Effect of Pregnane X Receptor on CYP3A29 Expression in Porcine Alveolar Macrophages during Mycoplasma hyopneumoniae Infection

Simple Summary In the currently intense production process, infection of swine with Mycoplasma pneumonia is common in pig farms around the world, and reduction in the feeding efficiency and the growth rate of sick pigs causes considerable economic losses to the pig-rearing industry. Our study aimed to determine the molecular mechanism by which Mycoplasma hyopneumoniae induces inflammation in pigs. Our study showed that Mycoplasma hyopneumoniae can regulate the expression of CYP3A29 by upregulating PXR during the inflammatory response induced in porcine alveolar macrophages. These findings may provide useful information for breeding pigs that are resistant to disease. Abstract Mycoplasma hyopneumoniae (M. hyopneumoniae, Mhp) is the causative agent of mycoplasma pneumonia of swine (MPS). M. hyopneumoniae infection causes inflammation in pigs and leads to considerable economic losses in the pig industry. Pregnane X receptor (PXR) is a pluripotent gene regulatory protein that plays an important role in regulating cytochrome P-450 (CYP) in pigs in the context of inflammatory responses, drug metabolism, homeostasis, etc. We previously reported that cytochrome P450 3A29 (CYP3A29) expression was significantly upregulated in pigs infected with M. hyopneumoniae compared with healthy control pigs. This experiment mainly focused on identifying the role of PXR in the regulation of CYP3A29 and inflammatory factors after M. hyopneumoniae infection by establishing pig alveolar macrophage (PAM) cells in which PXR was overexpressed or silenced. Our results showed that the overexpression of PXR could significantly improve the protein and the mRNA expression levels of CYP3A29 with and without M. hyopneumoniae infection in PAM cells. After the expression of PXR was inhibited, protein and mRNA expression levels of CYP3A29 were significantly reduced with and without M. hyopneumoniae infection in PAM cells. Moreover, PXR can regulate the mRNA expression levels of IL-6 and IL-8 during M. hyopneumoniae infection of PAM cells. In conclusion, these results suggest that PXR positively regulates CYP3A29 expression during the inflammatory response caused by M. hyopneumoniae infection.


Introduction
Mycoplasma pneumonia of swine, commonly known as swine asthma, is a chronic respiratory infection in pigs that is caused by M. hyopneumoniae and spreads due to close contact with infected individuals [1]. Sick pigs exhibit slow growth and low feed conversion

Transfection
Cells transfected with the pcDNA3.1-PXR plasmid were used as the experimental group, and cells transfected with the empty pcDNA3.1 vector were used as the negative control group. The cells were transfected with Lipofectamine TM 3000 (Invitrogen, Carlsbad, CA, USA) using 2.5 g plasmid per well for 36 h when the cell density reached 60%. RPMI-1640 medium containing 10% fetal bovine serum was used in the transfection process. In the interference experiment, the same method was used to transfect the porcine PXR interfering fragment siRNA.

M. hyopneumoniae Infection of PAM 3D4/21 Cells
Infection was carried out in a 6-well plate at a density of 2 × 10 5 cells/well. Two milliliters of 10 6 CCU/mL M. hyopneumoniae strain J was added to each well of the infected group, and 2 mL of RPMI 1640 medium containing 10% fetal bovine serum was added to each well of the control group. The M. hyopneumoniae cultures were centrifuged at 12,000× g for 20 min, washed with sterile phosphate buffered saline (PBS) twice, and resuspended with antibiotic-free RPMI 1640 medium, and samples were collected 24 h after infection [18]. Infection was confirmed by PCR amplification of the P36 gene, which encodes a membrane protein of M. hyopneumoniae, as previously described [19].

