Small Intestinal Pro-Inflammatory Immune Responses Following Campylobacter Jejuni Infection of Secondary Abiotic IL-10-/- Mice Lacking Nucleotide-Oligomerization-Domain-2.

Host immune responses are crucial for combating enteropathogenic infections including Campylobacter jejuni. Within 1 week following peroral C. jejuni infection, secondary abiotic IL-10-/- mice develop severe immunopathological sequelae affecting the colon (ulcerative enterocolitis). In the present study, we addressed whether pathogen-induced pro-inflammatory immune responses could also be observed in the small intestines dependent on the innate receptor nucleotide-oligomerization-domain-protein 2 (Nod2). Within 7 days following peroral infection, C. jejuni stably colonized the gastrointestinal tract of both IL-10-/- mice lacking Nod2 (Nod2-/- IL-10-/-) and IL-10-/- controls displaying bloody diarrhea with similar frequencies. Numbers of apoptotic and regenerating epithelial cells increased in the small intestines of C. jejuni-infected mice of either genotype that were accompanied by elevated ileal T and B lymphocyte counts. Notably, ileal T cell numbers were higher in C. jejuni-infected Nod2-/- IL-10-/- as compared to IL-10-/- counterparts. Furthermore, multifold increased concentrations of pro-inflammatory cytokines including IFN-γ, TNF, and MCP-1 could be measured in small intestinal ex vivo biopsies derived from C. jejuni-infected mice of either genotype. In conclusion, C. jejuni-induced pro-inflammatory immune responses affected the small intestines of both Nod2-/- IL-10-/- and IL-10-/- mice, whereas ileal T lymphocyte numbers were even higher in the former.

Introduction

Host immune responses are pivotal for combating enteropathogenic infections. The nucleotide-binding oligomerization domain (Nod)-like receptors constitute an important family of intracellular pattern recognition receptors that regulate host immunity by sensing microbial products and damage-associated factors [1]. Among these, Nod2 is expressed by innate (including monocytes, macrophages, and dendritic cells) and adaptive (such as T lymphocytes) immune cell populations [2, 3] and is activated by muramyl dipeptide (MDP), a constituent of bacterial peptidoglycans [4]. Activation of the Nod2 signaling pathway confers resistance against a broad variety of bacterial species including enteropathogens [1, 5–7]. Among these, Campylobacter jejuni are colonizing domestic and wild animals as commensals. Humans, however, become infected via the food chain by consumption of contaminated livestock animals or surface water [8, 9]. Depending on the virulence of the acquired strain and the host immune status, infected individuals present with symptoms of varying degree. Whereas most patients display rather mild malaise and watery diarrhea, others suffer from severe ulcerative enterocolitis with fever, abdominal cramps, and inflammatory, bloody diarrhea [10, 11]. Despite the progressively increasing prevalence of human campylobacteriosis cases, molecular and cellular events involved in disease development are not fully understood. For a long time, in vivo studies have been hampered by a lack of suitable animal models. We have recently shown that secondary abiotic mice generated by broad-spectrum antibiotic treatment (virtually lacking an intestinal microbiota) developed wasting non-selflimiting ulcerative enterocolitis within 1 week following peroral C. jejuni infection mimicking key features of campylobacteriosis in severely immunocompromized patients [12-16]. Importantly, the large intestinal immunopathology was mediated by distinct innate immune receptors such as Toll-like receptor (TLR) -2 and -4 [12, 17]. However, pathogen-induced inflammatory responses in the small intestines have not been reported so far.

Given the colon as predilection site of C. jejuni infection, we addressed in the present study 1) whether C. jejuni-induced inflammatory sequelae could also be observed in the small intestines of secondary abiotic IL-10–/– and, if so, 2) whether Nod2 was involved in mediating small intestinal immunopathology.

