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Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis
Author’s Accepted Manuscript Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis Wenyao Zhang, Xuezhong Li, Tong Xu, Ma Mengru, Yong Zhang, Ming-Qing Gao www.elsevier.com/locate/yexcr
PII: DOI: Reference:
S0014-4827(16)30307-X http://dx.doi.org/10.1016/j.yexcr.2016.09.016 YEXCR10345
To appear in: Experimental Cell Research Received date: 27 June 2016 Revised date: 11 September 2016 Accepted date: 24 September 2016 Cite this article as: Wenyao Zhang, Xuezhong Li, Tong Xu, Ma Mengru, Yong Zhang and Ming-Qing Gao, Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis, Experimental Cell Research, http://dx.doi.org/10.1016/j.yexcr.2016.09.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis Wenyao Zhanga, Xuezhong Lia, Tong Xua, Mengru, Maa, Yong Zhanga,b and Ming-Qing Gaoa,b a
College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China b
*Corresponding author: E-mail: [email protected] (MQ. Gao) or [email protected] (Y. Zhang).
Abbreviations: LPS, lipopolysaccharide; LTA, lipoteichoic acid; S. aureus, Staphylococcus aureus; E. coli, Escherichia coli; BMEs, bovine mammary gland epithelial cells; BMFs, bovine mammary stromal fibroblasts; ECM, extracellular matrix; TNF-α, tumor necrosis factor-alpha; CCK-8, Cell Counting Kit-8; IL-6, interleukin6; IL-8, interleukin- 8; CXCL2, Chemokine (C-X-C motif) ligand 2; CCL5, Chemokine (C-C motif) ligand 5; α-SMA, α-smooth muscle actin; PDGFR-α, growth factor receptor α; PDGFR-β, growth factor receptor β; EGFR, epidermal growth factor receptor; GSK-3β, Glycogen synthase kinase-3β; HMGB1, high mobility group box-1 protein; ERK, extracellular signal-regulated kinase
Abstract Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. Keywords: mastitis; fibroblasts; epithelial cells; lipopolysaccharide; lipoteichoic acid; bovine
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1. Introduction Mastitis is one of the most critical diseases in dairy production. Mastitis is a mammary gland inflammation caused by changes in metabolism, physiological trauma, or contagious or environmental pathogenic microorganisms.[1] Invasive pathogens are the main causes of mastitis. Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are two of the most important pathogens causing bovine mastitis.[2] Lipopolysaccharides (LPS), the major structural elements of the E. coli cell membrane, trigger strong immune responses. Lipoteichoic acid (LTA), an important cell wall component of S. aureus, causes septic shock and multiple organ apoptosis. Therefore, these two important antigens might be used as conditional factors to induce inflammatory responses. Neutrophils are the first line of defense against bacterial invasion in the bovine mammary gland.[2] During bacterial invasion, bovine mammary gland epithelial cells (BMEs) initiate neutrophil recruitment through inflammatory and immune responses, including increased pro-inflammatory cytokine secretion.[3, 4] Tumor necrosis factor alpha (TNF-α) is the main pro-inflammatory cytokine producing inflammatory responses and promoting the release of various inflammatory mediators by multifarious cell types.[5] TNF-α induces fibroblasts to produce granulocyte macrophage colony stimulating factors and promotes granulocyte and macrophage progenitor maturation.[6] Proteins of TNF and the TNF receptor family play important roles in the control of cell death, proliferation, autoimmunity, the function of immune cells, and the organogenesis of lymphoid organs.[7] Dairy cow mammary glands mainly consist of BMEs and bovine mammary stromal fibroblasts (BMFs). BMEs play an important role in inducing a relevant innate immune response in mammary glands, act as sentinels, and signal invading mastitis-causing pathogens.[2] In bovine mastitis, bacterial infection first occurs in the epithelial cells of mammary gland crypts.[1, 2, 8] During the early stages of infection, epithelial cells release immunological factors to prevent the production and development of inflammation and also release inflammatory mediators to affect adjacent cells.[1, 2, 8]. Fibroblasts are important stromal cells which activate and interact with infiltrating immune cells in a dynamic and site-specific manner. Fibroblasts also define the external tissue features, provide positional memory, and regulate the switch from resolving to persistent inflammation.[9] The regional identity of fibroblasts can be modified by inflammation.[10-13] Therefore, targeting fibroblasts and the stromal microenvironment are likely important methods in treating inflammation. Toll-like receptor 2 recognizes LTA, whereas toll-like receptor 4 recognizes LPS.[14, 15] Meanwhile, ligand binding activates a range of signaling molecules and releases transcription factors.[16, 17] Released transcription factors mediate cytokine, chemokine, and growth factor expression levels.[18] Inflammatory mediators of abnormal expression induces inflammatory cell repair. Many abnormal and uncontrolled repair mechanisms may lead to fibrosis and extracellular matrix (ECM) deposition. During the early phase of bovine mastitis, inflammatory mediators released from epithelial cells stimulate fibroblasts and other stromal cells.[19] Activated fibroblasts are also key inflammation mediators and mediate the 3
development of inflammation in bovine mastitis. Better knowledge of inflammatory epithelial cell reactions to BMFs could eventually lead to novel therapeutic approaches to combat the spread of inflammation during the early phase of bovine mastitis. With that aim in mind, this study used an in vitro model of mastitis to investigate the mechanism of inflammatory epithelial cell effects on BMF. The proliferation, apoptosis, migration, secretion of inflammatory cytokines, and ECM deposition of BMFs cultured in conditioned medium from LPS- or LTA- induced inflammatory BMEs were investigated. 2. Materials and methods 2.1 Cell Isolation and Culture BMEs and BMFs were obtained from Holstein cow mammary tissues as previously described.[20] Mammary tissues were individually sampled from eight Holstein dairy cows. All mammary glands were healthy with no obvious evidence of infection. Fresh tissues were digested in a solution of Enzyme Cocktail (ISU ABXIS, Seoul, Korea) and incubated at 37 °C overnight in a humidified 5% CO2 incubator. Digested tissue was filtered and centrifuged to yield pellets containing epithelial organoids and supernatants containing fibroblasts. The isolated epithelial cells and fibroblasts were seeded on cell culture dishes and cultured in complete DMEM/F12 medium (GIBCO BRL , Life Technologies, Burlington, ON) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 μg/ml streptomycin (Gibco BRL, Grand island, NY) at 37 °C in an incubator with 5% CO2. Cells were passaged by digestion with 0.15% trypsin and 0.02% EDTA. BMEs and BMFs were almost pure after three to four separation and culture passages. By the fifth passage, extracted BMEs or BMFs were used as cell pools in all subsequent experiments. Both BMEs and BMFs were prepared by mixing equivalent amounts of cells from all individuals. All reagents were purchased from Sigma-Aldrich Canada (Oakville, ON), unless otherwise stated. 2.2 Establishment of Inflammatory Cell Model An inflammatory cell model of mastitis was established with LPS- or LTA-treated BMEs. Tumor necrosis factor-alpha (TNF-α) released in the medium and mRNA expression in the treated cells were utilized as indicators for LPS- or LTA-induced immune responses. Before stimulation with LPS and LTA, confluent BMEs monolayers were cultured in fresh medium without FBS for 24 h at 37 °C. Then, the medium was replaced with complete medium containing LPS or LTA at final concentrations of 10 ng/μl or 20 ng/μl according to recommended concentrations. After stimulation for 3 h, 6 h, 12 h, and 24 h with LPS or LTA, cell culture supernatants were discarded and replaced with fresh medium without FBS. The culture was incubated again for 24 h. Finally, cell culture supernatants were collected for TNF-α secretion measurement and BMEs were harvested for RNA extraction and TNF-α real-time PCR analysis. BMEs cultured in media without FBS and without stimulation were designated as controls. The inflammatory cell model of mastitis was established under the shortest treatment period exhibiting significantly upregulated TNF-α expression in BMEs. BMEs treated with LPS and LTA were designated as LPS-BMEs and LTA-BMEs, respectively. 2.3 Indirect co-culture experiments 4
