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(2021) 52:58Gondaira et al. Vet RCH ARTICLEOpen AccessInnate immune response in bovineneutrophils stimulated with Mycoplasma bovisSatoshi Gondaira1, Koji Nishi1, Jumpei Fujiki2, Hidetomo Iwano2, Reina Watanabe1, Ayako Eguchi1, Yuki Hirano3,Hidetoshi Higuchi1* and Hajime Nagahata1,4AbstractMycoplasma bovis (M. bovis) is a significant worldwide pathogen of cattle. Neutrophils have an important role in theinnate immune response during infection with M. bovis. However, even though neutrophils accumulate in M. bovisinfection, the interaction of M. bovis and neutrophils has not been fully elucidated. We attempted to elucidate theinnate immune response of neutrophils stimulated with M. bovis and evaluate the transcriptome and functional analysis of bovine neutrophils stimulated with M. bovis. Proinflammatory cytokines, such as inducible nitric oxide (iNOS),which was the most increased gene in transcriptome analysis, were increased in quantitative polymerase chain reaction analysis of bovine neutrophils stimulated with live or heat-killed M. bovis. Nitric oxide and intracellular reactiveoxygen species production of neutrophils stimulated with M. bovis was significantly increased. Neutrophils stimulatedwith M. bovis showed an increased ratio of nonapoptotic cell death compared to unstimulated controls. We demonstrated that neutrophil extracellular traps (NETs) formation was not recognized in neutrophils stimulated with live M.bovis. However, heat-killed M. bovis induced NETs formation. We also showed the interaction with M. bovis and bovineneutrophils regarding proinflammatory cytokine gene expression and functional expression related to NETs formation. Live and killed M. bovis induced innate immune responses in neutrophils and had the potential to induce NETsformation, but live M. bovis escaped NETs.Keywords: Mycoplasma bovis, Bovine, Neutrophils, Innate immune, Transcriptome analysisIntroductionMycoplasmas are classified under the class Mollicutes,which do not have a cell wall and are cause widespreadinfections of eukaryotes in nature [1]. Mycoplasma bovis(M. bovis) is a significant worldwide pathogen of cattle[2, 3] and is known to cause pneumonia, arthritis, andmastitis [2, 4], resulting in calf mortality, weight loss insurviving calves, and decreased milk production in dairycows [2, 5], which all contribute to significant economiclosses [2, 6].*Correspondence: [email protected] Health Laboratory, Department of Veterinary Medicine, Schoolof Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido069‑8501, JapanFull list of author information is available at the end of the articleNeutrophils have an important role in infectious diseases at the front line. In M. bovis infection, neutrophilsconstitute the major accumulation of cells at an infectionsite [7]. M. bovis reportedly suppressed the production ofreactive oxygen species (ROS) in the immune response ofneutrophils [8]. ROS is the major innate immune factorof neutrophils to pathogens and is required for neutrophil extracellular traps (NETs) formation [9]. ROS andinducible nitric oxide (iNOS) are involved in the pathogenesis of Mycoplasma pneumoniae calves [10], andnitric oxide (NO) also triggers and enhances the releaseof NETs [11]. M. bovis was considered to escape the hostimmune response, and we previously reported that M.bovis escaped bovine NETs following the degradation ofnucleic acid [12]. The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing,adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party materialin this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If materialis not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds thepermitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Gondaira et al. Vet Res(2021) 52:58Inflammatory cytokines have an important role in theinnate immune responses of neutrophils, such as recruitment, activation, and NETs formation [13]. The suppression