Yersinia pseudotuberculosis Anti-Inflammatory Components Reduce Trinitrobenzene Sulfonic Acid-Induced Colitis in the Mouse

Abstract

Rectal instillation of trinitrobenzene sulfonic acid (TNBS) induces acute colitis in the mouse. We tested the efficacy of Yersinia pseudotuberculosis anti-inflammatory components in preventing TNBS-triggered colitis. Animals were orally inoculated with virulence-attenuated Yersinia cells (a phoP mutant) prior to TNBS administration. Under these experimental conditions, colonic lesions and tumor necrosis factor alpha mRNA levels were significantly reduced.

Crohn's disease and ulcerative colitis are the major human forms of chronic inflammatory bowel disease (IBD), an important public health problem in Western countries that affects 1 in 1,000 individuals (12, 16). This condition is believed to be caused by a dysfunction of the mucosal immune system driven by the normal intestinal flora: the result is an inappropriate activation of the mucosal immune response, with overproduction of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) (9, 16). The production of TNF-α and other proinflammatory cytokines requires activation of the nuclear factor κB (NF-κB) pathway, together with stress-activated kinases of the p38 mitogen-activated protein kinase (p38 MAPK) family and c-Jun NH₂-terminal kinases (JNK) (10, 19, 29). In patients with IBD, there are several indications that the pathways which regulate cytokine production are abnormally active (20, 23, 27). This finding may be due (at least in part) to impaired expression of the peroxisome proliferator-activator receptor γ (8), a nuclear factor which is highly expressed by epithelial cells and that can be regulated by microorganisms present in the intestinal lumen (7). Since mesalamine, corticosteroids, immunosuppressive drugs, and (as shown more recently) TNF-α-specific monoclonal antibodies fail to induce or maintain remission in about 30% of patients with IBD (11), inhibitors of the NF-κB, p38, and JNK MAPK pathways (as well as activators of peroxisome proliferator-activator receptor γ) are viewed as potential therapeutic agents.

The enteroinvasive bacterium Yersinia pseudotuberculosis synthesizes virulence factors encoded by a 70-kb plasmid, pYV. Some of these factors (designated Yops [Yersinia outer proteins]) are delivered into the cytosol of macrophages, epithelial cells, and endothelial cells via a type III secretion-translocation apparatus (for a review, see reference 2). The ubiquitin-like protease YopJ (known as YopP in the other enteropathogenic yersinia, Yersinia enterocolitica) has received particular attention: it has the ability to down-regulate the MAPK and NF-κB pathways in infected cells by preventing the activation (via phosphorylation) of MAPK kinases and IKKβ (IκB kinase β) (for a review, see reference 17). This results in reduced release of TNF-α by macrophages (11) and of interleukin-8 (IL-8) by epithelial and endothelial cells (4, 22). It has further been reported that two other pYV-encoded factors counteract the host's inflammatory response: the GTPase-activating protein YopE and the tyrosine phosphatase YopH both down-regulate IL-8 production in Yersinia-infected epithelial cells by blocking the NF-κB and MAPK pathways (G. I. Viboud, S. Shun Kin So, and J. B. Bliska, Abstr. 103rd Gen. Meet. Am. Soc. Microbiol., abstr. D-099, p. 220, 2003; 26). YopE suppresses NF-κB activation as efficiently as YopJ, whereas YopH has only a weak inhibitory effect on this signaling pathway. YopE potently inhibits the activation of JNK and (to a lesser extent) extracellular signaling-regulated kinase, whereas YopH prevents activation of the two MAPKs (Viboud et al., Abstr. 103rd Gen. Meet. Am. Soc. Microbiol.) and also blocks (via phosphatidylinositol 3-kinase activation) the expression of monocyte chemoattractant protein 1 by macrophages in vitro (21). Additionally, it has been reported that LcrV (V antigen, part of the translocation apparatus of the type III secretion system) induces anti-inflammatory cytokine IL-10 release in macrophages in vitro through an as yet unknown signaling pathway (24). Taken together, these observations suggest that Yersinia components might be potential drugs for the treatment of patients with IBD. Demonstration of their efficacy in an experimental model of colitis mimicking human IBD is, however, a prerequisite for further development.