Real-Time Fluorescence Quantitative PCR
Total RNA was extracted from PAM 3D4/21 cells using the HP Total RNA Kit (Omega Bio-Tek, Norcross, GA, USA) according to the instructions. The total RNA concentration was measured by a spectrophotometer. The PrimeScript TM RT reagent Kit (Takara, Otsu, Shiga, Japan) was used for reverse transcription. qPCR was conducted on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex Taq TM (Tli RNaseH Plus) (Takara, Otsu, Shiga, Japan). The total amplification reaction system contained 20 µL, and the amplification was carried out at 95 • C for 2 min, followed by 40 cycles of 95 • C for 15 s, 60 • C for 30 s, and 72 • C for 30 s. HPRT1 was used as the internal reference. Primer sequences are shown in Table 1.

Western Blot
The total protein was extracted by radio immunoprecipitation assay (RIPA) buffer, and the sample concentration was determined by a bicinchoninic acid (BCA) protein assay (Pierce, Thermo Scientific, Waltham, MA, USA). The supernatant was absorbed, mixed with 5× SDS, and denatured at 99 • C for 10 min. SDS-PAGE was performed by adding 30 µg of protein per well to a 10% gel and then transferring the protein to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% bovine serum albumin (BSA) in 0.1% Tween 20 for 2 h. Then, a rabbit anti-PXR antibody (1:500 dilution) and a mouse anti-pig CYP3A29 antibody (1:1000 dilution) were prepared by diluting the antibodies in 5% BSA; then, the diluted antibodies were incubated with the membranes in the refrigerator at 4 • C overnight. Then, the membranes were washed 3 to 4 times with tris buffered saline tween (TBST), and diluted goat anti-rabbit and goat anti-mouse (1:1000 dilution) antibodies were prepared as secondary antibodies. The membranes were incubated at room temperature for 2 h and washed with TBST. The bands were visualized and quantified with the Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA) and quantified by a DNR Bio Imaging System (DNR Industries, Anaheim, CA, USA).

Enzyme-Linked Immunosorbent Assay ELISA
Porcine IL-6 ELISA Kit (Absin, Shanghai, China) and Porcine IL-8 ELISA Kit (Jingmei, Yancheng, Jangsu, China) were used to detect the protein expression of IL-6 and IL-8 in the cell culture supernatant of the PAM 3D4/21 cells, respectively.

Statistical Analysis
This experiment used the 2 − Ct method to analyze the data, and HPRT1 was used as the reference gene. GraphPad Prism was used to generate the graphs and conduct the analyses, and a t-test was performed. p < 0.05 is indicated by *; p < 0.01 is indicated by **; p < 0.001 is indicated by ***; and NS indicates no significant difference. Image J was used to detect the gray values of the western blot bands so that the data could be quantitatively analyzed.  [20]. Infection was confirmed by PCR amplification of the P36 gene [19]. P36 is an immunodominant protein encoded by the genome of M. hyopneumoniae, and p36 proteins have been shown to be specific for M. hyopneumoniae [21]. A single 427-bp band of P36 was detected by agarose gel electrophoresis, indicating that M. hyopneumoniae had successfully infected the PAM 3D4/21 cells. Therefore, infection with 100 MOI M. hyopneumoniae for 24 h was used in the subsequent experiments.
We then examined the effect of M. hyopneumoniae infection on the expression of IL-6 and IL-8. The results showed that the mRNA expression of IL-6 ( Figure 1B) and IL-8 ( Figure 1C

M. hyopneumoniae Infection Increased the Expression of CYP3A29 and PXR in PAM 3D4/21 Cells
To investigate whether the expression of CYP3A29 and PXR was altered in response to M. hyopneumoniae infection, we detected mRNA and protein expression levels of PXR and CYP3A29. The results showed that the mRNA levels of both PXR ( Figure 2A

M. hyopneumoniae Infection Increased the Expression of CYP3A29 and PXR in PAM 3D4/21 Cells
To investigate whether the expression of CYP3A29 and PXR was altered in response to M. hyopneumoniae infection, we detected mRNA and protein expression levels of PXR and CYP3A29. The results showed that the mRNA levels of both PXR (