of NO production and increased gene expression ofinflammatory cytokines, such as IL-12 and TNF-α, as aresponse of neutrophils to M. bovis, have been previouslyreported [14]. However, the mechanisms of the neutrophil immune response involved in M. bovis comprehensive gene expression have not been fully elucidated.We attempted to elucidate the innate immune responseof neutrophils stimulated with M. bovis, determininggene expression related to the innate immune responsethrough comprehensive gene expression analysis, and todetermine whether M. bovis is capable of inducing NETsformation.Page 2 of 11Japan) and 10% fetal bovine serum (FBS). Isolated neutrophils from five individual cows were used immediately. Neutrophils (concentration of 1   107 cells in 3 mLRPMI 1640 medium) were incubated in the presence oflive or heat-killed M. bovis at a multiplicity of infection(MOI) of 1000:1 for 3 and 6 h at 37 C and 5% CO2 in60-mm dishes (Asahi Glass, Tokyo, Japan). The numberof bacteria in the milk of M. bovis mastitis was of 109 to 1011 CFU and the number of somatic cell counts was 1 067to 10 cells/mL [15]. Thus, MOI of 1000 is a sufficientlyreasonable number of bacteria, taking into account theactual infection.RNA extractionThe bacterial strain used in this study was M. bovis(PG45: ATCC 25,523), grown in modified pleuropneumonia-like organisms (PPLO) medium (Kanto Kagaku,Tokyo, Japan) at 37 C for 48 h. M. bovis was obtainedby centrifugation (16 000 g for 40 min) and then washedwith phosphate-buffered saline (PBS). The bacteria werethen suspended in PBS to a cell density of 1 08 colonyforming units per milliliter (CFU/mL), and the suspension was stored at 70 C until used.Total RNA (TRNA) extracted from neutrophils wasobtained using the PureLink RNA mini kit (Ambion, TX,USA). DNAse digestion was performed using TURBODNA-free DNAse (Ambion). TRNA was quantified viaspectrophotometry using a BioSpec-nano (Shimadzu,Kyoto, Japan). cDNA was synthesized from 1 μg TRNAwith ReverTra Ace reverse transcriptase (Toyobo, Osaka,Japan) and oligo dT primers (Toyobo). For each reaction, a parallel negative control reaction was performedin the absence of reverse transcriptase and analyzed viathe β-actin band using polymerase chain reaction (PCR)and 1.5% agarose gels stained with ethidium bromide andvisualized on an ultraviolet transilluminator.Bovine neutrophilsMicroarray experiment and analysisBlood samples (20 mL) were collected in evacuationtubes containing sodium heparin (Terumo, Tokyo, Japan)from eleven clinically healthy primiparous Holstein cowsin mid-lactation with no history of M. bovis infection.Three cows were used for microarray analysis and fivecows were used for validation experiments using realtime PCR, detection of apoptotic cells, quantity of NO,and ROS production. Another three cows were usedfor the observation of NETs formation. The experimental protocol was approved by the Institutional AnimalCare and Use Committee of Rakuno Gakuen University.Neutrophils were isolated by centrifugation on a Lympholyte device (Cedarlane, Ontario, Canada) accordingto the manufacturer’s protocol. Cells were separated bycentrifugation (300 g for 30 min), and neutrophils weretransferred to a sterile tube (Becton Dickinson, Tokyo,Japan) and washed with cold PBS. Neutrophil viability was assessed using an AO/PI cell viability kit (LogosBiosystems, Gyeonggi, Korea) and Luna-FL (Logos Biosystems). Neutrophil ratios in polymorphonuclear leukocytes (PMNL) were obtained following Diff-Quickstaining (Sysmex, Hyogo, Japan). Neutrophils were suspended in Hank’s balanced salt solution or RPMI 1640medium with L-glutamine (Sigma-Aldrich Corp., Tokyo,Six microarray (three stimuli and three control) data forthe neutrophils stimulated with M. bovis for 3 h wereprovided by Takara Bio, Inc. (Siga, Japan). The geneexpression dataset was obtained using an Agilent singlecolor microarray platform (4 44 K bovine gene expression array, grid ID 023,647). Samples were