Capsular Polysaccharide of Mycoplasma ovipneumoniae Induces Sheep Airway Epithelial Cell Apoptosis via ROS-Dependent JNK/P38 MAPK Pathways

In an attempt to better understand the pathogen-host interaction between invading Mycoplasma ovipneumoniae (M. ovipneumoniae) and sheep airway epithelial cells, biological effects and possible molecular mechanism of capsular polysaccharide of M. ovipneumoniae (CPS) in the induction of cell apoptosis were explored using sheep bronchial epithelial cells cultured in air-liquid interface (ALI). The CPS of M. ovipneumoniae was first isolated and purified. Results showed that CPS had a cytotoxic effect by disrupting the integrity of mitochondrial membrane, accompanied with an increase of reactive oxygen species and decrease of mitochondrial membrane potential (ΔΨm). Of importance, the CPS exhibited an ability to induce caspase-dependent cell apoptosis via both intrinsic and extrinsic apoptotic pathways. Mechanistically, the CPS induced extrinsic cell apoptosis by upregulating FAS/FASL signaling proteins and cleaved-caspase-8 and promoted a ROS-dependent intrinsic cell apoptosis by activating a JNK and p38 signaling but not ERK1/2 signaling of mitogen-activated protein kinases (MAPK) pathways. These findings provide the first evidence that CPS of M. ovipneumoniae induces a caspase-dependent apoptosis via both intrinsic and extrinsic apoptotic pathways in sheep bronchial epithelial cells, which may be mainly attributed by a ROS-dependent JNK and p38 MAPK signaling pathways.


Introduction
Mycoplasma ovipneumoniae (M. ovipneumoniae, MO) has been identified as a pathogenic agent of chronic nonprogressive infection of pneumonia in sheep and goats [1][2][3][4]; the nature between a protective and a pathological host response of MO infection currently remains largely unknown. In this regard, the role of capsular polysaccharide (CPS), a major active component for cellular adherence, invasion, immune modulation, and virulence of M. ovipneumoniae remains undefined [5], although several lines of evidences have shown that M. ovipneumoniae is able to produce polysaccharide capsules for facilitating its adherence to ciliated epithelium [3,6]. In this context, the respiratory epithelium is responsible for facilitating key defense mechanism and acting as first line of the immune system in response to a pathogen infection, including M. ovipneumoniae, for which tract epithelial cells serve as portals for bacteria entering hosts. Together with the pivotal role of CPS in the adherence of M. ovipneumoniae to host cells, it is therefore of importance to characterize biological functions and underlying mechanisms of immune responses of the CPS in respiratory epithelial cells.
Apoptosis is an active form of programmed cell death that plays a crucial role in the development and maintenance of organism homeostasis by eliminating of damaged or redundant cells [7][8][9]. In this context, organisms can employ antioxidant defense system to counteract oxidative stress and further prevent oxidative damage [10]. A compelling body order to maximize yield of polysaccharide, glucose was added in the culture medium with a final concentration of 10%; the Mycoplasma cells were cultured at 37 ∘ C for two or three days after the medium color was changed from red to yellow; the cells were then collected by centrifugation at 12,000 for 30 min at 4 ∘ C. The preparation of CPS was performed as described previously with modifications [6]. Briefly, the cell pellet was washed three times with phosphate buffered saline (PBS, pH = 7.4) containing 10% glucose prior to the isolation/extraction procedure, in order to minimize potential contaminants. Mycoplasma polysaccharides were extracted using 60 ∘ C preheated phenol for 30 min with constantly stirring. The resulting extracts in the aqueous phase were dialyzed in cellulose membrane tubing (exclusion limit 3500 Da), prior to be further treated with DNase I, RNase, and pronase K for removal of nucleic acids and protein contaminations, which was confirmed by a spectrophotometric assay in terms of determining 260/280 ratio. The extracts were then subjected to phenol/chloroform extraction (1 : 1) using a Phase-lock gel (Tiangen, Beijing, China), followed by being extracted with an equal volume of chloroform in order to remove possible lipophilic components of cytoplasmic membranes. The upper aqueous phase was subsequently collected and dialyzed against distilled water and PBS, followed by being precipitated with 4 equivalent volume of cold ethanol containing 3 M sodium acetate at 4 ∘ C overnight. The result was crude polysaccharides, which was dissolved in distilled water and then lyophilized using a vacuum freeze dryer. For purification of CPS, above crude CPS was dissolved in H 2 O and applied to a 1.0 by 5 cm DEAE-cellulose anionexchange chromatography column, and fractions were eluted with a linear gradient of 0-1.0 M NaCl at a flow rate of 0.5 mL/min. Thirty-milliliter fractions were equally collected into 100 consecutive tubes. The content of polysaccharide of effluents was then detected by an improved phenolsulfuric acid colorimetric method at 490 nm [16]. As a result, purified CPS-containing factions were collected, lyophilized, and stored at −80 ∘ C until further use.