We investigated the effects of inflammatory epithelial cells induced by LPS or LTA on BMFs with an indirect co-culture model utilizing conditioned medium. In brief, LPS-BMEs or LTA-BMEs were rinsed with PBS. Fresh serum-free DMEM/F12 medium was then added to the culture dishes. Cells were cultured for another 24 h at 37°C under 5% CO2. Then, the medium was collected and cells debris was moved by centrifuge. Finally, the medium was used at a 9:1 ratio with fresh DMEM/F12 and 10% FBS as the conditioned medium. BMFs cultured in conditioned medium collected from un-stimulated BMEs were taken as control. For indirect co-culture, BMFs were cultured in LPS-BME- or LTA-BME-conditioned media. After day 3 of culture, cell culture supernatants were collected and BMFs were harvested for analysis. 2.4 RNA Extraction and Real-time PCR Total RNA was isolated from cells washed twice with PBS. Cells were re-suspended in ice-cold TriZol solution (TransGene, Shanghai, China). Cells were passed through a ribonuclease-free 20-gauge needle 10 times for cell disruption. Total mRNA extraction was performed with the RNA Easy Kit (TransGene) according to the manufacturer’s instructions. Total mRNA concentration was measured with a spectrophotometer (ND 2.0; NanoDrop Technologies, Wilmington, Delaware), and 1 μg of RNA was reverse-transcribed into cDNA with the TransScript II First-Strand cDNA Synthesis SuperMix (TransGene). Primers were designed based on sequences from the National Center for Biotechnology Information Database. Specificity was determined with Primer-BLAST. The primers are listed in Supplementary Table S1. All PCR primers were synthesized by Sangon Biotech (Shanghai, China). Real-time PCR was performed with an iQ5 light cycler (Bio-Rad, Hemel Hempstead, UK) using TransStart Probe qPCR SuperMix (TransGene) in 25-μl reactions containing 1 μmol/L concentrations of each forward and reverse primer (Sangon Biotech). PCR conditions were as follows: activation at 95 °C for 30 seconds, 40 cycles of denaturation at 95 °C for 5 seconds, and annealing/extension at 60 °C for 30 seconds. Melt curves were generated to estimate primer specificities. A standard curve was generated with reference cDNA to determine the starting quantity of mRNA in each sample. The expression of each target gene was normalized to that of GAPDH, the reference gene. Comparative Ct method was employed to quantify normalized target gene expression relative to the calibrator. Data were expressed as relative gene expression = 2-ΔΔCt. 2.5 Cell Viability Assay Cell viability was determined with CCK-8 kits (Beyotime, Shanghai, China) according to the manufacturer’s protocol. BMFs were evenly seeded into 96-well plates with 100 μl complete medium at a density of approximately 2x103 cells/well and incubated for 24 h. Then, the complete medium was replaced with BME-, LPS-BME-, or LTA-BME-conditioned medium. BMFs were then cultured in conditioned medium for 0, 1, 2, and 3 days. Before the cell viability assay, the medium was replaced with 100 μl fresh medium containing 10 μl of CCK-8 solution and incubated for an additional 3 h at 37 °C in a humidified incubator. The absorbance of samples in triplicate wells was measured at 450 nm wavelength with a microplate reader (Bio-Rad, Hercules, CA) at indicated time points. Cell proliferation rate was calculated by the formula [(ODn−ODblank)−(OD0−ODblank)]/(OD0−ODblank)×100, where ODn is 5
the optical density value at the indicated day after culture, OD0 is the optical density value at 0 day, and ODblank is the optical density value of the wells without cells. BME-conditioned medium was designated as the control. 2.6 Western Blotting Protein lysates from cells were prepared with PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Inc., Gyeonggi-do, Korea) according to the manufacturer’s instructions. Lysates were precipitated at 1200 rpm for 10 min. Total protein content was determined with Bradford Easy Protein Quantitative Kit (TransGene). Equal amounts of protein extracts in lysis buffer were subjected to SDS-PAGE on 4%–12% polyacrylamide gels (German, Sigma-Aldrich) then transferred to a nitrocellulose membrane. Resolved proteins were blotted onto PVDF transfer membranes and blocked with 10% non-fat milk in TBST. Membranes were incubated with anti-α-SMA (Abcam, Cambridge, MA, UK), anti-vimentin (Bioss, Beijing, China), anti-fibronectin (Bioss), anti-collagen I (Abcam), anti-E-cadherin (Abcam), anti-β-catenin (Abcam), anti-N-cadherin (Bioss), and anti-GAPDH (TransGene) antibodies at 4 °C overnight. Membranes were washed 3 times with TBST for 5 min before incubation with HRP-conjugated secondary antibodies. Finally, immunoreactive proteins were visualized with an enhanced chemiluminescence detection kit (Beyotime). 2.7 ELISA We detected BMF-secreted TNF-α, IL-6, IL-8, CXCL2, and CCL5 in the medium by ELISA. BMFs were cultured in fresh serum-free medium for another 24 h after being cultured in LPS-BME- or LTA-BME- conditioned media for 3 days. Then, the medium was collected and cells debris was removed by centrifuge. TNF-α, IL-6, IL-8, CXCL2, and CCL5 secreted in the medium were measured with corresponding ELISA kits (Huzhen Biological Technology Co., LTD, Shanghai, China) according to the manufacturer’s instructions. 2.8 Flow Cytometry BMF apoptotic rate was determined by flow cytometric techniques with a commercial apoptosis assay kit (TransGene). Co-cultured BMFs were harvested and uniformly re-suspended in 100 μl pre-cooled Annexin V Binding buffer prior to the addition of 5 μl Annexin V-FITC and 5 μl PI. Samples were gently mixed and incubated for 15 min under dark conditions and ambient temperature. Finally, cells were transferred to tubes containing an additional 300 μl of Annexin V binding buffer and analyzed by flow cytometry within an hour. 2.9 Cell Migration Assay BMFs were routinely cultured and grown to approximate confluence. Then, the BMF monolayer was wounded by a single scratch with a pipette tip. Media were replaced by conditioned medium from LPS-BME, LTA-BME and BME (control) conditioned media. Cells were incubated for 24 h to allow cell growth and wound healing. Cell migration capacity to fill the wounded area with cells was assessed by photographing the wound. 2.10 Statistical Analysis 6
