Porcine alveolar macrophages (PAM, swine 3D4/21 cell lines) were cultured in Roswell Park Memorial Institute 1640 Medium (RPMI 1640) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified 5% CO₂ incubator. The medium used for maintenance of PAMs was RPMI-1640 (Life Technology, USA) supplemented with 2% heated-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Escherichia coli strains DH5a and BL21 (DE3) were cultured in Luria Bertani (LB) medium supplemented with ampicillin (100 mg/mL) or kanamycin (50 mg/mL) when needed. M. hyopneumoniae strain 168 was isolated from the Er-hua-nian pig with typical clinical and pathogenic characteristics of mycoplasmal pneumonia of swine (Liu et al., 2011), which was kindly provided by Guoqing Shao (Jiangsu Academy of Agricultural Sciences, Nanjing, China). The strain 168 was grown in KM₂ cell-free medium (modified Friis’ medium supplemented with 20% pig serum) in a humidified 5% CO₂ incubator at 37°C.

The P68 (pET30a-mhp390) and P97 expression plasmids were prepared in our laboratory as described previously (Liu et al., 2019b). The cDNA of NOD1 was cloned from swine PK15 cells and inserted into pCMV-HA vector (Clontech) to generate HA-NOD1 expression plasmid (EcoRI and XhoI restriction sites, primers listed in Table S1 ), which expressed HA-tagged proteins. The constructed plasmids were verified by nucleotide sequencing analysis. The commercial antibodies used in this study included polyclonal antibody against LC3 purchased from Proteintech (Cat No. 14600-1-AP, China). NOD1 monoclonal antibody was purchased from Santa Cruz (Cat No. sc-398696, USA). Anti-β-actin was obtained from Beyotime (Cat No. AF0003, China). Anti-mhp390 or anti-mhp384 polyclonal antibody was produced in rabbits through immunization with mhp390 or mhp384 protein respectively in our laboratory (Liu et al., 2019b).

The plasmids pET30a-mhp390 and pET30a-NOD1 (EcoRI and XhoI restriction sites, primers listed in Table S1 ) were transformed into E. coli BL21 (DE3) to express the recombinant protein. The resultant recombinant protein tagged with 6×His was purified with Ni-NTA agarose (Bio-Rad, Shanghai, China). The concentration of the purified protein was measured by using the BCA Protein Assay Kit (Beyotime, China).

PAM cells grown in 6-well plates (Corning, Inc., USA) were infected with M. hyopneumoniae (10⁷ Color Change Unit, CCU) or mock-infected. Total RNA was isolated at the indicated time points using the TRIzol reagent (Invitrogen). Then, the RNA was reverse transcribed into cDNA through TransScript One Step RT-PCR SuperMix (Trans, Beijing, China). qRT-PCR was performed using PerfectStart green qPCR super Mix (TransGen Biotech, China) in the LightCycler 96 (Roche Molecular Biochemicals). The PCR conditions were as follows: pre-denaturing at 95°C for 5 min, denaturing at 95°C for 15 s, annealing at 58°C for 30 s, extension at 72°C for 20 s, and a total of 40 cycles of amplification. The fold change in gene expression relative to the normal was calculated using the delta delta cycles to threshold (ΔΔCt) method. Primers ( Table S1 ) were designed with the Primer Express software (version 3.0; Applied Biosystems, CA). The experiments were performed independently for three times.

Transient transfection was performed by using Lipofectamine 2000 (Invitrogen). PAM cells were seeded on 6/12-well plates and cultured until the cells reached approximately 70%–80% confluence, and then transfected with the indicated plasmids or siRNA. For each transfection, 3 ng of the HA-NOD1 expression plasmid or 40 pM of siRNA was used. Cell extract was collected at 6, 9, 12, and 24 h post infection (hpi).

The siRNA targeting porcine NOD1 or negative control siRNA was synthesized by GenePharma (China). Knockdown of endogenous NOD1 in PAM cells was carried out by transfection of NOD1 siRNA. Transient transfection of siRNA was performed by using lipofectamine 2000 (Vazyme, China) according to the manufacturer’s instructions. The siRNA sequences used were as follows: si2815-NOD1, sense 5’-CAGACGUUGAAACACUUAUTT-3’, antisense 5’-AUAAGUGUUUCAACGUCUGTT-3’; Negative control, sense 5’-UUCUUCGAACGUGUCACGUTT-3’, antisense 5’-ACGUGACACGUUCGG AGAATT-3’.

