Distinct roles of intracellular heat shock protein 70 in maintaining gastrointestinal homeostasis

Abstract

The inducible heat shock protein 70 (Hsp70) is both cytoprotective and immunomodulatory, potentially accounting for its critical role in maintaining gastrointestinal homeostasis. When levels are reduced in conditions like inflammatory bowel diseases (IBD), loss of function contributes to the severity and chronicity of these diseases, although through which cell types and mechanisms remains unclear. Here, the role of Hsp70-mediated intestinal epithelial protection and immune regulation in experimental colitis was examined by using a villin promoter-driven Hsp70 transgene in the 2,4,6-trinitrobenzene sulfonic acid (TNBS) and dextran sodium sulfate (DSS) models and in IL-10/Hsp70 double knockout (IL10^-/-/Hsp70^-/-) mice. In addition, Hsp70-mediated IL-10 production and immune protection were investigated using a CD45RB^high transfer model and measuring colonic and immune cell cytokine expression during colitis. We found that the epithelial-specific expression of Hsp70 transgene attenuated DSS-induced colitis in Hsp70^-/- mice by protecting tight junctions (TJ) and their interaction with the TJ-associated protein ZO-1. In the TNBS colitis and CD45RB^high model, Hsp70 carried out its intracellular anti-inflammatory function by maintaining IL-10 production. Impaired ERK phosphorylation, but not p38 or JNK phosphorylation pathways, was associated with decreased IL-10 production in Hsp70-deficient cells. Together, these actions can be leveraged in the context of cellular specificity to develop complementary strategies that can lead to reduction in mucosal injury and immune activation in colonic colitis development. NEW & NOTEWORTHY Using four different experimental colitis models, we filled an important gap in knowledge by defining essential roles of intracellular heat shock protein 70 in different cell types in maintaining intestinal integrity and immune regulation. These findings are relevant to human inflammatory bowel diseases and represent potential avenues for developing therapeutic strategies, not only to counter the destructive processes of inflammation but also to promote tissue healing and prevent complications frequently associated with chronic intestinal inflammation.

Keywords: gastrointestinal homeostasis; heat shock protein; inflammatory bowel diseases.

Fig. 1.

Epithelial specific expression of heat shock protein-70 (Hsp70) cannot protect Hsp70^−/− mouse from 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. A: immunostaining of Hsp70 in mouse colon (dark brown staining). Hsp70 expression was observed only in epithelium and not in lamina propria cells of Hsp70^−/−/V-TG mice. Scale bars represent 100 μm. B: representative colonic histological features of TNBS-induced colitis in WT, Hsp70^−/−, and Hsp70^−/−/V-TG mice. There were extensive inflammatory cell infiltration and marked colonic mucosal destruction in Hsp70^−/− and Hsp70^−/−/V-TG mice. Scale bars represent 50 μm. C and D: TNBS induces severe colitis in Hsp70^−/− and Hsp70^−/−/V-TG mice, but not in WT mice. Hsp70^−/− and Hsp70^−/−/V-TG mice lost significantly more body weight and had significantly higher histological scores than WT mice. No differences were found between Hsp70^−/− and Hsp70^−/−/V-TG mice. E–H: colonic cytokine expression was measured with and without TNBS treatment. The highest levels of the proinflammatory cytokines IFNγ, IL-12p40, and IL-6 were found in the colonic mucosa of Hsp70^−/− and Hsp70^−/−/V-TG mice with active colitis. In contrast, IL-10 levels were significantly lower in Hsp70^−/− and Hsp70^−/−/V-TG mice after TNBS challenge. No differences of cytokine levels were found between Hsp70^−/− and Hsp70^−/−/V-TG mice. Bars represent means ± SD. Data were analyzed by one-way ANOVA with Bonferroni’s multiple comparisons test. *P < 0.05, **P < 0.01; n ≥ 6 in each group.

Fig. 2.