Results were reported as mean ± s.d. All data were obtained from at least three independent experiments. All statistical analyses were performed with ANOVA (SPSS11.5 software). P < 0.05 was considered statistically significant. 3. Results 3.1 Establishment of Inflammatory Cell Model of Mastitis TNF-α was used as an index of BME inflammatory responses to LPS or LTA. We incubated BMEs with the recommended LPS or LTA concentrations for several different treatment periods. We found that TNF-α gene expression was significantly upregulated in BMEs challenged with 10 ng/μl LPS for 3 h or with 20 ng/μl LTA for 12 h (Fig. 1a). TNF-α secretion peaked at 3 h after treatment with 10 ng/μl LPS or at 12 h after treatment with 20 ng/μl LTA (Fig. 1b). Hence, LPS-treated BMEs (LPS-BMEs) and LTA-treated BMEs (LTA-BMEs) under the previously mentioned treatment conditions were used to mimic the bovine mammary cell gland epithelial cells infected by E. coli and S. aureus. 3.2 Inflammatory Epithelial Cells Activated BMFs. We previously reported that stromal fibroblasts derived from bovine mammary glands with mastitis were activated and displayed inflammation-specific changes.[20] The expression levels of α-smooth muscle actin (α-SMA) and vimentin, two indicators of activated fibroblasts, were examined in BMFs cultured in LPS-BME- and LTA-BME-conditioned media to validate whether fibroblast activation was directly achieved by infected epithelial cells during mastitis. We found that both LPS-BMEs and LTA-BMEs upregulated BMF α-SMA and vimentin expression levels (Figs. 2a and b), indicating that inflammatory epithelial cells activated BMFs. 3.3 Inflammatory Epithelial Cells Induced the Expression of Inflammatory Mediators in BMFs Fibroblasts have been suggested as vital immumoregulatory cells regulating immune response.[21] We examined the expression levels of several inflammatory mediators, including TNF-α, IL-6, IL-8, CXCL2, and CCL5 at both gene and protein levels to investigate if inflammatory epithelial cells induced fibroblast inflammatory response (Fig. 3). Results showed that both LPS-BMEs and LTA-BMEs upregulated mRNA expression levels of all the selected genes. Meanwhile, LPS-BMEs enhanced protein secretion of inflammatory mediators. However, LTA-BMEs only promoted TNF-α and CCL5 protein secretion in BMFs, indicating that a different mechanism may mediate interactions between mammary gland epithelial cells and fibroblasts in mastitis caused by E. coli and S. aureus. 3.4 Inflammatory Epithelial Cells Induced ECM Protein Expression in BMFs Fibroblasts are primarily responsible for ECM protein synthesis in tissue and play a critical role during repair in many organs.[22] We analyzed type I collagen and fibronectin expression levels by Western blotting and RT-PCR to determine whether inflammatory epithelial cells induced ECM deposition by BMFs in mammary glands with mastitis. Results showed that LTA-BMEs enhanced type I collagen and fibronectin mRNA (Fig. 4a) and protein expression levels (Fig. 4b). Although LPS-BMEs did not upregulate fibronectin gene 7
expression, it regulated type I collagen and fibronectin protein expression levels in BMFs. 3.5 Inflammatory Epithelial Cells Inhibited BMF growth We analyzed proliferation and apoptosis to investigate whether epithelial cells infected by bacteria affected the growth of stromal fibroblasts in bovine mammary glands with mastitis. Proliferation was measured and cell proliferation curves were generated. Results showed that on days 2 and 3, the proliferation rates of BMF cells cultured in conditioned medium from both LPS-BMEs and LTA-BMEs were lower compared to those of BMF cells cultured in control medium (Fig. 5a). However, we did not find a significant difference in cell apoptotic rates between all groups (Fig. 5b). We analyzed the gene expression levels of platelet-derived growth factor receptor (PDGFR) α, PDGFR-β, and epidermal growth factor receptor (EGFR) in BMFs by real-time PCR to explore possible signal molecules mediating the conditioned medium’s inhibitory effects on BMFs. As seen in Fig. 5c, the expression levels of all examined genes were downregulated in BMFs cultured in LPS-BME- or LTA-BME-conditioned media compared with the control medium. These results suggested that EGFR, PDGFR-α, and PDGFR-β contributed to the effect of inflammatory epithelial cells on stromal fibroblast proliferation. 3.6 Stimulation of BMFs Migration by Inflammatory Epithelial Cells The effect of inflammatory epithelial cells on BMF migration was analyzed with a scratch-recovery assay in a co-culture model. The scratched area in the BMF monolayer cultured in LPS-BME- or LTA-BME-conditioned media completely cicatrized at 18 h, whereas the scratched area in the control group completely closed at 24 h. Photomicrographs of scratched areas 12 h after scratching are presented in Fig. 6a and show that scratched areas in the control group were larger than in BMFs cultured in LPS-BME- or LTA-BME-conditioned media. This result indicated that inflammatory epithelial cells promoted BMF migration. The expression levels of several selected genes associated with cell migration were analyzed, including WNT1, E-cadherin, β-catenin, N-cadherin, GSK-3β, and HMGB1. We found that both LPS- and LTA-infected BMEs downregulated E-cadherin and GSK-3β mRNA expression levels and upregulated WNT1, β-catenin, and N-cadherin mRNA expression levels in BMFs. HMGB1 gene expression increased in BMFs cultured in LPS-BME-conditioned medium, but not in BMFs cultured in LTA-BME-conditioned medium. We also examined β-catenin, E-cadherin, and N-cadherin protein expression levels by Western blotting. Their protein expression levels were consistent with gene expression levels (Fig. 6b). 4. Discussion Epithelial cells are the first cell barriers resisting bacterial infections in mammary glands. When the organism cannot resist bacterial infections because of reduced immunity, epithelial cells are first infected by pathogenic bacteria. The infected cells release a large number of inflammatory mediators, including TNF-α, IL-6, and IL-8, which affect adjacent cells[23]. The present study investigated whether stromal fibroblasts responded to inflammatory epithelial cells in bovine mammary glands with mastitis. An inflammatory cell model of bovine mastitis was established by challenging epithelial cells with the gram-negative and gram-positive bacterial cell wall components LPS and LTA. BMF immune 8
responses to inflammatory epithelial cells were investigated with an in vitro co-culture model in which BMFs were cultured in LPS-BME- and LTA-BME-conditioned media. Our results showed that inflammatory epithelial cells affected BMF characteristics, decreasing proliferation capacity, enhancing migration ability, increasing expression of inflammatory mediators, and elevating deposition of ECM proteins. Changes in BMF proliferation and migration abilities mediated some signal molecules, such as PDGFR, EGFR, or components of WNT signal pathway. S. aureus and E. coli infections result in the upregulation of various pro-inflammatory cytokines.[24] TNF-α, a potent pro-inflammatory cytokine, exerts pleiotropic functions in immunity and control of cell proliferation, differentiation and apoptosis.[25] TNF-α expression has been observed in inflammatory response in different tissues, including mastitis.[26] Chu X et al. reported that TNF-α is the earliest and primary endogenous mediator of inflammatory response.[27] Therefore, we used TNF-α as an indicator to investigate whether BMEs produced inflammatory responses after LPS or LTA treatment. Peak cytokine secretion in gram-negative infection occurred within 1 hour to 5 hours after bacterial challenging, whereas time to peak secretion was extended significantly after gram-positive challenging.[28] In the current study, we found that TNF-α protein secretion and mRNA expression levels in BMEs peaked at 3 h after treatment with 10 ng/μl LPS at or at 12 h after treatment with 20 ng/μl LTA. Thus, we successfully established an inflammatory epithelial cell model of mastitis by challenging BMEs with LPS and LTA. In mastitis, inflammatory epithelial cells engage in reciprocal molecular dialogues with surrounding stromal cells, including inflammatory cells, vascular cells, and fibroblasts, resulting in the production of stromal-derived inflammation-aiding factors, such as growth factors, chemokines, cytokines, and proteases.[29] We found that inflammatory epithelial cells, regardless of treatment with LPS or LTA, activated BMFs, as indicated by upregulated mRNA and protein expression levels of α-SMA, an indicator of active fibroblasts.[30, 31] In addition, pathogen invasion of host cells commonly involves the rearrangement of the host cell’s actin cytoskeleton. α-SMA is an important fibroblast actin cytoskeleton gene and is upregulated in inflammatory response development.[32] Vimentin is another important actin component of the cell cytoskeleton. We found that vimentin was also upregulated in BMFs co-cultured with LPS-BMEs or LTA-BMEs. We examined the expression levels of several inflammation-related cytokines and chemokines, including TNF-α, IL-6, IL-8, CXCL2, and CCL5, in BMFs cultured in LPS-BME- or LTA-BME-conditioned media. We found that LPS-BMEs promoted the secretion of all these mediators in BMFs, whereas LTA-BMEs only enhanced the secretion of TNF-α and CCL5, and had no effects on the secretions of IL-6, IL-8, and CXCL2 from BMFs, indicating that E. coli and S. aureus cause mastitis via different mechanisms. For instance, LPS and LTA can combine with TLR4 and TLR2 separately and promote pro-inflammatory cytokine expression to activate inflammatory response.[29] Although LPS-BMEs and LTA-BMEs showed differential induction of selected inflammatory mediators at a protein secretion level in BMFs, we can conclude that inflammatory epithelial cells treated by LPS and LTA induced the secretion of some certain inflammation-related proteins. Chronic and subclinical mastitis are associated with mammary gland fibrosis.[33] Fibrosis is associated with major alterations in ECM quantity and composition.[34-36] We found that 9