Cell extracts were prepared by adding 200 μL 2 × lysis buffer A (LBA) (65 mM Tris–HCl [pH 6.8], 4% sodium dodecyl sulfate, 3% DL-dithiothreitol, and 40% glycerol) and sonicated. The extracts were lysed by boiling in sodium dodecylsulphate (SDS) sample buffer (2% SDS, 60 mM Tris-HCl [pH 6.8], 10% glycerol, 0.001% bromophenolblue, and 0.33% β-mercaptoethanol), and then loaded on acrylamide sodium dodecyl sulphate polyacrylamide (SDS-PAGE) gel. The separated proteins were transferred onto PVDF membranes (Millipore, Billerica, MA). The resultant membranes were blocked with 5% (w/v) dried skim milk in tris-buffered saline containing 0.05% (v/v) Tween 20 (TBST) and incubated with primary antibodies. The antibodies used in western blot assay were anti-NOD1 monoclonal antibody (Santa Cruz, USA), and anti-mhp384 polyclonal antibody (Liu et al., 2019b). β-actin was detected with an anti-beta-actin monoclonal antibody (MAb) (Beyotime, China) to serve as the loading control. Secondary antibodies, horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG antibody, were used at a dilution of 1:4000, and the protein bands were visualized through enhanced chemiluminescence (ECL) reagents (Thermo, USA) following the manufacturer’s instructions. The signal was acquired by scanning the membranes, while the number of pixels per surface area was quantified by using ImageJ software.

The role of mhp390 in M. hyopneumoniae adherence was evaluated through flow cytometry. PAM cells were seeded in 24-well plates, and then rMhp390 was incubated with PAM cells at 37°C for 30 min. Each well was washed for 5 times to remove the unbound protein. The bound rMhp390 was labeled green through anti His-tag mouse mAb and goat anti-mouse IgG-FITC. M. hyopneumoniae adhesin P97R1 and BSA served as the positive and negative control, respectively. The fluorescence intensity of PAM cells was evaluated with flow cytometry (BD C6 plus flow cytometer).

For the rMhp390 inhibition assay, M. hyopneumoniae was pre-blocked with two-fold serial dilution of rMhp390, which corresponded to 1.25–10.0 μg/ml of protein. BSA (10 μg/mL) and 1 mL of RPMI 1640 alone were used as the negative and blank controls, respectively. For the rMhp390 antiserum inhibition assay, M. hyopneumoniae was incubated with anti-rMhp390 serum diluted from 1:50 to 1:1000 before infection. The mixed negative serum (unimmunized rabbit serum, 1:50 dilution) was used as the negative control. The cells were evaluated with flow cytometry (BD C6 plus flow cytometer) and the adhesion ability of M. hyopneumoniae was evaluated based on the fluorescence intensity of PAM cells. These experiments were performed independently for three times.

PAM cells were propagated in RPMI-1640 on microscope coverslips in 6-well plates (Corning, Inc., USA). M. hyopneumoniae was cultured in a 5% CO₂ atmosphere at 37°C for 42 h and fluorescently labeled by incubation with 5 μM CFDA-SE at 37°C for 30 min (avoid light). The bacteria were washed three times to remove excess CFDA-SE. PAM cells (10⁶) were treated with fluorescently labeled M. hyopneumoniae (10⁸ CCU) or PBS at 37°C. The cells were washed with PBS for three times and fixed with 4% neutralized paraformaldehyde for 15 min at room temperature. Then, the cells were blocked with 1% (w/v) BSA in PBS (pH 7.4) overnight at 4°C. Endogenous NOD1 was detected by incubation with mouse anti-NOD1 mAb (Santa Cruz, USA) at 37°C for 1 h, and then overlain with Cy3-conjugated affinipure goat anti-mouse IgG (H+L) diluted at 1:100 (Proteintech, China) at 37°C for 1 h. 4,6-Diamidino-2-phenylindole (DAPI) (Beyotime, China) was used to label the cell nuclei. The cells were washed with PBS between each step. Immunofluorescence was detected with a Zeiss LSM900 laser scanning confocal microscope (Zeiss LSM 900 with Airyscan 2, Germany).