Identification and Validation of the pcDNA3.1-PXR Recombinant Plasmid and Efficiency of PXR Overexpression and Interference
The PXR recombinant plasmids were identified by restriction analysis and sequencing. Analysis of two fragments released from the recombinant plasmids by digestion with homologous restriction enzymes (EcoR I and Sal I) revealed that the PXR gene was correctly inserted. The clone was further confirmed by sequencing.
The efficiency of PXR overexpression and silencing was analyzed by assessing the expression of PXR mRNA and protein. The PXR mRNA and protein levels in PAM 3D4/21 cells were significantly increased after transfection with the pcDNA3.1-PXR recombinant plasmid for 36 h ( Figure 3A,B). However, the PXR mRNA and protein levels in PAM 3D4/21 cells were significantly decreased after transfection with PXR-SUS-992 for 36 h ( Figure 3C,D). The data are presented as the mean ± SD. The experiment was repeated three independent times. *** p < 0.001 compared to the uninfected group. The PXR recombinant plasmids were identified by restriction analysis and sequencing. Analysis of two fragments released from the recombinant plasmids by digestion with homologous restriction enzymes (EcoR I and Sal I) revealed that the PXR gene was correctly inserted. The clone was further confirmed by sequencing.

PXR Regulates the
The efficiency of PXR overexpression and silencing was analyzed by assessing the expression of PXR mRNA and protein. The PXR mRNA and protein levels in PAM 3D4/21 cells were significantly increased after transfection with the pcDNA3.1-PXR recombinant plasmid for 36 h ( Figure 3A,B). However, the PXR mRNA and protein levels in PAM 3D4/21 cells were significantly decreased after transfection with PXR-SUS-992 for 36 h ( Figure 3C,D).

PXR can Promote CYP3A29 Expression during M. hyopneumoniae Infection
To further explore whether PXR regulates the expression of CYP3A29 during M. hyopneumoniae infection, mRNA and protein expression of PXR and CYP3A29 were analyzed by qPCR and western blot. The results showed that the overexpression of PXR in normal PAM 3D4/21 cells significantly increased mRNA ( Figure 4A) and protein ( Figure 4B) levels of CYP3A29. PXR was overexpressed in PAM 3D4/21 cells infected with M. hyopneumoniae, and mRNA ( Figure 4C) and protein ( Figure 4D) levels of CYP3A29 showed the same trend. When compared Figure 4A,B with Figure 4C,D, it was found that the promotion effect of overexpression of PXR on CYP3A29 with M. hyopneumoniae was lower than that without M. hyopneumoniae infection. However, silencing of PXR in normal PAM 3D4/21 cells significantly decreased mRNA ( Figure 5A) and protein ( Figure 5B) levels of CYP3A29. After transfection of PXR-sus-992 into PAM 3D4/21 cells infected with M. hyopneumoniae, mRNA ( Figure 5C) and protein ( Figure 5D) levels of CYP3A29 were also significantly reduced. Comparing Figure 5A,B with Figure 5C,D, it was found that the inhibiting effect of silencing of PXR on opneumoniae infection, mRNA and protein expression of PXR and CYP3A29 were analyzed by qPCR and western blot. The results showed that the overexpression of PXR in normal PAM 3D4/21 cells significantly increased mRNA ( Figure 4A) and protein ( Figure 4B) levels of CYP3A29. PXR was overexpressed in PAM 3D4/21 cells infected with M. hyopneumoniae, and mRNA ( Figure 4C) and protein ( Figure 4D) levels of CYP3A29 showed the same trend. When compared Figure 4A,B with Figure 4C,D, it was found that the promotion effect of overexpression of PXR on CYP3A29 with M. hyopneumoniae was lower than that without M. hyopneumoniae infection. However, silencing of PXR in normal PAM 3D4/21 cells significantly decreased mRNA ( Figure 5A) and protein ( Figure 5B) levels of CYP3A29. After transfection of PXR-sus-992 into PAM 3D4/21 cells infected with M. hyopneumoniae, mRNA ( Figure 5C) and protein ( Figure 5D) levels of CYP3A29 were also significantly reduced. Comparing Figure 5A,B with Figure 5C,D, it was found that the inhibiting effect of silencing of PXR on CYP3A29 with M. hyopneumoniae was higher than that without M. hyopneumoniae infection. It shows that CYP3A29 further increased after M. hyopneumoniae infection.  The data are presented as the mean ± SD. The experiment was repeated three independent times. * p < 0.05; ** p < 0.01; *** p < 0.001.

