Susan L. Tonkonogy

Lactobacillus plantarum 299V in the treatment and prevention of spontaneous colitis in interleukin-10-deficient mice.

Interleukin (IL)-10-deficient (IL-10−/−) mice develop colitis under specific pathogen-free (SPF) conditions and remain disease free if kept sterile (germ free [GF]). We used four different protocols that varied the time-points of oral administration of Lactobacillus plantarum 299v (L. plantarum) relative to colonization with SPF bacteria to determine whether L. plantarum could prevent and treat colitis induced by SPF bacteria in IL-10−/− mice and evaluated the effect of this probiotic organism on mucosal immune activation. Assessment of colitis included blinded histologic scores, measurements of secreted colonic immunoglobulin isotypes, IL-12 (p40 subunit), and interferon (IFN)-&ggr; production by anti-CD3-stimulated mesenteric lymph node cells. Treating SPF IL-10−/− mice with L. plantarum attenuated previously established colonic inflammation as manifested by decreased mucosal IL-12, IFN-&ggr;, and immunoglobulin G2a levels. Colonizing GF animals with L. plantarum and SPF flora simultaneously had no protective effects. Gnotobiotic IL-10−/− mice monoassociated with L. plantarum exhibited mild immune system activation but no colitis. Pretreatment of GF mice by colonization with L. plantarum, then exposure to SPF flora and continued probiotic therapy significantly decreased histologic colitis scores. These results demonstrate that L. plantarum can attenuate immune-mediated colitis and suggest a potential therapeutic role for this agent in clinical inflammatory bowel diseases.

IL-2-deficient mice raised under germfree conditions develop delayed mild focal intestinal inflammation

Interleukin-2 (IL-2) amplifies immune stimuli and influences B cell differentiation. IL-2-deficient mice spontaneously develop intestinal inflammation if raised under specific pathogen-free (SPF) conditions. We quantitatively determined the aggressiveness and kinetics of gastrointestinal and hepatic inflammation in the presence or absence of viable bacteria in IL-2-deficient mice. Breeding colonies were maintained under SPF and germfree (GF) conditions. Intestinal tissues, serum, and mesenteric lymph nodes were obtained from mice at different ages for blind histological scoring, immunoglobulin measurements, mucosal T cell infiltration, and cytokine secretion. GF IL-2 -/- mice developed mild, focal, and nonlethal intestinal inflammation with delayed onset, whereas the more aggressive inflammation in SPF IL-2 -/- mice led to their death between 28 and 32 wk. Periportal hepatic inflammation was equal in the presence or absence of bacterial colonization. Intestinal immunoglobulin secretion decreased significantly by 13 wk of age in IL-2 -/- mice in both GF and SPF environments. In contrast to other genetically engineered rodents, IL-2 -/- mice develop mild focal gastrointestinal and active portal tract inflammation in the absence of viable bacteria.Interleukin-2 (IL-2) amplifies immune stimuli and influences B cell differentiation. IL-2-deficient mice spontaneously develop intestinal inflammation if raised under specific pathogen-free (SPF) conditions. We quantitatively determined the aggressiveness and kinetics of gastrointestinal and hepatic inflammation in the presence or absence of viable bacteria in IL-2-deficient mice. Breeding colonies were maintained under SPF and germfree (GF) conditions. Intestinal tissues, serum, and mesenteric lymph nodes were obtained from mice at different ages for blind histological scoring, immunoglobulin measurements, mucosal T cell infiltration, and cytokine secretion. GF IL-2 -/- mice developed mild, focal, and nonlethal intestinal inflammation with delayed onset, whereas the more aggressive inflammation in SPF IL-2 -/- mice led to their death between 28 and 32 wk. Periportal hepatic inflammation was equal in the presence or absence of bacterial colonization. Intestinal immunoglobulin secretion decreased significantly by 13 wk of age in IL-2 -/- mice in both GF and SPF environments. In contrast to other genetically engineered rodents, IL-2 -/- mice develop mild focal gastrointestinal and active portal tract inflammation in the absence of viable bacteria.

Enterococcus faecalis metalloprotease compromises epithelial barrier and contributes to intestinal inflammation.