The binding kinetics of rMhp390 and rNOD1 was monitored with a biolayer interferometry on ForteBio Octet Red96 instrument (ForteBio, USA). rMhp390 was labeled with biotin through LinKine™ biotin labeling kit (Abbkine, China) and immobilized on the streptavidin (SA) biosensors. After equilibrium in PBST (PBS containing 0.02% (v/v) Tween20) for 300 s, association between rMhp390 and rNOD1 was performed for 300 s. Dissociation was carried out in PBST buffer for 600 s. The kinetic dissociation rate constant (K_D) was obtained using a 1:1 binding model. The association and dissociation constants were obtained using the DataAnalysisHT software (ForteBio, USA) version 12.0.

Statistical analysis was performed with the two-way analysis of variance test using the GraphPad PRISM software (version 8.4.3 for Windows; www.graphpad.com/) to evaluate the potential differences among different groups. Data were presented as mean ± standard deviation (SD). P-value < 0.05 was considered as significant; P-value < 0.01 was considered as highly significant; P-value < 0.001 was considered as extremely significant; and ns represents no difference.

PAM cells are highly permissive for M. hyopneumoniae infection (Bai et al., 2013; Liu et al., 2017). To investigate the expression kinetics of NOD1 after challenge with M. hyopneumoniae, PAM cells were infected with M. hyopneumoniae (100 μL, 10⁸ CCU). The infected cells were collected at 0, 6, 12, 24, 36 hpi, and then subjected to western blotting and qRT-PCR using the primers listed in Table S1 . As shown in Figure 1A , M. hyopneumoniae infection steadily increased the expression of NOD1 protein to nearly 1.69 folds at 6 hpi. A similar increase in the mRNA expression of NOD1 was observed after M. hyopneumoniae infection, with a maximum level of 4.19 folds at 6 hpi ( Figure 1B ).

siRNA-mediated interference was performed to determine whether the silencing of NOD1 affects the M. hyopneumoniae-induced innate immune signal pathway. As shown in Figure 2A , the knockdown efficiency of the NOD1-specific si2815 was over 50% at the protein level, while the mRNA knockdown efficiency of si2815 was about 30% at 24 h post transfection (hpt) ( Figure 2B ). No obvious interference of NOD1 was found by using si41 or si2684. Thus, NOD1 si2815 was used for further experiments in this study.

It is well known that the cytosolic receptor NOD1 can sense bacterial peptidoglycan and activate the NF-κB signaling pathway through its adaptor protein, the receptor-interacting protein 2 (RIP2), which will lead to the production of multiple inflammatory cytokines (Jing et al., 2014). Therefore, we investigated whether interference of NOD1 blocks the activation of RIP2 expression in PAM cells upon M. hyopneumoniae infection. As shown in Figure 2C , the mRNA of RIP2 was dramatically decreased in NOD1 siRNA-treated cells compared with that in the NC siRNA cells at 12 hpi.

To study the involvement of the NOD1-mediated innate immune signaling pathway in M. hyopneumoniae infection, we firstly determined the mRNA levels of pro-inflammatory cytokines in PAMs after M. hyopneumoniae infection by qRT-PCR using primers listed in Table S1 . As shown in Figures 3A, B , the mRNA expression of both TRIF and MYD88 was significantly up-regulated with a maximum increase at 12 hpi. In addition, M. hyopneumoniae infection remarkably enhanced the mRNA expression of TNF-α ( Figure 3C ).

Then, PAM cells were transfected with si2815 or the negative control (NC). At 24 hpt, the transfected cells were stimulated by mock infection or M. hyopneumoniae infection. Furthermore, the mRNA expression of cytokines was determined in M. hyopneumoniae-infected cells. As expected, the mRNA of TRIF, MYD88 and TNF-α significantly decreased in NOD1 si2815 cells compared with that in the NC siRNA cells after M. hyopneumoniae challenge ( Figure 3 ). Taken together, these results suggested that NOD1 silencing blocks the activation of pro-inflammatory cytokines triggered by M. hyopneumoniae.

