Bovine Macrophages Research Articles

Analysis of mRNA and circRNA Expression Profiles of Bovine Monocyte-Derived Macrophages Infected With Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the pathogen of Johne’s disease (paratuberculosis), which mainly causes chronic infectious granulomatous enteritis in ruminants and has brought huge economic losses to animal husbandry. As a specific intracellular pathogen, when MAP invades the body, it is internalized by macrophages where it is able to replicate by inhibition of the phagosome maturation, escaping the host immune system and surviving, which leads to the spread of the disease. More recent studies have shown that circRNA is involved in many pathological and physiological processes of the body as the molecular sponge of miRNA, the scaffold of RNA binding protein and having the characteristic of being able to translate into protein. In this study, the mRNA and circRNA expression profiles of MAP-infected bovine monocyte-macrophages and uninfected bovine cells were analyzed by high-throughput sequencing. A total of 618 differentially expressed mRNA were screened out, including 322 upregulated mRNA and 296 downregulated mRNA. In addition, the analysis of circRNA differential expression profile showed 39 differentially expressed genes including 12 upregulated and 27 downregulated genes. Moreover, differential genes belonging to cytokine activity, chemokine activity, inflammatory reaction, apoptosis, and other functional groups related to macrophage immune response were significantly enriched in Gene Ontology (GO). Multiple signal pathways including NF-κB, MAPK, Toll-like receptor, IL-17, JAK-STAT, and other signaling pathways related to activating macrophage immune response were significantly enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG). In addition, RT-qPCR technology verified the accuracy of the mRNA sequencing results. In this study, we have obtained the transcriptome information of mRNA and circRNA of bovine monocyte-macrophage infected with MAP. These results will provide data support for the further study of mRNA–miRNA–circRNA network and immune escape mechanism of MAP and will enrich the knowledge of the molecular immune mechanisms of Johne’s disease as well.

Forsythin Inhibits Β-Hydroxybutyrate-Induced Oxidative Stress in Bovine Macrophages by Regulating P38/Erk, Pi3k/Akt, and Nrf2/Ho-1 Signaling Pathways

Forsythin Inhibits Β-Hydroxybutyrate-Induced Oxidative Stress in Bovine Macrophages by Regulating P38/Erk, Pi3k/Akt, and Nrf2/Ho-1 Signaling Pathways

Effect of Experimental Foot and Mouth Disease Virus Infection on the Activation and Polarization of Bovine Monocyte-Derived Macrophages 

Effect of Experimental Foot and Mouth Disease Virus Infection on the Activation and Polarization of Bovine Monocyte-Derived Macrophages 

Lipidomics revealed the global lipid responses of primary bovine alveolar macrophages to infections of Mycobacterium tuberculosis and Mycobacterium bovis

Mycobacterium tuberculosis (M. tuberculosis (MTB) and M. bovis (MB) of the Mycobacterium tuberculosis complex (MTBC) are the causative agents of the notorious infectious disease tuberculosis (TB) in a range of mammals, including bovine and human. The lipid composition of MTB/MB performed imperative function as invading host macrophage. However, the detailed variations in lipid compositions of MTB and MB were unknown, while the responses relevant to lipid metabolisms in MTB/MB-infected host were also unclear. In the present study, a dual-Lipidomics were used to elucidate the differences in lipid composition of MTB and MB and responses in lipid metabolisms of primary bovine alveolar macrophages infected by MTB/MB. The Lipidomics showed significant differences in lipid composition, especially differences in levels of Glycerophospholipids, Sterol Lipids, Fatty Acyls and Polyketides between these two mycobacterium species. Meanwhile, both MTB and MB could invoke various responses of lipid metabolisms in host macrophages. An infection of MTB mainly induced the increases of Polyketides and Glycerophospholipids in macrophages, whereas an MB infection induced the increases of Glycerophospholipids and Sterol. Furthermore, TAG 13:0–18:5–18:5 of MTB and PC (16:1(9E)/0:0), PI(20:2(11Z,14Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), 4, 6-Decadiyn-1-ol isovalerate and LacCer (d18:1/24:1(15Z)) of MB were identified to cause variations in lipid metabolisms of macrophages, respectively. From these data, we proposed that the differential compositions of lipid compositions in MTB and MB could successfully colonize in macrophage by different mechanisms. MTB could promote the formation of foam cells of macrophage for its colonization and development, while MB mainly suppresses the macrophage autophagy to escape the immune responses of host.

