Cytolethal Distending Toxin Promotes Helicobacter cinaedi-Associated Typhlocolitis in Interleukin-10-Deficient Mice

Helicobacter cinaedi colonizes a wide host range, including rodents, and may be an emerging zoonotic agent. Colonization parameters, pathology, serology, and inflammatory responses to wild-type H. cinaedi (WT_Hc) were evaluated in B6.129P2-IL-10^tm1Cgn (IL-10^−/−) mice for 36 weeks postinfection (WPI) and in C57BL/6 (B6) mice for 12 WPI. Because cytolethal distending toxin (CDT) may be a virulence factor, IL-10^−/− mice were also infected with the cdtB_Hc and cdtB-N_Hc isogenic mutants and evaluated for 12 WPI. Consistent with other murine enterohepatic helicobacters, WT_Hc did not cause typhlocolitis in B6 mice, but mild to severe lesions developed at the cecocolic junction in IL-10^−/− mice, despite similar colonization levels of WT_Hc in the cecum and colon of both B6 and IL-10^−/− mice. WT_Hc and cdtB mutants also colonized IL-10^−/− mice to a similar extent, but infection with either cdtB mutant resulted in attenuated typhlocolitis and hyperplasia compared to infection with WT_Hc (P < 0.03), and only WT_Hc infection caused dysplasia and intramucosal carcinoma. WT_Hc and cdtB_Hc mutant infection of IL-10^−/− mice elevated mRNA expression of tumor necrosis factor alpha, inducible nitric oxide synthase, and gamma interferon in the cecum, as well as elevated Th1-associated serum immunoglobulin G2a^b compared to infection of B6 mice (P < 0.05). Although no hepatitis was noted, liver samples were PCR positive at various time points for WT_Hc or the cdtB_Hc mutant in approximately 33% of IL-10^−/− mice and in 10 to 20% of WT_Hc-infected B6 mice. These results indicate that WT_Hc can be used to model inflammatory bowel disease in IL-10^−/− mice and that CDT contributes to the virulence of H. cinaedi.The association of Helicobacter cinaedi infection with a variety of human diseases has received increasing attention in recent years. H. cinaedi was first isolated from homosexual men suffering from enteritis, proctitis, or proctocolitis (51). H. cinaedi was subsequently isolated from immunocompromised patients afflicted with meningitis, bacteremia, cellulitis, septic arthritis, and enteritis (2) and a neonate clinically ill with septicemia and meningitis (39), as well as from immunocompetent patients with metabolic disease (36). There are several reports of H. cinaedi isolation from various clinically healthy animal hosts, including dogs, cats, foxes, and hamsters (52). H. cinaedi has also been isolated from the inflamed colon, mesenteric lymph node, and liver of a rhesus monkey with chronic idiopathic colitis and hepatitis and from a baboon with hepatitis, as well as from feces of clinically normal, captive rhesus monkeys (11, 15, 18). Because H. cinaedi has been isolated from intestinal flora of normal hamsters and identified by PCR-based assays with wild rodents, rodents may be a zoonotic reservoir for humans (6, 47). Although H. cinaedi is the most commonly reported enterohepatic helicobacter isolated from humans, the pathogenic properties of this organism in humans or animals have not been thoroughly investigated (13).H. cinaedi and other enterohepatic helicobacters produce a cytolethal distending toxin (CDT), which arrests cell cycle progression and causes cell death in vitro (49). CDT activity also has been associated with a variety of other gram-negative bacterial pathogens that cause human diseases affecting mucosal surfaces, such as chancroid, periodontitis, and gastroenteritis (7, 48, 56). CDT from Escherichia coli, Campylobacter jejuni, Haemophilus ducreyi, Actinobacillus actinomycetemcomitans, and enterohepatic Helicobacter species irreversibly blocks the cell cycle at the G₂/M phase of growth in a wide range of cultured cells. CDT consists of three polypeptide subunits encoded by the closely linked cdtA, cdtB, and cdtC genes. cdtB is an enzymatically active subunit which functions as a nuclease that damages DNA and triggers cell cycle arrest. cdtA and cdtC are heterodimeric subunits required for the delivery of cdtB (5, 7, 48, 55, 56). In addition to inhibiting cell cycle progression, it has been reported that C. jejuni CDT directly mediates the release of proinflammatory interleukin-8 (IL-8) from intestinal epithelial cells, suggesting that CDT has a role in the inflammatory response to mucosal infections (24).IL-10 is an anti-inflammatory cytokine that inhibits the production of inflammatory cytokines in vitro and in vivo (38, 45). IL-10^−/− mice spontaneously develop enterocolitis when housed in conventional animal facilities but not in germfree conditions, suggesting that gut flora are critical for development of inflammation in this model (29, 46). It has been reported that Helicobacter bilis, Helicobacter hepaticus, Helicobacter typhlonius, and Helicobacter trogontum persistently colonize the lower bowel of mice and induce proliferative typhlocolitis in immune-dysregulated mice, including IL-10^−/− mice (14, 26, 30, 31, 34, 54). In this study, we used the IL-10^−/− mouse model to evaluate the pathogenic potential of H. cinaedi and the role of CDT in inducing gastrointestinal disease.