Fig 2: Representative images of CXCL5 immunostaining in lung tissue at 1 month after exposure to CL-PAA. A control lung (HE staining), B 1.0 mg CL-PAA-exposed lung (HE staining), C control lung (CXCL5 immunostaining), D 1.0 mg CL-PAA-exposed lung (CXCL5 immunostaining). Positive cells of CXCL5 immunostaining on 1.0 mg CL-PAA-exposed lung were mainly macrophages (blue arrow heads) (internal scale bar = 250 µm for all)

Fig 3: Activation of inflammatory signals including CCL28 and CXCL5 in aged stCldn18–/– mice. (A) Expression levels of IL-1ß, TNF-a, and CXCL1 in the stomach from stCldn18+/+ (n = 8, 4, 11, 10) and stCldn18–/– mice (n = 14, 4, 13, 16) (8, 40, 60, and 100 w.o.) quantified by qRT-PCR. Gene expressions were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Results are expressed as mean ± SD. Comparisons between 2 groups were performed using Student’s t test, and differences with P < .05 were considered statistically significant. *P < .05; **P < .01. (B) Immunofluorescence micrographs for CXCL5 (green) co-stained with a-catenin (red) and DAPI (blue) in the stomach from stCldn18+/+ mice and stCldn18–/– mice (8, 40, 60, and 100 w.o.). CXCL5 signals were clearly observed in the stomach of stCldn18–/– mice after middle-age (40, 60, and 100 w.o.) (arrows). High-magnification images are shown in insets. Representative images from at least 3 independent experiments are shown. Scale bars = low magnification: 50 µm, high magnification: 10 µm. (Right) Expression level of CXCL5 in the stomach from stCldn18+/+ (n = 8, 4, 12, 10) and stCldn18–/– mice (n = 6, 4, 13 16) (8, 40, 60, and 100 w.o.) quantified by qRT-PCR. Gene expressions were normalized to GAPDH. Results are expressed as mean ± SD. Comparisons between 2 groups were performed using Student’s t test, and differences with P < .05 were considered statistically significant. **P < .01. (C) Expression levels of STAT3, IKKß, and NF-?B in the stomach from stCldn18+/+ (n = 4, 4, 10, 10) and stCldn18–/– mice (n = 6, 4, 12, 16) (8, 40, 60, and 100 w.o.) quantified by qRT-PCR. Gene expressions were normalized to GAPDH. Results are expressed as mean ± SD. Comparisons between 2 groups were performed using Student’s t test, and differences with P < .05 were considered statistically significant. *P < .05; **P < .01. (D) (Left) Immunofluorescence micrographs for CCL28 (green), a lymphocyte chemoattractant and maintainer of chronic inflammation, co-stained with a-catenin (red) and DAPI (blue) in the stomach from stCldn18+/+ and stCldn18–/– mice (10, 40, 60, and 100 w.o.). CCL28 signals were clearly observed in the stomach of the stCldn18–/– mice after middle-age (40, 60, and 100 w.o.) (arrows). High-magnification images are shown in insets. Representative images from at least 3 independent experiments are shown. Scale bars = low magnification: 50 µm, high magnification: 10 µm. (Right) Expression level of CCL28 in the stomach from stCldn18+/+ (n = 8, 4, 12, 10) and stCldn18–/– (n = 10, 4, 13 16) mice (8, 40, 60, and 100 w.o.) quantified by qRT-PCR. Gene expressions were normalized to GAPDH. Results are expressed as mean ± SD. Comparisons between 2 groups were performed using Student’s t test, and differences with P < .05 were considered statistically significant. **P < .01. (E) Expression levels of CXCL5, IKKß, NF?B, and ASCL2 in the stomach from stCldn18+/+ and stCldn18–/– mice (around 50 w.o.) after POL7085 or cimetidine treatment, quantified by qRT-PCR (n: stCldn18–/– = 7, 5, 6, given saline, POL7085, and cimetidine, respectively). Gene expressions were normalized to GAPDH. Results are expressed as mean ± SD. Comparisons between 2 groups were performed using Student’s t test, and differences with P < .05 were considered statistically significant. *P < .05; **P < .01.

Fig 4: No tumor formation in young Wnt1-Tg stCldn18–/– DM mice. (A) Hematoxylin and eosin–stained images of the stomach from stCldn18+/+, Wnt1-Tg, stCldn18–/–, and Wnt1-Tg mice crossed with stCldn18–/– DM mice at a young age (8 w.o.). No obvious difference was detected in the stomachs between stCldn18–/– and DM mice at the young age. Representative images from at least 3 independent experiments are shown. Scale bars = 1 mm. (B) Expression levels of IL-1β, TNF-α, CXCL1, CCL28, and CXCL5 in the stomach from stCldn18+/+, Wnt1-Tg, stCldn18–/–, and DM mice at a young age (8 w.o.) quantified by qRT-PCR (n = 8, 4, 10, 11). No significant differences were detected between the stomachs of stCldn18–/– and DM mice at the young age. Gene expressions were normalized to GAPDH. Results are expressed as mean ± SD. Comparisons among more than 3 groups were performed using the Kruskal-Wallis 1-way analysis of variance on ranks (P < .01) followed by the Steel-Dwass test. *P < .05. (C) Immunofluorescence micrographs for CD3 or CXCL5 (green) co-stained with ZO-2 (red) and DAPI (blue) in the stomach from stCldn18+/+, Wnt1-Tg, stCldn18–/–, and DM mice at a young age (8 w.o.). High-magnification images are shown in insets. No significant differences were detected between the stomachs of stCldn18–/– and DM mice at the young age. Representative images from at least 3 independent experiments are shown. Scale bars = low magnification: 100μm, high magnification: 10 μm.

Fig 5: Histological findings following intratracheal instillation (hematoxylin and eosin [HE] and Masson trichrome [MT] staining). Histological findings at (A) 3 days following the instillation (HE and MT staining), (B) 1 week following the instillation (HE and MT staining), (C) 1 month following the instillation (HE and MT staining), (D) 3 months following the instillation (HE and MT staining), (E) 6 months following the instillation (HE and MT staining). (a, d, g) One of the specimens from the group that received distilled water as a control. (b, e, h) One of the specimens from the group that received 0.2 mg of cross-linked polyacrylic acid (CL-PAA) as a low dose. (c, f, i) One of the specimens from the group that received 1.0 mg of CL-PAA as a high dose. Red arrowheads and black arrowheads show neutrophils and macrophages, respectively, in HE staining. Black arrows show collagen fibers in MT staining (internal scale bars in A–E = 250 µm). (F) Histological findings at 3 days, 1 month, and 6 months following the instillation. Red arrows show type II alveolar epithelium-like cells. There was almost no proliferation of alveolar type II epithelial cells at 6 months (internal scale bars in F, 50 µm). (G) Ashcroft scale at each time. Each data are presented as mean ± SE (*P < .05, **P < .01, ‡P < .05, and ‡‡P < .01). One-way ANOVA followed by Tukey's test was used to appropriately detect differences among the exposed groups. (H) Representative images of CXCL5 immunohistochemical staining in lung tissue at 1 month after intratracheal instillation of distilled water as a control group or 1.0 mg CL-PAA as a high-dose group. (a) control group (HE staining), (b) high-dose group (HE staining), (c) control group (CXCL5 immunostaining), (d) high-dose group (CXCL5 immunostaining). Positive cells of CXCL5 immunohistochemical staining on the high-dose group were mainly macrophages (blue arrowheads) (internal scale bars in H, 250 µm).