In Vitro ALI Culture of Sheep Bronchial Epithelium and
Infection of M. ovipneumoniae. This study was approved by the ethics committee for the care and use of animals at Ningxia University. Primary culture of sheep bronchial epithelial cells was prepared as previously reported in our lab [14]. Briefly, bronchial epithelial cells were obtained from 1.5to 2.0-year-old Tan sheep. Bronchus was washed and digested overnight at 4 ∘ C using DMEM/F12 medium (1 : 1) containing 5% FBS and 0.1% DNaseI. Cells were then collected by centrifugation at 800 ×g for 5 min at 4 ∘ C. Subsequently, fibroblast cells were removed via adherence in basic media, while nonadherent cells were collected and cultivated in collagencoated Millicell insert membrane using 5% FBS/BEGM medium at 37 ∘ C for 24-48 h. The BEGM was then replaced by Ultroser G medium (USG) (F12/MEM 1 : 1, +Supplements Mix +2% USG) to establish an air-liquid interface (ALI) culture system. After 4-6 weeks of cultivation, epithelial cells were well-differentiated. For a 2.4 cm diameter Millicell insert membrane, normally 1 × 10 7 well-differentiated epithelial cells were determined [17]. For infection or treatment, Oxidative Medicine and Cellular Longevity 3 M. ovipneumoniae cells or CPS were suspended or diluted in 2% USG medium and applied on the apical surface of ALI epithelial cells for infection with indicated time periods; equal volume of 2% USG medium was used as untreated control. Volumes of 0.5 mL and 0.1 mL were employed for covering wells with diameters of 2.4 cm and 1.2 cm, respectively.

Cell Viability (CCK) Assays and Lactate Dehydrogenase
(LDH) Assays. Cell viability was determined by Cell Counting Kit-8 (CCK-8) assay according to the manufacturer's instruction. In brief, cells (5 × 10 3 cells/well) were seeded in 96-well microplates and cultured overnight at 37 ∘ C. The cells were then treated with M. ovipneumoniae or CPS for indicated time periods. Subsequently, the CCK-8 solution (10 L) was added to each well and incubated for additional 4 h. Absorbance at 450 nm was measured using an ELISA reader (Bio-Rad Laboratories, Richmond, CA, USA). The death of ALI epithelial cells was determined by a LDH assay. Briefly, cells cultured in 1.2 cm diameter inserts were pretreated with or without N-acetyl-cysteine (NAC, ROS scavenger) (10 mM) for 2 h, followed by exposure to CPS (100 ng/mL), MO (multiplicity of infection, MOI = 30), or H 2 O 2 (1 mmol/L) for 48 h. Media were used for analysis using a LDH Cytotoxicity Assay Kit according to the manufacturer's instruction. Subsequently, the absorbance at 450 nm was measured using a plate reader. Untreated ALI cells were served as a blank control. The cytotoxicity was determined according to the following formula: cytotoxicity (%) = OD (tested sample − control)/OD (positive control − control) × 100%. The positive control was provided in the kit.