Methods

Generation of secondary abiotic mice and C. jejuni infection

Female IL-10–/– mice and IL-10–/– mice lacking Nod2 (Nod2–/– IL-10–/–) mice (all in C57BL/6j background) were reared and kept within the same specific pathogen-free (SPF) unit of the Forschungseinrichtungen für Experimentelle Medizin (FEM, Charité – University Medicine Berlin). To counteract physiological colonization resistance and assure stable intestinal colonization of the pathogen [18], secondary abiotic (i.e., gnotobiotic) mice virtually lacking an intestinal microbiota were generated by broad-spectrum antibiotic treatment for 8 weeks as described previously [18, 19]. Three days prior infection, the antibiotic cocktail was withdrawn and replaced by autoclaved tap water. Mice were then perorally infected with 109 colony forming units (CFU) of viable C. jejuni strain 81-176 in a volume of 0.3 ml phosphate buffered saline (PBS; Gibco, Life Technologies, UK) on two consecutive days (days 0 and 1) by gavage as described earlier [18]. To prevent mice from contaminations, animals were continuously maintained in a sterile environment (autoclaved food and drinking water or sterile antibiotic cocktail) and handled under strict aseptic conditions.

Clinical conditions

To assess clinical signs of C. jejuni-induced infection on a daily basis, a standardized cumulative clinical score (maximum 12 points), addressing the occurrence of blood in feces (0: no blood; 2: microscopic detection of blood by the Guajac method using Haemoccult, Beckman Coulter/PCD, Germany; 4: macroscopic blood visible), diarrhea (0: formed feces; 2: pasty feces; 4: liquid feces), and the clinical aspect (0: normal; 2: ruffled fur, less locomotion; 4: isolation, severely compromized locomotion, pre-final aspect) was used as described earlier [12, 20, 21].

Sampling procedures and immunohistochemistry

Mice were sacrificed at day 7 p.i. by isofluran treatment (Abbott, Germany). Ileal ex vivo biopsies were asserved under sterile conditions and collected from each mouse in parallel for microbiological, histopathological, immunohistopathological, and immunological analyses.

In situ immunohistochemical analysis of ileal paraffin sections was performed as described elsewhere [14, 20–22]. Primary antibodies against cleaved caspase-3 (Asp175, Cell Signaling, USA, 1:200), Ki67 (TEC3; Dako, Denmark; 1:100), CD3 (#N1580; Dako; 1:10), FOXP3 (FJK-16s; eBioscience, Germany; 1:100), and B220 (eBioscience; 1:200) were used. For each animal, the average number of positively stained cells within at least six high power fields (HPF, 0.287 mm2, 400× magnification) were determined microscopically by a blinded independent investigator.

Quantitative analysis of bacterial colonization

Viable C. jejuni were quantitatively assessed in feces over time p.i. or at time of necropsy (i.e., day 7 p.i.) in luminal samples taken from the gastrointestinal tract (i.e., stomach, duodenum, ileum, and colon), dissolved in sterile PBS and serial dilutions streaked onto Karmali- and Columbia-Agar supplemented with 5% sheep blood (Oxoid, Germany) for 2 days at 37 °C under microaerobic conditions using CampyGen gas packs (Oxoid). The respective weights of fecal or gastrointestinal luminal samples were determined by the difference of the sample weights before and after asservation. The detection limit of viable pathogens was ≈100 CFU per g.

Cytokine detection in supernatants of small intestinal ex vivo biopsies

Ileal ex vivo biopsies were cut longitudinally and washed in PBS. Strips of approximately 1 cm2 of small intestinal tissues were placed into 24-flat-bottom well culture plates (Nunc, Germany) containing 500 μl serum-free RPMI 1640 medium (Gibco, Life Technologies, UK) supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml; PAA Laboratories, Germany). After 18 h at 37 °C, culture supernatants were tested for IFN-γ, TNF, MCP-1, and IL-6 by the Mouse Inflammation Cytometric Bead Assay (CBA; BD Biosciences, Germany) on a BD FACS-Canto II flow cytometer (BD Biosciences). Nitric oxide (NO) was measured by the Griess reaction as described earlier [19].