Establishment of an Efficient Immortalization Strategy Using HMEJ-Based bTERT Insertion for Bovine Cells.

Immortalized cell lines have been used in a wide range of applications in research on immune disorders and cellular metabolic regulation due to the stability and uniformity of their cellular characteristics. At present, the investigation into molecular functions and signaling pathways within bovine cells remains largely limited by the lack of immortalized model cells. Current methods for immortalizing bovine cells are mainly restricted to the ectopic expression of human telomerase reverse transcriptase (hTERT) through transient transfection or virus-mediated delivery, which have defects in efficiency and reliability. In this study, we identified bovine TERT (bTERT) as a novel potent biofactor for immortalizing bovine cells with great advantages over hTERT, and established an efficient and easily manipulated strategy for the immortalization of bovine primary cells. Through the homology-mediated end-joining-based insertion of bTERT at the ROSA26 locus, we successfully generated immortalized bovine fetal fibroblast cell lines with stable characteristics. The observed limitation of this strategy in immortalizing bovine bone marrow-derived macrophages was attributed to the post-translational modification of bTERT, causing inhibited nuclear localization and depressed activity of bTERT in this terminally differentiated cell. In summary, we constructed an innovative method to achieve the high-quality immortalization of bovine primary cells, thereby expanding the prospects for the future application of immortalized bovine model cell lines.

Histophilus somni stimulates bovine monocyte-derived macrophages to release microparticles that increase fibrin clot formation in vitro

Histophilus somni is a Gram-negative coccobacillus that causes diffuse vasculitis and intravascular thrombosis that can lead to multiple organ failure in cattle. Macrophages are important cellular mediators of fibrin deposition and removal at sites of inflammation. It has become evident that macrophages and other cells release microparticles (MPs) that have an array of biological activities, including pro-coagulant activity. We sought to determine whether monocyte-derived macrophages exposed to H. somni in vitro release MPs that activate the clotting cascade in a manner that could lead to thrombus formation. Bovine monocyte-derived macrophages were incubated with H. somni (at a 10:1 ratio) in RPMI with 10% heat inactivated fetal bovine serum for 6 h at 37 °C with 5 % CO2. Membrane-shed MPs were isolated from the conditioned media, washed twice with Ca2+ and Mg2+ free HBSS, and pro-coagulant activity assessed by a one-step plasma clotting assay. We observed greater pro-coagulant activity for MPs from H. somni stimulated macrophages than from unstimulated controls. Microparticle pro-coagulant activity was inhibited by addition of an anti-tissue factor antibody. We also observed co-localization of fluorescein-labeled H. somni cells and annexin V staining as evaluated by confocal microscopy. These results demonstrate that exposure to H. somni cells causes bovine monocyte-derived macrophages to release MPs that contain tissue factor, the first such report for bovine macrophages. We infer that if similar events occur in vivo they could amplify thrombus formation in bovine histophilosis.

Complement-containing small extracellular vesicles from adventitial fibroblasts induce proinflammatory and metabolic reprogramming in macrophages.

Pulmonary hypertension (PH) is a severe cardiopulmonary disease characterized by complement-dependent, fibroblast-induced perivascular accumulation and proinflammatory activation of macrophages. We hypothesized that, in PH, nanoscale-sized small extracellular vesicles (sEVs), released by perivascular/adventitial fibroblasts, are critical mediators of complement-dependent proinflammatory activation of macrophages. Pulmonary adventitial fibroblasts were isolated from calves with severe PH (PH-Fibs) and age-matched controls (CO-Fibs). PH-Fibs exhibited increased secretion of sEVs, compared with CO-Fibs, and sEV biological activity was tested on mouse and bovine bone marrow–derived macrophages (BMDMs) and showed similar responses. Compared with sEVs derived from CO-Fibs, sEVs derived from PH-Fibs (PH-Fib-sEVs) induced augmented expression of proinflammatory cytokines/chemokines and metabolic genes in BMDMs. Pharmacological blockade of exosome release from PH-Fibs resulted in significant attenuation of proinflammatory activation of BMDMs. “Bottom-up” proteomic analyses revealed significant enrichment of complement and coagulation cascades in PH-Fib-sEVs, including augmented expression of the complement component C3. We therefore examined whether the PH-Fib-sEV–mediated proinflammatory activation of BMDMs was complement C3 dependent. Treatment of PH-Fibs with siC3-RNA significantly attenuated the capacity of PH-Fib-sEVs for proinflammatory activation of BMDMs. PH-Fib-sEVs mediated proglycolytic alterations and complement-dependent activation of macrophages toward a proinflammatory phenotype, as confirmed by metabolomic studies. Thus, fibroblast-released sEVs served as critical mediators of complement-induced perivascular/microenvironmental inflammation in PH.

