MSC-ACE2 Ameliorates Streptococcus uberis-Induced Inflammatory Injury in Mammary Epithelial Cells by Upregulating the IL-10/STAT3/SOCS3 Pathway

Abstract

In the dairy industry, Streptococcus uberis (S. uberis) is one of the most important pathogenic bacteria associated with mastitis in milk-producing cows, causing vast economic loss. To date, the only real effective method of treating and preventing streptococcal mastitis is antimicrobial therapy. In many inflammatory diseases, mesenchymal stem cells (MSCs) and angiotensin-converting enzyme 2 (ACE2) play an anti-inflammatory and anti-injurious role. Accordingly, we hypothesized that MSCs overexpressing ACE2 (MSC-ACE2) would ameliorate the inflammatory injury caused by S. uberis in mammary epithelial cells more efficiently than MSC alone. By activating the transcription 3/suppressor of cytokine signaling 3 (IL-10/STAT3/SOCS3) signaling pathway, MSC-ACE2 inhibited the NF-κB, MAPKs, apoptosis, and pyroptosis passways. Moreover, MSC-ACE2 overturned the downregulation of Occludin, Zonula occludens 1 (ZO-1), and Claudin-3 expression levels caused by S. uberis, suggesting that MSC-ACE2 promotes the repair of the blood-milk barrier. MSC-ACE2 demonstrated greater effectiveness than MSC alone, as expected. Based on these results, MSC-ACE2 effectively inhibits EpH4-Ev cell's inflammatory responses induced by S. uberis, and would be an effective therapeutic tool for treating streptococcal mastitis.

Keywords: MSC-ACE2; S. uberis; blood-milk barrier; inflammatory injury; mammary epithelial cells; pyroptosis.

Figure 1

Effect of MSC-ACE2 on the secretion level of inflammatory mediators in EpH4-Ev cells. (A-C) Detection of relative transcript levels of TNF-α, IL-1β, and IL-6 in EpH4-Ev cells by qPCR. TNF-α, tumor necrosis factor-α; IL-Iβ, interleukin-Iβ; IL-6, interleukin-6. (D-F) Detection of TNF-α, IL-1β, and IL-6 concentration in cell culture supernatants by ELISA. Experiments were repeated three times and data were presented as the mean ± SEM (n = 4). ^* P < 0.05 vs. EpH4-Ev; ^# P < 0.05 vs. S. uberis; ^$P < 0.05 vs. MSC; ^& P < 0.05 vs. MSC-GFP.

Figure 2

Effect of MSC-ACE2 on NAGase activity and bacterial load in EpH4-Ev cells. (A) Detection of NAGase activity in cell culture supernatant according to kit instructions. (B)The number of S. uberis colonies in EpH4-Ev cells. Experiments were repeated three times and data were presented as the mean ± SEM (n = 4). ^* P < 0.05 vs. EpH4-Ev; ^# P < 0.05 vs. S. uberis; ^$P < 0.05 vs. MSC; ^& P < 0.05 vs. MSC-GFP.

Figure 3

The effects of MSC-ACE2 on EpH4-Ev cells’ secretion of Ang II, Ang-(1–7). (A) Detection of Ang II concentration in cell culture supernatants by ELISA. (B) Detection of Ang-(1–7) concentration in cell culture supernatants by ELISA. Experiments were repeated three times and data were presented as the mean ± SEM (n = 4). ^* P < 0.05 vs. EpH4-Ev; ^# P < 0.05 vs. S. uberis; ^$P < 0.05 vs. MSC; ^& P < 0.05 vs. MSC-GFP.

Figure 4

MSC-ACE2 inhibited EpH4-Ev cells apoptosis induced by S. uberis. (A) Apoptosis kit to detect apoptosis rate. (B) Total apoptosis ratio of EpH4-Ev cells. (C) Detection of relative protein expression levels of Caspase-3, Bax, and Bcl-2 by Western blot. (D) Statistics of the Caspase-3, Bax, and Bcl-2 Western blot results. Experiments were repeated three times and data were presented as the mean ± SEM (n = 3). ^* P < 0.05 vs. EpH4-Ev; ^# P < 0.05 vs. S. uberis; ^$P < 0.05 vs. MSC; ^& P < 0.05 vs. MSC-GFP.

