In this study, we described whole transcriptomic genes changes in bovine endometrial stromal cells treated with LPS compared with control group by RNA-seq. Stromal cells are exposed to an inflammatory environment when epithelial cells are disrupted (Figure 6), inflammation on endometrial stromal cells is activated (Figure 7 ) [1, 10]. Results indicate that more than 97% of clean tags of RNA-seq results mapped to the genome and PCA was low, which proved our data could be used for functional analyses.
LPS binds to lipoprotein binding protein (LBP), which presents it to CD14 receptor. This induces CD14 to present LPS-LBP complex to MD-2 (myeloid differentiation factor 2) [17, 18]. This, in turn, promotes dimerization of toll-like receptor 4 (TLR4)/MD-2, activating two downstream signaling pathways including MyD88-dependent pathway (MyD88-) and TRIF-dependent pathway. The former triggers NF-κB and mitogen-activated protein kinase (MAP kinase) signaling and induces inflammatory cytokines, and the latter causes the induction of type I interferons (IFN) through interferon regulatory factor 3 (IRF3) activation and inflammatory cytokines through NF-κB activation [17]. Our data presents evidence for the activation of the MyD88-independent pathway as LPS upregulated inflammatory cytokines, such as IL6, IL1A, CXCL8 and activated NF-κB pathway (Additional file 2: Table S1 and Additional file 4: Table S3). Bovine endometrial epithelial and stromal cells express the TLR4/CD14/MD2 receptor complex. We found increased expression levels of CD14 but constant levels of TLR4 or MyD88 in our study in which bovine stromal cells were treated for 12 h with 0.5 µg/mL LPS (Additional file 2: Table S1), consistent with previous studies that exposed mixed bovine epithelial and stromal endometrial cells to 100 ng/ml LPS for 6 h [19]. In addition, LPS upregulated TLR4 only in bovine endometrial epithelial cells which were treated with 1 µg/mL LPS for 6 h and 12h, and the expression was greatest at 12h [20].
After the membrane surface receptor is recognized, intracellular inflammation is recognized and intracellular signaling cascades are activated by LPS, which is mediated by innate pattern recognition receptors (PRR) including TLR, retinoic acid-inducible gene-I-like (RIG-I-like) receptors, NOD-like receptors, and C-type lectin receptors [21]. These lead to the expression of inflammatory mediators and contribute to the clearance of pathogens [22]. Our data further confirm these findings because LPS triggers NOD-like receptor signaling pathway, TLR signaling pathway, and C-type lectin receptor signaling pathway in bovine endometrial stromal cells (Additional file 4, Table S3). In addition, our RNA-seq results also demonstrate the upregulation of expression of inflammatory and chemotactic cytokines related genes, such as IL6, IL1A, IL2RB, CCL2, CCL5, CXCL5, CXCL8, CXCL2, CX3CL1, and CCL20 (Additional file 2: Table S1). This is also consistent with previous studies that mRNA of proinflammatory cytokines IL1A, IL1B, IL6, TNF, and expression of chemokines IL8 and CXCL5 increased in endometrial epithelial cells during the estrous cycle and subclinical or clinical endometritis [23, 24]. The expression of these cytokines was also upregulated in only epithelial cells, only stromal cells, and mixed epithelial and stromal cells treated with LPS in vitro [12, 19, 25]. On the other hand, activation of PRR also triggers many signal transduction pathways through one or more of the IRF family of transcription factors, leading to the expression of IFNs [26]. LPS upregulated the expression IFN-stimulated genes (ISG) including RSAD2, MX2, OAS1Y, ISG15, and BST2 in mixed epithelial and stromal cells [19]. In the present study, our results show that LPS upregulated the expression of ISG15 and IRF1, 3.71 fold and 2.36 fold, respectively (Additional file 2: Table S1).