Flow
Cytometric Analysis of Apoptosis. The 4-week ALI cultures were infected with MO or treated with CPS as described above. The cultures were washed three times in PBS at 37 ∘ C prior to be disassociated with trypsin, subsequently harvested by centrifugation, and finally resuspended in 1x binding buffer (10 mM HEPES/NaOH, 140 mM, NaCl, 2.5 mM CaCl 2 , pH 7.4) Annexin V-FITC/propidium iodide (PI) double-staining assay was detected by flow cytometry using Apoptosis Detection Kit according to the manufacturer's instruction. The data was then analyzed using a flow cytometry system (CyFlow5 Cube 15, USA).

Mitochondrial Membrane Potential (MMP)
Assay. JC-1 is a common fluorescent cationic probe to detect the disruption of mitochondrial membrane potential. Mechanistically, JC-1 forms red fluorescence at high mitochondrial membrane potential, while green fluorescence indicates a lower membrane potential. Cells (1 × 10 7 cells/well) were incubated with 10 M JC-1 staining at 37 ∘ C for 30 min and then the mitochondrial potential was detected using a CyFlow Cube 15 and analyzed by FCS Express 5.0 software.
After rinsing with PBS twice, cells (1 × 10 7 cells/well) were subsequently collected and treated in PBS containing 10 M DCFH-DA at 37 ∘ C for 20 min. Fluorescence intensity was measured at 488 nm/525 nm (excitation/emission wavelengths) by a CyFlow Cube 15 system. Data from at least three independent experiments were analyzed with FCS Express 5.0 software.

Western
Blotting Analysis. 6-well ALI inserts were infected with indicated conditions for 48 h. Cells were washed with cold PBS twice and lysed in RIPA buffer containing protease/phosphatase inhibitors (Beyotime, Beijing, China) overnight at 4 ∘ C. Protein concentrations were then determined by BCA protein assay. The protein samples were resolved by 6-15% SDS-PAGE and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% nonfat milk in TBST at room temperature for 2-4 h, followed by incubation with appropriate specific primary antibodies to proteins of interest overnight at 4 ∘ C. After extensive washing, membranes were probed with appropriate HRPlabelled secondary antibodies for 1-2 h at room temperature. Blots were finally developed and visualized using an ECL Advanced Western Blot Detection Kit. The specific protein blots were semiquantified by densitometric analysis using Image J software.

Statistical Analysis.
All data in this study were expressed as the mean ± SD from data of at least three independent experiments. SPSS statistics 17.0 (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. Statistical differences between groups were performed using one-way analysis of variance (ANOVA) followed by post hoc Tukey's test. A value < 0.05 was considered statistically significant. The column was first eluted with distilled water, followed by eluting with a linear concentration gradient of NaCl from 0 to 1.0 mol/L. extraction of crude polysaccharides. In the present study, the crude capsular polysaccharide of M. ovipneumoniae has been successfully isolated by a phenol extraction and ethanol precipitation method. The average yield of crude capsular polysaccharides was 4.658 ± 0.662% of wet weight of bacteria. There were no detectable nucleic acid and protein contamination as determined by a ratio of absorption at 260 and 280 nm.

Isolation and
To obtain high-purity of polysaccharide products for biofunctional studies, a subsequent purification was performed by a high performance liquid chromatography (HPLC) using a cellulose DEAE-52 column. Figure 1 showed the curve of capsular polysaccharide detected by an improved phenolsulfuric acid colorimetric method [16]. The curve displayed a main peak of CPS between fractions 46 to 72 following a linear elution. The eluents of this peak were collected and lyophilized. The average recovery rate of the purified CPS was 62.26 ± 5.002% of the crude CPS in this study. This purified CPS was used for subsequent biofunctional analysis.