both LPS- and LTA-challenged epithelial cells promoted the synthesis of type I collagen and fibronectin, which may explain how persistent inflammation in the mammary gland leads to fibrosis in cow udders, consequentially decreasing milk production. We found that inflammatory epithelial cells inhibited BMF proliferation. This result differed from our previous finding that stromal fibroblasts isolated from bovine mammary glands with mastitis grew faster than those isolated from normal mammary glands.[37] Stromal fibroblasts in mammary glands with mastitis are possibly affected by other stromal cells, such as macrophages and mastocytes, and invasive pathogens, except for inflammatory epithelial cells. Researchers have proved that EGFR and PDGFR participate in cell proliferation regulation via the Ras/Raf/MEK/ERK pathway.[38, 39] Here, we found that inhibited BMF proliferation was accompanied by EGFR, PDGFR-α, and PDGFR-β downregulation. Both proliferation and apoptosis should be considered to predict cell growth.[40] Our finding demonstrated that decreased BMF proliferation rate was not accompanied by changes in apoptosis rate. The overexpression of inflammatory mediators, growth factors, and cytokines could promote fibroblast migration.[41, 42] In our study, we found that inflammatory epithelial cells promoted expression levels of TNF-α, IL-8, and IL-6 and enhanced the migration ability of BMFs. Although we did not directly find evidence that TNF-α, IL-8, and IL-6 mediated the enhanced migration ability of BMFs, it was previously reported that TNF-α, IL-8, and IL-6 promoted migration of fibroblasts.[43-47] Stortelers C et.al suggested that LTA stimulated Wnt/β-catenin signaling in fibroblasts.[48] In our study, we found that both LPS-BMEs and LTA-BMEs inhibited mRNA expression of E-cadherin and GSK-3β and promoted BMF mRNA expression of N-cadherin, Wnt, and β-catenin in BMFs. All these molecules are key components or regulators of the classical Wnt/β-catenin signaling pathway that participates in the regulation of cell-cell adhesion. Wnt may inactivate GSK-3β, upregulating β-catenin expression and inducing cell migration.[49, 50] E-cadherin is also an important cell-cell adhesion molecule[51, 52]. Suppressed E-cadherin expression plays a fundamental role in increased cell migration.[53] β-catenin is an essential component of adherent junctions, where it links E-cadherin and α-catenin and modulates cell-cell adhesion and cell migration. N-cadherin may promote migration by forming labile cellular adhesions.[54] Our data indicated that the Wnt signal pathway may play important roles in BMF migration induced by inflammatory epithelial cells. HMGB1 may also induce increased cytokine production and molecule adhesion via activating NF-κB translocation. HMGB1 overexpression also induced cell migration in inflammatory response.[55] We found that LPS-BMEs upregulated HMGB1 gene expression in BMFs, whereas LTA-BMEs did not affect its expression, suggesting mammary stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes in mammary glands with mastitis. However, LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. This work provides evidence supporting BMFs’ important role in the induction of inflammatory responses from epithelial cells during bovine mastitis. Our results suggest that mammary gland fibroblasts play crucial roles in bovine mastitis. Further in vitro and in vivo studies to understand the complex cross-talk between BMFs and BMEs are clearly necessary to exploit them as valid targets for novel mastitis therapies. 10
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Figure captions Fig. 1. TNF-α mRNA expression and secretion levels in LPS- or LTA-treated mammary epithelial cells. After epithelial cells were treated with 10 ng/μl LPS for 3 h or 20 ng/μl LTA for 12 h, media were replaced with fresh media without FBS. Cells were cultured for another 24 h. TNF-α mRNA expression in treated cells was analyzed by real-time PCR (a), and TNF-α protein secretion in the medium was measured with an ELISA kit (b). Untreated epithelial cells were used as control. GAPHD was used as an internal control for real-time PCR. Data were expressed as the mean ± s.d. *p<0.01 vs. control. Fig. 2. BMF activation after co-culture with inflammatory epithelial cells. α-SMA and vimentin expression levels in BMFs cultured in LPS-BME- or LTA-BME-conditioned media were analyzed by real-time PCR (a) and Western blot analysis (b). BMFs cultured in growth medium were used as control. GAPHD was used as an internal control. Data were expressed as the mean ± s.d. *p<0.01 vs. control. Fig. 3. Inflammatory mediator expression in LPS-BME- or LTA-BME-stimulated BMFs. (a) TNF-α, CCL5, CXCL2, IL-6, and IL-8 gene expression levels in BMFs were determined by real-time PCR. TNF-α (b), CCL5 (c), CXCL2 (d), IL-6 (e), and IL-8 (f) protein secretion by BMFs were measured with corresponding ELISA kits. Data were expressed as the mean ± s.d.*p<0.01 vs. control. Fig. 4. ECM protein expression in LPS-BME- or LTA-BME-stimulated BMFs. (a) Fibronectin and type I collagen gene expression levels in BMFs were determined by real time real time PCR. GAPDH was used as internal control. Data were expressed as the mean ± s.d.*p<0.01 vs. control. (b) Fibronectin and collagen type I protein expression levels in BMFs were detected by Western blot. Fig. 5. Inflammatory epithelial cells inhibited BMF growth. (a) LPS-BME- or LTA-BME-stimulated BMF proliferation curve, *p<0.01 vs. LPS-BMEs and LTA-BMEs. Cell proliferation rate was calculated by the formula [(ODn-ODblank)-(OD0-ODblank)]/ (OD0-ODblank)×100, where ODn is the optical density value at indicated day after culture, OD0 is the optical density value at 0 day, and ODblank is the optical density value of wells without 14
cells. (b) BMF apoptosis stimulated by stimulated by LPS-BMEs or LTA-BMEs. Data represented one of three independently repeated experiments. (c) PDGF-α, PDGF-β, and EGFR gene expression levels in BMFs were determined by real- time PCR. All data were expressed as the mean ± s.d.*p<0.01 vs. control. Fig. 6. Inflammatory epithelial cells promoted BMF migration. (a) Phase-contrast microscopy showed an obvious reduction in wound width among all groups. However, the size of the scratched area in the control group was larger than in BMFs cultured in LPS-BME or LTA-BME-conditioned medium. (b) WNT1, GSK-3β, β-catenin, N-cadherin, E-cadherin, and HMGB1 gene expression levels in BMFs were determined by real-time PCR. Data were expressed as the mean ± s.d. *p<0.01, **p<0.05 vs. control. (c) E-cadherin, β-catenin, and N-cadherin protein expression levels in BMFs were detected by Western blot.
Highlights ►Inflammatory BMEs affect the properties of BMFs during mastitis. ►BMEs inhibited the proliferation and promoted the migration of BMFs. ►BMEs enhanced secretion of inflammatory mediators and deposition of ECM in BMFs. ►Changes of the properties of BMFs were mediated by specific signal molecules.
