Fig 1.
Consequences of neutrophil depletion with α -Ly6G on luminal pathogen loads and epithelial cell expulsion.
A) Scheme summarizing the experimental setup of Panels B-E. Streptomycin pretreated C57BL/6 mice were infected orally with 5x107 CFU of wt S.Tm (SL1344) for 3 days. One group (control) treated with the vector (PBS; black symbols) and the second group with α-Ly6G (orange symbols) intraperitoneally (I.P.). B) Relative ranks of the tagged S.Tm strain abundances in feces at day 3 p.i.. C) Evenness score at day 3 p.i. D) Representative micrographs of cecal tissue sections, stained for epithelial marker EpCam and Salmonella LPS. Lu. = Lumen. White arrows point at expelled epithelial cells. Scale bar = 50 μm. E) Microscopy-based quantification of expelled IECs per 63x field of view (i.e., cells/high power field; hpf). Each data point is the average of 5 fields of view (FOV) per section. Lines or Upper ends of the bars indicate the median. Dotted lines indicate the detection limit. Panels B-C) Pooled from 2 independent experiments for each group: control (n = 11 mice) and neutrophil depletion (n = 7 mice). Panels D-E) Pooled from 3 independent experiments for each group: control (n = 8 mice) and neutrophil depletion (n = 9 mice). Two-tailed Mann Whitney-U tests were used to compare two groups in each panel. p<0.01 (**).
Fig 2.
Microscopy analysis of the time course of neutrophil infiltration into the gut lumen (18h p.i., 24h p.i., and 72h p.i.).
A) Representative micrographs of cecal tissue sections from mice infected with S.Tm for 18h, for 1 day (24h), or for 3 days stained for neutrophil marker Ly6B.2 and Salmonella LPS. Lu. = Lumen. Ep. = Epithelium. White arrows indicate S.Tm associated with the epithelium. Scale bar = 50 μm. B-D) Microscopy-based quantification of B) S.Tm associated with the epithelium at day 1 p.i.. (total 24 FOVs from 6 mice; filled symbols; neutrophils per field≤15 vs empty symbols; neutrophils per field>15). C) neutrophils per 63x field of view (each data point is the average of 5 FOVs per section of the same mouse). D) average number of S.Tm associated with the epithelium. Upper ends of the bars indicate the median. Dotted lines indicate the detection limit. Panels C-D) Pooled from 2 or 3 independent experiments for each group: day 1 p.i. (n = 6 mice) and day 3 p.i. (n = 9 mice). Two-tailed Mann Whitney-U tests were used to compare two groups in each panel. P≥0.05 not significant (ns), p<0.05 (*), p<0.01 (**), p<0.001 (***).
Fig 3.
Microscopy analysis of intraluminal NETs at day 3 p.i.
A) Experimental scheme for Panels B-D. Streptomycin pretreated C57BL/6 mice were infected orally with 5x107 CFU of wt S.Tm for 3 days. One group (control from Fig 1B and 1C) treated with the vector (PBS; black symbols) and the second group with PAD4 inhibitor (GSK484; purple symbols) intraperitoneally (I.P.). B) Representative micrographs of cecal tissue sections from mice infected with S.Tm, taken at 3 days p.i., stained for NET marker cit-His3 and Salmonella LPS. Lu. = Lumen. Ep. = Epithelium. Yellow arrows indicate S.Tm associated with the NET marker cit-His3. White dotted lines indicate the epithelial barrier. Scale bar = 50 μm. C) Representative micrographs of cecal tissue sections, stained for epithelial marker EpCam and Salmonella LPS. Lu. = Lumen. White arrows point at expelled epithelial cells. Scale bar = 50 μm. D) Microscopy-based quantification of IECs per 63x field of view (i.e., cells/high power field; hpf). Each data point is the average of 5 fields of view (FOV) per section. Two-tailed Mann Whitney-U tests were used to compare two groups in each panel. p≥0.05 not significant (ns).
Fig 4.
Investigation of the contribution of wt pathogen loads in the gut tissue to epithelial cell loss.
A) Experimental scheme. Streptomycin pretreated C57BL/6 mice were infected orally with 5x107 CFU of wt S.Tm for 3 days. Mice were divided into two groups: 1) without gentamicin, 2) with gentamicin in drinking water starting from day 1 p.i.. Total S.Tm pathogen loads B) in cecal content, C) in the cecal tissue, and D) mLN at day 3 p.i. in each group. E) Microscopy-based quantification of S.Tm cells residing in the lamina propria. F) Representative micrographs of cecal tissue sections, stained for epithelial marker EpCam and Salmonella LPS. Lu. = Lumen. Ep. = Epithelium. White arrows point at expelled epithelial cells. Scale bar = 50 μm. G) Microscopy-based quantification of luminal IECs per 63x field of view (i.e., cells/high power field; hpf). Each data point is the average of 5 fields of view (FOV) per section. Upper ends of the bars indicate the median. Panels B-G) Pooled from total 2 independent experiments; group-1 (n = 5 mice), group-2 (n = 5 mice). Two-tailed Mann Whitney-U tests were used to compare two indicated groups in each panel. p≥0.05 not significant (ns), p<0.01 (**).