Figure 5

MSC-ACE2 ameliorated S. uberis-induced Eph4-Ev cells pyroptosis. (A) Detection of relative transcript levels of ASC and IL-18 by qPCR (n = 4). (B) Detection of relative protein expression levels of NLRP3, cleaved-Caspase-1 (p20), ASC, cleaved GSDMD, and cleaved-IL-Iβ by Western blot. (C) Statistics of NLRP3, cleaved-Caspase-1 (p20), ASC, cleaved GSDMD, and cleaved-IL-Iβ Western blot results (n = 3). Experiments were repeated three times and data were presented as the mean ± SEM. ^* P < 0.05 vs. EpH4-Ev; ^# P < 0.05 vs. S. uberis; ^$P < 0.05 vs. MSC; ^& P < 0.05 vs. MSC-GFP.

Figure 6

Effect of MSC-ACE2 on IL-10/STAT3/SOCS3 signaling pathway. (A) Detection of relative protein expression levels of IL-10, phosphorylation levels of STAT3, STAT3, and SOCS3 by Western blot. (B-D) Statistics of the IL-10, phosphorylation levels of STAT3, STAT3, and SOCS3 Western blot results (n = 3). (E) Detection of relative transcript levels of IL-10 and SOCS3 in EpH4-Ev cells by qPCR (n = 4). Experiments were repeated three times and data were presented as the mean ± SEM. ^* P < 0.05 vs. EpH4-Ev; ^# P < 0.05 vs. S. uberis; ^$P < 0.05 vs. MSC; ^& P < 0.05 vs. MSC-GFP.

Figure 7

Effect of MSC-ACE2 on NF-κB and MAPKs signaling pathways. (A) Detection of relative protein expression levels of p65 and p-p65 by Western blot. (B) Statistics of p65 and p-p65 Western blot results. (C) Detection of relative protein expression levels of p38, ERK, JNK, and p-p38, p-ERK, p-JNK by Western blot. (D-F) Statistics of p38, ERK, JNK, and p-p38, p-ERK, p-JNK Western blot results. Experiments were repeated three times and data were presented as the mean ± SEM (n = 3). ^* P < 0.05 vs. EpH4-Ev; ^# P < 0.05 vs. S. uberis; ^$P < 0.05 vs. MSC; ^& P < 0.05 vs. MSC-GFP.

Figure 8

MSC-ACE2 reversed the S. uberis-induced downregulation of the expression abundance of blood-milk barrier-associated proteins. (A) Detection of relative transcript levels of ZO-1, Occludin, and Claudin-3 in EpH4-Ev cells by qPCR (n = 4). (B) Detection of relative protein expression levels of ZO-1, Occludin, Claudin-3 by Western blot; (C) Statistics of the ZO-1, Occludin, Claudin-3 Western blot results (n = 3). Experiments were repeated three times and data were presented as the mean ± SEM. ^* P < 0.05 vs. EpH4-Ev; ^# P < 0.05 vs. S. uberis; ^$P < 0.05 vs. MSC; ^& P < 0.05 vs. MSC-GFP.

Figure 9

Schematic representation of MSC-ACE2 ameliorate S. uberis-induced Inflammatory Injury in Mammary Epithelial Cells by upregulating the IL-10/STAT3/SOCS3 Pathway (Charting with BioRender.com software). Treatment of EpH4-Ev cells with S. uberis at MOI = 10 for 3 h caused EpH4-Ev cells pyroptosis, activated MAPKs/NF-κB signaling pathways, promoted the release of inflammatory factors TNF-α, IL-6, IL-Iβ, and IL-18, induced apoptosis, and disrupted the blood-milk barrier. Co-culture with MSC, MSC-GFP or MSC-ACE2 activated the IL-10/STAT3/SOCS3 signaling pathway and inhibited MAPKs/NF-κB, apoptosis, and pyroptosis pathways. Furthermore, MSC-ACE2 reversed the S. uberis-induced downregulation of Occludin, ZO-1, and Claudin-3 expression levels and promoted blood-milk barrier repair. MSC-ACE2 had a better effect than MSC alone.