PXR can Promote the Expression of IL-6 and IL-8 during M. hyopneumoniae Infection
Our previous research showed that M. hyopneumoniae infection increased the expression of PXR, IL-6, and IL-8. To prove whether PXR could affect the expression of IL-6 and IL-8 during M. hyopneumoniae infection, we detected the expression of IL-6 and IL-8 after the overexpression or silencing of PXR. The results showed that the overexpression of PXR without M. hyopneumoniae infection significantly increased the mRNA levels of IL-6 and IL-8 ( Figure 6A). The silencing of PXR without M. hyopneumoniae infection decreased the mRNA levels of both IL-6 and IL-8 ( Figure 6B). Moreover, the overexpression of PXR with M. hyopneumoniae infection significantly increased the mRNA levels of IL-6 and IL-8 The protein expression of CYP3A29 was measured by western blot. The data are presented as the mean ± SD. The experiment was repeated three independent times. * p < 0.05; ** p < 0.01; *** p < 0.001.

PXR can Promote the Expression of IL-6 and IL-8 during M. hyopneumoniae Infection
Our previous research showed that M. hyopneumoniae infection increased the expression of PXR, IL-6, and IL-8. To prove whether PXR could affect the expression of IL-6 and IL-8 during M. hyopneumoniae infection, we detected the expression of IL-6 and IL-8 after the overexpression or silencing of PXR. The results showed that the overexpression of PXR without M. hyopneumoniae infection significantly increased the mRNA levels of IL-6 and IL-8 ( Figure 6A). The silencing of PXR without M. hyopneumoniae infection decreased the mRNA levels of both IL-6 and IL-8 ( Figure 6B). Moreover, the overexpression of PXR with M. hyopneumoniae infection significantly increased the mRNA levels of IL-6 and IL-8 ( Figure 6C). The silencing of PXR with M. hyopneumoniae infection decreased the mRNA levels of IL-6 and IL-8 ( Figure 6D). Subsequently, we used ELISA to detect the protein expression of IL-6 and IL-8 in the cell culture supernatant of the PAM 3D4/21 cells. The results showed that the overexpression of PXR without M. hyopneumoniae infection increased the protein expression of IL-8, but the protein expression of IL-6 did not change significantly ( Figure 7A). The silencing of PXR without M. hyopneumoniae infection decreased the protein expression of IL-6 and IL-8 ( Figure 7B). Moreover, the overexpression of PXR with M. hyopneumoniae infection increased the protein expression of IL-8, but the protein expression of IL-6 did not change significantly ( Figure 7C). The silencing of PXR with M. hyopneumoniae infection decreased the protein expression of IL-6 and IL-8 ( Figure 7D). The data are presented as the mean ± SD. The experiment was repeated three independent times. * p < 0.05; ** p < 0.01; *** p < 0.001. Subsequently, we used ELISA to detect the protein expression of IL-6 and IL-8 in the cell culture supernatant of the PAM 3D4/21 cells. The results showed that the overexpression of PXR without M. hyopneumoniae infection increased the protein expression of IL-8, but the protein expression of IL-6 did not change significantly ( Figure 7A). The silencing of PXR without M. hyopneumoniae infection decreased the protein expression of IL-6 and IL-8 ( Figure 7B). Moreover, the overexpression of PXR with M. hyopneumoniae infection increased the protein expression of IL-8, but the protein expression of IL-6 did not change significantly ( Figure 7C). The silencing of PXR with M. hyopneumoniae infection decreased the protein expression of IL-6 and IL-8 ( Figure 7D).