BACKGROUND & AIMS Matrix metalloproteases (MMPs) mediate pathogenesis of chronic intestinal inflammation. We characterized the role of the gelatinase (GelE), a metalloprotease from Enterococcus faecalis, in the development of colitis in mice. METHODS Germ-free, interleukin-10-deficient (IL-10(-/-)) mice were monoassociated with the colitogenic E faecalis strain OG1RF and isogenic, GelE-mutant strains. Barrier function was determined by measuring E-cadherin expression, transepithelial electrical resistance (TER), and translocation of permeability markers in colonic epithelial cells and colon segments from IL-10(-/-) and TNF(ΔARE/Wt) mice. GelE specificity was shown with the MMP inhibitor marimastat. RESULTS Histologic analysis (score 0-4) of E faecalis monoassociated IL-10(-/-) mice revealed a significant reduction in colonic tissue inflammation in the absence of bacteria-derived GelE. We identified cleavage sites for GelE in the sequence of recombinant mouse E-cadherin, indicating that it might be degraded by GelE. Experiments with Ussing chambers and purified GelE revealed the loss of barrier function and extracellular E-cadherin in mice susceptible to intestinal inflammation (IL-10(-/-) and TNF(ΔARE/Wt) mice) before inflammation developed. Colonic epithelial cells had reduced TER and increased translocation of permeability markers after stimulation with GelE from OG1RF or strains of E faecalis isolated from patients with Crohns disease and ulcerative colitis. CONCLUSIONS The metalloprotease GelE, produced by commensal strains of E faecalis, contributes to development of chronic intestinal inflammation in mice that are susceptible to intestinal inflammation (IL-10(-/-) and TNF(ΔARE/Wt) mice) by impairing epithelial barrier integrity.

Helicobacter hepaticus Does Not Induce or Potentiate Colitis in Interleukin-10-Deficient Mice

ABSTRACT Helicobacter hepaticus has been reported to induce colitis, hepatitis, and hepatocellular carcinoma in several different murine models. The aim of this study was to determine if H. hepaticus will cause colitis in monoassociated mice lacking the interleukin-10 gene (IL-10−/− mice) and potentiate colitis in specific-pathogen-free (SPF) IL-10−/− mice. Germfree IL-10−/− mice on either a mixed (C57BL/6 × 129/Ola) or inbred (129/SvEv) genetic background were monoassociated with H. hepaticus ATCC 51448 by oral feeding and rectal enemas. In a second experiment, germfree IL-10−/− mice were colonized with stool from SPF mice that harbored or did not harbor endogenous H. hepaticus. After 7 to 9 weeks of colonization, weight loss and mortality were assessed, the colon was isolated for histology and IL-12 secretion, and mesenteric lymph node cells were assessed for T-cell activation markers. It was found that IL-10−/− mice monoassociated with H. hepaticus for up to 16 weeks showed almost no histologic colitis or increased IL-12 production. SPF IL-10-knockout mice had no significant difference in weight loss, mortality rate, histologic scores, colonic IL-12 secretion, or T-cell activation with or withoutH. hepaticus. We conclude that H. hepaticusdoes not induce or potentiate disease in our IL-10−/−mice and therefore is not required to induce colitis in genetically susceptible hosts.

Dual-association of gnotobiotic Il-10−/− mice with 2 nonpathogenic commensal bacteria induces aggressive pancolitis

Background: Monoassociating gnotobiotic IL‐10‐deficient (−/−) mice with either nonpathogenic Enterococcus faecalis or a nonpathogenic Escherichia coli strain induces T‐cell‐mediated colitis with different kinetics and anatomical location (E. faecalis: late onset, distal colonic; E. coli: early onset, cecal). Hypothesis: E. faecalis and E. coli act in an additive manner to induce more aggressive colitis than disease induced by each bacterial species independently. Methods: Germ‐free (GF) inbred 129S6/SvEv IL‐10−/− and wildtype (WT) mice inoculated with nonpathogenic E. faecalis and/or E. coli were killed 3–7 weeks later. Colonic segments were scored histologically for inflammation (0 to 4) or incubated in media overnight to measure spontaneous IL‐12/IL‐23p40 secretion. Bacterial species were quantified by serial dilution and plated on culture media. Mesenteric lymph node (MLN) CD4+ cells were stimulated with antigen‐presenting cells pulsed with bacterial lysate (E. faecalis, E. coli, Bacteroides vulgatus) or KLH (unrelated antigen control). IFN‐&ggr; and IL‐17 levels were measured in the supernatants. Results: Dual‐associated IL‐10−/− (but not WT) mice developed mild‐to‐moderate pancolitis by 3 weeks that progressed to severe distal colonic‐predominant pancolitis with reactive atypia and duodenal inflammation by 7 weeks. NF‐&kgr;B was activated in the duodenum and colon in dual‐associated IL‐10−/− × NF‐&kgr;BEGFP mice. The aggressiveness of intestinal inflammation and the degree of antigen‐specific CD4+ cell activation were greater in dual‐ versus monoassociated IL‐10−/− mice. Conclusion: Two commensal bacteria that individually induce phenotypically distinct colitis in gnotobiotic IL‐10−/− mice act additively to induce aggressive pancolitis and duodenal inflammation. (Inflamm Bowel Dis 2007)