Supervivencia intracelular de cepas de Mycobacterium bovis de alta y baja frecuencia probado en un modelo de macrófagos bovinos

Bovine tuberculosis is a disease caused by Mycobacterium bovis that affects cattle and other species, including humans. Mycobacterium bovis resides mainly in macrophages, so bacilli survival within macrophages is related to virulence. Isolation and strain identification are important for disease control. However, little is known about virulence of the circulating strains in cattle populations. Therefore, the aim of this study was to compare the intracellular survival of Mycobacterium bovis strains with high and low frequency genotypes in cattle in Mexico. Four high frequency genotypes and four low frequency genotypes were identified and subjected to intracellular survival assays in bovine macrophages. Results showed that the phagocytosis proportion was approximately 63 % for all strains. There were no significant differences in the average Colony Forming Units (CFUs) in phagocytosis and survival between the high and low frequency groups; however, when the CFU average of phagocytosis was compared with the survival, significant differences were found in both groups. In intracellular growth, a significant difference was observed between low and high frequency strains, and between low frequency strains. Finally, the intracellular growth average of the groups was analyzed showing no significant difference. These results suggest that the frequency of the genotype in cattle population is not related to the intracellular survival and the virulence of the M. bovis strains.

Host defense against Neospora caninum infection via IL-12p40 production through TLR2/TLR3-AKT-ERK signaling pathway in C57BL/6 mice

Neospora caninum is an intracellular parasite which can cause neosporosis and significant economic losses in both dairy and beef industries worldwide. A better understanding of the immune response by host cells against N. caninum could help to design better strategies for the prevention and treatment of neosporosis. Although previous studies have shown TLR2/TLR3 were involved in controlling N. caninum infection in mice, the precise mechanisms of the AKT and MAPK pathways controlled by TLR2/TLR3 to regulate N. caninum-induced IL-12p40 production and the role of TLR2/TLR3 in anti-N. caninum infection in bovine macrophages remain unclear. In the present study, TLR2−/− mice displayed more parasite burden and lower level of IL-12p40 production compared to TLR3−/− mice. N. caninum could activate AKT and ERK signaling pathways in WT mouse macrophages, which were inhibited in TLR2−/− and TLR3−/− mouse macrophages. In N. caninum-infected WT mouse macrophages, AKT inhibitor or AKT siRNA could decrease the phosphorylation of ERK. AKT or ERK inhibitors reduced the production of IL-12p40 and increased the number of parasites. The productions of ROS, NO, and GBP2 were significantly reduced in TLR2−/− and TLR3−/− mouse macrophages. Supplementation of rIL-12p40 inhibited N. caninum proliferation and rescued the productions of IFN-γ, NO, and GBP2 in WT, TLR2−/−, and TLR3−/− mouse macrophages. In bovine macrophages, the expressions of TLR2, TLR3, and IL-12p40 mRNA were significantly enhanced by N. caninum, and N. caninum proliferation was inhibited by TLR2/TLR3 agonists. Taken together, the proliferation of N. caninum in mouse macrophages was controlled by the TLR2/TLR3-AKT-ERK signal pathway via increased IL-12p40 production, which in turn lead to the productions of NO, GBP2, and IFN-γ during N. caninum infection. And in bovine macrophages, TLR2 and TLR3 contributed to inhibiting N. caninum proliferation via increased IL-12p40 production.

