2,4,6-trinitro-benzene sulfonic acid (TNBS) induces intestinal inflammation in zebrafish larvae. (A) Experimental outline. Tg(lyz:DsRed2, cldn15la:GFP) zebrafish larvae were exposed to TNBS (50 µg/ml) or dextran sodium sulphate (DSS) (0.5%) from 72 hours post-fertilization (hpf) until 120 hpf. (B) Confocal microscopy images of DSS, TNBS and untreated (UT) Tg(lyz:DsRed2, cldn15la:GFP) larvae at 5× magnification at 120 hpf. Green fluorescence marks intestinal epithelial cells. Red fluorescence marks neutrophils. Scale bars: 100 µm. (C,D) Quantification of DsRed2⁺ cells in the intestine (C) and mean GFP⁺ intensity (sum of pixel intensities per number of pixels) (D). n=5, one experiment. Each data point represents one 120 hpf zebrafish larva. (E) Violin plots showing the relative expression of proinflammatory cytokines il1b, tnfa, il17a/f3 and il22 analyzed by qPCR in whole larvae at 120 hpf following exposure to TNBS (70 µg/ml). n=24-25, nine experiments. Data show transcript levels as arbitrary units (A.U.) with respect to eef1a1l1 (indicated as ef1a). Each dot represents a pool of ten zebrafish larvae. The black line represents the median. N.S., not significant; *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA with Fisher's LSD test was used in C; unpaired Student's t-test was used in D and E.

Perfluorooctanesulfonic acid (PFOS) exacerbates intestinal-specific expression of proinflammatory cytokines during TNBS-induced inflammation in zebrafish larvae. (A) Zebrafish larvae were exposed to TNBS (70 µg/ml) and/or PFOS, perfluorooctanoic acid (PFOA) or perfluorohexanesulfonic acid (PFHxS) (200 nM) from 72 hpf until 120 hpf. Violin plots showing the relative expression of il1b, tnfa, il17a/f3 and il22 in whole larvae at 120 hpf following exposure to TNBS and PFOS, PFOA or PFHxS. n=7-9, three experiments. Each dot represents a pool of ten zebrafish larvae. Values for the UT and TNBS groups for the experiment with each individual polyfluoroalkyl substance (PFAS) in A have previously been presented as a pool in Fig. 1D. (B) Panel of PFASs tested. (C,D) Scheme of dissection of intestines from zebrafish larvae (C, left) and violin plots showing relative expression of an intestine-specific gene, cldn15la (C, right), and proinflammatory cytokines, tnfa (D, left) and il1b (D, right), analyzed by qPCR in dissected intestines or carcasses at 120 hpf following exposure to TNBS (70 µg/ml) and PFOS (200 nM). n=11-12, four experiments. Data show transcript levels as A.U. with respect to eef1l1a1. Each dot represents a pool of ten intestines or carcasses. The black line represents the median. *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA with Fisher's LSD test for all plots, except in C, for which an unpaired Student's t-test was used.

PFOS increases neutrophil recruitment to the intestine during TNBS-induced intestinal inflammation in zebrafish larvae and mice. (A) Experimental outline. Tg(lyz:DsRed2) zebrafish larvae were exposed to TNBS (50 µg/ml) and PFOS (200 nM) from 72 hpf until 120 hpf. (B) High-resolution light-sheet microscopy images of TNBS+PFOS and untreated lysC:DsRed2 larvae at 5× magnification at 120 hpf. The intestine is marked in yellow and outlined in white. Red fluorescence marks neutrophils. Scale bars: 100 µm. (C) Quantification of DsRed2⁺ cells in the intestine. n=32-35, five experiments. Each data point represents one 120 hpf zebrafish larva. The black line represents the median. (D) Experimental outline. Mice received a single dose of TNBS (1%, in 50% ethanol, intrarectally administered) and four doses of PFOS (10 mg/kg/total dose, orally gavaged). (E) Flow cytometry analysis of neutrophils isolated from the colon lamina propria. CD45⁺ cells were gated out for analysis of Ly6C and Ly6G. Neutrophils are gated as Ly6C^int and Ly6G⁺. n=5-11, three experiments. (F) Violin plots showing neutrophil frequencies out of CD45⁺ cells and absolute numbers. (G) Body weight loss curves from mice treated with TNBS and PFOS. (H) Representative H&E microscopy images of distal colons from mice treated with TNBS and PFOS. Scale bars: 50 µm. (I) Violin plots showing histological scores quantified from H&E staining of colon sections as shown in H. n=5-11, three experiments. *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA with Fisher's LSD test.

PFOS exposure during colitis leads to increased systemic levels and intestinal permeability in mice. (A) Violin plots represent normalized PFOS levels detected in serum (middle) and liver (right) at day 3 after TNBS administration, following the experimental outline shown in the scheme (left). Values have been calculated as fold change of the average concentration detected in the PFOS group of each experiment. Values for vehicle and TNBS groups were not detected (nd). n=8-9, three experiments. (B) Intestinal permeability to 4 kDa FITC-Dextran measured in serum at 4 h after oral gavage at day 1 following TNBS administration and PFOS (5 mg/kg/total dose), following the experimental outline shown in the scheme. Data shown in violin plots relative to the average of the values of the vehicle group in each experiment. n=7-9, three experiments. (C) Mice received a single dose of TNBS (1%, in 50% ethanol, intrarectally administered), four doses of PFOS (10 mg/kg/total dose, orally gavaged) and 500 µg per dose of anti-Ly6G monoclonal antibody every other day, following the experimental outline shown in the scheme. Intestinal permeability measured in serum at 4 h after oral gavage at day 3 post-TNBS administration. Data shown in violin plots are relative to the average of the values of the vehicle group in each experiment. n=5-7, three experiments. The black line represents the median. N.S., not significant; *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA with Fisher's LSD test.

Effects on T-cell homeostasis upon PFOS exposure are neutrophil dependent in colitic mice. (A) Experimental outline. Mice received a single dose of TNBS (2.5%, in 50% ethanol, intrarectally administered), five doses of PFOS (10 mg/kg/total dose, orally gavaged) and 500 µg per dose of anti-Ly6G monoclonal antibody every other day. (B,C) Violin plots showing the absolute numbers of CD45⁺ (B) and CD4⁺ (C) T cells in spleen analyzed by flow cytometry. n=3-8, four experiments. (D-G) Flow cytometry analysis of FOXP3⁺ (D,E) and RORγt⁺ (F,G) CD4⁺ T cells in the spleen, following neutrophil depletion. Violin plots represent the absolute numbers and frequencies of these populations out of CD45⁺ cells. n=3-8, four experiments. The black line represents the median. NS, not significant; *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA with Fisher's LSD test.

PFOS exposure during intestinal inflammation enhances neutrophil-mediated damage and leads to CD4⁺ T-cell expansion in the periphery. Scheme showing the proposed model in which PFOS exposure during ongoing intestinal inflammation leads to increased neutrophil recruitment, impairs epithelial barrier function and results in increased PFOS bioavailability and CD4⁺ T-cell numbers systemically.