M2a macrophages facilitate resolution of chemically-induced colitis in TLR4-SNP mice

Abstract

Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, impacts millions of individuals worldwide and severely impairs the quality of life for patients. Dysregulation of innate immune signaling pathways reduces barrier function and exacerbates disease progression. Macrophage (Mφ) signaling pathways are potential targets for IBD therapies. While multiple treatments are available for IBD, (i) not all patients respond, (ii) responses may diminish over time, and (iii) treatments often have undesirable side effects. Genetic studies have shown that the inheritance of two co-segregating SNPs expressed in the innate immune receptor, TLR4, is associated with human IBD. Mice expressing homologous SNPs ("TLR4-SNP" mice) exhibited more severe colitis than WT mice in a DSS-induced colonic inflammation/repair model. We identified a critical role for M2a "tissue repair" Mφ in the resolution of colitis. Our findings provide insight into potential development of novel therapies targeting Mφ signaling pathways that aim to alleviate the debilitating symptoms experienced by individuals with IBD.

Keywords: DSS-induced colitis; IL-4Rα; M2a macrophages; PPARγ; TLR4 SNPs; rosiglitazone.

Fig 1

TLR4-SNP mice are more sensitive to DSS-induced colonic inflammation than C57BL/6J mice. (A) Schematic of the DSS experimental set-up. Experimental mice were given 3% DSS in their drinking water beginning on Day 0 and were switched to regular drinking water on Day 8, with fresh DSS water replaced on Days 2, 4, and 6. Control mice received regular drinking water throughout the experiment. On alternating days, stool consistency and fecal occult blood were assessed. Mice were euthanized and colons were resected on Day 7 or on Day 14. (B) Mean ± SEM colonic symptom scores (stool consistency score + fecal occult blood score) over time in control and DSS-treated WT (C57BL/6J) and TLR4-SNP mice (males and females combined). (C) Comparison of male only (left panel, blue graph) and female only (right panel, pink graph), control and DSS-treated, WT vs TLR4-SNP mouse responses. Two-way ANOVA with Sidak post-hoc multiple comparisons test. **P < 0.01, ***P < 0.001, ****P < 0.0001. On the female graph, # indicates days in which 3% DSS-treated female TLR4-SNP mice were determined to have significantly lower colonic symptom scores compared to 3% DSS-treated male TLR4-SNP mice (Student’s t-test). Results are derived from five independent experiments in which, together, control WT (n = 14; 6 males); control TLR4-SNP (n = 18; 11 males); 3% DSS WT Days 0–7 (n = 57; 30 males), Days 7–14 (n = 31; 17 males); 3% DSS TLR4-SNP Days 0–7 (n = 58; males), Days 7–14 (n = 32; 18 males). (D) Colonic permeability in WT vs TLR4-SNP male mice measured by the concentration of 4 kDa fluorescein isothiocyanate (FITC)-dextran in sera 4 h after administration of FITC-dextran. One-tailed Student’s t-test at each time point. Results are derived from four independent experiments in which, together, control WT (n = 10); control TLR4-SNP (n = 12); 3% DSS WT, Day 7 (n = 18), Day 14 (n = 15); 3% DSS TLR4-SNP Day 7 (n = 16), Day 14 (n = 17). (E) Representative colons from DSS-treated WT and TLR4-SNP mice at Day 14 (left panel). Mean colon lengths (showing individual responses) at Days 7 and 14 in control and DSS-treated WT and TLR4-SNP mice: males and females combined (center panel) and males vs females (right panel). Two-way ANOVA with Sidak (males and females combined) or Tukey (males vs females) post-hoc multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Results are derived from five independent experiments in which, together, control WT (n = 14; 6 males); control TLR4-SNP (n = 18; 11 males); 3% DSS WT, Day 7 (n = 26; 13 males), Day 14 (n = 31; 17 males); 3% DSS TLR4-SNP, Day 7 (n = 26; 13 males), Day 14 (n = 32; 18 males).