Milk lymphocyte profile and macrophage functions: new insights into the immunity of the mammary gland in quarters infected with Corynebacterium bovis

BackgroundsThe present study explored the viability of bovine milk macrophages, their intracellular production of reactive oxygen and nitrogen species (RONS), and their phagocytosis of Staphylococcus aureus, as well as the profile of lymphocytes, from healthy udder quarters and udder quarters infected by Corynebacterium bovis. The study included 28 healthy udder quarters from 12 dairy cows and 20 udder quarters infected by C. bovis from 10 dairy cows. The percentages of macrophages and lymphocytes were identified by flow cytometry using monoclonal antibodies. Macrophage viability, RONS production, and S. aureus phagocytosis were evaluated by flow cytometry.ResultsMilk samples from quarters infected with C. bovis showed a lower percentage of macrophages but an increased number of milk macrophages per mL and a higher percentage of macrophages that produced intracellular RONS and phagocytosed S. aureus. No effect of C. bovis infection on macrophage viability was found. Udder quarters infected by C. bovis showed a higher percentage of T cells and CD4+ T lymphocytes, but no effect was found on the percentage of CD8+ CD4− T, CD8− CD4− T, or B lymphocytes.ConclusionsThus, our results corroborate, at least in part, the finding that intramammary infections by C. bovis may offer protection against intramammary infections by major pathogens.

The Regulation of Staphylococcus aureus-Induced Inflammatory Responses in Bovine Mammary Epithelial Cells.

Mastitis, an inflammatory disease, causes severe economic loss in the dairy industry, which is mainly infected by bacteria. Staphylococcus aureus (S. aureus), the major pathogenic microorganism, derived from lipoteichoic acid (LTA) has been identified to activate inflammatory responses, but the cellular or intercellular regulatory mechanism is unclear. This study mainly focused on the effects of LTA in bovine mammary epithelial cells (Mac-T) and elaborated the regulation of microRNAs (miRNAs). The results showed that LTA enhanced the messenger RNA (mRNA) expression and production of tumor necrosis factor α (TNF-α) and interleukin (IL)-6. Furthermore, LTA could activate Toll-like receptor (TLR)2/MyD88-mediated phosphoinositide 3-kinase (PI3K)/AKT pathway, and TLR2 plays a pivotal role in LTA-induced inflammatory responses. The results of qRT-PCR showed that miRNA levels increased and reached the highest at 3 h and then gradually decreased over time in Mac-T cells. In exosomes, the levels of 11 and three miRNAs were upregulated and downregulated at 24 h, respectively. In addition, miR-23a showed the highest increase in Mac-T cells treated with LTA and targeted PI3K to regulate inflammatory responses. Furthermore, Mac-T cell-derived exosomes were identified to play a cell–cell communication by promoting M1 polarization of bovine macrophages. In summary, our study demonstrated that LTA could activate inflammatory responses via TLR2/MyD88/PI3K/AKT signaling pathway, and miR-23a inhibited it by targeting PI3K. Furthermore, we found that Mac-T cell-derived exosomes might be associated with inflammatory responses by promoting M1 polarization of bovine macrophages.

Effect of the deletion of lprG and p55 genes in the K10 strain of Mycobacterium avium subspecies paratuberculosis

The lprG-p55 operon of Mycobacterium tuberculosis, M. bovis and M. avium strain D4ER has been identified as a virulence factor involved in the transport of toxic compounds. LprG is a lipoprotein that modulates the host immune response against mycobacteria, whereas P55 is an efflux pump that provides resistance to several drugs. In the present study we search for, and characterize, lprg and p55, putative virulence genes in Mycobacterium avium subsp. paratuberculosis (MAP) to generate a live-attenuated strain of MAP that may be useful in the future as live-attenuated vaccine. For this purpose, we generated and evaluated two mutants of MAP strain K10: one mutant lacking the lprG gene (ΔlprG) and the other lacking both genes lprG and p55 (ΔlprG–p55). None of the mutant strains showed altered susceptibility to first-line and second-line antituberculosis drugs or ethidium bromide, only the double mutant had two-fold increase in clarithromycin susceptibility compared with the wild-type strain. The deletion of lprG and of lprG-p55 reduced the replication of MAP in bovine macrophages; however, only the mutant in lprG-p55 grew faster in liquid media and showed reduced viability in macrophages and in a mouse model. Considering that the deletion of both genes lprG-p55, but not that of lprG alone, showed a reduced replication in vivo, we can speculate that p55 contributes to the survival of MAP in this animal model.

