Lactobacillus reuteri Alleviates Hyperoxia-Induced BPD by Activating IL-22/STAT3 Signaling Pathway in Neonatal Mice

Abstract

Bronchopulmonary dysplasia (BPD) is the most common chronic respiratory disease in preterm infants. Little is known about the regulatory effect of lung Lactobacillus and its mechanism in BPD. This study explored the effect of L. reuteri on hyperoxia-induced mice lung injuries and examined whether L. reuteri played a role via the IL-22/STAT3 pathway. We found that the intranasal administration of L. reuteri and its tryptophan metabolite indole-3-aldehyde (3-IAld) ameliorated hyperoxia-induced mice lung BPD-like changes, deceased proinflammatory cytokines (IL-1β, IL-6, and TNF-α), and increased the levels of surfactant-associated protein C (SPC), aquaporin 5 (AQP5), and vascular endothelial growth factor receptor 2 (VEGFR2, also known as FLK-1). Furthermore, L. reuteri and 3-IAld increased the expression of IL-22. IL-22 was also confirmed to ameliorate hyperoxia-induced mice lung pathological changes, and the protective effects of L. reuteri could be inhibited by anti-IL-22 neutralizing antibody. Finally, we confirmed STAT3 activation by IL-22 in MLE-12 cells. In summary, our study confirmed L. reuteri alleviated hyperoxia-induced lung BPD-like changes in mice by activating the IL-22/STAT3 signaling pathway via IL-22 production. Probiotics Lactobacillus is a potential treatment for hyperoxia-induced lung injury in newborns.

Keywords: IL-22/STAT3 signaling pathway; L. reuteri; bronchopulmonary dysplasia; lung microbiota.

Figure 1

L. reuteri ameliorates hyperoxia-induced BPD-like changes and lung inflammation in mice. (A) H&E staining of lung tissue from the control, hyperoxia, hyperoxia+L, and hyperoxia+3-IAld groups (400×). (B) MLI and RAC analyses were performed to evaluate alveolarization in each group. The MLI and RAC values represent the average of five visual fields in the same section (n = 5). (C) Comparison of body weight was performed on P17 (n = 15). (D) ELISAs of IL-1β, IL-6, and TNF-α levels in each group (n = 6). The data are presented as means ± SDs. All the statistical analyses were performed by ordinary one-way ANOVA following normality and homogeneity tests. ⁣^∗p < 0.05, ⁣^∗∗p < 0.01, ⁣^∗∗∗p < 0.001, ⁣^∗∗∗∗p ≤ 0.0001. BPD, bronchopulmonary dysplasia; ELISAs, enzyme-linked immunosorbent assays; MLI, mean linear intercept; RAC, radical alveolar count.

Figure 2

L. reuteri increases the expression of SPC, AQP5, FLK-1, and VEGF in mice. (A) Western blot analysis of SPC, AQP5, and FLK-1 expression in the control, hyperoxia, hyperoxia+L, and hyperoxia+3-IAld groups (n = 3). (B) qRT-qPCR was used to assess the relative mRNA expression of SPC, AQP5, and VEGF in each group (n = 5). The data are presented as means ± SDs. All the statistical analyses were performed by ordinary one-way ANOVA following normality and homogeneity tests. ⁣^∗p < 0.05, ⁣^∗∗p < 0.01,⁣^∗∗∗p < 0.001, ⁣^∗∗∗∗p ≤ 0.0001. 3-IAld, indole-3-aldehyde; AQP5, aquaporin 5; SPC, surfactant-associated protein C; VEGF, vascular endothelial growth factor.

Figure 3

L. reuteri and 3-IAld upregulate IL-22 expression in mice. ELISA of IL-22 expression levels in mice treated with PBS, L. reuteri or 3-IAld under normoxia (21% O₂) or hyperoxia (85% O₂) (n = 6). The statistical analyses were performed by ordinary two-way ANOVA. ⁣^∗p < 0.05, ⁣^∗∗p < 0.01,⁣^∗∗∗p < 0.001, ⁣^∗∗∗∗p ≤ 0.0001. 3-IAld, indole-3-aldehyde; ELISAs, enzyme-linked immunosorbent assay.