Effects of CPS on Cell Viability in ALI Cultures of Sheep
Bronchial Epithelial Cells. In order to evaluate biological effects of CPS and M. ovipneumoniae in sheep bronchial epithelial cells, their effect on cell viability was first determined by a Cell Counting Kit-8 (CCK-8) assay. Results showed that both CPS and M. ovipneumoniae significantly inhibited the cell proliferation in a time-and dose-dependent manner, with an exception of no difference between cells exposed to 100 ng/ml and 1 g/ml of CPS for 36 h and 48 h, respectively ( Figure 2(a)). In agreement with results of the CCK assay, the LDH assay showed a similar result, and there was no significant statistical difference between cells treated with 100 ng/ml and 1.0 g/ml of CPS for 48 h (Figure 2(b)). Moreover, to assess whether the CPS-induced cell death was dependent on ROS production, cells were pretreated with NAC, a ROS scavenger prior to CPS treatment. The result showed pretreatment with NAC significantly suppressed both CPS-and MO-mediated cytotoxicity, suggesting that ROS might play key roles involved in CPS-and MO-induced cell death (Figure 2(b)). As the preferable effect of CPS on cell viability was observed at 100 ng/mL, the concentration of 100 ng/mL CPS was chosen for the following experiments.

CPS-Induced Cell Apoptosis in ALI Cultures of Sheep Bronchial Epithelial Cells.
Apoptosis is a process of programmed cell death that occurs in multicellular organisms [7]. In present study, a flow cytometry assay following Annexin V/PI double-staining was used to evaluate whether CPS-and MO-induced cell deaths were relevant to apoptosis. As expected, a significantly increased apoptotic cell fraction was observed in cells treated with CPS or M. ovipneumoniae infection compared to untreated cells (Figure 3(a)). The percentage of CPS-induced apoptotic cells was increased from 15.26% to 19.86%, while the fraction of M. ovipneumoniaetreated apoptotic cells rose to 21.54%, suggesting that both CPS and MO remarkably induced a cell apoptosis progression. Unexpectedly, it is worth noting that pretreatment with NAC could not apparently inhibit the CPS-induced cell apoptosis, although NAC could dramatically alleviate cell apoptosis induced by a M. ovipneumoniae infection. These results indicated that the CPS-induced cell apoptosis might be not mainly attributed by ROS production. As caspase-3 and its downstream substrate, PARP, play an active role in the early apoptotic events, and a cleaved form of PARP has been considered as an indicative of functional caspase activation [18].

ROS Generation and Oxidative Stress Induced by CPS in ALI Cultures of Sheep Bronchial Epithelial Cells.
There is now widespread and strong evidence implicating that ROS and oxidative stress play key roles in mediating the mitochondrial dysfunction [19,20]. Accumulation of excessive ROS can directly interact with cellular components, such as DNA and protein, thereby causing oxidative stress and subsequent cell death [13]. With this in mind, we further investigated whether the elevated ROS level of CPS-or MO-treated cells served as a cause of apoptosis induced by these insults. FACS analysis revealed that both CPS and M. ovipneumoniae could induce a notably increased ROS production compared to controls, as measured by an oxidant-sensitive probe, CM-H 2 DCFDA. These effects were almost completely abolished by scavenging ROS with NAC, indicative of a potential role of CPS and M. ovipneumoniae in ROS production in sheep bronchial epithelial cells (Figures 4(a) and 4(b)). Protective antioxidants form the first line of defense against ROS by reacting directly with free radicals, or by enhancing the bioactivity and expression of various antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione synthetase (GSS) [21]. With this in mind, to further explore whether CPS could induce oxidative stress in ALI Oxidative Medicine and Cellular Longevity