processedfor Agilent microarrays, and data were normalized asdescribed previously [16]. We used t-tests to identify significant gene expression differences (P 0.025) betweensamples. In a further filtering step, we selected only geneswith a fold change of 2. The gene annotation used wasbioDBnet [17]. Heat map analysis was done using R version 3.6.1, and gene ontology enrichment was done usingthe BioMart enrichment tool [18]. The whole dataset isavailable publicly from the ArrayExpress database (accession number E-MTAB-9022).Materials and methodsBacterial strainsQuantitative reverse transcription PCR (qPCR) analysisThe reaction was performed using a Thunderbird SYBRqPCR mix (Toyobo) and a CFX real-time PCR system(Bio-Rad Laboratories, Hercules, CA, USA). Information on the primers is depicted in Additional file 1. Weused the melting curve analysis to evaluate each primerpair for specificity to ascertain that only one product

Gondaira et al. Vet Res(2021) 52:58Page 3 of 11was amplified. We performed a Basic Logical Alignment Search Tool (BLAST) search to confirm that theprimer sequences amplified only the target gene of interest. Thermal cycling consisted of initial denaturation at95 C for 5 min, followed by 40 cycles of denaturation at95 C for 15 s, annealing at 60 C for 30 s, and extensionat 72 C for 30 s. The melting temperature of the PCRproduct was determined by melting curve analysis, whichwas performed by heating the PCR product from 55 Cto 95 C and monitoring the fluorescence change every0.5 C. β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) were used asreference genes [19, 20].CA, USA), and viewed using a fluorescence microscope(Nikon, Tokyo, Japan). Three bovine neutrophil studieswere performed individually.Detection of apoptotic cells and quantity of NO and ROSproductionWe investigated gene expression in neutrophils stimulated with live M. bovis using an Agilent Bovine GeneExpression Microarray. Statistical analysis revealedthat 61 genes in neutrophils stimulated with live M.bovis were significantly increased and 30 significantlydecreased (P 0.025 with twofold increase) comparedto unstimulated neutrophils (Figure 1A and Additionalfile 3). Expression gene patterns with significant differences were visualized using a heat map (Figure 1B). TheM. bovis stimulated and unstimulated groups showedsimilar expression patterns. The gene set related to function did not significantly recognize change, but that ofthe immune system and carbohydrate metabolic processes were increased (Figure 1C and Additional file 2).To validate these results, genes related to the immunesystem were quantified using real-time PCR (Figure 2).Inducible NO synthase (iNOS), interleukin 36A (IL-36A),chemokine C-X-C motif ligand 2 (CXCL2), and signalinglymphocytic activation molecule (SLAM) family member7 (SLAMF7) mRNA expression in neutrophils after 3 hof stimulation with M. bovis were significantly (P 0.01)increased compared to unstimulated controls. Basic leucine zipper transcription factor ATF-like (BATF) andSLAM family member 1 (SLAMF1) mRNA expressionalso were significantly increased (P 0.05). C–C motifchemokine ligand 24 (CCL24) was significantly decreased(P 0.01) compared to unstimulated controls.To evaluate NO production and the ratio of apoptosiscells in neutrophils stimulated with M. bovis, neutrophils(2   105 cells/200 μL RPMI 1640 medium in 96-well tissue culture plates) were incubated in the presence of liveM. bovis at an MOI of 1000 (10 μL) for 1, 3, and 6 h at37 C and under 5% CO2. The NO production and ratioof apoptosis cell were measured using a Muse NO kit orMuse Annexin V and dead cell kit (Millipore, Darmstadt,Germany) and Muse cell analyzer (Millipore) accordingto the manufacturer’s protocol. To measure the quantityof intracellular ROS in neutrophils stimulated with M.bovis, neutrophils (2   105 cells/1 mL RPMI 1640 mediumin a 3 cm dish) were stimulated with 10 μL conteining liveM. bovis and/or phorbol