Epithelial specific expression of Hsp70 protects Hsp70^−/− mice from dextran sodium sulfate (DSS)-induced colitis. A and B: DSS induces severe colitis in Hsp70^−/− mice, but not in WT and Hsp70^−/−/V-TG mice. Compared with Hsp70^−/− mice, Hsp70^−/−/V-TG mice exhibited less body weight loss and had significantly lower histological colitis scores. However, Hsp70^−/−/V-TG mice still had more body weight loss and higher colitis score than WT mice. C: representative colonic histological features of DSS-induced colitis in WT, Hsp70^−/−, and Hsp70^−/−/V-TG mice. All 3 groups exhibited mucosal destruction and inflammatory cell infiltration after DSS treatment. Scale bars represent 50 μm. D–G: cytokine levels measured in colonic mucosa among the 3 groups of mice with and without DSS treatment. No significant differences in cytokine levels were detected among WT, Hsp70^−/−, and Hsp70^−/−/V-TG mice at baseline. After DSS treatment, both Hsp70^−/− and Hsp70^−/−/V-TG mice showed significantly elevated IFNγ, TNFα, and IL-6 compared with WT mice. No differences of IFNγ, IL-10, and IL-6 were detected between Hsp70^−/− and Hsp70^−/−/V-TG mice. TNFα was significantly lower in Hsp70^−/−/V-TG compared with that in Hsp70^−/−. IL-10 was significantly lower in Hsp70^−/− and Hsp70^−/−/V-TG mice compared with that in WT mice after DSS treatment. Bars represent means ± SD. Data were analyzed by one-way ANOVA with Bonferroni’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001; n ≥ 6 in each group.

Fig. 3.

Hsp70 maintains tight junction through interaction with ZO-1. A: increased colonic permeability is detected in Hsp70^−/− mice after DSS treatment. At days 4 and 10, Hsp70^−/− mice exhibited significantly higher colonic leakage than both WT and Hsp70^−/−/Villin TG mice. There was no significant difference of colonic permeability between WT and Hsp70^−/−/V-TG mice at day 4, but difference was significant at day 10 after DSS treatment. B: immunoprecipitation shows Hsp70 binds to ZO-1 directly. C: colocalization of Hsp70 and ZO-1. Hsp70 is stained in red and ZO-1 is stained in green. Scale bars represent 100 μm. D: expression pattern of ZO-1 in colonic epithelium was characterized and found to be significantly disrupted and irregular in Hsp70^−/− mice compared with those in WT and IL10^−/−/Hsp70^−/−/V-TG mice. ZO-1 is stained in green and cell nucleus in blue. Scale bars represent 50 μm. Bars represent means ± SD. Data were analyzed by one-way ANOVA with Bonferroni’s multiple comparisons test. **P < 0.01, ***P < 0.001, ****P < 0.0001; n ≥ 6 in each group.

Fig. 4.

CD4⁺CD45RB^high adoptive transfer colitis. A: change of body weight over 12-wk period after CD45RB^high adoptive transfer. Equal numbers of CD45RB^high cells from WT mice were intraperitoneally injected into Rag^−/− mice. Two weeks later, mice were divided into 2 groups randomly, and equal amounts of CD45RB^low cells from WT and Hsp70^−/− mice were intraperitoneally injected into Rag^−/− mice, respectively. Mice were followed for another 10 wk before tissue collection. B: representative colonic histological features of CD4⁺CD45RB^high adoptive transfer colitis from 2 groups. Scale bars represent 50 μm. C: Rag^−/− mice receiving CD45RB^low cells from Hsp70^−/− mice have significant higher colitis scores than mice receiving CD45RB^low cells from WT mice. D: cytokine levels between 2 groups of mice. Mice receiving CD45RB^low cells from Hsp70^−/− mice had significant higher IFNγ, TNFα, and IL-6. E: IL-10 expression in CD4⁺ T cells collected from Rag^−/− mice after adoptive transfer. Mesenteric lymph nodes were collected from mice, and IL-10 expression in CD4+ T cells was measured by flow cytometry. Significantly lower expression of IL-10 production was observed in Rag^−/− mice receiving CD45RB^low cells from Hsp70^−/− mice. Bars represent means ± SD. Data were analyzed by 2-tailed Student’s t-tests. **P < 0.01, ***P < 0.001; n = 6 in each group.

Fig. 5.