CPS Triggered an Intrinsic Apoptotic Pathway in Sheep
Bronchial Epithelial Cells. Apoptosis is a highly regulated and controlled process, which can be initiated through two pathways: mitochondria-mediated (intrinsic) and celldeath-receptor-mediated (extrinsic) apoptotic pathways [7,22]. To further interrogate the mechanism underpinning processes of CPS-induced apoptosis, several apoptosisassociated parameters widely implicated in these two pathways were examined. The cell membrane potential (ΔΨ ) is a key index to evaluate the integrity of mitochondrial membrane. Disruption of ΔΨ by several factors, for instance, excessive ROS generation, can cause cell damage [23]. JC-1, a fluorescent probe, was thus applied to observe whether there was loss in ΔΨ using a flow cytometry-based assay. Indeed, an exposure to CPS or M. ovipneumoniae alone for 48 h resulted in an evident elevation in percentages of cells with green fluorescence to more than 200% as compared with the control (Figure 5 Meanwhile, an enhanced activity of caspase-9, an initiator caspase linking to the intrinsic apoptotic pathway, was also observed ( Figure 5(h)). This finding suggested that pretreatment with NAC could alleviate CPS or bacteria-induced intrinsic apoptosis in part, indicating that a ROS-dependent mitochondrial-mediated (intrinsic) apoptosis might be involved in the CPS-and M. ovipneumoniae-induced apoptosis in sheep bronchial epithelial cells.

CPS Triggered an Extrinsic Apoptotic Pathway in Sheep
Bronchial Epithelial Cells. We next sought to investigate whether an extrinsic apoptotic pathway was involved in the cell apoptosis induced by CPS or bacteria cells of M. ovipneumoniae. Apoptosis can be initiated by ligation of death receptors on cell surface to their cognate death receptor ligands. It is well known that FAS-associated death domain (FADD) protein is a key adaptor in the death-receptor-mediated apoptosis [24]. In this context, the death receptor FAS is activated upon the binding of FAS ligand (FASL) and cytoplasmic FADD via its death domain (DD), which in turn further recruits and activates procaspase-8 to initiate an induction of apoptosis [24,25]. examined. Of interest, a significant upregulation of above extrinsic apoptotic proteins was determined in both CPS-and M. ovipneumoniae-treated cells, and a pretreatment of NAC led to almost completely abolishing of the induced expression of these proteins, particularly the cleaved form of caspase-8, except the FASL, which was slightly increased in the CPStreated group (Figures 6(a)-6(e)). In consistence, biological activity assays further confirmed an enhanced activation of caspase-8 activity in the CPS or M. ovipneumoniae-treated cells, which could be inhibited by NAC (Figure 6(f)). Taken together, these data clearly suggest that the extrinsic apoptotic signaling is also involved in the CPS/M. ovipneumoniaeinduced apoptosis in sheep bronchial epithelial cells.

Activation of the MAPK Signaling Pathway in Sheep Bronchial Epithelial Cells in Response to CPS Stimulation.
In order to explore a potential mechanism underlying CPSinduced cell apoptosis, we next examined the activation of mitogen-activated protein kinase (MAPK) signaling in sheep epithelial cells, since evidence has recently revealed that MAPK signaling acts as a key signal transduction pathway in regulating cell inflammation and apoptosis. To this end, ALI cultures were pretreated with NAC for 2 h, followed by exposing to CPS or M. ovipneumoniae cells for 48 h and cell lysates were then harvested for Western blot analysis. Compared to the control, the phosphorylation of ERK1/2, JNK/c-Jun, and P38 was remarkably upregulated after treatments of CPS and bacterial cells (    Unexpectedly, a pretreatment of ERK inhibitor U0126 failed to inhibit the CPS-induced activation of ERK1/2 and caspase-3 (Figures 8(a) and 8(b)). In contrast, a pretreatment of either JNK signaling inhibitor SP600125 (Figures 8(c) and 8(d)) or p38 signaling inhibitor SB203580 (Figures 8(e) and 8(f)) showed a significantly downregulated CPS-and M. ovipneumoniae-induced phosphorylation of JNK/c-Jun or p38, with a remarkable decrease of the activity of caspase-3. Taken together, these results imply that CPS-induced apoptosis may be mainly attributed to an activation of JNK and p38, but not ERK1/2 of the MAPK pathways, although all three MAPK members including ERK1/2, JNK, and P38 can be activated by stimuli of CPS and M. ovipneumoniae in sheep bronchial epithelial cells.