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S0014-4827(16)30307-X http://dx.doi.org/10.1016/j.yexcr.2016.09.016 YEXCR10345
To appear in: Experimental Cell Research Received date: 27 June 2016 Revised date: 11 September 2016 Accepted date: 24 September 2016 Cite this article as: Wenyao Zhang, Xuezhong Li, Tong Xu, Ma Mengru, Yong Zhang and Ming-Qing Gao, Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis, Experimental Cell Research, http://dx.doi.org/10.1016/j.yexcr.2016.09.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis Wenyao Zhanga, Xuezhong Lia, Tong Xua, Mengru, Maa, Yong Zhanga,b and Ming-Qing Gaoa,b a
College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China b
*Corresponding author: E-mail: [email protected] (MQ. Gao) or [email protected] (Y. Zhang).
Abbreviations: LPS, lipopolysaccharide; LTA, lipoteichoic acid; S. aureus, Staphylococcus aureus; E. coli, Escherichia coli; BMEs, bovine mammary gland epithelial cells; BMFs, bovine mammary stromal fibroblasts; ECM, extracellular matrix; TNF-α, tumor necrosis factor-alpha; CCK-8, Cell Counting Kit-8; IL-6, interleukin6; IL-8, interleukin- 8; CXCL2, Chemokine (C-X-C motif) ligand 2; CCL5, Chemokine (C-C motif) ligand 5; α-SMA, α-smooth muscle actin; PDGFR-α, growth factor receptor α; PDGFR-β, growth factor receptor β; EGFR, epidermal growth factor receptor; GSK-3β, Glycogen synthase kinase-3β; HMGB1, high mobility group box-1 protein; ERK, extracellular signal-regulated kinase
Abstract Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. Keywords: mastitis; fibroblasts; epithelial cells; lipopolysaccharide; lipoteichoic acid; bovine
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1. Introduction Mastitis is one of the most critical diseases in dairy production. Mastitis is a mammary gland inflammation caused by changes in metabolism, physiological trauma, or contagious or environmental pathogenic microorganisms.[1] Invasive pathogens are the main causes of mastitis. Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are two of the most important pathogens causing bovine mastitis.[2] Lipopolysaccharides (LPS), the major structural elements of the E. coli cell membrane, trigger strong immune responses. Lipoteichoic acid (LTA), an important cell wall component of S. aureus, causes septic shock and multiple organ apoptosis. Therefore, these two important antigens might be used as conditional factors to induce inflammatory responses. Neutrophils are the first line of defense against bacterial invasion in the bovine mammary gland.[2] During bacterial invasion, bovine mammary gland epithelial cells (BMEs) initiate neutrophil recruitment through inflammatory and immune responses, including increased pro-inflammatory cytokine secretion.[3, 4] Tumor necrosis factor alpha (TNF-α) is the main pro-inflammatory cytokine producing inflammatory responses and promoting the release of various inflammatory mediators by multifarious cell types.[5] TNF-α induces fibroblasts to produce granulocyte macrophage colony stimulating factors and promotes granulocyte and macrophage progenitor maturation.[6] Proteins of TNF and the TNF receptor family play important roles in the control of cell death, proliferation, autoimmunity, the function of immune cells, and the organogenesis of lymphoid organs.[7] Dairy cow mammary glands mainly consist of BMEs and bovine mammary stromal fibroblasts (BMFs). BMEs play an important role in inducing a relevant innate immune response in mammary glands, act as sentinels, and signal invading mastitis-causing pathogens.[2] In bovine mastitis, bacterial infection first occurs in the epithelial cells of mammary gland crypts.[1, 2, 8] During the early stages of infection, epithelial cells release immunological factors to prevent the production and development of inflammation and also release inflammatory mediators to affect adjacent cells.[1, 2, 8]. Fibroblasts are important stromal cells which activate and interact with infiltrating immune cells in a dynamic and site-specific manner. Fibroblasts also define the external tissue features, provide positional memory, and regulate the switch from resolving to persistent inflammation.[9] The regional identity of fibroblasts can be modified by inflammation.[10-13] Therefore, targeting fibroblasts and the stromal microenvironment are likely important methods in treating inflammation. Toll-like receptor 2 recognizes LTA, whereas toll-like receptor 4 recognizes LPS.[14, 15] Meanwhile, ligand binding activates a range of signaling molecules and releases transcription factors.[16, 17] Released transcription factors mediate cytokine, chemokine, and growth factor expression levels.[18] Inflammatory mediators of abnormal expression induces inflammatory cell repair. Many abnormal and uncontrolled repair mechanisms may lead to fibrosis and extracellular matrix (ECM) deposition. During the early phase of bovine mastitis, inflammatory mediators released from epithelial cells stimulate fibroblasts and other stromal cells.[19] Activated fibroblasts are also key inflammation mediators and mediate the 3
development of inflammation in bovine mastitis. Better knowledge of inflammatory epithelial cell reactions to BMFs could eventually lead to novel therapeutic approaches to combat the spread of inflammation during the early phase of bovine mastitis. With that aim in mind, this study used an in vitro model of mastitis to investigate the mechanism of inflammatory epithelial cell effects on BMF. The proliferation, apoptosis, migration, secretion of inflammatory cytokines, and ECM deposition of BMFs cultured in conditioned medium from LPS- or LTA- induced inflammatory BMEs were investigated. 2. Materials and methods 2.1 Cell Isolation and Culture BMEs and BMFs were obtained from Holstein cow mammary tissues as previously described.[20] Mammary tissues were individually sampled from eight Holstein dairy cows. All mammary glands were healthy with no obvious evidence of infection. Fresh tissues were digested in a solution of Enzyme Cocktail (ISU ABXIS, Seoul, Korea) and incubated at 37 °C overnight in a humidified 5% CO2 incubator. Digested tissue was filtered and centrifuged to yield pellets containing epithelial organoids and supernatants containing fibroblasts. The isolated epithelial cells and fibroblasts were seeded on cell culture dishes and cultured in complete DMEM/F12 medium (GIBCO BRL , Life Technologies, Burlington, ON) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 μg/ml streptomycin (Gibco BRL, Grand island, NY) at 37 °C in an incubator with 5% CO2. Cells were passaged by digestion with 0.15% trypsin and 0.02% EDTA. BMEs and BMFs were almost pure after three to four separation and culture passages. By the fifth passage, extracted BMEs or BMFs were used as cell pools in all subsequent experiments. Both BMEs and BMFs were prepared by mixing equivalent amounts of cells from all individuals. All reagents were purchased from Sigma-Aldrich Canada (Oakville, ON), unless otherwise stated. 2.2 Establishment of Inflammatory Cell Model An inflammatory cell model of mastitis was established with LPS- or LTA-treated BMEs. Tumor necrosis factor-alpha (TNF-α) released in the medium and mRNA expression in the treated cells were utilized as indicators for LPS- or LTA-induced immune responses. Before stimulation with LPS and LTA, confluent BMEs monolayers were cultured in fresh medium without FBS for 24 h at 37 °C. Then, the medium was replaced with complete medium containing LPS or LTA at final concentrations of 10 ng/μl or 20 ng/μl according to recommended concentrations. After stimulation for 3 h, 6 h, 12 h, and 24 h with LPS or LTA, cell culture supernatants were discarded and replaced with fresh medium without FBS. The culture was incubated again for 24 h. Finally, cell culture supernatants were collected for TNF-α secretion measurement and BMEs were harvested for RNA extraction and TNF-α real-time PCR analysis. BMEs cultured in media without FBS and without stimulation were designated as controls. The inflammatory cell model of mastitis was established under the shortest treatment period exhibiting significantly upregulated TNF-α expression in BMEs. BMEs treated with LPS and LTA were designated as LPS-BMEs and LTA-BMEs, respectively. 2.3 Indirect co-culture experiments 4