Discussion
Although mycoplasma pneumonia of swine (MPS) is a serious threat to pig production, the mechanism by which the inflammatory response to M. hyopneumoniae is regulated is rarely studied. It was reported that M. hyopneumoniae infection could cause inflammation in live pigs [22] and induce the secretion of IL-6 and IL-8 from PAM 3D4/21 cells [23], which is consistent with our research. In our study, CYP3A29 and PXR were upregulated after M. hyopneumoniae infection in PAM 3D4/21 cells. CYP3A29, an important cytochrome P450 (CYP) enzyme, is one of the key candidate genes involved in the regulation of inflammation induced by M. hyopneumoniae infection [9]. However, it was not clear how M. hyopneumoniae regulates CYP3A29-induced inflammation. It is well known that the activation of nuclear receptors can increase the expression and the activity of CYPs [24]. PXR is the major transcriptional regulator of cytochrome P450 [13], and it mediates responses to various xenobiotics and endogenous chemicals [25]. Studies have found that PXR can positively regulate the interferon-mediated expression of CYP3A29 in pig hepatocytes [14,15], and cecropin B inhibits the expression of CYP3A29 by regulating RXR-α and PXR in pig liver cells [26]. It is speculated that PXR may regulate the expression of CYP3A29 during mycoplasma infection. In our study, the overexpression and the silencing of PXR increased and decreased the expression of CYP3A29 in normal PAM 3D4/21 cells, respectively. In addition, M. hyopneumoniae-infected normal PAM 3D4/21 cells significantly increased gene and protein expression levels of CYP3A29, while the silencing of PXR decreased the expression of CYP3A29, and the overexpression of PXR increased the expression of CYP3A29. Our study is consistent with previous studies, and based on the pathogen infection pattern, our study validated that PXR could upregulate the expression of CYP3A29 during M. hyopneumoniae infection in PAM cells.