Antigen-Presenting Cell Production of IL-10 Inhibits T-Helper 1 and 17 Cell Responses and Suppresses Colitis in Mice

BACKGROUND & AIMS Mice that are deficient in interleukin (IL)-10 develop colitis, mediated by T-helper (Th)1 and Th17 cells, and IL-10-producing regulatory T (Treg) cells suppress colitis, implicating IL-10 in maintaining mucosal homeostasis. We assessed the relative importance of immunoregulatory IL-10 derived from T cells or from antigen presenting cells (APCs) in development of intestinal inflammation. METHODS CD4(+) cells from germ-free (GF) or specific pathogen-free (SPF) IL-10(-/-) or wild-type mice were injected into IL-10(-/-), Rag2(-/-) mice or Rag2(-/-) mice that express IL-10. After 6-8 weeks, we evaluated inflammation, spontaneous secretion of cytokines from colonic tissue, and mRNA levels of the transcription factor T-bet and the immunoregulatory cytokine transforming growth factor (TGF)-β. CD4(+) T cells were co-cultured with bacterial lysate-pulsed APCs and assayed for cytokine production, FoxP3 expression, and TGF-β-mediated Smad signaling. RESULTS CD4(+) cells from GF or SPF IL-10(-/-) or wild-type mice induced more severe colitis and higher levels of inflammatory cytokines in IL-10(-/-), Rag2(-/-) mice than in IL-10-replete, Rag2(-/-) mice. Co-cultures of IL-10(-/-) or wild-type CD4(+) T cells plus bacterial lysate-pulsed APCs from IL-10(-/-) mice contained more interferon (IFN)-γ, IL-12/23p40, and IL-17 than co-cultures of the same T cells plus APCs from wild-type mice. CD11b(+) APCs were required for these effects. Blocking IL-10 receptors increased production of IFN-γ and IL-12/23p40 whereas exogenous IL-10 suppressed these cytokines. IL-10-producing APCs induced TGF-β-mediated, retinoic acid-dependent, differentiation of FoxP3(+) Treg cells, whereas blocking the retinoic acid receptor, in vitro and in vivo, reduced proportions of FoxP3(+) Treg cells. CONCLUSIONS IL-10 produced by APCs regulates homeostatic T-cell responses to commensal bacteria.

A reverse transcription-polymerase chain reaction technique to detect feline cytokine genes

The ability to detect feline cytokine expression would allow further characterization of the feline immune system. Bioassays are currently available for the measurement of feline IL2, IL6 and TNF alpha but not for other biologically important cytokines. To detect the expression of other cytokines, a reverse transcription-polymerase chain reaction (RT-PCR) procedure was developed. Since feline cytokine gene sequences other than TNF alpha were not available, mammalian DNA and mRNA sequences for IL2, IFN gamma, IL4, IL6, IL10, IL12 and beta-actin, obtained from the Genbank database were compared and oligonucleotide primers chosen from consensus sequences. To validate the cytokine and beta-actin primers, peripheral blood mononuclear cells from specific pathogen free (SPF) cats were cultured in the presence of Con A for various periods of time (0-72 h). RNA was collected, reverse transcribed into cDNA, and the cDNA was amplified by PCR with each set of cytokine primer pairs. RT-PCR products were hybridized with specific 32P end-labeled internal oligonucleotide probes and then analyzed with the AMBIS imaging system to determine the kinetics of cytokine mRNA production. The beta-actin signal was used to control for sample to sample variation in the quantity of mRNA and variation in the RT and PCR reactions. Peak mRNA expression for most cytokines was found to occur between 2 to 4 h of Con A stimulation. mRNA expression was correlated with cytokine bioactivity for IL2 and IL6. Peak IL2 bioactivity occurred after 8 h of Con A stimulation, 4 h after the mRNA expression had peaked. Although IL6 mRNA expression peaked between 2 and 4 h of stimulation, bioactivity was not detected until 8 h of stimulation and continued to increase over the next 24-48 h.

Reduced ratio of protective versus proinflammatory cytokine responses to commensal bacteria in HLA-B27 transgenic rats