We investigated the effects of inflammatory epithelial cells induced by LPS or LTA on BMFs with an indirect co-culture model utilizing conditioned medium. In brief, LPS-BMEs or LTA-BMEs were rinsed with PBS. Fresh serum-free DMEM/F12 medium was then added to the culture dishes. Cells were cultured for another 24 h at 37°C under 5% CO2. Then, the medium was collected and cells debris was moved by centrifuge. Finally, the medium was used at a 9:1 ratio with fresh DMEM/F12 and 10% FBS as the conditioned medium. BMFs cultured in conditioned medium collected from un-stimulated BMEs were taken as control. For indirect co-culture, BMFs were cultured in LPS-BME- or LTA-BME-conditioned media. After day 3 of culture, cell culture supernatants were collected and BMFs were harvested for analysis. 2.4 RNA Extraction and Real-time PCR Total RNA was isolated from cells washed twice with PBS. Cells were re-suspended in ice-cold TriZol solution (TransGene, Shanghai, China). Cells were passed through a ribonuclease-free 20-gauge needle 10 times for cell disruption. Total mRNA extraction was performed with the RNA Easy Kit (TransGene) according to the manufacturer’s instructions. Total mRNA concentration was measured with a spectrophotometer (ND 2.0; NanoDrop Technologies, Wilmington, Delaware), and 1 μg of RNA was reverse-transcribed into cDNA with the TransScript II First-Strand cDNA Synthesis SuperMix (TransGene). Primers were designed based on sequences from the National Center for Biotechnology Information Database. Specificity was determined with Primer-BLAST. The primers are listed in Supplementary Table S1. All PCR primers were synthesized by Sangon Biotech (Shanghai, China). Real-time PCR was performed with an iQ5 light cycler (Bio-Rad, Hemel Hempstead, UK) using TransStart Probe qPCR SuperMix (TransGene) in 25-μl reactions containing 1 μmol/L concentrations of each forward and reverse primer (Sangon Biotech). PCR conditions were as follows: activation at 95 °C for 30 seconds, 40 cycles of denaturation at 95 °C for 5 seconds, and annealing/extension at 60 °C for 30 seconds. Melt curves were generated to estimate primer specificities. A standard curve was generated with reference cDNA to determine the starting quantity of mRNA in each sample. The expression of each target gene was normalized to that of GAPDH, the reference gene. Comparative Ct method was employed to quantify normalized target gene expression relative to the calibrator. Data were expressed as relative gene expression = 2-ΔΔCt. 2.5 Cell Viability Assay Cell viability was determined with CCK-8 kits (Beyotime, Shanghai, China) according to the manufacturer’s protocol. BMFs were evenly seeded into 96-well plates with 100 μl complete medium at a density of approximately 2x103 cells/well and incubated for 24 h. Then, the complete medium was replaced with BME-, LPS-BME-, or LTA-BME-conditioned medium. BMFs were then cultured in conditioned medium for 0, 1, 2, and 3 days. Before the cell viability assay, the medium was replaced with 100 μl fresh medium containing 10 μl of CCK-8 solution and incubated for an additional 3 h at 37 °C in a humidified incubator. The absorbance of samples in triplicate wells was measured at 450 nm wavelength with a microplate reader (Bio-Rad, Hercules, CA) at indicated time points. Cell proliferation rate was calculated by the formula [(ODn−ODblank)−(OD0−ODblank)]/(OD0−ODblank)×100, where ODn is 5
the optical density value at the indicated day after culture, OD0 is the optical density value at 0 day, and ODblank is the optical density value of the wells without cells. BME-conditioned medium was designated as the control. 2.6 Western Blotting Protein lysates from cells were prepared with PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Inc., Gyeonggi-do, Korea) according to the manufacturer’s instructions. Lysates were precipitated at 1200 rpm for 10 min. Total protein content was determined with Bradford Easy Protein Quantitative Kit (TransGene). Equal amounts of protein extracts in lysis buffer were subjected to SDS-PAGE on 4%–12% polyacrylamide gels (German, Sigma-Aldrich) then transferred to a nitrocellulose membrane. Resolved proteins were blotted onto PVDF transfer membranes and blocked with 10% non-fat milk in TBST. Membranes were incubated with anti-α-SMA (Abcam, Cambridge, MA, UK), anti-vimentin (Bioss, Beijing, China), anti-fibronectin (Bioss), anti-collagen I (Abcam), anti-E-cadherin (Abcam), anti-β-catenin (Abcam), anti-N-cadherin (Bioss), and anti-GAPDH (TransGene) antibodies at 4 °C overnight. Membranes were washed 3 times with TBST for 5 min before incubation with HRP-conjugated secondary antibodies. Finally, immunoreactive proteins were visualized with an enhanced chemiluminescence detection kit (Beyotime). 2.7 ELISA We detected BMF-secreted TNF-α, IL-6, IL-8, CXCL2, and CCL5 in the medium by ELISA. BMFs were cultured in fresh serum-free medium for another 24 h after being cultured in LPS-BME- or LTA-BME- conditioned media for 3 days. Then, the medium was collected and cells debris was removed by centrifuge. TNF-α, IL-6, IL-8, CXCL2, and CCL5 secreted in the medium were measured with corresponding ELISA kits (Huzhen Biological Technology Co., LTD, Shanghai, China) according to the manufacturer’s instructions. 2.8 Flow Cytometry BMF apoptotic rate was determined by flow cytometric techniques with a commercial apoptosis assay kit (TransGene). Co-cultured BMFs were harvested and uniformly re-suspended in 100 μl pre-cooled Annexin V Binding buffer prior to the addition of 5 μl Annexin V-FITC and 5 μl PI. Samples were gently mixed and incubated for 15 min under dark conditions and ambient temperature. Finally, cells were transferred to tubes containing an additional 300 μl of Annexin V binding buffer and analyzed by flow cytometry within an hour. 2.9 Cell Migration Assay BMFs were routinely cultured and grown to approximate confluence. Then, the BMF monolayer was wounded by a single scratch with a pipette tip. Media were replaced by conditioned medium from LPS-BME, LTA-BME and BME (control) conditioned media. Cells were incubated for 24 h to allow cell growth and wound healing. Cell migration capacity to fill the wounded area with cells was assessed by photographing the wound. 2.10 Statistical Analysis 6
Results were reported as mean ± s.d. All data were obtained from at least three independent experiments. All statistical analyses were performed with ANOVA (SPSS11.5 software). P < 0.05 was considered statistically significant. 3. Results 3.1 Establishment of Inflammatory Cell Model of Mastitis TNF-α was used as an index of BME inflammatory responses to LPS or LTA. We incubated BMEs with the recommended LPS or LTA concentrations for several different treatment periods. We found that TNF-α gene expression was significantly upregulated in BMEs challenged with 10 ng/μl LPS for 3 h or with 20 ng/μl LTA for 12 h (Fig. 1a). TNF-α secretion peaked at 3 h after treatment with 10 ng/μl LPS or at 12 h after treatment with 20 ng/μl LTA (Fig. 1b). Hence, LPS-treated BMEs (LPS-BMEs) and LTA-treated BMEs (LTA-BMEs) under the previously mentioned treatment conditions were used to mimic the bovine mammary cell gland epithelial cells infected by E. coli and S. aureus. 3.2 Inflammatory Epithelial Cells Activated BMFs. We previously reported that stromal fibroblasts derived from bovine mammary glands with mastitis were activated and displayed inflammation-specific changes.