The microbiota mediates pathogen clearance from the gut lumen after non-typhoidal Salmonella diarrhea

Abstract

Many enteropathogenic bacteria target the mammalian gut. The mechanisms protecting the host from infection are poorly understood. We have studied the protective functions of secretory antibodies (sIgA) and the microbiota, using a mouse model for S. typhimurium diarrhea. This pathogen is a common cause of diarrhea in humans world-wide. S. typhimurium (S. tm(att), sseD) causes a self-limiting gut infection in streptomycin-treated mice. After 40 days, all animals had overcome the disease, developed a sIgA response, and most had cleared the pathogen from the gut lumen. sIgA limited pathogen access to the mucosal surface and protected from gut inflammation in challenge infections. This protection was O-antigen specific, as demonstrated with pathogens lacking the S. typhimurium O-antigen (wbaP, S. enteritidis) and sIgA-deficient mice (TCRβ(-/-)δ(-/-), J(H) (-/-), IgA(-/-), pIgR(-/-)). Surprisingly, sIgA-deficiency did not affect the kinetics of pathogen clearance from the gut lumen. Instead, this was mediated by the microbiota. This was confirmed using 'L-mice' which harbor a low complexity gut flora, lack colonization resistance and develop a normal sIgA response, but fail to clear S. tm(att) from the gut lumen. In these mice, pathogen clearance was achieved by transferring a normal complex microbiota. Thus, besides colonization resistance ( = pathogen blockage by an intact microbiota), the microbiota mediates a second, novel protective function, i.e. pathogen clearance. Here, the normal microbiota re-grows from a state of depletion and disturbed composition and gradually clears even very high pathogen loads from the gut lumen, a site inaccessible to most "classical" immune effector mechanisms. In conclusion, sIgA and microbiota serve complementary protective functions. The microbiota confers colonization resistance and mediates pathogen clearance in primary infections, while sIgA protects from disease if the host re-encounters the same pathogen. This has implications for curing S. typhimurium diarrhea and for preventing transmission.

Figure 1. S. tm ^att infection yields ‘asymptomatic excretors’ and elicits O-antigen specific immunity.

A. Time course of fecal S. tm ^att shedding. Sm-treated mice were infected with S. tm ^att and fecal Salmonella shedding was monitored (5<n<48 individual mice per time point). Medians are shown in black; orange and red: 5%, 25%, 75% and 95% quantiles. Dashed red lines: weighted linear regression on the 5% and 95% quantiles. B. H&E stained cross-section of the cecum at day 3 (left) and day 44 (right) post S. tm ^att infection. Enlarged section (white box) is shown in inset. Scale bar: 50µm. C. S. tm ^att loads in cecum (left panel; Log₁₀ cfu/g), spleen (red) and MLN (blue; both Log₁₀ cfu/organ; 2^nd panel) and cecal mucosa inflammation (3^rd panel) at the indicated times post S. tm ^att infection. ‘Asymptomatic excretors’ (4^th panel) are defined as showing a pathological score ≤3 while shedding ≥10⁵ cfu/g S. tm ^att.(green box). Each dot represents an individual mouse between day 3 and 60 S. tm ^att immunization. Black dotted line: detection limit.

Figure 2. S. tm ^att induces O-antigen specific mucosal protection.

