Survey of small intestinal and systemic immune responses following murine Arcobacter butzleri infection

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
G0184/12). Animal welfare was monitored twice daily by assessment of clinical conditions.

Mice

IL-10
^?/?
mice (in C57BL/6j background, B6) were bred and maintained in the facilities of the
“Forschungseinrichtungen für Experimentelle Medizin” (FEM, Charité-Universitätsmedizin,
Berlin, Germany) under specific pathogen-free (SPF) conditions.

Gnotobiotic IL-10
^?/?
mice (with a virtually depleted gastrointestinal microbiota) were generated following
broad-spectrum antibiotic treatment as described earlier 31], 36]. In brief, mice were transferred to sterile cages and treated by adding ampicillin/sulbactam
(1 g/L; Pfizer, Berlin, Germany), vancomycin (500 mg/L; Hexal, Holzkirchen, Germany),
ciprofloxacin (200 mg/L; Hexal), imipenem (250 mg/L; Fresenius Kabi, Graz, Austria),
and metronidazole (1 g/L; Braun, Melsungen, Germany) to the drinking water ad libitum
starting at 3 weeks of age immediately after weaning and continued for 3 months before
the infection experiment 33]. Three days prior infection, the antibiotic cocktail was replaced by sterile tap
water (ad libitum). Mice were continuously kept in a sterile environment (autoclaved
food and tap water) and handled under strict antiseptic conditions.

Arcobacter butzleri infection of mice

Gnotobiotic IL-10
^?/?
mice were perorally infected with approximately 10
⁹
viable CFU of two different Arcobacter butzleri strains either (CCUG 30485 or C1 strain, respectively) by gavage in a total volume
of 0.3 mL phosphate buffered saline (PBS) on two consecutive days (day 0 and day 1).
Naive age- and sex-matched gnotobiotic IL-10
^?/?
mice served as uninfected controls.

The A. butzleri reference strain CCUG 30485 was initially isolated from a fecal sample derived from
a diarrheal patient 35], whereas the C1 strain was isolated from fresh chicken meat 19]. Both A. butzleri strains were grown on Karmali-Agar (Oxoid, Wesel, Germany) for 2 days at 37 °C under
microaerobic conditions using CampyGen gas packs (Oxoid).

Clinical score

To assess clinical signs of A. butzleri 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, Krefeld,
Germany; 4: overt 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 compromised locomotion, pre-final aspect) was used 31].

Sampling procedures

Mice were sacrificed by isoflurane treatment (Abbott, Greifswald, Germany) on day
6 or day 16 p.i.. Tissue samples from MLNs, spleen, and ileum were removed under sterile
conditions. Absolute small intestinal lengths were determined by measuring the distances
from the transition of the stomach to the duodenum to the very distal terminal ileum
by a ruler. Ileal ex vivo biopsies from each mouse were collected in parallel for
immunohistochemical, microbiological, and immunological analyses. Immunohistopathological
changes were determined in ileal samples immediately fixed in 5 % formalin and embedded
in paraffin. Sections (5 ?m) were stained with HE or respective antibodies for in
situ immunohistochemistry as described earlier 33].

Histopathological grading of small intestinal lesions

To evaluate the severity of small intestinal histopathological lesions, an established
scoring scheme 44] with minor modifications was applied. In detail, the composition of the immune cell
infiltrates (0: none; 1: mononuclear cells; 2: mononuclear cell dominated, fewer neutrophils;
3: neutrophil dominated, fewer mononuclear cells), quantity of the immune cell infiltrates
(0: none; 1: mild; 2: moderate; 3: severe), vertical extent of inflammation (0: none;
1: mucosa; 2: mucosa and submucosa; 3: transmural), and horizontal extent of inflammation
(0: no; 1: focal; 2: multifocal; 3: multifocal-coalescent; 4: diffuse) were assessed.
The cumulative histologic scoring ranged from 0 to 13 for ileal samples.

Immunohistochemistry

In situ immunohistochemical analysis of ileal paraffin sections was performed as described
previously 30], 31], 45]–47]. Primary antibodies against cleaved caspase-3 (Asp175, Cell Signaling, Beverly, MA,
USA, 1:200), Ki67 (TEC3, Dako, Denmark, 1:100), CD3 (#N1580, Dako, 1:10), FOXP3 (FJK-16 s,
eBioscience, San Diego, CA, USA, 1:100), B220 (eBioscience, 1:200), and F4/80 (# 14-4801,
clone BM8, eBioscience, 1:50), were used. For each animal, the average number of positively
stained cells within at least six high power fields (HPF, 0.287 mm
²
, 400× magnification) were determined microscopically by a double-blinded investigator.

Quantitative analysis of Arcobacter butzleri

Viable A. butzleri were detected in feces or at time of necropsy (day 6 or 16 p.i.) in luminal samples
taken from the duodenum, ileum or colon, dissolved in sterile PBS and cultured in
serial dilutions on Karmali- and Columbia-Agar supplemented with 5 % sheep blood (Oxoid)
in parallel for 2 days at 37 °C under microaerobic conditions using CampyGen gas packs
(Oxoid). To quantify bacterial translocation, MLNs and spleen (?1 cm
²
) were homogenized in 1 mL sterile PBS, streaked onto Karmali-Agar and cultivated
accordingly. The respective weights of fecal or tissue samples were determined by
the difference of the sample weights before and after asservation. The detection limit
of viable pathogens was ?100 colony forming units (CFU) per g.

Cytokine detection in culture supernatants of ex vivo biopsies taken from ileum, mesenteric
lymphnodes and spleen

Ileal ex vivo biopsies were cut longitudinally and washed in PBS. Spleen, MLNs or
strips of approximately 1 cm
²
ileum were placed in 24-flat-bottom well culture plates (Nunc, Wiesbaden, Germany)
containing 500 ?L serum-free RPMI 1640 medium (Gibco, life technologies, Paisley,
UK) supplemented with penicillin (100 U/mL) and streptomycin (100 µg/mL; PAA Laboratories).
After 18 h at 37 °C, culture supernatants were tested for IFN-?, TNF, MCP-1, IL-6 and
IL-12p70 by the Mouse Inflammation Cytometric Bead Assay (CBA; BD Biosciences, San
Jose, CA, USA) on a BD FACSCanto II flow cytometer (BD Biosciences). Nitric oxide
(NO) was determined by Griess reaction as described earlier 36].

Statistical analysis

Medians and levels of significance were determined using Mann–Whitney test (GraphPad
Prism v5, La Jolla, CA, USA). Two-sided probability (P) values ?0.05 were considered significant. Experiments were reproduced twice.