myristate acetate (PMA; SigmaAldrich Corp.) or PBS for control at an MOI of 1000 for30 min at 37 C and 5% CO2. After that, intracellular ROSproduction was detected using the Muse Oxidative stresskit and Muse cell analyzer (Millipore) according to themanufacturer’s protocol.Observation of NETs formationNeutrophils (concentration of 1   106 cells suspended in100 μL RPMI medium with 10% FBS) were seeded ontoglass coverslips treated with 0.001% poly-l-lysine (Matsunami glass, Tokyo, Japan) and placed in a 35 mm dish(Iwaki, Shizuoka, Japan). Cells were incubated for 1 hat 37 C in 5% CO2. Neutrophils were incubated withPMA for 30 min to induce NETs formation (or with PBSfor control), and then, 107 CFU octadecyl rhodamine Bchloride (Sigma-Aldrich Corp.) labeled live or heat-killedM. bovis (or with PBS for control) were added and incubated for 30 min at 37 C under a 5% C O2. Neutrophilswere washed with PBS and stained with 4,6-diamidino2-phenylindole, dilactate (DAPI) for 15 min (Dojindo,Tokyo, Japan). Coverslips were washed with PBS, coatedwith Fluoromount (Diagnostic Biosystems, Pleasanton,Statistical analysisData from five cows were expressed as mean standarderror (SE). The Kruskal–Wallis test was performed forcomparison between groups, Steel test for multiple comparisons, and Welch’s t-test for paired groups using Ekuseru-Toukei 2010 for Windows (Social Survey ResearchInformation, Tokyo, Japan). In all cases, P 0.05 was considered statistically significant.ResultsMicroarray analysisQuantification of proinflammatory cytokine mRNAexpressionExpression of proinflammatory cytokine, IL-1β, IL-6,tumor necrosis factor α (TNF-α), IL-8, IL-12, and interferon γ (IFN-γ) mRNA in neutrophils stimulated withlive or heat-killed M. bovis at 3 and 6 h was evaluatedby qPCR (Figure 3). These cytokines showing mRNAexpression in neutrophils stimulated with live M. bovisfor 3 h were significantly (IL-1β and IL-12, P 0.05;TNF-α and IL-8, P 0.01) increased compared to

Gondaira et al. Vet Res(2021) 52:58Page 4 of 1180UBA7USP14TUBB2BRRAS260RGS19CDAUBXN8gene number40PRDX1USP3820INOSCRTC2PGHS-2up regulate0down regulateM. GALT5AFLCNPRKCSHNEU1CORO1AIL36ABATF TNFAIP8L2CCL24immune system processGPN2USP14RIN2Uncharacterized ponse to stressendoplasmic reticulumis 3Cbov2catabolic processM.vi sM.bo1vi sM.bo32c ontrolc ontrol1c ontrol-1.0carbohydrate metabolic processBFigure 1 Microarray analysis in neutrophils stimulated with M. bovis. Bovine neutrophils were evaluated at 3 h after stimulation with M. bovisin three cows. A Number of significantly (t-tests, P 0.025 and twofold change) downregulated or upregulated mRNA. B Heat map analysis ofgenes with significantly (t-tests, P 0.025 and twofold change) different gene expression levels by microarray analysis in neutrophils stimulatedwith M. bovis. C Gene ontology enrichment analysis of genes with significantly (t-tests, P 0.025 and twofold change) different gene expressionlevels by microarray analysis in neutrophils stimulated with M. bovis.

Gondaira et al. Vet Res(2021) 52:58Page 5 of 11Figure 2 Validation for the microarray analysis of mRNA expression in neutrophils stimulated with M. bovis. Bovine neutrophils wereevaluated at 3 h after stimulation with M. bovis in three cows. Validation of mRNA expression for immune response-related factors confirmed bymicroarray analysis; significant difference at *P 0.05 or **P 0.01 compared to unstimulated controls.

Gondaira et al. Vet Res(2021) 52:58Page 6 of 5Fold Time (hours)Figure 3 mRNA expression of proinflammatory cytokine related genes after stimulation with M. bovis. Bovine neutrophils were evaluatedat 3 or 6 h after stimulation with live or heat-killed M. bovis in five cows. The mRNA expression of IL-1β, IL-6, TNF-α, IL-8, IL-12, and IFN-γ wasdetermined by qPCR and expressed as a fold increase, as described in the Materials and methods. The data were expressed in five cows; significantdifference at *P 0.05 or **P 0.01 compared to unstimulated controls.

