored higher Escherichia coli loads in their colon as compared to respective controls. Following oral infection, C. jejuni 43431 ATCC readily colonized the intestines of CAF and ob/ob mice, whereas GB mice fed with a standard chow (MUD) eradicated the pathogen within days. Furthermore, live C. jejuni translocated into mesenteric lymph nodes of CAF, but not MUD mice. Strikingly, stably infected animals developed enterocolitis as indicated by increased numbers of immune and apoptotic cells in the colon in situ. We conclude that a specific human diet and obesity render mice susceptible to C. jejuni infection. The corresponding murine models are excellently suited for the study of C. jejuni pathogenesis and will help to get further insights into interplays between C. jejuni, microbiota, diet, obesity and immunity.