Discussion
Although mycoplasma pneumonia of swine (MPS) is a serious threat to pig production, the mechanism by which the inflammatory response to M. hyopneumoniae is regulated is rarely studied. It was reported that M. hyopneumoniae infection could cause inflammation in live pigs [22] and induce the secretion of IL-6 and IL-8 from PAM 3D4/21 cells [23], which is consistent with our research. In our study, CYP3A29 and PXR were upregulated after M. hyopneumoniae infection in PAM 3D4/21 cells. CYP3A29, an important cytochrome P450 (CYP) enzyme, is one of the key candidate genes involved in the regulation of inflammation induced by M. hyopneumoniae infection [9]. However, it was not clear how M. hyopneumoniae regulates CYP3A29-induced inflammation. It is well known that the activation of nuclear receptors can increase the expression and the activity of CYPs [24]. PXR is the major transcriptional regulator of cytochrome P450 [13], and it mediates responses to various xenobiotics and endogenous chemicals [25]. Studies have found that PXR can positively regulate the interferon-mediated expression of CYP3A29 in pig hepatocytes [14,15], and cecropin B inhibits the expression of CYP3A29 by regulating RXR-α and PXR in pig liver cells [26]. It is speculated that PXR may regulate the expression of CYP3A29 during mycoplasma infection. In our study, the overexpression and the silencing of PXR increased and decreased the expression of CYP3A29 in normal PAM 3D4/21 cells, respectively. In addition, M. hyopneumoniae-infected normal PAM 3D4/21 cells significantly increased gene and protein expression levels of CYP3A29, while the silencing of PXR decreased the expression of CYP3A29, and the overexpression of PXR increased the expression of CYP3A29. Our study is consistent with previous studies, and based on the pathogen infection pattern, our study validated that PXR could upregulate the expression of CYP3A29 during M. hyopneumoniae infection in PAM cells.
In our study, we also found that PXR can regulate the mRNA expression of IL-6 and IL-8. The silencing of PXR in PAM 3D4/21 cells can reduce the mRNA expression of IL-6 and IL-8. When PAM 3D4/21 cells were infected with M. hyopneumoniae, the mRNA expression levels of IL-6 and IL-8 increased, while the mRNA expression levels of IL-6 and IL-8 decreased after PXR silencing. In addition, the mRNA expression levels of IL-6 and IL-8 increased after the overexpression of PXR. Previous studies using atrazine to treat quail hearts found that CYP3A4 expression induced by the PXR/CAR pathway and NF-κB pathway upregulates IL-6 and IL-8 expression [27]. IL-6 can upregulate the expression of CYP2C33 through CAR/RXRα heterodimers in primary porcine hepatocytes [28]. IL-6 is an important proinflammatory cytokine that has been shown to affect CYP450 expression in humans [29]. Because there are many members of the CYP450 family, their biological functions may be different. The expression of CYP3A29 varies among different tissues and ages and is influenced by various factors, such as drugs and inflammatory stimuli [30][31][32]. The role of PXR in pigs may be more complex, and PXR may react differently to ligands in human and other animal models than it does in pigs [33,34]. For instance, our previous study suggested that CYP1A1 can inhibit the inflammatory response caused by M. hyopneumoniae infection through the PPAR-γ signaling pathway in PAM cells [23]. Therefore, our results suggest that, during the inflammatory response to M. hyopneumoniae infection in pigs, PXR may regulate the expression of the proinflammatory cytokines IL-6 and IL-8 by regulating the expression of CYP3A29.
In the cell culture supernatant of the PAM 3D4/21 cells, the protein expression of IL-8 showed the same trend as the mRNA expression. These results indicate that PXR does have a regulatory effect on IL-8 during M. hyopneumoniae infection. The protein expression of IL-6 did not change significantly in the overexpression of PXR with and without M. hyopneumoniae infection. The protein expression of IL-6 was down-regulated after PXR silencing with and without M. hyopneumoniae infection. These results suggested that PXR overexpression had a higher regulation effect on IL-6 at mRNA level than at protein level in PAM cells. Moreover, during the inflammatory response caused by M. hyopneumoniae infection, the expression of inflammatory factors may also have other regulatory effects on molecular pathways. Studies have reported that many pathways are involved in regulating the expression of inflammatory cytokines mediated by mycoplasmal lipopeptide Pam2CGDPKHPKSF (FSL-1) in pig monocyte-derived macrophages (MDMs); these pathways include the TNF signaling pathway, the cytokine-cytokine receptor interaction pathway, the Toll-like receptor signaling pathway, the janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, the chemokine signaling pathway, the nucleotide oligomerization domain (NOD)-like receptor signaling pathway, and the NF-κB signaling pathway [35]. Furthermore, previous reports have shown that activated PXR inhibits the activity of NF-κB and the expression of the NF-κB target gene IL-6 [36]. The nuclear factor NF-κB family is a key regulator of inflammatory, innate immune, and adaptive immune responses [37,38]. Therefore, the expression of the proinflammatory cytokines IL-6 and IL-8 may be regulated by multiple pathways. The relationship between cytochrome P450 enzymes and inflammation should be further studied and will be helpful for the treatment of inflammation. Future studies should investigate the relationship between the NF-κB pathway and PXR during the inflammatory response to M. hyopneumoniae infection. Further elucidation of the molecular mechanism by which mycoplasma causes inflammation in the body is necessary.

Conclusions
In conclusion, our study found that PXR regulates the expression of CYP3A29 during the course of M. hyopneumoniae infection and inflammatory response through inflammatory factors IL-6 and IL-8. This study provides a theoretical molecular basis for the treatment of MPS and for the breeding of pigs resistant to this disease.

Conflicts of Interest:
The authors declare no conflict of interest.