Integrative genomics of the mammalian alveolar macrophage response to intracellular mycobacteria

BackgroundBovine TB (bTB), caused by infection with Mycobacterium bovis, is a major endemic disease affecting global cattle production. The key innate immune cell that first encounters the pathogen is the alveolar macrophage, previously shown to be substantially reprogrammed during intracellular infection by the pathogen. Here we use differential expression, and correlation- and interaction-based network approaches to analyse the host response to infection with M. bovis at the transcriptome level to identify core infection response pathways and gene modules. These outputs were then integrated with genome-wide association study (GWAS) data sets to enhance detection of genomic variants for susceptibility/resistance to M. bovis infection.ResultsThe host gene expression data consisted of RNA-seq data from bovine alveolar macrophages (bAM) infected with M. bovis at 24 and 48 h post-infection (hpi) compared to non-infected control bAM. These RNA-seq data were analysed using three distinct computational pipelines to produce six separate gene sets: 1) DE genes filtered using stringent fold-change and P-value thresholds (DEG-24: 378 genes, DEG-48: 390 genes); 2) genes obtained from expression correlation networks (CON-24: 460 genes, CON-48: 416 genes); and 3) genes obtained from differential expression networks (DEN-24: 339 genes, DEN-48: 495 genes). These six gene sets were integrated with three bTB breed GWAS data sets by employing a new genomics data integration tool—gwinteR. Using GWAS summary statistics, this methodology enabled detection of 36, 102 and 921 prioritised SNPs for Charolais, Limousin and Holstein-Friesian, respectively.ConclusionsThe results from the three parallel analyses showed that the three computational approaches could identify genes significantly enriched for SNPs associated with susceptibility/resistance to M. bovis infection. Results indicate distinct and significant overlap in SNP discovery, demonstrating that network-based integration of biologically relevant transcriptomics data can leverage substantial additional information from GWAS data sets. These analyses also demonstrated significant differences among breeds, with the Holstein-Friesian breed GWAS proving most useful for prioritising SNPS through data integration. Because the functional genomics data were generated using bAM from this population, this suggests that the genomic architecture of bTB resilience traits may be more breed-specific than previously assumed.

Early Immune Response and Cytokine Induction of PBMC‐derived Bovine Macrophages to Infection with Rift Valley Fever Virus in the Presence and Absence of Culex tarsalis Mosquito Saliva

Rift Valley fever virus (RVFV) is a zoonotic arbovirus in the family Phenuiviridae. In Africa, outbreaks of Rift Valley fever (RVF) usually occur following periods of heavy rainfall that prompt population surges in mosquito vectors. RVF causes significant mortality in ruminants, especially in ovine fetuses and neonates (up to 100%), while human infections cause mild febrile illness to hemorrhagic fever and death. Macrophages play a critical role as phagocytes in the innate immune system and produce cytokines that stimulate the adaptive immune system. As in other arboviral diseases, macrophages and dendritic cells are thought to be early infection targets of RVFV, enabling the virus to suppress the immune response. To further investigate their role in RVF early immune response and cytokine induction, bovine peripheral blood mononuclear cell-derived macrophages were infected with RVFV MP-12 (an attenuated vaccine strain) in the presence and absence of Culex tarsalis saliva. We hypothesized that in macrophages treated with Culex saliva and RVFV will have increased transcription of Th2-associated cytokines compared to those infected with virus only due to immunomodulatory proteins present in mosquito saliva. The macrophage lineage of the cells and RVFV infection were confirmed by dual label immunofluorescence for IBA-1 (macrophage marker) and RVFV nucleoprotein. Total RNA was extracted from 3 biological replicates of macrophages treated respectively with saliva only, MP-12 only, MP-12 and saliva at 0, 8 and 24 hours post-infection (hpi) as well as untreated controls. RNA was reverse-transcribed and the resulting cDNA analyzed by qPCR of four housekeeping genes (ActinB, H3F3, PPIA, YWHAZ), 6 early response genes (TLR3, TLR7, TLR8, RIG1, MDA5, IFNa) and 9 cytokine genes (IL4, IL6, IL10, IL12b, IL18, IFNg, TNFa, IL1a, IL1b). Relative gene expression (RGE) was determined by the method described by Vandesompele which uses multiple reference genes for normalization and does not assume equal PCR efficiencies for the reference genes and genes of interest. RGEs for each target were log-transformed and analyzed with two-way ANOVAs and Tukey's post-hoc test with p-value correction for multiple tests to evaluate statistical differences between treatments and time points. There was no statistical increase in transcription of Th2-associated cytokines when bovine macrophages were infected with MP-12 in the presence of saliva at any of the time points when compared to virus only. In contrast, significant differences in gene expression were present when infected samples were compared to uninfected controls. At 8 hpi, transcription was significantly different than controls for all virus infected samples for TLR3, RIG1, MDA and IFNa as well as IL6, IL10, IL12b, IL18, TNFa, IL1a and IL1b. This trend continued at 24 hpi with the exception of IL12b. While Culex saliva may not enhance suppression of the early immune response in MP-12 infection, it may still have a role in virulent RVFV strain infections.

