microbiota is less severe, the results raise the hypothesis that intestinal bacteria potentiate the development of indomethacin- induced mucosal lesions. Thus, dysmotility induced by gluten sensitization [11] or indomethacin [36], and/or the ability of indomethacin to induce small intestinal dysbiosis [36,37] may facilitate bacterial translocation. Due to a severely impaired intestinal barrier in both gluten-sensitized and indomethacin- treated mice, increased permeation of luminal bacteria may disturb the natural commensal homeostasis in the gut promoting a pro-inflammatory response. SPF mice have been shown to be systemically ignorant to their intestinal microbiota due to the geographic and functional separation between the mucosal and systemic immune system by the mesenteric lymph nodes (MLN) [17,38]. Our results show, however, that a low level of systemic priming against intestinal microbiota occurs in SPF mice treated with either gluten or indomethacin alone. Gluten-sensitized mice, in which barrier function is further perturbed by indomethacin treatment, show dramatic systemic priming to their intestinal microbiota. These data therefore imply that gluten-sensitization, in combination with indomethacin treatment, results in decreased mucosal containment of the commensal flora. NSAIDs have been shown to reduce the phagocytic properties of macrophages [39]. Thus, we acknowledge that it is possible that in addition to changes in intestinal barrier function, indomethacin may have a dual effect by inhibiting macrophage function, allowing the persistence of live bacteria, and facilitating a systemic immune response against intestinal microbiota. F4/80+ cell counts in the lamina propria of gluten plus indomethacin-treated mice were significantly increased (Figure S8, Protocol S1), however macro- phage function was not assessed. The exact identities of the commensals to which gluten and indomethacin-treated mice are systemically primed in this model are not yet known but the absence of IgM binding to Salmonella, known to be absent from the commensal flora of our mice, strongly suggests the specificity of the IgM antibodies against commensal flora in our mice (Figure S7). The clinical relevance of the loss of systemic ignorance against the intestinal microbiota remains to be established, however, systemic priming to the commensal flora represents a significant shift in the normal relationship between host and commensal bacteria [40]. Consequently, this may indicate a novel mechanism that could contribute to the progression of disease in a gluten-sensitive host. On the other hand, specific IgM against flora may be part of a protective mechanism mounted by the immune system to limit subsequent translocation and widespread inflammation. Addition- al host factors, such as an underlying immune dysbalance, may play a role in determining whether this mechanism will become maladaptive and contribute to widespread inflammation. A recent epidemiological study has determined that consumption of non- steroidal anti-inflammatory drugs (NSAIDs) is a risk factor for the development of irritable bowel syndrome [41]. No epidemiological studies to date have investigated whether a history of NSAID consumption is also a risk factor for the protean clinical expression in gluten sensitivity. Although the role of the intestinal microbiota in other chronic diseases of the gut is clearly established [for review see 42] little is known about the role of abnormal immune responses to commensals in gluten and other food intolerances. Recent findings, however, report presence of rod-shaped bacteria in the mucosa of active and non-active celiac patients but not in healthy controls [43]. A study in patients with CD revealed the presence of serological responses to microbial antigens, such as anti-Saccharo- myces cerevisiae, anti-I2 (Pseudomonas fluorescens) and anti-ompW, compared to healthy controls. Interestingly, microbial seropositiv- ity was also present in gluten-sensitive patient with no evidence of NSAID and Gluten Sensitivity active CD. However, increasing age was associated with sero- reactivity for anaerobic bacteria, possibly reflecting exposure to different environmental antigens with longer duration of disease [44]. The disappearance of anti-Saccaromyces cervisiae-antibodies (ASCA) after a gluten-free diet suggests that healing of mucosal lesions is related to microbial sero-markers [45]. However, a causal relationship between gut dysfunction, symptoms and microbial sero-responses in CD remains to be determined. It is possible that accumulated bacterial products have a bystander effect and lower the threshold for immune cell activation [46,47]. To this respect, a study in DQ8 mice has shown that oral challenge with Lactobacillus casei at the time of mucosal sensitization with gliadin and cholera toxin exacerbates the Th1 response induced in the model [48]. Thus, it is possible that dysbiosis or shifts in the composition of the intestinal microbiota at the time of gluten sensitization, and not necessarily the presence of a pathogen, contribute to enhance gluten-induced immune responses. An altered microbiota compo- sition has been reported in patients with CD compared to healthy controls [49,50]. In this study, we observed significant alterations in the composition of the small intestinal microbiota in gluten- sensitized mice treated with indomethacin. It is unclear, however, if these changes are primary or secondary to the functional gut abnormalities observed in the model [11]. In conclusion, our findings suggest that an environmental alteration of the intestinal barrier plays a critical role in determining host immune responses to gluten and intestinal microbiota antigens. Bystander luminal antigens such as compo- nents of the intestinal microbiota may contribute to enhance inflammatory responses to dietary antigens such as gluten. This mechanism may become important in genetically predisposed hosts with longstanding barrier abnormalities. The results warrant further investigations on the interactions between host genotype, diet, and intestinal microbiota. Materials and Methods Animals All experiments were conducted with approval from the McMaster University Animal Care Committee. Male transgenic mice expressing HLA-DQ8 genes (HLA-DQA1*0301; HLA- DQb1*0302) in the absence of endogenous mouse class II genes or HLA-DQ8/HCD4 double transgenic mice were used [8,51]. The mice were bred in a conventional specific pathogen free colony (SPF) at McMaster University and maintained for at least 2 generations prior to breeding on a gluten-free diet (Bio-Serv, New Jersey). Mice were used at the age of 8-14 weeks. Male C57BL/6 mice were purchased from Taconic (Hudson, NY, USA) (supplementary data). Sensitization protocol and indomethacin treatment All mice were continuously fed with a gluten-free diet and water available ad libitum. Mice were sensitized by injecting intraperitoneally (ip) 500 mg of gluten (Sigma-Aldrich, Ontario) dissolved in 0.02 mM acetic acid in 50 ml of Complete Freund’s Adjuvant (CFA, Sigma-Aldrich, Ontario). One week after sensitization, gluten challenge was performed 3 times on a weekly basis by intragastric gavage, for 7 weeks, using 2 mg of gluten dissolved in 0.02 mM acetic acid. Indomethacin was administered by gavage (Ovation Pharmaceu- ticals, Ontario) (3.5 mg/kg) 24 hours before the gluten challenge. Control groups consisted of a) non-sensitized mice (CFA only) subsequently gavaged with rice cereal (2 mg/0.02 mM acetic acid), b) gluten-sensitized mice subsequently gavaged with gluten PLoS ONE | www.plosone.org 8 July 2009 | Volume 4 | Issue 7 | e6472