Fig 2

TLR4-SNP mice develop exacerbated colonic histopathology by Day 14. (A) Representative H&E-stained images of control and DSS-treated WT and TLR4-SNP distal 1 cm colon (DC) sections at Days 7 and 14 (males and females). In the TLR4-SNP Male Day 14 image, the red line indicates the region of extensive inflammatory cells in the mucosa; red arrowhead indicates inflammatory cells infiltrating into the submucosa. (B) Histology scores of H&E-stained DC sections from control and DSS-treated WT vs TLR4-SNP mice at Days 7 and 14 (showing individual responses): males and females combined (top panel) and males vs females (bottom panel). Two-way ANOVA with Sidak post-hoc multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Results were derived from five independent experiments in which, together, control WT (n = 14; 6 males); control TLR4-SNP (n = 18; 11 males); 3% DSS WT, Day 7 (n = 26; 13 males), Day 14 (n = 31; 17 males); 3% DSS TLR4-SNP, Day 7 (n = 26; 13 males), Day 14 (n = 32; 18 males). (C) Measurements of histologic region sizes (mucosa depth, submucosal height, muscularis externa height) from control and experimental H&E-stained DC sections at Days 7 and 14. Two-way ANOVA with Tukey’s post-hoc multiple comparisons test. *P < 0.05; **P < 0.01; ****P < 0.0001. Results are derived from five independent experiments in which, together, control WT (n = 14; 6 males); control TLR4-SNP (n = 18; 11 males); 3% DSS WT Day 7 (n = 26; 13 males), Day 14 (n = 27; 13 males); 3% DSS TLR4-SNP Day 7 (n = 26; 13 males), Day 14 (n = 27; 13 males). (D) Additional microscopic images were taken of the identical tissue sections shown in Fig. 2A (DSS, male WT vs TLR4-SNP, Day 14) at both 10× and 100× magnification to better illustrate the observed inflammatory differences between the two mouse strains and to allow for quantification of the infiltrating cell types as shown in Fig. 2E. (E) Number of granulocytes (left panel) and monocytes (right panel) in 100× high power H&E-stained DC sections of WT and TLR4-SNP mice. Two-way ANOVA with Sidak’s post-hoc multiple comparisons test. ***P < 0.001; ****P < 0.0001. Data are derived from two independent experiments, n = 5 mice/strain for each time point.

Fig 3

IL-4Rα^−/− mice are more sensitive to DSS-induced colitis than WT BALB/cByJ WT mice. (A) qRT-PCR was carried out on MC sections for Il4ra gene expression in control and DSS-treated WT and IL-4Rα^−/− mice to confirm genotype. (B) Colonic symptom scores (mean ± SEM) over time in control and DSS-treated WT vs IL-4Rα^−/− mice (left panel, responses of males and females mice combined; right panel, comparison of male vs female responses to DSS). Two-way ANOVA with Sidak’s post-hoc multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0001. (C) Colon lengths at Day 14 in control and experimental WT vs IL-4Rα^−/− mice. Far left panel, representative DSS-treated WT and IL-4Rα^−/− colons at Day 14. Middle panel, male and female responses combined. Two-way ANOVA with Sidak post-hoc multiple comparisons test. Far right panel, DSS-treated male vs female IL-4Rα^−/− mice. Unpaired two-tailed Student’s t-test. (D) Representative H&E-stained images of DC sections from control and DSS-treated WT and IL-4Rα^−/− mice at Day 14. (E) Histology scores of H&E-stained DC sections from control and DSS-treated WT and IL-4Rα^−/− mice at Day 14 (left panel, males and females combined; right panel, male vs female, DSS-treated IL-4Rα^−/− mice). Two-way ANOVA with Sidak post-hoc multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Right panel: Histology scores at Day 14 in male vs female DSS-treated IL-4Rα^−/− mice. Unpaired two-tailed Student’s t-test. Results are derived from three independent experiments in which, together, control WT (n = 10; 5 males), control IL-4Rα^−/− (n = 13; 5 males); 3% DSS WT Day 14 (n = 10; 5 males); 3% DSS IL-4Rα^−/− Day 14 (n = 23; 13 males).