Diverging in vitro inflammatory responses toward Streptococcus uberis in mouse macrophages either preconditioned or continuously treated with β-hydroxybutyrate

Hyperketonemia is a common condition in early-lactation dairy cows that has been associated with an increase in the risk of infectious disease. Recent mouse studies have elucidated an anti-inflammatory effect of the ketone body β-hydroxybutyrate (BHB). Therefore, the objective of this study was to determine whether BHB altered inflammatory responses in macrophages challenged with the common mastitis pathogen Streptococcus uberis. A secondary objective was to determine whether the inflammatory response to the S. uberis challenge was dependent on whether BHB was present in the medium during the challenge (i.e., preconditioned vs. continuous treatment). Two cell culture experiments were conducted. In the first experiment, mouse macrophages (RAW 264.7 line) were preconditioned with BHB (0, 0.6, 1.2, and 1.8 mM) for 24 h; the medium was then replaced with a standard cell culture medium, and the cells were challenged or not with S. uberis for an additional 6 h. In the second experiment, a similar protocol was used; however, cells were preconditioned with BHB (0, 0.6, 1.2, and 1.8 mM) for 24 h, the medium was replaced with fresh medium containing the same concentration of BHB, and cells were either challenged or not with S. uberis for 6 h. In both experiments, relative transcript abundance of cell membrane receptors (Tlr2 and Gpr109a), cytokines (Il1b, Il10, Tnf, and Tgfb1), and chemokines (Cxcl2 and Ccl5) were determined using quantitative real-time PCR and normalized against the geometric mean of Hprt and B2m. Data were analyzed using a linear mixed model, and orthogonal contrasts were conducted to examine the effect of S. uberis challenge and BHB treatment. Streptococcus uberis activated the macrophages, noted by greater transcript abundance of analyzed genes. Intriguingly, in both experiments, the S. uberis challenge increased expression of Gpr109a, which encodes a receptor that is ligated by BHB. Paradoxically, preconditioning macrophages with BHB increased transcript abundance of the immunosuppressive cytokine Tgfb1 and increased that of the neutrophil chemoattractant Cxcl2. Preconditioning decreased Tlr2 and tended to decrease Il10 transcript abundance. In opposition to the preconditioning experiment, continuous treatment of BHB during the S. uberis challenge linearly increased abundance of Tlr2 and Il10 transcripts. Continuous BHB treatment also increased expression of Il1b. In conclusion, BHB treatment altered macrophage inflammatory responses during an S. uberis challenge; however, the direction of this response was dependent on whether BHB was added to the medium during the S. uberis challenge. Future studies should be conducted using bovine macrophages and in vivo approaches to examine BHB effects during an S. uberis challenge.

Macrophage-specific responses to human- and animal-adaptedtubercle bacilli reveal pathogen and host factors drivingmultinucleated cell formation.