Discussion
In the present study, we describe the isolation and purification of the capsular polysaccharide (CPS) of M. ovipneumoniae for the first time and substantially evaluate the effects and possible molecular mechanism in cell apoptosis of sheep bronchial epithelial cells. Our results demonstrate that the CPS had a cytotoxic effect of decreasing cell viability with an ability to induce ROS production, which in turn disrupted the integrity of mitochondrial membrane, along with a reduction of ΔΨ and an increased expression of proapoptotic proteins.  to Western blotting analysis using indicated antibodies. The blots were probed for -actin as a loading control. Results showed that NAC significantly suppressed CPS-induced phosphorylation of ERK1/2, JNK/c-Jun, and P38. (b-e) Protein ratio of interest. protein/ -actin was calculated following ImageJ densitometric analysis. These bar graphs shown here were representative of three independent experiments with similar results. Compared to non-CPS or MO treated control, * < 0.05 and * * < 0.01; compared to non-NAC treated group, < 0.05 and < 0.01.
Mechanistically, the CPS-induced cell apoptosis was induced by mechanisms involved in caspase-dependent intrinsic and extrinsic apoptotic pathways. Moreover, the CPS-induced intrinsic cell apoptosis was ROS-dependent and mediated by MAPK signaling pathways. In this regard, the JNK and p38 but not the ERK signaling of the MAPK pathway might be the main intrinsic apoptotic pathways that contributed to the CPS-induced apoptosis in these cells.
Apoptosis is an active form of programmed cell death that plays a vital role in the maintenance and development of organism homeostasis [8,9,26]. It allows cells to undergo a highly regulated form of death in response to various proapoptotic stimuli. Evidence has demonstrated that ROS are generated from both mitochondria and NADPH oxidases [7,27], which has been well defined to regulate various signal transduction pathways including inflammation and apoptosis. Thus, in this study we tentatively explored the potential mechanism that CPS induces apoptotic process via the generation of ROS in ALI cultures of sheep bronchial epithelial cells, and an elevated production of ROS was found in cells exposed to the CPS and M. ovipneumoniae bacteria cells.
It is well known that an activation of caspase represents a crucial step in the induction of apoptosis, and the cleavage of poly ADP-ribose polymerase (PARP) by caspase-3 is also considered to be one of the hallmarks of apoptosis [18]. FACS analysis showed an increased proportion of apoptotic cells in CPS-and M. ovipneumoniae-treated cells compared to untreated cells, which was consistent with the results from Western blotting, from which the cleaved-caspase-3 and cleaved-PARP1 were increased in both CPS-and M. ovipneumoniae-treated cells. Of particular interest, the caspase-3, one of the most widely studied and the most key apoptosis effectors [9], was strikingly activated following a stimulation of CPS and M. ovipneumoniae cells, thereby suggesting that CPS-and M. ovipneumoniae-induced cell toxicity were at least in part related to an apoptotic progression. In order to determine the effects of ROS in regulating apoptosis, a ROS scavenger, NAC (N-Acetyl-L-cysteine), was used to further clarify the underlying mechanism. Results showed that a pretreatment of NAC could dramatically downregulate the generation of ROS induced by CPS and M. ovipneumoniae, which resulted in an increase in CAT, SOD 2 , and GSS levels.
Interestingly, cytotoxicity of CPS was still observed in treated cells even if NAC pretreatment could scavenge most of ROS production, suggesting that there may exist other proapoptotic mechanisms apart from the ROS-induced oxidative damage of epithelial cells. Collectively these data suggest that ROS might be an essential player in apoptosis of cells in response to M. ovipneumoniae infection; however, its precise role in CPS-induced apoptosis needs further investigation.
