Progranulin Improves Acute Lung Injury through Regulating the Differentiation of Regulatory T Cells and Interleukin-10 Immunomodulation to Promote Macrophage Polarization

Abstract

Progranulin (PGRN), which plays an anti-inflammatory role in acute lung injury (ALI), is promising as a potential drug. Studies have shown that regulatory T cells (Tregs) and interleukin- (IL-) 10 can repress inflammation and alleviate tissue damage during ALI. In this study, we built a lipopolysaccharide- (LPS-) induced ALI mouse model to illustrate the effect of PGRN on regulation of Treg differentiation and modulation of IL-10 promoting macrophage polarization. We found that the proportion of Tregs in splenic mononuclear cells and peripheral blood mononuclear cells was higher after treatment with PGRN. The increased proportion of Tregs after PGRN intratracheal instillation was consistent with the decreased severity of lung injury, the reduction of proinflammatory cytokines, and the increase of anti-inflammatory cytokines. In vitro, the percentages of CD4⁺CD25⁺FOXP3⁺ Tregs from splenic naïve CD4⁺ T cells increased after PGRN treatment. In further research, it was found that PGRN can regulate the anti-inflammatory factor IL-10 and affect the polarization of M1/M2 macrophages by upregulating IL-10. These findings show that PGRN likely plays a protective role in ALI by promoting Treg differentiation and activating IL-10 immunomodulation.

Figure 1

PGRN has an anti-inflammatory role in LPS-induced ALI. C57BL/6 mice were randomly divided into WT, WT+LPS, WT+LPS+PGRN, and PGRN-deficient (PGRN^−/−) mice with a C57/BL6 background which were randomly divided into the PGRN^−/−+LPS and PGRN^−/−+LPS+PGRN groups (n = 5/group). (a) The lungs from each experimental group were processed for histological examination after H&E staining. Compared with the WT group, thickened alveolar wall, alveolar hemorrhage and collapse, and inflammatory cell in filtration were less severe and were treated with PGRN 30 min after LPS challenge. The trend was the same in the PGRN^−/− group. (b) PGRN alleviates pulmonary edema in the LPS-induced mouse model. Pulmonary edema was measured by lung W/D weight ratio. (c) PGRN plays a potential anti-inflammatory role in the LPS-induced ALI mouse model. IL-1β, IL-6, IL-10, IL-17A, TNF-α, and CXCL1 expression levels in sera of mice were detected using a Mouse Cytokine/Chemokine Magnetic Bead Panel Kit. PGRN downregulated the production of proinflammatory cytokines/chemokines in plasma, including IL-1β, IL-6, IL-17A, TNF-α, and CXCL1, and upregulated the expression of the anti-inflammatory cytokine IL-10 in plasma in all experimental groups. ns: not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 by the one-way ANOVA followed by the Tukey Post Hoc test comparing the WT, WT+LPS, WT+LPS+PGRN, PGRN^−/−+LPS, and PGRN^−/−+LPS+PGRN groups.

Figure 2

PGRN ameliorated LPS-induced ALI through antiapoptosis, inflammatory infiltration, and neutrophil aggregation. C57BL/6 mice were randomly divided into WT, WT+LPS, WT+LPS+PGRN, and PGRN-deficient (PGRN^−/−) mice with a C57/BL6 background which were randomly divided into the PGRN^−/−+LPS and PGRN^−/−+LPS+PGRN groups (n = 5/group). (a) PGRN had an antiapoptotic effect on the lungs in the LPS-induced ALI mouse model. Apoptosis was detected by TUNEL staining, and strongly positive apoptosis appeared in the lungs of the WT+LPS and PGRN^−/−+LPS groups compared with the WT group. Apoptosis was reduced in mice treated with PGRN. (b) PGRN exerted a protective effect on the LPS-induced ALI mouse model, at least in part by IL-10 immune modulation. The expression of IL-10 in the lung tissues increased significantly in the WT+LPS and PGRN^−/−+LPS groups compared to the WT group. After the intervention of PGRN, IL-10 expression declined significantly. (c) PGRN reduced the level of MPO-producing neutrophils and played a protective role in the LPS-induced ALI mouse model. (d) PGRN reduced the total number of PMNs in BALF in the LPS-induced mouse models. ns: not significant; ∗∗p < 0.01and ∗∗∗∗p < 0.0001 by one-way ANOVA followed by the Tukey Post Hoc test comparing the WT, WT+LPS, WT+LPS+PGRN, PGRN^−/−+LPS, and PGRN^−/−+LPS+PGRN groups. Representative data are shown.

Figure 3

PGRN treatment increased CD4⁺CD25⁺Foxp3⁺ Treg proportions significantly in PBMCs and splenic MNCs of the WT+LPS and PGRN^−/−+LPS groups. (a, b) CD4⁺CD25⁺Foxp3⁺ Tregs in PBMCs and splenic MNCs were detected by flow cytometry, and the proportions were analysed using FlowJo. ns: not significant; ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001 by one-way ANOVA followed by Tukey Post Hoc test comparing the WT, WT+LPS, WT+LPS+PGRN, PGRN^−/−+LPS, and PGRN^−/−+LPS+PGRN groups. In each group, n = 5; three replicate experiments were performed three times, and the results were in good agreement. Representative data are shown.

Figure 4

PGRN treatment reduced M1 macrophage proportions but increased M2 macrophage proportions in the lungs of the LPS-induced ALI mouse model and showed a protective role in ALI. Macrophages were fixed and immunolabelled with a pan-macrophage marker (F4/80: green fluorescence) and strong indicators of M1 (iNOS: red fluorescence) and M2 (CD206: pink fluorescence) phenotypes. Five 400-fold fields of positive cells were counted and analysed. ns: not significant; ∗∗p < 0.01 and ∗∗∗∗p < 0.0001 by one-way ANOVA followed by the Tukey Post Hoc test comparing the WT, WT+LPS, WT+LPS+PGRN, PGRN^−/−+LPS, and PGRN^−/−+LPS+PGRN groups. In each group, n = 5; three replicate experiments were performed three times, and the results were in good agreement. Representative data are shown.

Figure 5

PGRN promotes Treg differentiation of naive CD4⁺ T cells in vitro and improves the polarization of M1/M2 macrophages. (a) PGRN significantly promotes the differentiation of CD4⁺CD25⁺Foxp3⁺ Tregs from CD4⁺ naïve T cells. CD4⁺ naïve T cells were sorted from the spleens of wild-type C57BL/6 mice and cultured. Tregs were detected by flow cytometry on day 3 and analysed by FlowJo. ∗∗p < 0.01 by Student's t test comparing controls and the PGRN intervention group. (b) PGRN and IL-10 can affect the polarization of M1/M2 macrophages. M1/M2 types were examined by flow cytometry on day 3 and analysed by FlowJo. ∗∗∗∗p < 0.0001 by one-way ANOVA followed by the Tukey Post Hoc test comparing the control, LPS, LPS+IL-10, LPS+PGRN, and LPS+IL-10+PGRN groups. In each group, n = 5; three replicate experiments were performed three times, and the results were in good agreement. Representative data are shown.