The Mycobacterium tuberculosis complex (MTBC) is a group of related pathogens that cause tuberculosis (TB) in mammals. MTBC species are distinguished by their ability to sustain in distinct host populations. While Mycobacterium bovis (Mbv) sustains transmission cycles in cattle and wild animals and causes zoonotic TB, M. tuberculosis (Mtb) affects human populations and seldom causes disease in cattle. The host and pathogen determinants underlying host tropism between MTBC species are still unknown. Macrophages are the main host cell that encounters mycobacteria upon initial infection, and we hypothesised that early interactions between the macrophage and mycobacteria influence species-specific disease outcome. To identify factors that contribute to host tropism, we analysed blood-derived primary human and bovine macrophages (hMϕ or bMϕ, respectively) infected with Mbv and Mtb. We show that Mbv and Mtb reside in different cellular compartments and differentially replicate in hMϕ whereas both Mbv and Mtb efficiently replicate in bMϕ. Specifically, we show that out of the four infection combinations, only the infection of bMϕ with Mbv promoted the formation of multinucleated giant cells (MNGCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bMϕ promote macrophage multinucleation. Importantly, we extended our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNGCs. Our findings implicate MNGC formation in the contrasting pathology between Mtb and Mbv for the bovine host and identify MPB70 from Mbv and extracellular vesicles from bMϕ as mediators of this process.

Novel Secreted Protein of Mycoplasma bovis MbovP280 Induces Macrophage Apoptosis Through CRYAB.

Mycoplasma bovis causes important diseases and great losses on feedlots and dairy farms. However, there are only a few measures to control M. bovis-related diseases. As in other mycoplasma species, this is predominantly because the virulence related factors of this pathogen are largely unknown. Therefore, in this study, we aimed to identify novel virulence-related factors among the secreted proteins of M. bovis. Using bioinformatic tools to analyze its secreted proteins, we preliminarily predicted 39 secreted lipoproteins, and then selected 11 of them for confirmation based on SignalP scores >0.6 or SceP scores >0.8 and conserved domains. These 11 genes were cloned after gene modification based on the codon bias of Escherichia coli and expressed. Mouse antiserum to each recombinant protein was developed. A western blotting assay with these antisera confirmed that MbovP280 and MbovP475 are strongly expressed and secreted proteins, but only MbovP280 significantly reduced the viability of bovine macrophages (BoMac). In further experiments, MbovP280 induced the apoptosis of BoMac treated with both live M. bovis and MbovP280 protein. The conserved coiled-coil domain of MbovP280 at amino acids 210–269 is essential for its induction of apoptosis. Further, immunoprecipitation, mass spectrometry, and coimmunoprecipitation assays identified the anti-apoptosis regulator αB-crystallin (CRYAB) as an MbovP280-binding ligand. An αβ-crystallin knockout cell line BoMac-cryab−, Mbov0280-knockout M. bovis strain T9.297, and its complemented M. bovis strain CT9.297 were constructed and the apoptosis of BoMac-cryab− induced by these strains was compared. The results confirmed that CRYAB is critical for MbovP280 function as an apoptosis inducer in BoMac. In conclusion, in this study, we identified MbovP280 as a novel secreted protein of M. bovis that induces the apoptosis of BoMac via its coiled-coil domain and cellular ligand CRYAB. These findings extend our understanding of the virulence mechanism of mycoplasmal species.