Mitochondria are a main generator of ROS, which play an essential role in the oxidative stress-triggered signaling pathway during the process of apoptosis [28][29][30]. The integrity of mitochondrial membrane serves as the basis for mitochondria function, which can be mainly regulated by different Bcl-2 family members including proapoptotic proteins, such as Bax, and antiapoptotic proteins, such as Bcl-2 and Bcl-xl [31,32]. Previous studies have documented that Bax, the most important proapoptotic protein, can induce the translocation of cytochrome C from mitochondria to cytoplasm. The released cytochrome C subsequently leads to the release of apoptosis inducing factor (AIF), which in turn activates caspase cascades, finally resulting in cell apoptosis [33,34]. In this signal cascade, caspases are key regulators in the apoptotic pathway, which generally consist of the upstream initiators, such as caspase-8, -9, and -10 and the downstream effectors such as caspase-3, -6, and -7 [9]. In line with this notion, we firstly confirmed that mitochondria were involved in CPS-and M. ovipneumoniaeinduced cell apoptosis. The results showed that an exposure of CPS or an infection of M. ovipneumoniae could markedly upregulate the percentage of apoptotic cells compared to the control. In contrast, an elimination of ROS with NAC led a partial suppression of apoptotic effects induced by CPS and M. ovipneumoniae. Immunoblotting analysis further revealed that the occurrences of apoptosis induced by both CPS and M. ovipneumoniae were linked to an upregulation of proapoptotic protein Bax, cytochrome C, and AIF in ALI cells. Furthermore, a scavenging ROS by NAC could alleviate the CPS-and M. ovipneumoniae-induced mitochondrial damage, indicating that the intrinsic apoptosis pathway might be at least in part involved in the finally apoptotic death of ALI cells. Notably, inconsistent with results from other studies, the expression of Bcl-2 in the present study was evidently upregulated in response to CPS-or M. ovipneumoniae stimulation, suggesting that Bcl-2 might play a protective role in these cells in response to excessive oxidative stress, therefore rescuing cells from cell death [31]. Another possible reason might be that the Bax could antagonize the antiapoptotic effects of Bcl-2 and Bcl-xl via a binding to one another depending on a BH 3 domain, thereby promoting apoptosis [35].
To further elucidate the role of CPS in M. ovipneumoniaeinduced apoptosis, we examined the effects of CPS in FAS receptor-mediated extrinsic apoptotic pathway. Results showed that the expressions of FAS-associated proteins including FADD, FAS, FASL, caspase-8, and cleaved-caspase-8 were extremely upregulated in response to CPS and M. ovipneumoniae treatments. This effect could be completely inhibited by ROS scavenger NAC, indicating that an increased transcriptional activity of FAS-associated proteins was downstream of ROS signaling. The result was further confirmed by detecting the activation of caspase-8, an upstream initiator of caspase-3 involved in extrinsic apoptotic pathway [36,37]. Interestingly, our results also showed that the CPS and M. ovipneumoniae-mediated extrinsic apoptotic signaling were much more activated relative to intrinsic apoptotic signaling suggesting that an extrinsic apoptotic signaling might contribute significantly to CPS-induced epithelial cell apoptosis in this study.
Given the fact that both intrinsic and extrinsic apoptotic pathways are involved in the CPS of M. ovipneumoniae-induced apoptosis in ALI cultures of sheep bronchial epithelial cells, the underlying mechanisms by which CPS inhibited ALI cells were thus further explored. The MAPKs signaling is believed to be involved in the regulation of several signal transduction pathways including cell proliferation, migration, differentiation, autophagy, and apoptosis [8,38]. MAPKs are evolutionarily conserved Ser/Thr protein kinases that are divided into at least three distinct groups in mammals, including the extracellular signal regulated kinase (ERK), the Jun N-terminal kinases (JNK), and the P38 MAPKs [39]. Mechanistically, upon an activation of this signaling cascade, ERK usually plays an antiapoptotic role, while JNK and P38 exert proapoptotic effects during an apoptotic process [40,41]. In this context, the dynamic equilibrium