[20] The expression levels of α-smooth muscle actin (α-SMA) and vimentin, two indicators of activated fibroblasts, were examined in BMFs cultured in LPS-BME- and LTA-BME-conditioned media to validate whether fibroblast activation was directly achieved by infected epithelial cells during mastitis. We found that both LPS-BMEs and LTA-BMEs upregulated BMF α-SMA and vimentin expression levels (Figs. 2a and b), indicating that inflammatory epithelial cells activated BMFs. 3.3 Inflammatory Epithelial Cells Induced the Expression of Inflammatory Mediators in BMFs Fibroblasts have been suggested as vital immumoregulatory cells regulating immune response.[21] We examined the expression levels of several inflammatory mediators, including TNF-α, IL-6, IL-8, CXCL2, and CCL5 at both gene and protein levels to investigate if inflammatory epithelial cells induced fibroblast inflammatory response (Fig. 3). Results showed that both LPS-BMEs and LTA-BMEs upregulated mRNA expression levels of all the selected genes. Meanwhile, LPS-BMEs enhanced protein secretion of inflammatory mediators. However, LTA-BMEs only promoted TNF-α and CCL5 protein secretion in BMFs, indicating that a different mechanism may mediate interactions between mammary gland epithelial cells and fibroblasts in mastitis caused by E. coli and S. aureus. 3.4 Inflammatory Epithelial Cells Induced ECM Protein Expression in BMFs Fibroblasts are primarily responsible for ECM protein synthesis in tissue and play a critical role during repair in many organs.[22] We analyzed type I collagen and fibronectin expression levels by Western blotting and RT-PCR to determine whether inflammatory epithelial cells induced ECM deposition by BMFs in mammary glands with mastitis. Results showed that LTA-BMEs enhanced type I collagen and fibronectin mRNA (Fig. 4a) and protein expression levels (Fig. 4b). Although LPS-BMEs did not upregulate fibronectin gene 7
expression, it regulated type I collagen and fibronectin protein expression levels in BMFs. 3.5 Inflammatory Epithelial Cells Inhibited BMF growth We analyzed proliferation and apoptosis to investigate whether epithelial cells infected by bacteria affected the growth of stromal fibroblasts in bovine mammary glands with mastitis. Proliferation was measured and cell proliferation curves were generated. Results showed that on days 2 and 3, the proliferation rates of BMF cells cultured in conditioned medium from both LPS-BMEs and LTA-BMEs were lower compared to those of BMF cells cultured in control medium (Fig. 5a). However, we did not find a significant difference in cell apoptotic rates between all groups (Fig. 5b). We analyzed the gene expression levels of platelet-derived growth factor receptor (PDGFR) α, PDGFR-β, and epidermal growth factor receptor (EGFR) in BMFs by real-time PCR to explore possible signal molecules mediating the conditioned medium’s inhibitory effects on BMFs. As seen in Fig. 5c, the expression levels of all examined genes were downregulated in BMFs cultured in LPS-BME- or LTA-BME-conditioned media compared with the control medium. These results suggested that EGFR, PDGFR-α, and PDGFR-β contributed to the effect of inflammatory epithelial cells on stromal fibroblast proliferation. 3.6 Stimulation of BMFs Migration by Inflammatory Epithelial Cells The effect of inflammatory epithelial cells on BMF migration was analyzed with a scratch-recovery assay in a co-culture model. The scratched area in the BMF monolayer cultured in LPS-BME- or LTA-BME-conditioned media completely cicatrized at 18 h, whereas the scratched area in the control group completely closed at 24 h. Photomicrographs of scratched areas 12 h after scratching are presented in Fig. 6a and show that scratched areas in the control group were larger than in BMFs cultured in LPS-BME- or LTA-BME-conditioned media. This result indicated that inflammatory epithelial cells promoted BMF migration. The expression levels of several selected genes associated with cell migration were analyzed, including WNT1, E-cadherin, β-catenin, N-cadherin, GSK-3β, and HMGB1. We found that both LPS- and LTA-infected BMEs downregulated E-cadherin and GSK-3β mRNA expression levels and upregulated WNT1, β-catenin, and N-cadherin mRNA expression levels in BMFs. HMGB1 gene expression increased in BMFs cultured in LPS-BME-conditioned medium, but not in BMFs cultured in LTA-BME-conditioned medium. We also examined β-catenin, E-cadherin, and N-cadherin protein expression levels by Western blotting. Their protein expression levels were consistent with gene expression levels (Fig. 6b). 4. Discussion Epithelial cells are the first cell barriers resisting bacterial infections in mammary glands. When the organism cannot resist bacterial infections because of reduced immunity, epithelial cells are first infected by pathogenic bacteria. The infected cells release a large number of inflammatory mediators, including TNF-α, IL-6, and IL-8, which affect adjacent cells[23]. The present study investigated whether stromal fibroblasts responded to inflammatory epithelial cells in bovine mammary glands with mastitis. An inflammatory cell model of bovine mastitis was established by challenging epithelial cells with the gram-negative and gram-positive bacterial cell wall components LPS and LTA. BMF immune 8
responses to inflammatory epithelial cells were investigated with an in vitro co-culture model in which BMFs were cultured in LPS-BME- and LTA-BME-conditioned media. Our results showed that inflammatory epithelial cells affected BMF characteristics, decreasing proliferation capacity, enhancing migration ability, increasing expression of inflammatory mediators, and elevating deposition of ECM proteins. Changes in BMF proliferation and migration abilities mediated some signal molecules, such as PDGFR, EGFR, or components of WNT signal pathway. S. aureus and E. coli infections result in the upregulation of various pro-inflammatory cytokines.[24] TNF-α, a potent pro-inflammatory cytokine, exerts pleiotropic functions in immunity and control of cell proliferation, differentiation and apoptosis.[25] TNF-α expression has been observed in inflammatory response in different tissues, including mastitis.[26] Chu X et al. reported that TNF-α is the earliest and primary endogenous mediator of inflammatory response.[27] Therefore, we used TNF-α as an indicator to investigate whether BMEs produced inflammatory responses after LPS or LTA treatment. Peak cytokine secretion in gram-negative infection occurred within 1 hour to 5 hours after bacterial challenging, whereas time to peak secretion was extended significantly after gram-positive challenging.[28] In the current study, we found that TNF-α protein secretion and mRNA expression levels in BMEs peaked at 3 h after treatment with 10 ng/μl LPS at or at 12 h after treatment with 20 ng/μl LTA. Thus, we successfully established an inflammatory epithelial cell model of mastitis by challenging BMEs with LPS and LTA. In mastitis, inflammatory epithelial cells engage in reciprocal molecular dialogues with surrounding stromal cells, including inflammatory cells, vascular cells, and fibroblasts, resulting in the production of stromal-derived inflammation-aiding factors, such as growth factors, chemokines, cytokines, and proteases.[29] We found that inflammatory epithelial cells, regardless of treatment with LPS or LTA, activated BMFs, as indicated by upregulated mRNA and protein expression levels of α-SMA, an indicator of active fibroblasts.[30, 31] In addition, pathogen invasion of host cells commonly involves the rearrangement of the host cell’s actin cytoskeleton. α-SMA is an important fibroblast actin cytoskeleton gene and is upregulated in inflammatory response development.[32] Vimentin is another important actin component of the cell cytoskeleton. We found that vimentin was also upregulated in BMFs co-cultured with LPS-BMEs or LTA-BMEs. We examined the expression levels of several inflammation-related cytokines and chemokines, including TNF-α, IL-6, IL-8, CXCL2, and CCL5, in BMFs cultured in LPS-BME- or LTA-BME-conditioned media. We found that LPS-BMEs promoted the secretion of all these mediators in BMFs, whereas LTA-BMEs only enhanced the secretion of TNF-α and CCL5, and had no effects on the secretions of IL-6, IL-8, and CXCL2 from BMFs, indicating that E. coli and S. aureus cause mastitis via different mechanisms. For instance, LPS and LTA can combine with TLR4 and TLR2 separately and promote pro-inflammatory cytokine expression to activate inflammatory response.[29] Although LPS-BMEs and LTA-BMEs showed differential induction of selected inflammatory mediators at a protein secretion level in BMFs, we can conclude that inflammatory epithelial cells treated by LPS and LTA induced the secretion of some certain inflammation-related proteins. Chronic and subclinical mastitis are associated with mammary gland fibrosis.[33] Fibrosis is associated with major alterations in ECM quantity and composition.[34-36] We found that 9