Fig 4

DSS-treated TLR4-SNP mice exhibit reduced M2a markers in the colon and reduced β-hydroxybutyrate (β-HB) levels in the sera. (A) M2a gene expression measured in the MC of male WT vs TLR4-SNP mice, comparing control, Day 9, and Day 11 samples by qRT-PCR. One-way ANOVA with Sidak multiple comparisons test. *P < 0.05; **P < 0.01; ****P < 0.0001. Data are derived from two independent experiments. In total, control WT n = 11; control TLR4-SNP n = 15; WT Day 9 n = 13, Day 11 n = 13; TLR4-SNP Day 9 n = 15, Day 11 n = 15. (B) M2a (Ym1, Arg1) protein production measured in DC samples in WT vs TLR4-SNP mice, comparing control and Day 11 samples. Data are representative of results from two independent experiments totaling control WT n = 5; control TLR4-SNP n = 5; WT Day 11 n = 9; TLR4-SNP Day 11 n = 9. (C) M2a (Mrc1, PPARγ) protein production measured in DC samples in WT vs TLR4-SNP mice, comparing control and Day 11 samples. Data representative of results from two independent experiments totaling control WT n = 5; control TLR4-SNP n = 5; WT Day 11 n = 9; TLR4-SNP Day 11 n = 9. (D) β-HB concentration measured in the sera of control and DSS-treated, WT vs TLR4-SNP male mice at Days 9 and 11. Two-way ANOVA with Sidak multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are derived from three independent experiments in which, together, control WT n = 12; control TLR4-SNP n = 13; WT Day 11 n = 10; TLR4-SNP Day 11 n = 10.

Fig 5

PPARγ^cKO mice are more sensitive to DSS-induced colitis than WT mice. (A) PPARγ protein expression in thioglycollate-elicited macrophages from WT and PPARγ^cKO mice in the absence or presence of IL-4 (24 h) to confirm genotype. (B) Colonic symptom scores (Mean ± SEM) in control and DSS-treated WT vs PPARγ^cKO mice (responses of male and female mice combined). Two-way ANOVA with Sidak’s post-hoc multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0001. (C) Representative colon lengths at Day 14 in control and DSS-treated WT vs PPARγ^cKO mice. Left panel, gross image of representative DSS-treated WT and PPARγ^cKO colons on Day 14. Right panel, male and female responses combined. Two-way ANOVA with Sidak’s post-hoc multiple comparisons test. ****P < 0001. (D) Left panel: representative H&E-stained images of DC sections. Right panel: histology scores of H&E-stained DC sections from control and DSS-treated WT and PPARγ^cKO mice on Day 14, males and females combined. Two-way ANOVA with Sidak post-hoc multiple comparisons test. ****P < 0001. Results were derived from three independent experiments in which in total control WT (n = 5), control PPARγ^cKO (n = 5), 3% DSS WT (n = 16 males), 3% DSS PPARγ^cKO (n = 12; 8 males). Although the early kinetics of DSS-induced disease were delayed in PPARγ^cKO compared to WT mice, we observed deaths in four males and one female, suggesting that myeloid PPARγ plays a key role in tissue repair.

Fig 6

Administration of rosiglitazone, a PPARγ agonist ligand, ameliorates DSS-induced colonic damage in TLR4-SNP mice. Male TLR4-SNP mice were administered DSS starting on Day 0. On Days 2–7, mice were administered either saline or rosiglitazone (25 mg/kg) once daily i.p. (A) Average colonic symptom score over time in saline- and rosiglitazone-treated male TLR4-SNP mice. Two-way ANOVA with Sidak’s multiple comparisons; *P < 0.05. (B) Colon lengths at Day 14 in saline- and rosiglitazone-treated TLR4-SNP mice. Unpaired two-tailed Students t-test. ***P < 0.001. (C) Representative H&E-stained images of DC sections from saline- and rosiglitazone-treated TLR4-SNP mice at Day 14. (D) High-power image of submucosal infiltrating inflammatory cells in a representative DSS- and saline-treated TLR4-SNP mouse on Day 14. The infiltrating cells are predominantly mononuclear with occasional granulocytic cells (red arrows). (E) Right panel: histology scores of H&E-stained DC sections from saline- and rosiglitazone-treated TLR4-SNP mice at Day 14. Unpaired two-tailed Students t-test; **P < 0.01. Results were derived from three independent experiments. In total, TLR4-SNP saline n = 17; TLR4-SNP rosiglitazone n = 19. (F) Ym1 (Chil3) protein expression at Day 11 in DC of DSS-treated TLR4-SNP mice therapeutically administered rosiglitazone or saline. Data are representative of results from two independent experiments totaling TLR4-SNP rosiglitazone-treated n = 9; TLR4-SNP saline-treated n = 8.