Local immune responses are activated which result in expression of proinflammatory cytokines when the uterus is exposed to bacteria. This is followed by production of antimicrobial peptides (AMP) and acute phase proteins by epithelial and innate immune cells [10]. Many of AMP genes are expressed in bovine endometrium, including lingual antimicrobial peptide (LAP), tracheal antimicrobial peptide (TAP), and some β-defensins [10, 27, 28]. Defensins are small cationic polypeptides, which have antimicrobial and immunomodulatory properties and are secreted by many cell types [29]. Some studies have identified an increase in expression levels of TAP, LAP, DEFB1, and DEFB5 in endometrium of cows with serious inflammation or following LPS treatment in vitro [20, 25, 30]. Other studies showed LAP, TAP, neutrophil β-defensins (BNBD4, DEFB5) were all upregulated in bovine epithelial cells but not stromal cells with LPS [8, 10]. In the present study, we found DEFB10 was upregulated 2.79 fold in bovine stromal cells with LPS (Additional file 2: Table S1)..
Cascade reaction of complement signals also plays a key role in immune defense, and complement activation lead to opsonization of pathogens and their removal by phagocytes as well as cell lysis [31]. Some studies pointed out that complement factors C2 and CFB increased in primary cultures of mixed bovine epithelial and stromal endometrial cells with LPS [19]. Our results agreed with previous studies and identified that the expression of other complement genes were upregulated, such as C3, C1S, C1R, CF1, and C4A (Additional file 2: Table S1). LPS can change expression level of many genes involved in cell adhesion, such as VCAM1, SELP, CADM2, HEPACAM, and SDK2 (Additional file 2: Table S1). Leucocytes attach to cell adhesion molecules (CAM) on endothelial cells and are involved in inflammation and immune function [32]. Each CAM has an inherent effect on immune response process, such as vascular cell adhesion molecule-1 (VCAM-1), which promotes firm binding of T cells and induces trans-migration [33] In addition, there were reports in the 80s that CAMs may play a role in inherited inflammatory disease, which is characterized by deficient leucocyte CAMs [34].
This study also proves that that LPS can alter other gene expression levels in stromal cells, such as hyaluronan synthase 2 (HAS2), which showed 3.36 fold increase in expression. It belongs to the hyaluronan (HA) family, holds extracellular matrix (ECM) polysaccharides and has multiple functions, such as tissue repair and promoting the expression of inflammation-related genes including TNF, IL12, IL1B, and MMPs [35]. Six-transmembrane epithelial antigen of prostate 4 (STEAP4), which showed 81.57 fold increase in expression, is an anti-inflammatory protein induced by inflammatory signals such as TNFα and IL6 [36-38]. Notably, mice with STEAP4-knockout are susceptible to insulin resistance, hyperglycemia, and inflammation [39].
In brief, we identified a total of 366 DEGs in bovine endometrial stromal cells in LPS-induced group as compared to the control group. We analyzed the interactions between LPS and stromal cells including interaction of LPS with surface receptors of stromal cells and intracellular signaling cascades activated by PRRs. These indicate that stromal cells play a key role in bovine endometrium reaction to foreign intruders by initiating or inhibiting biochemical and molecular signals. In addition, our results identified many important immune-related genes and signaling pathways, especially, IL6, IL1A, IL2RB, CCL2, CCL5, CXCL5, CXCL8, CXCL2, and CX3CL1, and TNF signaling pathway, NF-κB signaling pathway. Our results also expound the cellular processes in the endometrium and may serve as a diagnostic norm of defining and detecting endometritis, which may assist in development of more targeted and effective vaccines and drugs for the prevention and treatment of bovine endometritis. Furthermore, our analysis paves the way for the future elucidation of molecular mechanism of microbial invasion and host cell response. In our next study, we will continue to analyze and validate immune-related proteins and KEGG signaling pathways by western blot and compare genes and proteins in stromal cells treated with LPS in vitro with stromal cells from bovine endometritis with subclinical or clinical disease. These can better elucidate characteristics of bovine endometritis.