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IL-6 Stimulates Intestinal Epithelial Proliferation and Repair after Injury

Kuhn, Kristine A., Manieri, Nicholas A., Liu, Ta-Chiang, Stappenbeck, Thaddeus S.·2014·PLoS ONE
DOI 10.1371/journal.pone.0114195PMC PMC4257684
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Figure 1 IL-6 was induced with the initiation of colitis in dnKO mice. Antibiotic pretreated dnKO and IL-10rb+/- littermate control mice were co-housed with non-antibiotic treated mice to induce colitis in dnKO mice. From individual mice, colons and sera were harvested with no co-housing (baseline, day zero) and every three days after co-housing. IL-6 mRNA and protein expression was analyzed by ELISA (A) and in situ hybridization (B), respectively. Two experiments were performed with a total of 10–14 mice/group/time point. (A) Plot of the average ± SEM IL-6 protein (pg/ml) in the sera over time for each group of mice. A student's t-test was used to determine statistical significance for each time point; *, p<0.05; **, p<0.0001. (B) Representative images of IL-6 in situ hybridization (red staining, arrowheads) are shown for days 0, 3 and 6 post co-housing. Bars = 500 µm.
Figure 1 IL-6 was induced with the initiation of colitis in dnKO mice. Antibiotic pretreated dnKO and IL-10rb+/- littermate control mice were co-housed with non-antibiotic treated mice to induce colitis in dnKO mice. From individual mice, colons and sera were harvested with no co-housing (baseline, day zero) and every three days after co-housing. IL-6 mRNA and protein expression was analyzed by ELISA (A) and in situ hybridization (B), respectively. Two experiments were performed with a total of 10–14 mice/group/time point. (A) Plot of the average ± SEM IL-6 protein (pg/ml) in the sera over time for each group of mice. A student's t-test was used to determine statistical significance for each time point; *, p<0.05; **, p<0.0001. (B) Representative images of IL-6 in situ hybridization (red staining, arrowheads) are shown for days 0, 3 and 6 post co-housing. Bars = 500 µm.CC BY · PubMed Central
Figure 2 Inhibition of IL-6 resulted in more severe colitis and inhibition of intestinal epithelial proliferation. Colitis was induced in dnKO and IL-10rb+/- littermate controls by co-housing. On day zero and three times weekly, mice were injected intraperitoneally with 500 µg of either anti-IL-6 mAb or control IgG mAb. Two independent experiments were performed with 8-9 mice/group. (A) Plot of the average percent of starting weight ± SEM shown for indicated groups of mice. Mice were weighed every three days. (B) Representative H+E stained sections of descending colons at day 9 post co-housing. Bars = 500 µm. Black dotted lines outline remaining crypts in the dnKO anti-IL-6 mAb treated mouse histology. (C) Graph of the average number of descending colonic crypts per high-powered field ± SEM. (D) At day 9 post-co-housing, mice were injected with BrdU one hour before sacrifice. Representative colonic sections stained with mAb to BrdU and detected with fluorescently conjugated antibodies were shown. The white dotted lines delineate crypts. Bars = 100 µm. (E) Graph of the average number ± SEM of BrdU positive cells per crypt. (F) Graph of the average ± SEM number of apoptotic bodies/crypt. One-way analysis of variance: (A) F = 3.5, P<0.05 (for day 9 weights); (C) F = 57.36, P<0.0001; (E) F = 17.92, P<0.0001; (F) F = 10.87, P<0.0001. Means with different letters are significantly different by Bonferroni's multiple comparison test.