To assess the role of NOD1 in M. hyopneumoniae proliferation, we evaluated the yield of M. hyopneumoniae in PAM cells transfected with hemagglutinin (HA)-NOD1-expressing plasmid ( Figure 4A ). At 24 hpt, the transfected cells were infected with equal amounts of M. hyopneumoniae. The abundance of mhp384 protein from M. hyopneumoniae, which can indirectly indicate the amount of M. hyopneumoniae, was then determined at 24 hpi. The results demonstrated that overexpression of NOD1 significantly suppressed the proliferation of M. hyopneumoniae compared with the HA infected group ( Figure 4B ). In contrast, the abundance of mhp384 was slightly increased in NOD1 knockdown cells compared with that in the NC siRNA cells after M. hyopneumoniae challenge ( Figure 4C ).

The adhesion of M. hyopneumoniae to alveolar macrophages was investigated through fluorescence microscopy. Briefly, the host NOD1 was stained red by mouse anti-NOD1 mAb and goat anti-mouse IgG-Cy3, while the M. hyopneumoniae cells were labeled with CFDA-SE (green). As shown in Figure 5 , M. hyopneumoniae infection up-regulated the expression of NOD1 compared with the mock infection control. M. hyopneumoniae cells were adhered and ingested into the alveolar macrophages and localized to regions enriched in NOD1, as indicated by the merged yellow signal. These observations suggested an intimate association between M. hyopneumoniae and host NOD1.

The binding ability of mhp390 to alveolar macrophages was determined by using flow cytometry. Briefly, PAM cells were seeded in 24-well plates, and then infected with rMhp390. The unbound protein was removed, and the bound rMhp390 was labeled green through anti His-tag mouse mAb and goat anti-mouse IgG-FITC. M. hyopneumoniae adhesin P97R1 and BSA were used as positive and negative control, respectively. The results demonstrated that mhp390 has a strong adhesive capacity to alveolar macrophages ( Figure 6A ). In the positive control group, P97R1 showed a 3.65-fold adherence activity relative that of the mock group.

We further investigated the role of mhp390 in M. hyopneumoniae adhesion through flow cytometry. The adhesion of M. hyopneumoniae to PAM cells was first tested. Briefly, M. hyopneumoniae was cultured in a 5% CO₂ atmosphere at 37°C for 48 h and fluorescently labeled by incubation with CFDA-SE. Then, the bacteria were washed to remove excess CFDA-SE. PAM cells (10⁶) were infected with fluorescently labeled M. hyopneumoniae at 37°C for 30 min at a multiplicity of infection (MOI) of 1:100. The cells were evaluated with flow cytometry and the adhesion ability of M. hyopneumoniae was evaluated based on the fluorescence intensity of PAM cells.

Subsequently, the inhibitory effect of both rMhp390 and antiserum on the adhesion of M. hyopneumoniae to PAM cells was assayed. To check the binding inhibition effect of rMhp390, M. hyopneumoniae was pre-blocked with two-fold serial dilution of rMhp390. The adhesion rate between the M. hyopneumoniae and alveolar macrophages was determined. With increasing rMhp390 concentration, more significant and dose-dependent inhibition of the binding between M. hyopneumoniae and alveolar macrophage was observed ( Figure 6B ). In contrast, no significant inhibition effect was found in the BSA and mock groups. As shown in Figure 6C , the binding of M. hyopneumoniae to PAM cells was effectively inhibited by antiserum directed against rMhp390 in a dose-dependent manner, whereas the negative serum from mock-immunized rabbit did not affect M. hyopneumoniae binding.

To gain more insights into the mechanism for the activation of NOD1 signaling by M. hyopneumoniae, we immobilized rMhp390 on biosensors and analyzed its interaction with rNOD1 through biolayer interferometry (BLI, Octet. RED 96e). As shown in the association and dissociation kinetic chart ( Figure 6D ), rNOD1 was rapidly bound to but slowly dissociated from SA-biosensors coated with rMhp390. rMhp390 bound rNOD1 with a high affinity (K_D = 8.14 μM). In the negative control group, no interaction was observed, with no value of K_D.

To determine whether NOD1 plays a role in autophagy induction upon M. hyopneumoniae challenge, PAM cells were transfected with NOD1 siRNA or NC negative control siRNA before M. hyopneumoniae infection. Cell lysates were incubated with anti-LC3 antibody and analyzed through Western blotting. The LC3II/I ratio was determined by using ImageJ. As shown in Figure S1 , M. hyopneumoniae activated the autophagy of PAM cells, whereas NOD1 silencing did not impede the autophagy induction. The results suggested that M. hyopneumoniae may induce autophagy through some other pathways instead of the NOD1 signaling pathway.