Statistical analysis

Medians and levels of significance were determined using Mann–Whitney test (GraphPad Prism v5, La Jolla, CA, USA) as indicated. Two-sided probability (p) values ≤ 0.05 were considered significant. Experiments were repeated at least twice.

Ethics statement

All animal experiments were conducted according to the European Guidelines for animal welfare (2010/63/EU) with approval of the commission for animal experiments headed by the “Landesamt für Gesundheit und Soziales” (LaGeSo, Berlin, registration number G0135/10). Animal welfare was monitored twice daily by assessment of clinical conditions.

Results

Gastrointestinal colonization densities of C. jejuni following peroral infection of secondary abiotic IL-10–/– mice lacking Nod2

In order to overcome physiological colonization resistance provided by the complex conventional gastrointestinal microbiota, secondary abiotic IL-10–/– mice lacking Nod2 and IL-10–/– controls were generated by quintuple antimicrobial treatment. Following cessation of the antibiotic cocktail and a 3-day washout phase, mice were perorally infected with 109 CFU C. jejuni strain 81-176 by gavage. A kinetic survey of C. jejuni counts in fecal samples revealed that mice of either genotype could be stably colonized by the pathogen with median loads of approximately 109 CFU per gram (n.s., ). At day of necropsy (i.e., day 7 p.i.), Nod2–/– IL-10–/– and IL-10–/– mice harbored C. jejuni alongside the gastrointestinal tract with highest loads in the colonic lumen (i.e., approximately 109 CFU per gram), whereas 107 CFU per gram could be cultured from the ileal lumen of mice of either genotype (n.s.; ).

Clinical sequelae upon C. jejuni infection of secondary abiotic IL-10–/– mice lacking Nod2

As early as 4 days following C. jejuni infection, approximately 50% of mice of either genotype exhibited bloody diarrhea, whereas at day 7 p.i., fecal blood-positivity rates were 81.3% and 92.9% in Nod2–/– IL-10–/– and IL-10–/– mice, respectively (.

Small intestinal microscopic sequelae upon C. jejuni infection of secondary abiotic IL-10–/– mice lacking Nod2

Even though the large intestine is known to be the predilection site of C. jejuni-induced inflammation in secondary abiotic IL-10–/– mice (i.e., acute enterocolitis [18]), we next addressed whether C. jejuni infection was accompanied by distinct microscopic changes also within the small intestinal tract. Since apoptosis is regarded as diagnostic marker for histopathological grading of intestinal inflammation including campylobacteriosis [18], we stained ileal paraffin sections with caspase-3 antibodies. Remarkably, numbers of apoptotic epithelial cells more than doubled in ilea of both Nod2–/– IL-10–/– and IL-10–/– mice until day 7 p.i. (p < 0.001 and p < 0.01 versus naive controls, respectively; ). These C. jejuni-induced increases in apoptotic cells were accompanied by elevated numbers of Ki67+ cells in the ileal epithelium of mice of either genotype at day 7 p.i. (p < 0.05 versus naive; ), indicative for regenerative/proliferative responses counteracting small intestinal cell damage following enteropathogenic infection.

Small intestinal immune cell responses upon C. jejuni infection of secondary abiotic IL-10–/– mice lacking Nod2

Since recruitment of pro-inflammatory immune cell populations to the site of infection is one of the key features of campylobacteriosis [18], we next quantitatively assessed numbers of distinct immune cell subsets applying in situ immunohistochemical staining of ileal paraffin sections at day 7 p.i. Adaptive immune cells such as T and B lymphocytes had inceased in the small intestinal mucosa and lamina propria until day 7 following C. jejuni infection (p < 0.05–0.001; ), whereas elevated Treg numbers could be observed only in the small intestines of IL-10–/– mice lacking Nod2 at day 7 p.i. (p < 0.05; ). Remarkably, ileal T lymphocyte counts were higher in C. jejuni-infected Nod2–/– IL-10–/– as compared to IL-10–/– mice (p < 0.05; ). Notably, small intestinal innate immune cell populations such as macrophages and monocytes as well as neutrophils were virtually unaffected by C. jejuni infection of mice (not shown).