Germ‐free HLA‐B27 transgenic (TG) rats do not develop colitis, but colonization with specific pathogen‐free (SPF) bacteria induces colitis accompanied by immune activation. To study host‐dependent immune responses to commensal caecal bacteria we investigated cytokine profiles in mesenteric lymph node (MLN) cells from HLA‐B27 TG versus nontransgenic (non‐TG) littermates after in vitro stimulation with caecal bacterial lysates (CBL). Supernatants from CBL‐stimulated unseparated T‐ or B‐ cell‐depleted MLN cells from HLA‐B27 TG and non‐TG littermates were analysed for IFN‐γ, IL‐12, TNF, IL‐10 and TGF‐β production. Our results show that unfractionated TG MLN cells stimulated with CBL produced more IFN‐γ, IL‐12 and TNF than did non‐TG MLN cells. In contrast, CBL‐stimulated non‐TG MLN cells produced more IL‐10 and TGF‐β. T cell depletion abolished IFN‐γ and decreased IL‐12 production, but did not affect IL‐10 and TGF‐β production. Conversely, neither IL‐10 nor TGF‐β was produced in cultures of B cell‐depleted MLN. In addition, CD4+ T cells enriched from MLN of HLA‐B27 TG but not from non‐TG rats produced IFN‐γ when cocultured with CBL‐pulsed antigen presenting cells from non‐TG rats. Interestingly, IL‐10 and TGF‐β, but not IFN‐γ, IL‐12 and TNF were produced by MLN cells from germ‐free TG rats. These results indicate that the colitis that develops in SPF HLA‐B27 TG rats is accompanied by activation of IFN‐γ‐producing CD4+ T cells that respond to commensal bacteria. However, B cell cytokine production in response to components of commensal intestinal microorganisms occurs in the absence of intestinal inflammation.

Bifidobacterium animalis Causes Extensive Duodenitis and Mild Colonic Inflammation in Monoassociated Interleukin-10-Deficient Mice

Background: We recently showed that Bifidobacterium animalis is more prevalent within the colons of interleukin (IL)‐10‐deficient (−/−) mice than in wildtype (WT) animals colonized with the same specific pathogen‐free (SPF) fecal contents. Here we tested the ability of this organism to cause T‐cell‐mediated intestinal inflammation by introducing it into germ‐free (GF) IL‐10−/− mice. Methods: GF IL‐10−/− or WT mice were monoassociated with Bifidobacterium animalis subsp. animalis ATCC (American Type Culture Collection, Manassas, VA) 25527T or with B. infantis ATCC 15697T. Inflammation was measured by blinded histologic scores of the duodenum, cecum, and colon and by spontaneous secretion of IL‐12/IL‐23 p40 from colonic explants. Bacterial antigen‐specific CD4+ mesenteric lymph node (MLN) T‐cell recall responses were measured in response to antigen‐presenting cells (APC) pulsed with bacterial lysates. Results: B. animalis caused marked duodenal inflammation and mild colitis in monoassociated IL‐10−/− mice, whereas the intestinal tracts of WT animals remained free of inflammation. B. infantis colonization resulted in mild inflammation in the duodena of IL‐10−/− mice. CD4+ MLN T cells from B. animalis monoassociated IL‐10−/− mice secreted high levels of IFN‐&ggr; and IL‐17 in response to B. animalis lysate. B. animalis equally colonized the different intestinal regions of WT and IL‐10−/− mice. Conclusions: B. animalis, a traditional probiotic species that is expanded in experimental colitis in this model, induces marked duodenal and mild colonic inflammation and TH1/TH17 immune responses when introduced alone into GF IL‐10−/− mice. This suggests a potential pathogenic role for this commensal bacterial species in a susceptible host.

Dysregulated luminal bacterial antigen-specific T-cell responses and antigen-presenting cell function in HLA-B27 transgenic rats with chronic colitis

HLA‐B27/β2 microglobulin transgenic (TG) rats spontaneously develop T‐cell‐mediated colitis when colonized with normal commensal bacteria, but remain disease‐free under germ‐free conditions. We investigated regulation of in vitro T‐cell responses to enteric bacterial components. Bacterial lysates prepared from the caecal contents of specific pathogen‐free (SPF) rats stimulated interferon‐γ (IFN‐γ) production by TG but not non‐TG mesenteric lymph node (MLN) cells. In contrast, essentially equivalent amounts of interleukin‐10 (IL‐10) were produced by TG and non‐TG cells. However, when cells from MLNs of non‐TG rats were cocultured with TG MLN cells, no suppression of IFN‐γ production was noted. Both non‐TG and TG antigen‐presenting cells (APC) pulsed with caecal bacterial lysate were able to induce IFN‐γ production by TG CD4+ cells, although non‐TG APC were more efficient than TG APC. Interestingly, the addition of exogenous IL‐10 inhibited non‐TG APC but not TG APC stimulation of IFN‐γ production by cocultured TG CD4+ lymphocytes. Conversely, in the presence of exogenous IFN‐γ, production of IL‐10 was significantly lower in the supernatants of TG compared to non‐TG APC cultures. We conclude that commensal luminal bacterial components induce exaggerated in vitro IFN‐γ responses in HLA‐B27 TG T cells, which may in turn inhibit the production of regulatory molecules, such as IL‐10. Alterations in the production of IFN‐γ, and in responses to this cytokine, as well as possible resistance of TG cells to suppressive regulation could together contribute to the development of chronic colitis in TG rats.