Hsp70 regulates IL-10 production through ERK-phosphorylation. (A–D) In vitro stimulation assay. IL-10 expression between immune cells from WT and Hsp70^−/− mice was examined before and after stimulation. IL-10 level was significant lower in macrophage, dendritic cells, B cells and CD4⁺ T cells from Hsp70^−/− mice after stimulation (macrophage and dendritic cell were stimulated with LPS for 16 h; B cell and CD4⁺ T cell were stimulated with PMA/ionomycin for 24 h), but no differences in IL-10 were found between the two groups at baseline. (E) Western blot of ERK phosphorylation in macrophage and dendritic cells before and after LPS stimulation. ERK phosphorylation was decreased in cells from Hsp70^−/− mice after stimulation. (F) Western blot of ERK phosphorylation in B and CD4⁺ T cells before and after PMA/ionomycin stimulation. ERK phosphorylation was decreased in cells from Hsp70^−/− mice after stimulation. (G and H) Densitometry measurements show ERK phosphorylation is significantly decreased in macrophage, dendritic cells, B cells and CD4⁺ T cells from Hsp70^−/− mice after LPS and PMA/ionomycin stimulation, respectively. Bars represent mean ± SD. Data were analyzed by 2-tailed Student’s t tests. * P < 0.05, *** P < 0.001, n = 3 and experiment was performed by two times independently.

Fig. 6.

Expression of JNK and p38 in cells after stimulation. A: Western blot was used to compare expression of total and phosphorylation JNK and p38 in macrophage, dendritic cells, B cells, and CD4⁺ T cells before and after stimulation. B: densitometry of Western blot shows that phosphorylation of p38 and JNK was not affected by Hsp70 deficiency. Bars represent means ± SD. Data were analyzed by 2-tailed Student’s t tests; n = 3 in each group and experiment was performed 2 times independently.

Fig. 7.

ERK phosphorylation in mesenteric lymph nodes in TNBS- and DSS-treated mice. Mesenteric lymph nodes from mice with and without TNBS and DSS treatment were collected, respectively. ERK phosphorylation status was compared among groups by Western blot. No difference of ERK phosphorylation was detected in mice without colitis, but significantly lower levels of ERK phosphorylation were detected in mesenteric lymph nodes from Hsp70^−/− and Hsp70^−/−/V-TG mice compared with those from WT mice after TNBS (A and B) and DSS treatments (C and D), respectively. Bars represent means ± SD. Data were analyzed by one-way ANOVA with Bonferroni’s multiple comparisons test. *P < 0.05; n = 6 in each group, and experiment was performed 2 times independently.

Fig. 8.

NF-κB phosphorylation and expression of proinflammatory cytokines in B and T cells. A: NF-κB phosphorylation status in B and CD4⁺ T cells at baseline and after stimulation was examined by comparing p65 phosphorylation. Densitometry measurements showed NF-κB phosphorylation was significantly increased in cells collected from Hsp70^−/− mice compared with that in WT cells after stimulation. B: TNFα and IFNγ expression in B cells at baseline and after stimulation. No significant difference was detected between Hsp70^−/− and WT. C: TNFα and IFNγ expression in CD4⁺ T cells at baseline and after stimulation. No significant difference was detected between Hsp70^−/− and WT. Bars represent means ± SD. Data were analyzed by Student’s t-tests. *P < 0.05.

Fig. 9.

IL10/Hsp70 double knockout mice have increased penetrance of colitis. A: penetrance of spontaneous colitis in 6 mo period. Hsp70^−/− mice were bred with IL10^−/− mice to obtain IL10^−/−/Hsp70^−/− mice that had 100% penetrance of spontaneous colitis (10/10) at 6 mo old. Epithelial specific expression of Hsp70 in IL10^−/−/Hsp70^−/− double knockout mice could rescue the phenotype as IL10^−/−/Hsp70^−/−/V-TG mice and had the similar penetrance of colitis (2/7) as IL10^−/− mice (3/12). B: colitis scores compared among groups at 6 mo of age. C: representative colonic histological features of spontaneous colitis. Scale bars represent 50 μm. D–G: colonic mucosal TNFα, IFNγ, IL-6, and IL-17 were increased in those mice with colitis compared with mice without colitis from different groups. The level was not different between IL10^−/−/Hsp70^−/− mice with colitis and IL10^−/− mice with colitis. Box plot diagram shows minimum and maximum of all of cytokine expression; n ≥ 7 in each group, and data were analyzed by one-way ANOVA with Bonferroni’s multiple comparison tests.

Fig. 10.

Intracellular Hsp70 plays dual protective functions during colitis.