TRPV2-spike protein interaction mediates the entry of SARS-CoV-2 into macrophages in febrile conditions.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a novel strain of highly contagious coronaviruses that infects humans. Prolonged fever, particularly that above 39.5 °C, is associated with SARS-CoV-2 infection. However, little is known about the pathological effects of fever caused by SARS-CoV-2.Methods: Primary bovine alveolar macrophages (PBAMs), RAW264.7 mouse macrophages, and THP-1 human cells were transfected with plasmids carrying the genes encoding the SARS-CoV-2 spike (S) protein or receptor-binding domain (RBD). Proteins in the macrophages interacting with S-RBD at 39.5 °C or 37 °C were identified by immunoprecipitation-mass spectrometry. Glutathione S-transferase pulldown, surface plasmon resonance, and immunofluorescence were performed to evaluate the transient receptor potential vanilloid 2 (TRPV2) interaction with SARS-CoV-2-S-RBD at 39.5 °C. Using an RNA sequencing-based approach, cytokine gene expression induced by SARS-CoV-2 S transfection at 39.5 °C and 37.5 °C in primary alveolar macrophages was measured. Fluo-4 staining and enzyme-linked immunosorbent assays were used to assess the regulatory function of TRPV2 in intracellular Ca 2+ and cytokines under SARS-CoV-2-S-RBD at 39.5 °C. Additionally, cytokine release was examined after TRPV2 knockdown with shRNA oligonucleotides or inhibition using the SKF-96365 antagonist.Results: We identified an interaction between the primary alveolar macrophage receptor TRPV2 and S-RBD under febrile conditions. Febrile temperature promotes Ca2+ influx through SARS-CoV-2 infection in PBAMs, further activates the NF-κB p65 signaling pathway, and enhances the secretion of cytokines. Furthermore, knockdown or antagonist (with SKF-96365) of TRPV2 significantly decreased the release of cytokines that drive the inflammatory response.Conclusion: Collectively, our findings identified TRPV2 as a receptor of SARS-CoV-2 in conditions of febrile temperature, providing insight into critical interactions of SARS-CoV-2 with macrophages, as well as a useful resource and potential drug target for coronavirus disease 2019.

A Paradox in Bacterial Pathogenesis: Activation of the Local Macrophage Inflammasome Is Required for Virulence of Streptococcus uberis.

Streptococcus uberis is a common cause of intramammary infection and mastitis in dairy cattle. Unlike other mammary pathogens, S. uberis evades detection by mammary epithelial cells, and the host–pathogen interactions during early colonisation are poorly understood. Intramammary challenge of dairy cows with S. uberis (strain 0140J) or isogenic mutants lacking the surface-anchored serine protease, SUB1154, demonstrated that virulence was dependent on the presence and correct location of this protein. Unlike the wild-type strain, the mutant lacking SUB1154 failed to elicit IL-1β from ex vivo CD14+ cells obtained from milk (bovine mammary macrophages, BMM), but this response was reinstated by complementation with recombinant SUB1154; the protein in isolation elicited no response. Production of IL-1β was ablated in the presence of various inhibitors, indicating dependency on internalisation and activation of NLRP3 and caspase-1, consistent with inflammasome activation. Similar transcriptomic changes were detected in ex vivo BMM in response to the wild-type or the SUB1154 deletion mutant, consistent with S. uberis priming BMM, enabling the SUB1154 protein to activate inflammasome maturation in a transcriptionally independent manner. These data can be reconciled in a novel model of pathogenesis in which, paradoxically, early colonisation is dependent on the innate response to the initial infection.

Sodium Butyrate Alleviates Lipopolysaccharide-Induced Inflammatory Responses by Down-Regulation of NF-κB, NLRP3 Signaling Pathway, and Activating Histone Acetylation in Bovine Macrophages.

Sodium butyrate is the sodium salt of butyric acid, which possesses many biological functions including immune system regulation, anti-oxidant and anti-inflammatory ability. The present study was designed to elucidate the anti-inflammatory effects and mechanisms of sodium butyrate on lipopolysaccharide (LPS)-stimulated bovine macrophages. The effect of sodium butyrate on the cell viability of bovine macrophages was assayed by using the CCK-8 kit. Quantitative real-time PCR (qRT-PCR) was used to detect the gene expression of interleukin-6 (IL-6), interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2), and inducible Nitric Oxide Synthase (iNOS). NF-κB, NLRP3 signaling pathway, and histone deacetylase were detected by western blotting. The results showed that sodium butyrate had no significant effect on cell viability at 0–1 mM, and inhibited LPS-induced IL-6, IL-1β, COX-2, and iNOS expression. Moreover, sodium butyrate suppressed LPS (5 μg/ml)-stimulated the phosphorylation of IκB and p65, inhibited the deacetylation of histone H3K9, and has also been found to inhibit protein expression in NLRP3 inflammasomes. Thus, our finding suggested that sodium butyrate relieved LPS-induced inflammatory responses in bovine macrophage by inhibiting the canonical NF-κB, NLRP3 signaling pathway, and histone decetylation, which might be helpful to prevent cow mastitis.