A. ‘Immunization-challenge’ model. At day 0, sm-treated mice are infected and S. tm ^att (5×10⁷cfu; i.g.). They develop enteric pathology (red) that usually declines by day 14–20 p.i. (blue). Afterwards, the mice appear healthy. At day 40 p.i., mice are treated with ampicillin (20mg i.g.) and challenged with S. tm ^wt (or other strains; 200cfu i.g.). The degree of Salmonella-induced gut inflammation and pathogen loads are determined at day 1–5 post challenge. B. S. tm ^att immunized mice are protected in an O-antigen dependent fashion. S. tm ^att (filled symbols) or mock (open symbols) immunized mice were challenged with S. tm ^wt (black; n = 10), S. en ^wt (red circles; n = 5) or S. tm ^ΔO (blue; n = 5) at day 40 post immunization. At day 2 post challenge, challenge strain loads were assessed in the cecum content (left panel) and the MLN (middle panel). Dashed lines indicate detection limits. Right panels: inflammation of the cecal mucosa (score≤3 indicates no inflammation). Black bar: median; *p<0.05; **p<0.005; n.s. = not significant. H&E stained cross-sections of the cecum of S. tm ^att-immunized mice at day 2 post challenge with S. tm ^wt (upper panel), S. en ^wt (middle panel) and S. tm ^ΔO (lower panel). Enlarged section (white box) is shown in the lower panel. Scale bar: 50µm. C. Time course of the S. tm ^att specific humoral immune response. Antibodies directed against the surface of S. tm ^att in serum or gut wash of the S. tm ^att -immunized mice from Fig. 1C were analyzed by bacterial FACS (Materials and Methods; Fig.S1). The Y-axis shows S. tm ^att specifc Ig (relative fluorescence units; rfu), the X-axis different dilutions of serum or gut wash (1∶ 20, 1∶60, 1∶180) at different days post S. tm^att immunization. D. S. tm ^att-immunized mice mount an O-antigen specific antibody response. Serum and gut wash Ig from naïve and S. tm ^att infected mice (day 40 post infection) were analyzed by immunoblot against different bacterial lysates (S. tm ^ΔO; L. reuteri; E. faecalis; S. ent ^wt; E. coli; S. tm ^att; S. tm ^att digested with proteinase K; S. tm M933 [no flagella, no functional TTSS]; flagellin FliC). Ig was detected with the respective HRP-labeled secondary antibodies. The experiment is representative for 6 different animals.

Figure 3. IgA deficiency does not affect kinetics of pathogen clearance.

Time course of fecal S. tm ^att shedding in IgA^−/− and IgA-proficient littermates. IgA^−/− and IgA-proficient littermates were generated by crossing IgA^+/− mice in order to yield littermates with comparable gut flora. Sm-treated IgA^−/− (n = 10; white diamonds), IgA^+/− (n = 7; black circles) and wild type (n = 7; black circles) littermates were infected with S. tm ^att and fecal Salmonella shedding was monitored until day 60 post infection. Black bar: medians. Dashed line: detection limit. Statistics: comparison of fecal shedding by IgA^−/− vs. IgA^+/− and IgA^+/+ (n.s.: p>0.05).

Figure 4. Immunization elicits O-antigen specific sIgA which retards pathogen growth and restricts mucosal access.

A. sIgA retards pathogen growth and occludes mucosal access. Mice were immunized with S. tm ^att, S. en ^att or mock (n = 5 per group), treated with ampicillin and challenged for 1 day with a 1∶1 mixture S. tm ^avir(amp^R; pWKS30) and S. en ^avir (amp^R; pM979-GFP⁺). None of the mice developed gut inflammation. Left panel: competitive index (c.i.) of S. tm ^avir(amp^R; pWKS30) and S. en ^avir (amp^R; pM979-GFP⁺) in the cecal lumen. Right panels: Immunofluorescence microscopy of the cecal lumen (left panels) and the mucosal surface (right panels; actin brush boarder = blue) to detect S. tm ^avir (red α-S. tm LPS stain) and S. en ^avir (green GFP⁺). Scale bar: 10µm. B. sIgA blocks mucosal invasion by S. tm ^wt. C57Bl/6 (circles) or pIgR^−/− mice (diamonds) were immunized with mock (open symbols) or S. tm ^avir (closed symbols) and challenged with S. tm ^wt (black) or S. en ^wt (red). Bacterial loads in the cecal lumen (left) and Salmonella invasion into the mucosal tissue (middle) was determined at 2 days post challenge. Right panels: Immunofluorescence microscopy of GFP-expressing S. tm ^wt or S. en ^wt (green) in the cecal mucosa (red: ICAM-1, lamina propria; blue: Actin, epithelial brush border); White dotted lines mark epithelial-submucosal boarder; Scale bar: 10µm.

Figure 5. An O-antigen-specific sIgA response is insufficient to terminate S. tm ^att shedding by L-mice.