Fig 5.
Investigation of the role of intraluminal neutrophils in a mouse model with reduced systemic disease.
A) Experimental scheme for Panels B-F. Ampicillin pretreated C57BL/6 mice were infected orally with 5x107 CFU of S.TmssaV for 3 days. Mice were divided into four groups: 1) Control (I.P. PBS; black filled symbols) without gentamicin, 2) Neutrophil depletion (I.P. α-Ly6G; orange filled symbols) without gentamicin, 3) Control (I.P. PBS; black empty symbols) with gentamicin in drinking water starting from day 1 p.i., and 4) Neutrophil depletion (I.P. α-Ly6G; orange empty symbols) with gentamicin in drinking water starting from day 1 p.i. Total S.TmssaV pathogen loads B) in cecal content, C) in the cecal tissue at day 3 p.i. in each group. D) Quantification of gut inflammation by Lipocalin-2 levels in cecal content. E) Representative micrographs of cecal tissue sections, stained for epithelial marker EpCam and Salmonella LPS. Lu. = Lumen. Ep. = Epithelium. White arrows point at expelled epithelial cells. Scale bar = 50 μm. F) Microscopy-based quantification of luminal IECs per 63x field of view (i.e., cells/high power field; hpf). Each data point is the average of 5 fields of view (FOV) per section. Upper ends of the bars indicate the median. Panels B-F) Pooled from total 4 independent experiments; at least 2 for each group: Group-1 (n = 12 mice), group-2 (n = 9 mice), group-3 (n = 6 mice), group-4 (n = 6 mice). Two-tailed Mann Whitney-U tests were used to compare two indicated groups in each panel. p≥0.05 not significant (ns), p<0.05 (*), p<0.001 (***).
Fig 6.
Consequences of neutrophil depletion on epithelial health during S.TmssaV infection of germ-free mice.
A) Experimental scheme for Panels B-G. C57BL/6 germ-free mice were infected orally with 5x107 CFU of S.TmssaV for 2 or 3 days (mice were euthanized at either day according to the health status of the neutrophil-depleted mice). Mice were divided into 2 groups: 1) Control (I.P. PBS; black filled symbols), 2) Neutrophil depletion (I.P. α-Ly6G; orange filled symbols). Total S.TmssaV pathogen loads B) in cecal content and cecal tissue, C) in mLN and spleen (pooled data from day 2 and 3 p.i.) in each group. D) Quantification of mRNA expression levels in the cecal tissue by qRT-PCR, for genes involved in immune response to acute Salmonella infection and genes involved in tissue remodelling. Results are represented relative to β-actin mRNA levels. E) Representative micrographs of cecal tissue sections, stained for epithelial marker EpCam and Salmonella LPS. Lu. = Lumen. Ep. = Epithelium. L.P. = Lamina Propria. Yellow arrows point at regions with gaps in the epithelial barrier. Scale bar = 50 μm. Microscopy-based quantification of F) IECs remaining in the tissue per 63x field of view and G) Ki67+ cells per 63x field of view (i.e., cells/high power field; hpf). Each data point is the average of 5 fields of view (FOV) per section. Upper ends of the bars indicate the median. Data pooled from total 5 independent experiments. Data reported in Panel B-G is pooled from mice euthanized at day 2,3 and 4 p.i. as results were indistinguishable after day 2 p.i.. Panel C: control (n = 5 from day 2 and n = 4 from day 3), neutrophil depletion (n = 5 from day 2 and n = 5 from day 3). Panel D: control (n = 2 from day 2, n = 4 from day 3, and n = 2 from day 4), neutrophil depletion (n = 2 from day 2 and n = 5 from day 3). Panel F-G: control (n = 2 from day 2, n = 2 from day 3, and n = 2 from day 4), neutrophil depletion (n = 2 from day 2 and n = 3 from day 3). Two-tailed Mann Whitney-U tests were used to compare two indicated groups in each panel. p≥0.05 not significant (ns), p<0.05 (*), p<0.01 (**).
Table 1.
Primer Sequences used for real time qRT-PCR.