Gondaira et al. Vet Res(2021) 52:58Page 7 of 117*Nitric oxide expression (%)6controlM. bovis543*210136time (hour)ABFigure 4 Quantity of NO and ROS production in neutrophils stimulated with M. bovis. A Bovine neutrophils were evaluated at 1, 3, and 6 hafter stimulation with M. bovis in five cows. The ratio of NO production cells is shown. Data were expressed as means SE in five cows; significantdifference at *P 0.05 compared to unstimulated controls. B Neutrophils were incubated with M. bovis (MOI of 1000) and/or PMA for 30 min. Theratio of ROS production cells is shown. Data were expressed as means SE of five cows; significant difference at *P 0.05 compared to unstimulatedcontrols.unstimulated cells as were those stimulated with heatkilled M. bovis (IL-1β, TNF-α, IL-8, IL-12, and IFN-γ,P 0.05; IL-6, P 0.01). After 6 h of stimulation with M.bovis, IL-1β (live and heat-killed, P 0.05), IL-8 (heatkilled, P 0.05), IL-12 (live and heat-killed, P 0.05),and IFN-γ (live, P 0.01, and heat-killed, P 0.05) demonstrated significantly increased mRNA expression inneutrophils.

Gondaira et al. Vet Res(2021) 52:58Page 8 of 11Figure 5 Detection of apoptotic cells in neutrophils stimulated with M. bovis. Bovine neutrophils were evaluated at 1, 3, and 6 h afterstimulation with M. bovis in five cows. The ratio of positive or negative neutrophils in Annexin V and/or 7-AAD is shown. Annexin V-positive/negativecells were defined as apoptotic/nonapoptotic cells, and 7-AAD–positive/negative cells were defined as dead/live cells. Data were expressed asmeans SE in five cows; significant difference at *P 0.05 compared to unstimulated controls.Evaluation producing NO and intracellular ROSof neutrophils stimulated with M. bovisEvaluation of the apoptotic, nonapoptotic, dead, or livecells stimulated with M. bovisNO production of neutrophils stimulated with M. boviswas significantly increased (P 0.05) at 1 and 3 h compared to controls (Figure 4A). Intracellular ROS production of neutrophils stimulated with M. bovis, M. bovisand PMA, or PMA was significantly increased (P 0.05)compared to unstimulated controls (Figure 4B).The ratio of apoptotic cells in neutrophils stimulated withM. bovis at an MOI of 1000 for 1, 3, and 6 h is shownFigure 5. The ratios of Annexin-positive and 7-AAD–negative cells (early apoptosis) in bovine neutrophilswas significantly increased (P 0.05) at 3 (4.36%) and 6(9.92%) h compared to that at 1 h (2.54%). The ratios ofAnnexin-negative and 7-AAD–positive cells (dead cellsother than nonapoptotic cells) in bovine neutrophilsstimulated with M. bovis were significantly increased

Gondaira et al. Vet Res(2021) 52:58controlPage 9 of 11(P 0.05) at 3 (0.96%) and 6 (1.52%) h compared to that at1 h (0.20%). The ratios of Annexin-negative and 7-AAD–negative cells (live cells) in nonstimulated cells tendedto decrease in a time-dependent manner, and those ofAnnexin-positive and 7-AAD–positive cells (late apoptotic and necrotic cells) in neutrophils stimulated with M.bovis tended to increase in a time-dependent manner.Observation of NETs formationPMAliveM. boivskilledM. boivsFigure 6 Observation of NETs formation. Bovine neutrophilswere evaluated at 30 min after stimulation with live or heat-killedM. bovis (MOI of 100)/PMA in three cows (unstimulated controls,PMA-stimulated, live M. bovis-stimulated, and heat-killed M.bovis-stimulated). DNA was stained using DAPI, and M. bovis waslabeled with rhodamine. The white arrow indicates NETs formation.The white bar indicates 16 μm, and representative micrographs areshown.NETs formation in neutrophils stimulated with live orheat-killed M. bovis is shown in Figure 6. NETs formationwas detected in neutrophils stimulated with PMA as aninducer of NETs formation, whereas it was not observedin neutrophils stimulated with live M. bovis and unstimulated controls. However, in neutrophils stimulated withheat-killed M. bovis, NETs formation was recognized,and M. bovis was localized to be on