Figure 2 Inhibition of IL-6 resulted in more severe colitis and inhibition of intestinal epithelial proliferation. Colitis was induced in dnKO and IL-10rb+/- littermate controls by co-housing. On day zero and three times weekly, mice were injected intraperitoneally with 500 µg of either anti-IL-6 mAb or control IgG mAb. Two independent experiments were performed with 8-9 mice/group. (A) Plot of the average percent of starting weight ± SEM shown for indicated groups of mice. Mice were weighed every three days. (B) Representative H+E stained sections of descending colons at day 9 post co-housing. Bars = 500 µm. Black dotted lines outline remaining crypts in the dnKO anti-IL-6 mAb treated mouse histology. (C) Graph of the average number of descending colonic crypts per high-powered field ± SEM. (D) At day 9 post-co-housing, mice were injected with BrdU one hour before sacrifice. Representative colonic sections stained with mAb to BrdU and detected with fluorescently conjugated antibodies were shown. The white dotted lines delineate crypts. Bars = 100 µm. (E) Graph of the average number ± SEM of BrdU positive cells per crypt. (F) Graph of the average ± SEM number of apoptotic bodies/crypt. One-way analysis of variance: (A) F = 3.5, P<0.05 (for day 9 weights); (C) F = 57.36, P<0.0001; (E) F = 17.92, P<0.0001; (F) F = 10.87, P<0.0001. Means with different letters are significantly different by Bonferroni's multiple comparison test.CC BY · PubMed Central
Figure 3 IL-6 promoted intestinal epithelial proliferation in wound biopsy model. (A) WT mice were biopsy injured in the distal colon. Plot of the relative levels of IL-6 mRNA expression in the wound bed (relative to uninjured tissue) for various times after injury. N = 2–3 WT mice with a total of 4-6 wounds/time point. Data were shown as average ± SEM. One-way analysis of variance: F = 5.68, P<0.01 (B) Cartoon depicting the microanatomy of a wound at day six post-biopsy; AC = adjacent crypts (green area); WC = wound channels (blue area); WAE = wound-associated epithelium overlying the wound bed (pink area). (C) Colonic sections of wounds from IL-6+/− and IL-6-/- mice at day six post-injury stained with mAb to BrdU (labels S-phase cells, red), mAb to β-catenin (labels epithelium, green), and bis-benzimide (nuclei, blue). Bars = 500 µm. (D) Quantification of the number of BrdU positive cells/wound adjacent crypts. Data were graphed as average ± SEM. One way analysis of variance: F = 10.5, p<0.0001. Means with different letters are significantly different by Bonferroni's multiple comparison test.
Figure 3 IL-6 promoted intestinal epithelial proliferation in wound biopsy model. (A) WT mice were biopsy injured in the distal colon. Plot of the relative levels of IL-6 mRNA expression in the wound bed (relative to uninjured tissue) for various times after injury. N = 2–3 WT mice with a total of 4-6 wounds/time point. Data were shown as average ± SEM. One-way analysis of variance: F = 5.68, P<0.01 (B) Cartoon depicting the microanatomy of a wound at day six post-biopsy; AC = adjacent crypts (green area); WC = wound channels (blue area); WAE = wound-associated epithelium overlying the wound bed (pink area). (C) Colonic sections of wounds from IL-6+/− and IL-6-/- mice at day six post-injury stained with mAb to BrdU (labels S-phase cells, red), mAb to β-catenin (labels epithelium, green), and bis-benzimide (nuclei, blue). Bars = 500 µm. (D) Quantification of the number of BrdU positive cells/wound adjacent crypts. Data were graphed as average ± SEM. One way analysis of variance: F = 10.5, p<0.0001. Means with different letters are significantly different by Bonferroni's multiple comparison test.CC BY · PubMed Central
Figure 4 IL-6 inhibition after colitis induction did not inhibit epithelial proliferation but did not improve disease. Three days after induction of colitis in dnKO and IL-10rb+/- littermate controls, 500 µg anti-IL-6 antibody or control IgG was administered to mice three times weekly. Mice were sacrificed 9 days after induction of colitis. 6–8 mice per group in two independent experiments were analyzed. (A) Plot of the average percent of starting weight ± SEM shown for indicated groups of mice. Mice were weighed every three days. (B) Representative H+E-stained sections of descending colons from each group of mice. Bars = 500 µm. (C) Graph of the mean number of crypts/high-powered field ± SEM of the descending colon per animal. (D) Graph of the average number of BrdU+ cells/crypt ± SEM. One-way analysis of variance: (A) F = 4.88, P = 0.016; (C) F = 21.14, P<0.0001; and (D) F = 68.24, P<0.0001. Means with different letters are significantly different by Bonferroni's multiple comparison test.