between the opposing effects of ERK and JNK/P38 MAPK may be critical in determining whether cells survive or undergo apoptosis. Mounting evidences have demonstrated that ROS-induced apoptotic cell death is mainly mediated by JNK and P38 MAPK activation [42], both of which initiate mitochondria-dependent apoptosis through a caspases signaling pathway, partially owing to an enhanced transcription of proapoptotic genes, such as Bax [43,44]. In agreement with these notions, we showed that both CPS and M. ovipneumoniae stimuli resulted in an activation of all the three MAPK family members, but only the JNK and p38 pathways but not the ERK1/2 signaling participated in CPS-induced apoptosis. This observation is of particular mentioning to importance due to the fact that an inhibitor of ERK1/2 signaling is incapable of reducing the activation of caspase-3, indicating that CPS-induced apoptosis can be independent of ERK1/2 signaling and can progress alternatively through the JNK and p38 pathways. Of note, this observation was different from the M. ovipneumoniae bacteria-induced cell apoptosis determined in our recent study, in which M. ovipneumoniae was demonstrated to induce the cell apoptosis through an ERK-mediated MAPK pathway [15]. The discrepancy suggested distinct pathogenic roles of CPS of M. ovipneumoniae, as compared with its parent pathogen bacteria cells. The JNK is another key regulator in oxidative stressinduced apoptosis, as well as in the autophagy induction [45][46][47][48], and ROS have been reported to be a strong activator of JNK signaling and play a crucial role in JNK-dependent apoptosis and autophagy regulation [49]. Although our results showed that CPS-and M. ovipneumoniae-induced activation of JNK were accompanied by ROS production, it is noteworthy that NAC failed to alleviate the M. ovipneumoniae-induced phosphorylation of JNK, which contradicted the result of CPS, suggesting that ROS may be not the sole upstream activator of JNK signaling pathway in response to a M. ovipneumoniae infection. One possible explanation is that the activation of JNK participated in a crosstalk between apoptosis and autophagy triggered by M. ovipneumoniae via promoting autophagy and suppressing apoptosis in the presence of NAC. Consistently, both CPS and M. ovipneumoniae could induce an evident PARP-1 activation that was suggested to cause a prosurvival autophagy via the AMPK-mTOR signaling pathway [50]. This result therefore indicates that CPS-and M. ovipneumoniae-induced oxidative stress may be involved in the autophagy induction. As a consequence, it is reasonable to hypothesize that CPS-and M. ovipneumoniae-treatment simultaneously induce the progress of apoptosis and autophagy in ALI cultures of sheep bronchial epithelial cells, which requires further investigations. Taken together, we hypothesize that the ROS-mediated JNK/P38 MAPK signaling pathways may be one of the main routes contributing to CPS-induced apoptosis. These findings thus provide insight into the molecular pathways that lead to apoptosis of M. ovipneumoniae-infected respiratory tract epithelia ( Figure 9).

Conclusions
Collectively, this study reveals that ROS-regulated activation of JNK and p38 signaling may play key roles in sheep airway epithelial cells upon M. ovipneumoniae infection. Moreover, the capsular polysaccharide (CPS) of M. ovipneumoniae serves as an important virulence factor, contributing to M. ovipneumoniae-induced apoptosis through both ROSdependent-intrinsic and extrinsic apoptotic pathways. Our findings underline that CPS of M. ovipneumoniae may be exploited for acceleration of M. ovipneumoniae-induced oxidative stress and consequent disruption of homeostasis. Besides, considering that CPS may participate in autophagy and necrosis, whose activation in a ROS/JNK-dependent manner may serve as other critical mechanisms for cell death, whether CPS/M. ovipneumoniae is involved in above two processes remains unknown. Thus, further study on the regulation of autophagy and necrosis will help us establish a clearer understanding of the mechanism of CPS-and M. ovipneumoniae-induced cell death in sheep respiratory epithelial cells.