NETs.DiscussionWe studied the innate immunity of bovine neutrophilsto M. bovis, and we especially reported a transcriptomeanalysis of bovine neutrophils stimulated with M. bovisand its immune-related functional analysis. Proinflammatory cytokines, such as iNOS, IL-36A, and CXCL2mRNA, were increased in neutrophils stimulated with M.bovis as determined in the transcriptome analysis usingmicroarray and qPCR for validation. iNOS induced NOproduction with antibacterial activity and was related tothe formation of NETs [11]. Previous studies [14] did notshow the production of neutrophil NO upon M. bovisstimulation, which may be due to the use of differentMOIs and detection methods. CXCL2 is a chemokinerelated to priming of neutrophils [21] and enhancedantibacterial activity [22]. SLAMF1 and SLAMF7 affectthe development of T cells [23]. BATF was reported toregulate T and B lymphocytes in immune response anddifferentiation [24]. IL-1β, IL-6, and TNF-α are knownrapid transcription-genes, proinflammatory cytokines,and the peak time point in these genes was mainly 3 h.IL-1β- and IL-8-related migration and activation of neutrophils reportedly had an important role in maintaininginflammation in M. bovis infectious diseases. IL-12 andIFN-γ mainly reached peak levels at 6 h after stimulationwith M. bovis, which may be due to the activated neutrophils and a type 1 immune response [25]. The productionof IL-12 and TNF-α by M. bovis-stimulated neutrophilshas been also observed in a previous study [14], which isin line with our present results. In gene ontology enrichment analysis, most dynamically changed genes indicatedan immune response, and M. bovis induced an innateimmune response in bovine neutrophils. The carbohydrate metabolic process was the second largest geneset changed, and it includes the production of activated

Gondaira et al. Vet Res(2021) 52:58oxygen. Activated oxygen production in neutrophils stimulated with M. bovis was not recognized as reported previously [26, 27]. However, this study demonstrated thatintracellular production of activated oxygen was recognized. Mycoplasma species were known to have a resistance factor for activated oxygen [28, 29], which suggestedthat, although neutrophils stimulated with M. bovis produced activated oxygen, M. bovis attenuated extracellularactivated oxygen. Because ROS were required for NETsformation [9], M. bovis may have potential in inducingNETs formation. However, as ROS-independent NETsformation has been reported recently [30, 31], furtherstudies are needed on M. bovis-induced ROS production.The production of NO in neutrophils reportedly contributed to NETs formation [11]. In our study, neutrophilsstimulated with M. bovis in transcriptome analysis andqPCR analysis showed increased iNOS mRNA expression, and NO production was increased in neutrophilsstimulated with M. bovis. It had been reported that iNOSwas strongly expressed in lungs of calves with coagulativeand caseous necrosis lesion after infection with M. bovis[10]. Thus, it was suggested that the immune response ofneutrophils was involved in the pathogenesis in mycoplasma infectious disease. Neutrophils stimulated withM. bovis showed an increased ratio of nonapoptotic celldeath compared to unstimulated controls. It was considered that NETosis, which is cell death following NETs formation, contributed to nonapoptotic cell death. Mulongoet al. [32] and Maina et al. [33] reported that M. bovisdelayed the apoptosis of monocytes and macrophages,respectively. Instead of suppressing apoptosis in neutrophils, M. bovis may promote NETs formation. In thestudy by Jimbo et al., the production of elastase, whichis used as an index of NETs, was observed from neutrophils under stimulation with live M. bovis [14]. NETs formation was not observed in live bacteria stimulation, butit was observed in heat-killed bacteria stimulation. Thisresult cannot immediately indicate that M. bovis has notinduced NETs formation. This suggested that