A. L-mice do not clear S. tm ^att from the gut. L-mice (n = 15) were immunized with S. tm ^att (no streptomycin-treatment) and fecal shedding was monitored for 40 days (left panel). Gut inflammation was analyzed at day 2 and day 40 p.i. (5 mice per time point; right panel). B. and C. L-mice mount a pronounced adaptive mucosal immune response. Cecal tissue of naïve L-mice and L-mice at days two and 40 p.i. was stained by immunohistochemistry for Ly6G (granulocytes), IgA, CD4 (T-cells) and CD8 (T-cells) cellular markers. Quantitative data were from 15 randomly selected 40× high power fields (hpf) from 3–5 mice per group. Y-axis: average cell number per 40× hpf (C). D. Gene expression analysis of naïve and L-mice at day 40 post S. tm ^att immunization. Left: Numbers of differentially expressed genes in S. tm ^att-immunized L-mice. Middle: relative abundance of biological pathways significantly upregulated in S. tm ^att-immunized L-mice. Processes, p-values and characteristic examples are shown as a table (right).

Figure 6. O-antigen specific protection from enteropathogenesis of S. tm ^att-immunized L-mice.

S. tm ^att- (filled symbols) or mock- (open symbols) immunized mice were challenged for two days with S. tm ^wt (black; n = 10) or S. en ^wt (red circles; n = 5) at day 40 post immunization. Challenge-strain loads in the cecal content (left panel) and the MLN (middle panel) and cecal inflammation were assessed at day 2 post challenge. Dashed lines: detection limit or limit defining the absence of disease (pathological score ≤3). Black bars: median; *p<0.05; **p<0.005; n.s. = not significant. Right: H&E stained cross-sections of the cecum of S. tm ^att-immunized L-mice at day two post challenge with S. tm ^wt (upper panel) or S. en ^wt (lower panel). Scale bar: 50µm.

Figure 7. Transfer of a complex microbiota curbs S. tm ^att shedding by L-mice.

A. Exposure to conventional gut microbiota curbs pathogen shedding by ‘asymptomatic excretors’. L-mice were immunized with S. tm ^att. At day 40 p.i., donor animals with a conventional microbiota (C-mice) were added to one group (black circles; n = 17; S. tm ^att→L/C). The other group remained under hygienic isolation (open circles; n = 10; S. tm ^att→L). Fecal S. tm ^att shedding was monitored until day 83 p.i. Right panels show gut inflammation and organ loads at day 83 p.i.. B. and C. S. tm-specific Ig-response (serum IgA and IgG; IgA in gut wash; day 83 p.i.) in S. tm ^att→L and S. tm ^att→L/C mice was determined by bacterial FACS and by immunoblot as in Fig. 2A,B. D. A complex microbiota is transferred to L-mice by day 83 p. S. tm ^att immunization. Collectors' curves (CD = 0.05) were created for each mouse from the total number of filtered sequences for representative mice: S. tm ^att→L (d.2), S. tm ^att→L (d.40), S. tm ^att→L (d.83), S. tm ^att→L/C and 2 C-mice (donor). E. Phylotypes of mice shown in (D) (Clustering distance CD = 0.05) were sorted according to their family taxon (family level; x-axis) and average clustering was performed on Euclidean distances calculated between abundance profiles for each mouse and every time-point sampled. Red color indicates high abundance (Log2), yellow color low abundance.

Figure 8. Adoptive transfer of MLN cells isolated form mice that cleared S. tm ^att from the intestine into S. tm ^att→L mice (day 40 p.i.) does not induce S. tm ^att clearance.

L-mice were immunized with S. tm ^att. At day 40 p.i., animals with a conventional microbiota (C-mice) were added to one group (black circles; n = 5; S. tm ^att→L/C). The other group remained under hygienic isolation (open circles; n = 5; S. tm ^att→L). A. All mice were sacrificed at day 80 p.i. and S. tm ^att levels in the cecal content were defined. B. Total MLN cells from the two groups of mice were isolated and analyzed with respect to the expression of intracellular cytokines IL-17A (left) and IFNγ (right) in CD4⁺ T-cells at day 80 post S. tm ^att infection (% cytokine-expressing CD4⁺ T-cells). C. 3×10⁷ MLN cells of ‘donor’ mice S. tm ^att→L and S. tm ^att→L/C described above were adoptively transferred into another cohort of S. tm ^att→L mice (day 40 p.i.: = ‘aceptors’). Fecal S. tm ^att shedding was followed in the ‘acceptors’ until day 80 post S. tm ^att infection ( = day 40 post adoptive transfer, left). S. tm ^att levels in the MLN of ‘acceptors’ at day 80 post S. tm ^att infection ( = day 40 post adoptive transfer, left).