Small intestinal cytokine responses upon C. jejuni infection of secondary abiotic IL-10–/– mice lacking Nod2

We next measured pro-inflammatory cytokine secretion in supernatants of ileal ex vivo biopsies at day 7 p.i. Ileal IFN-γ, TNF, and MCP-1 concentrations were elevated in small intestines of C. jejuni-infected mice of either genotype (p < 0.05–0.001; ). Ileal secretion of nitric oxide was more pronounced in C. jejuni-infected IL-10–/– mice as compared to naive controls (p < 0.05; ), but did not reach statistical significance in Nod2–/– IL-10–/– mice (n.s., ). In both Nod2–/– IL-10–/– and IL-10–/– mice, a trend towards higher small intestinal IL-6 concentrations could be observed at day 7 p.i. (n.s.; ).

In summary, 1) C. jejuni is able to induce pro-inflammatory responses also in the small intestines of secondary abiotic IL-10–/– mice. 2) Nod2–/– IL-10–/– mice display higher ileal T cell numbers upon C. jejuni infection as compared to IL-10–/– controls.

Discussion

In the present study, we demonstrate for the first time that C. jejuni infection induces overt pro-inflammatory immune responses in the small intestines of IL-10–/– mice. So far, the colon has been known as predilection site of C. jejuni infection in the murine IL-10–/– infection model as shown previously by us and others. Within 2 to 35 days following C. jejuni infection, conventionally colonized IL-10–/– mice developed typhlocolitis affecting the colon and caecum including the ileocaecal junction [23-25]. Our group could further demonstrate that, even within less than 1 week following peroral C. jejuni infection, secondary abiotic IL-10–/– mice developed severe ulcerative enterocolitis with inflammatory, bloody diarrhea and succumbed to acute immunopathological sequelae [12-15]. As shown here, intestinal disease was not restricted to the colon, given that also the small intestines were affected upon C. jejuni infection as indicated by pronounced apoptosis in ileal epithelia and an enhanced influx of adaptive immune cells including T and B lymphocytes into the small intestinal mucosa and lamina propria that was accompanied by accelerated ileal secretion of pro-inflammatory cytokines such as IFN-γ, TNF, and MCP-1. The fact that immunopathological responses to C. jejuni infection of secondary abiotic IL-10–/– mice could additionally be observed in extra-intestinal organs including liver and kidney, but also in systemic compartments including spleen and serum [13], points towards a C. jejuni-induced systemic inflammatory response syndrome.

In the present study, we also addressed whether Nod2 was involved in mediating small intestinal pro-inflammatory immune responses upon C. jejuni infection. Whereas ileal apoptosis and pro-inflammatory cytokine secretion were Nod2-independent, higher numbers of T lymphocytes could be determined in the ileal mucosa and lamina propria of infected Nod2–/– IL-10–/– mice as compared to IL-10–/– controls. This is well in line with a C. jejuni-induced elevation of T cell counts in the large intestines of secondary abiotic IL-10–/– mice lacking Nod2 (submitted article) and Nod2–/– mice (article in revision). The same holds true for proliferating intestinal epithelial cells, given that higher Ki67+ cell numbers could be determined in small (present study) and large intestines (submitted article) of C. jejuni-infected secondary abiotic Nod2–/– IL-10–/– mice as well as in Nod2–/– mice (article in revision) as compared to respective infected controls. These results point towards more distinct regenerative properties of intestinal epithelia counteracting C. jejuni-induced cell damage in mice lacking Nod2.

Taken together, C. jejuni infection of secondary abiotic IL-10–/– mice resulted in pro-inflammatory responses in the small intestines, whereas increases in ileal T cell numbers were even more pronounced in IL-10–/– mice lacking Nod2.

In conclusion, not only large but also small intestinal changes need to be assessed during enteropathogenic infection studies employing IL-10–/– mice.