NETs weredegraded by M. bovis nuclease in live bacteria as reportedpreviously [26, 27], whereas in heat-killed M. bovis, thenuclease was inactivated by heat treatment and thusNETs formation was observed. The increased expressionof proinflammatory cytokine mRNA even after stimulation with heat-killed bacteria suggested that M. bovis hasthe potential to induce NETs formation even in killedbacteria.In conclusion, we demonstrated the innate immuneresponse gene expression of bovine neutrophils stimulated with M. bovis, its related function expression relatedto NETs formation, and interaction between M. bovis andbovine neutrophils.Page 10 of 11AbbreviationsM. bovis: Mycoplasma bovis; iNOS: Inducible nitric oxide; NETs: Neutrophilextracellular traps; ROS: Reactive oxygen species; PPLO: Pleuropneumonia-likeorganisms; PBS: Phosphate-buffered saline; CFU/mL: Colony-forming unitsper milliliter; FBS: Fetal bovine serum; MOI: Multiplicity of infection; TRNA:Total RNA; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; YWHAZ:Tryptophan 5-monooxygenase activation protein zeta polypeptide ; DAPI:4,6-Diamidino-2-phenylindole, dilactate ; IL-36A: Interleukin 36A; CXCL2:Chemokine C-X-C motif ligand 2 ; SLAMF7: Signaling lymphocytic activationmolecule family member 7; BATF: Basic leucine zipper transcription factor ATFlike; SLAMF1: Signaling lymphocytic activation molecule family member 1;CCL24: C–C motif chemokine ligand 24; TNF-α: Tumor necrosis factor α; IFN-γ:Interferon γ.Supplementary InformationThe online version contains supplementary material available at https:// doi. org/ 10. 1186/ s13567- 021- 00920-2.Additional file 1. Sequences of oligonucleotide primers.Additional file 2. The biological process GO term enrichment inbovine neutrophils stimulated with M. bovis. “Corrected P-Value” is correction for multiple testing.Additional file 3. Transcriptome analysis in neutrophils stimulatedwith M. bovis. Bovine neutrophils were evaluated by microarray analysisat 3 h after stimulation with M. bovis in three cows. After normalizationand gene annotation, fluorescence intensity in significantly (t-tests,P 0.025 and a twofold change) upregulated or downregulated geneswas shown for each cow (control as unstimulated control vs. Mb as M.bovis stimulation).AcknowledgementsThe authors would like to thank Enago for the English language review.Authors’ contributionsConceived and designed this study: SG, HI, JF, HH and HN. Performedthe experiments: SG, KN, JF, RW, AE and YH. Conducted the data analysis:SG. Wrote the paper: SG and HH. All authors read and approved the finalmanuscript.FundingThe authors have not declared a funding from any public or commercialagency.Availability of data and materialsAll datasets are presented in the paper or additional files supporting themanuscript.DeclarationsEthics approval and consent to participateAnimal experiments were approved by the Institutional Animal Care and UseCommittee of Rakuno Gakuen University (VH24A4).Competing interestsThe authors declare that they have no competing interests.Author details1Animal Health Laboratory, Department of Veterinary Medicine, Schoolof Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069‑8501,Japan. 2 Veterinary Biochemistry, Department of Veterinary Medicine, Schoolof Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069‑8501,Japan. 3 Animal Research Center, Agricultural Research Department, HokkaidoResearch Organization, Shintoku, Hokkaido 081‑0038, Japan. 4 Farm AnimalVeterinary Nursing Laboratory, Department of Veterinary Associated Science,Faculty of Veterinary Medicine, Okayama University, Imabari, Ehime 794‑8555,Japan.

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of apoptosis cell were measured using a Muse NO kit or Muse Annexin V and dead cell kit (Millipore, Darmstadt, Germany) and Muse cell analyzer (Millipore) according to the manufacturer’s protocol. To measure the quantity of intracellular ROS in neutrophils stimulated with M. bovis