In the mouse, rectal instillation of the hapten reagent 2,4,6-trinitrobenzene sulfonic acid (TNBS) triggers extensive necrosis of the colon within 5 days. This result is mediated by activation of the NF-κB pathway and leads to enhanced levels of TNF-α and IL-1β mRNA in the mucosa (6). Although this murine model has limitations, it does allow preclinical studies of new IBD drugs. We studied the impact of prior intestinal infection by Y. pseudotuberculosis on the development of TNBS-induced colitis. Since the wild type of this species is highly virulent in the mouse, we used a virulence-attenuated phoP mutant (32777ΔPhoPA; ΔphoPΩkan) (M. Marceau, F. Sebbane, F. Ewann, F. Collyn, B. Linder, M. A. Campos, J. A. Bengoechea, and M. Simonet, submitted for publication) which nevertheless retains production of pYV-encoded anti-inflammatory proteins. Mutation of the PhoP-PhoQ two-component regulatory system has been previously reported to increase by ∼100-fold the 50% lethal dose of pathogenic yersiniae (18; Marceau, unpublished results). We have shown that loss of a functional PhoP-PhoQ system does not impair yopJ, yopE, yopH, or lcrV expression as judged by RNA slot blot hybridization with specific DNA probes (Marceau, unpublished results). When our Y. pseudotuberculosis phoP mutant (Marceau et al., submitted) was inoculated orally in mice, it was still capable of colonizing the Peyer's patches and the colon wall and also translocated to the mesenteric lymph nodes and the spleen (Fig. 1). We then challenged C57BL/6 mice (which are more resistant than the BALB/c lineage to TNBS [15] and also to Yersinia infection [25]) with a sublethal intragastric dose of 10^7.7 32777ΔPhoPA cells. On day 5 following the bacterial challenge, infected animals and control animals were anesthetized for 90 to 120 min and then received an intrarectal administration of TNBS (150 mg/kg of body weight; 40 μl of a 1:1 mixture in 0.9% NaCl and 100% ethanol). Five days later, animals were sacrificed and the colon was evaluated under a dissecting microscope (magnification, ×5) for macroscopic lesions according to the Wallace criteria (28). This score rates macroscopic lesions over a 4-cm length of colon on a scale from 0 to 10, based on features reflecting inflammation, i.e., hyperemia, thickening of the bowel, and the extent of ulceration. Compared to the results for control mice (n = 7) given only TNBS, a significant decrease in the macroscopic lesion score (Wallace score) was observed in mice infected with Yersinia (n = 7) prior to TNBS administration (Fig. 2). We found an inverse relationship between the Wallace score and colonic colonization by Yersinia cells. Of the five individuals without colitis, four showed bacterial levels in the colon (over a 4-cm length) of 10^6.6 to 10^6.8 CFU (with just one showing 10^3.9 CFU), whereas for the two animals with colonic lesions scored at 1 and 3 on the Wallace scale, levels were 10^3.6 and 10^2.3 CFU, respectively. Suppression of TNBS-induced colitis was Yersinia specific since the Wallace score was not reduced when animals were challenged daily with 10⁷ Escherichia coli TG1 (instead of Y. pseudotuberculosis 32777ΔPhoPA) bacteria over 5 days to ensure persistence of this nonpathogenic strain in the intestinal tract (Fig. 2).

FIG. 1.

Growth of the PhoP-deficient mutant in mouse tissues. Eight-week-old female inbred C57BL/6 mice (Iffa Credo) were given an intragastric dose of 10^7.7 Y. pseudotuberculosis 32777ΔPhoPA resuspended in sterile distilled water (0.2 ml). Infected animals were kept in positive-pressure cabinets in a level 2 biosafety facility and had free access to regular rodent chow and tap water. Mice were sacrificed on day 5 after the intragastric challenge. The spleen, three Peyer's patches along a 10-cm length of the ileum, a 2-cm length of colon, and the entire chain of mesenteric lymph nodes were aseptically removed and homogenized in phosphate-buffered saline. Bacteria were counted by spreading dilutions of organ or tissue homogenate on Luria-Bertani agar containing 50 mg of kanamycin per liter since in strain 32777ΔPhoPA, phoP was replaced by a kanamycin resistance gene. Yersinia colonies were identified on the basis of colony morphology and urease production. Each data point shows bacterial counts in tissues from individual animals, and each bar represents the mean value for seven mice.

FIG. 2.

Macroscopic analysis of mouse colons on day 5 after intrarectal administration of TNBS in animals preinoculated with Yersinia or Escherichia cells and in noninoculated controls. Data from individual mice are shown (solid circles, controls given TNBS only; open circles, Yersinia-infected mice; solid squares, E. coli-infected mice), and bars indicate mean values. Results obtained in the three experimental groups were compared using the nonparametric Mann-Whitney test. NS, not significant.