Figure 4 IL-6 inhibition after colitis induction did not inhibit epithelial proliferation but did not improve disease. Three days after induction of colitis in dnKO and IL-10rb+/- littermate controls, 500 µg anti-IL-6 antibody or control IgG was administered to mice three times weekly. Mice were sacrificed 9 days after induction of colitis. 6–8 mice per group in two independent experiments were analyzed. (A) Plot of the average percent of starting weight ± SEM shown for indicated groups of mice. Mice were weighed every three days. (B) Representative H+E-stained sections of descending colons from each group of mice. Bars = 500 µm. (C) Graph of the mean number of crypts/high-powered field ± SEM of the descending colon per animal. (D) Graph of the average number of BrdU+ cells/crypt ± SEM. One-way analysis of variance: (A) F = 4.88, P = 0.016; (C) F = 21.14, P<0.0001; and (D) F = 68.24, P<0.0001. Means with different letters are significantly different by Bonferroni's multiple comparison test.CC BY · PubMed Central
Figure 5 Intraepithelial lymphocytes were a source of IL-6 early after injury. (A) Biopsy of the colon mucosa was performed in WT mice to create small wounds. IL-6 expression in the wound bed and adjacent tissue was evaluated by in situ hybridization one day after biopsy. Representative images were shown. Bars = 200 µm. Colored bars above wound images indicate areas of the wound bed as depicted in Figure 4B. (B) Co-localization by immunofluorescence was performed for IL-6 (red), CD3ε (green), and bis-benzimide (blue) on colon tissue from dnKO mice at day 6 after co-housing. Representative staining was shown at 63X. Bar = 200 µm. (C) Epithelial cells were harvested from dnKO mice on day 6 after co-housing, stained for T cell markers and IL-6, and assessed by flow cytometry. Representative dot plots were shown. (D, E) CD3+ CD4- CD8- IELs were harvested from WT mice and stimulated ex vivo with 10 ng/ml PMA and 1 µg/ml ionamycin for 5 hours. (D) RNA was collected and evaluated by qRT-PCR for IL-6 expression. (E) Culture supernatants were harvested and evaluated for secreted IL-6 by electrochemilluminescence. Data were shown as the average IL-6 expression or protein ± SEM. A paired student's t-test was used to determine significance; *, P<0.05.
Figure 5 Intraepithelial lymphocytes were a source of IL-6 early after injury. (A) Biopsy of the colon mucosa was performed in WT mice to create small wounds. IL-6 expression in the wound bed and adjacent tissue was evaluated by in situ hybridization one day after biopsy. Representative images were shown. Bars = 200 µm. Colored bars above wound images indicate areas of the wound bed as depicted in Figure 4B. (B) Co-localization by immunofluorescence was performed for IL-6 (red), CD3ε (green), and bis-benzimide (blue) on colon tissue from dnKO mice at day 6 after co-housing. Representative staining was shown at 63X. Bar = 200 µm. (C) Epithelial cells were harvested from dnKO mice on day 6 after co-housing, stained for T cell markers and IL-6, and assessed by flow cytometry. Representative dot plots were shown. (D, E) CD3+ CD4- CD8- IELs were harvested from WT mice and stimulated ex vivo with 10 ng/ml PMA and 1 µg/ml ionamycin for 5 hours. (D) RNA was collected and evaluated by qRT-PCR for IL-6 expression. (E) Culture supernatants were harvested and evaluated for secreted IL-6 by electrochemilluminescence. Data were shown as the average IL-6 expression or protein ± SEM. A paired student's t-test was used to determine significance; *, P<0.05.CC BY · PubMed Central
Figure 6 IL-6 expression was increased in human colons at sites of perforation. Tissue that was surgically resected from patients who suffered large bowel perforation was evaluated for IL-6 expression by in situ hybridization. Eleven cases were evaluated (8 males with trauma due to gun-shot wounds, ages 16–33; 1 female surgical trauma, age 45; and 2 females with diverticulitis, ages 72 and 79). (A) Representative staining from a patient with diverticulitis is shown at 20X and 100X, respectively. Bars = 100 µm. Arrows indicate IL-6+ cells with lymphocyte morphology. (B) Four high-powered fields with well-oriented crypts were evaluated for IL-6+ cells in the epithelial layer at the site of perforation and at the distal resection margin. The average ratio of IL-6+ cells in the perforation versus distal site ± SEM was shown. An unpaired student's t-test was used for statistical analysis; *, P = 0.02.
Figure 6 IL-6 expression was increased in human colons at sites of perforation. Tissue that was surgically resected from patients who suffered large bowel perforation was evaluated for IL-6 expression by in situ hybridization. Eleven cases were evaluated (8 males with trauma due to gun-shot wounds, ages 16–33; 1 female surgical trauma, age 45; and 2 females with diverticulitis, ages 72 and 79). (A) Representative staining from a patient with diverticulitis is shown at 20X and 100X, respectively. Bars = 100 µm. Arrows indicate IL-6+ cells with lymphocyte morphology. (B) Four high-powered fields with well-oriented crypts were evaluated for IL-6+ cells in the epithelial layer at the site of perforation and at the distal resection margin. The average ratio of IL-6+ cells in the perforation versus distal site ± SEM was shown. An unpaired student's t-test was used for statistical analysis; *, P = 0.02.CC BY · PubMed Central

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Metadata

Authors
Kuhn, K. A., Manieri, N. A., Liu, T.-C., & Stappenbeck, T. S.
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2014-12-05
Volume
9
Issue
12
Pages
e114195
ISSN
1932-6203
DOI
10.1371/journal.pone.0114195
PMC
PMC4257684
Cited by
254
References
3
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (unspecified)

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