Figure 4

IL-22 has a protective effect against hyperoxia-induced lung injury in mice. (A) H&E staining of lung tissue from the control, hyperoxia, and hyperoxia+IL-22 groups (400×). The MLI and RAC were measured to evaluate alveolarization in each group. The MLI and RAC values are the average of five visual fields in the same section (n = 5). (B) Comparison of mouse body weights at P17 showed that IL-22 treatment reduced weight loss (n = 14). (C) ELISAs were used to measure the expression levels of IL-1β, IL-6, and TNF-α in the control, hyperoxia, and hyperoxia+IL-22 groups (n = 6). (D) Western blot analysis of SPC, AQP5, and FLK-1 expression in the control, hyperoxia, and hyperoxia+IL-22 groups (n = 3). (E) RT-qPCR was used to determine the relative mRNA expression of SPC, AQP5, and VEGF in the control, hyperoxia, and hyperoxia+IL-22 groups (n = 5). The data are presented as means ± SDs. All the statistical analyses were performed by ordinary one-way ANOVA following normality and homogeneity tests. ⁣^∗p < 0.05, ⁣^∗∗p < 0.01, ⁣^∗∗∗p < 0.001, ⁣^∗∗∗∗p ≤ 0.0001. AQP5, aquaporin 5; ELISAs, enzyme-linked immunosorbent assays; MLI, mean linear intercept; RAC, radical alveolar count; SPC, surfactant-associated protein C; VEGF, vascular endothelial growth factor.

Figure 5

Anti-IL-22 neutralizing antibody inhibits the protective effect of L. reuteri on hyperoxia-induced lung injury in mice. (A) H&E staining of lung tissue from the control, hyperoxia, hyperoxia+L, and hyperoxia+L+anti-IL-22 groups (400×). Comparison of the MLI and RAC values for each group. The MLI and RAC values are the average of five visual fields in the same section (n = 5). (B) Comparison of the body weights of the mice in each group (n = 15). (C) Comparison of the expression levels of IL-1β, IL-6, and TNF-α in each group (n = 6). (D) Western blot analysis of SPC and FLK-1 expression in the control, hyperoxia, hyperoxia+L, and hyperoxia+L+anti-IL-22 groups (n = 3). (E) RT-qPCR results showing SPC and VEGF expression in the control, hyperoxia, hyperoxia+L, and hyperoxia+L+anti-IL-22 groups (n = 5). The data are presented as means ± SDs. All the statistical analyses were performed by ordinary one-way ANOVA following normality and homogeneity tests. ⁣^∗p < 0.05,⁣^∗∗p < 0.01, ⁣^∗∗∗p < 0.001, ⁣^∗∗∗∗p ≤ 0.0001. MLI, mean linear intercept; ns, not significant; RAC, radical alveolar count; SPC, surfactant-associated protein C.

Figure 6

IL-22 activates the pSTAT3 signaling pathway in MLE-12 cells. (A) Cell viability assessment of cells after treatment with different concentrations (0, 10, 30, 50, 70, and 100 ng/mL) of IL-22 (n = 7). (B) Western blot analysis of STAT3 and pSTAT3 expression in the normoxia, normoxia+IL-22, normoxia+IL-22+anti-IL-22, hyperoxia, hyperoxia+IL-22, and hyperoxia+IL-22+anti-IL-22 groups (n = 3). The data are presented as means ± SDs. All the statistical analyses were performed by ordinary one-way ANOVA following normality and homogeneity tests. ⁣^∗p < 0.05, ⁣^∗∗p < 0.01, ⁣^∗∗∗p < 0.001, ⁣^∗∗∗∗p ≤ 0.0001, ns, not significant.