TNF-α plays a key role in the pathophysiology of TNBS-induced colitis, as demonstrated by (i) the decrease in disease severity after the neutralization of the cytokine in vivo, (ii) the absence of colonic lesions in TNF-α knockout mice after intrarectal instillation of TNBS, and (iii) the development of lethal pan-colitis upon TNBS administration in TNF-α transgenic mice (14). To confirm and extend our previous results, we compared colon TNF-α mRNA levels measured in TNBS-treated mice preinfected with Yersinia (n = 9) to those of controls (n = 5). Total RNA was isolated from colonic biopsy samples with phenol and chloroform, and specific mRNAs were quantified by competitive reverse transcription-PCR (RT-PCR) as follows. Ten micrograms of DNA-free RNA extract was reverse transcribed into cDNA by using Moloney murine leukemia virus reverse transcriptase as previously detailed (5). cDNAs were further PCR amplified (40 cycles) in a Perkin-Elmer apparatus using custom-synthesized sense (s) and antisense (as) primers (Genset) specific for the genes coding for TNF-α (s, 5′-TCTCATCAGTTCTATGGCCC-3′; as, 5′-GGGAGTAGACAAGGTACAAC-3′) and β-actin (s, 5′-GGGTCAGAAGGATTCCTATG-3′; as, 5′-GGTCTCAAACATGATCTGGG-3′). Two linearized plasmid DNA competitors (pMus-3 and pQB3 for competition with TNF-α cDNA and β-actin cDNA, respectively) were added to the PCR mixture. After amplification, the DNA competitors and target cDNAs were separated by agarose gel electrophoresis, and cDNA quantification was performed by using an image analyzer (Gel Analyst; Clara Vision) as previously described (5). For each colonic sample, the results were expressed as the number of TNF-α mRNA molecules per 10⁹ mRNA molecules of internal control β-actin. Levels of TNF-α mRNA in each of the groups of mice are depicted in Fig. 3. As shown, we found a sixfold reduction (P < 0.001) in cytokine transcript synthesis in the colons of animals inoculated with Yersinia prior to TNBS administration.

FIG. 3.

Colonic TNF-α transcript levels on day 5 after intrarectal administration of TNBS in mice preinoculated with Yersinia cells and in noninoculated controls. Data from individual animals are shown with corresponding Wallace scores in parentheses (solid circles, controls given TNBS only; open circles, Yersinia-infected mice), and bars indicate mean values. Results obtained in the two experimental groups were compared using the nonparametric Mann-Whitney test.

This preliminary study shows that anti-inflammatory components produced by Y. pseudotuberculosis are effective in reducing experimental colitis in mice and thus appear to be promising candidates for use in IBD therapy. YopE, YopH, YopJ, and LcrV are likely to be the main (though perhaps not the only) effectors. In principle, this assertion could be proven by inoculating an isogenic yopEHJlcrV mutant prior to rectal instillation of TNBS and comparing the effects to those produced by the parental strain. However, such a mutant would almost certainly fail to colonize and persist in intestinal and lymph tissues, since YopE and YopH are necessary for this process (13). At present, IBD therapy necessitates lifelong maintenance drug administration. In the present work, anti-inflammatory bacterial products were given in a preventive mode before colitis induction so as to limit the potential translocation of Yersinia otherwise favored by alteration of the intestinal barrier after TNBS administration. We suggest that this new therapeutic strategy should be focused on maintenance rather than induction of IBD remission. Since cellular entry of Y. pseudotuberculosis anti-inflammatory effectors is required to inhibit the inflammatory response in the gut, we used live bacteria producing a type III secretion to reach the cytosol of intestinal cells. The development of microorganisms capable of efficiently delivering anti-inflammatory components represents a major advance in biological therapy for IBD. However, live, genetically modified bacteria are not approved for medical use, and concerns regarding the safety of this therapeutic option have not yet been sufficiently addressed. Progress in the future should lead to specific optimized mucosal delivery of Yersinia components into epithelial and subepithelial cells of the inflamed colon by using a replication-deficient adenovirus with genes coding for Yersinia anti-inflammatory products under the control of appropriate tissue-specific promoters. Polyclonal B lymphocyte activation (leading to autoantibody production) has been reported in a mouse inoculated with an extract of total Yops (3): this type of adverse effect may compromise the therapeutic use of anti-inflammatory Yersinia components, and it will be imperative to evaluate this phenomenon for each recombinant adenovirus.