both LPS- and LTA-challenged epithelial cells promoted the synthesis of type I collagen and fibronectin, which may explain how persistent inflammation in the mammary gland leads to fibrosis in cow udders, consequentially decreasing milk production. We found that inflammatory epithelial cells inhibited BMF proliferation. This result differed from our previous finding that stromal fibroblasts isolated from bovine mammary glands with mastitis grew faster than those isolated from normal mammary glands.[37] Stromal fibroblasts in mammary glands with mastitis are possibly affected by other stromal cells, such as macrophages and mastocytes, and invasive pathogens, except for inflammatory epithelial cells. Researchers have proved that EGFR and PDGFR participate in cell proliferation regulation via the Ras/Raf/MEK/ERK pathway.[38, 39] Here, we found that inhibited BMF proliferation was accompanied by EGFR, PDGFR-α, and PDGFR-β downregulation. Both proliferation and apoptosis should be considered to predict cell growth.[40] Our finding demonstrated that decreased BMF proliferation rate was not accompanied by changes in apoptosis rate. The overexpression of inflammatory mediators, growth factors, and cytokines could promote fibroblast migration.[41, 42] In our study, we found that inflammatory epithelial cells promoted expression levels of TNF-α, IL-8, and IL-6 and enhanced the migration ability of BMFs. Although we did not directly find evidence that TNF-α, IL-8, and IL-6 mediated the enhanced migration ability of BMFs, it was previously reported that TNF-α, IL-8, and IL-6 promoted migration of fibroblasts.[43-47] Stortelers C et.al suggested that LTA stimulated Wnt/β-catenin signaling in fibroblasts.[48] In our study, we found that both LPS-BMEs and LTA-BMEs inhibited mRNA expression of E-cadherin and GSK-3β and promoted BMF mRNA expression of N-cadherin, Wnt, and β-catenin in BMFs. All these molecules are key components or regulators of the classical Wnt/β-catenin signaling pathway that participates in the regulation of cell-cell adhesion. Wnt may inactivate GSK-3β, upregulating β-catenin expression and inducing cell migration.[49, 50] E-cadherin is also an important cell-cell adhesion molecule[51, 52]. Suppressed E-cadherin expression plays a fundamental role in increased cell migration.[53] β-catenin is an essential component of adherent junctions, where it links E-cadherin and α-catenin and modulates cell-cell adhesion and cell migration. N-cadherin may promote migration by forming labile cellular adhesions.[54] Our data indicated that the Wnt signal pathway may play important roles in BMF migration induced by inflammatory epithelial cells. HMGB1 may also induce increased cytokine production and molecule adhesion via activating NF-κB translocation. HMGB1 overexpression also induced cell migration in inflammatory response.[55] We found that LPS-BMEs upregulated HMGB1 gene expression in BMFs, whereas LTA-BMEs did not affect its expression, suggesting mammary stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes in mammary glands with mastitis. However, LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. This work provides evidence supporting BMFs’ important role in the induction of inflammatory responses from epithelial cells during bovine mastitis. Our results suggest that mammary gland fibroblasts play crucial roles in bovine mastitis. Further in vitro and in vivo studies to understand the complex cross-talk between BMFs and BMEs are clearly necessary to exploit them as valid targets for novel mastitis therapies. 10
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Figure captions Fig. 1. TNF-α mRNA expression and secretion levels in LPS- or LTA-treated mammary epithelial cells. After epithelial cells were treated with 10 ng/μl LPS for 3 h or 20 ng/μl LTA for 12 h, media were replaced with fresh media without FBS. Cells were cultured for another 24 h. TNF-α mRNA expression in treated cells was analyzed by real-time PCR (a), and TNF-α protein secretion in the medium was measured with an ELISA kit (b). Untreated epithelial cells were used as control. GAPHD was used as an internal control for real-time PCR. Data were expressed as the mean ± s.d. *p<0.01 vs. control. Fig. 2. BMF activation after co-culture with inflammatory epithelial cells. α-SMA and vimentin expression levels in BMFs cultured in LPS-BME- or LTA-BME-conditioned media were analyzed by real-time PCR (a) and Western blot analysis (b). BMFs cultured in growth medium were used as control. GAPHD was used as an internal control. Data were expressed as the mean ± s.d. *p<0.01 vs. control. Fig. 3. Inflammatory mediator expression in LPS-BME- or LTA-BME-stimulated BMFs. (a) TNF-α, CCL5, CXCL2, IL-6, and IL-8 gene expression levels in BMFs were determined by real-time PCR. TNF-α (b), CCL5 (c), CXCL2 (d), IL-6 (e), and IL-8 (f) protein secretion by BMFs were measured with corresponding ELISA kits. Data were expressed as the mean ± s.d.*p<0.01 vs. control. Fig. 4. ECM protein expression in LPS-BME- or LTA-BME-stimulated BMFs. (a) Fibronectin and type I collagen gene expression levels in BMFs were determined by real time real time PCR. GAPDH was used as internal control. Data were expressed as the mean ± s.d.*p<0.01 vs. control. (b) Fibronectin and collagen type I protein expression levels in BMFs were detected by Western blot. Fig. 5. Inflammatory epithelial cells inhibited BMF growth. (a) LPS-BME- or LTA-BME-stimulated BMF proliferation curve, *p<0.01 vs. LPS-BMEs and LTA-BMEs. Cell proliferation rate was calculated by the formula [(ODn-ODblank)-(OD0-ODblank)]/ (OD0-ODblank)×100, where ODn is the optical density value at indicated day after culture, OD0 is the optical density value at 0 day, and ODblank is the optical density value of wells without 14
cells. (b) BMF apoptosis stimulated by stimulated by LPS-BMEs or LTA-BMEs. Data represented one of three independently repeated experiments. (c) PDGF-α, PDGF-β, and EGFR gene expression levels in BMFs were determined by real- time PCR. All data were expressed as the mean ± s.d.*p<0.01 vs. control. Fig. 6. Inflammatory epithelial cells promoted BMF migration. (a) Phase-contrast microscopy showed an obvious reduction in wound width among all groups. However, the size of the scratched area in the control group was larger than in BMFs cultured in LPS-BME or LTA-BME-conditioned medium. (b) WNT1, GSK-3β, β-catenin, N-cadherin, E-cadherin, and HMGB1 gene expression levels in BMFs were determined by real-time PCR. Data were expressed as the mean ± s.d. *p<0.01, **p<0.05 vs. control. (c) E-cadherin, β-catenin, and N-cadherin protein expression levels in BMFs were detected by Western blot.
Highlights ►Inflammatory BMEs affect the properties of BMFs during mastitis. ►BMEs inhibited the proliferation and promoted the migration of BMFs. ►BMEs enhanced secretion of inflammatory mediators and deposition of ECM in BMFs. ►Changes of the properties of BMFs were mediated by specific signal molecules.
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