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The secretome of Staphylococcus aureus strains with opposite within-herd epidemiological behavior affects bovine mononuclear cell response

Abstract

Staphylococcus aureus modulates the host immune response directly by interacting with the immune cells or indirectly by secreting molecules (secretome). Relevant differences in virulence mechanisms have been reported for the secretome produced by different S. aureus strains. The present study investigated the S. aureus secretome impact on peripheral bovine mononuclear cells (PBMCs) by comparing two S. aureus strains with opposite epidemiological behavior, the genotype B (GTB)/sequence type (ST) 8, associated with a high within-herd prevalence, and GTS/ST398, associated with a low within-herd prevalence. PBMCs were incubated with different concentrations (0%, 0.5%, 1%, and 2.5%) of GTB/ST8 and GTS/ST398 secretome for 18 and 48 h, and the viability was assessed. The mRNA levels of pro- (IL1-β and STAT1) and anti-inflammatory (IL-10, STAT6, and TGF-β) genes, and the amount of pro- (miR-155-5p and miR-125b-5p) and anti-inflammatory (miR-146a and miR-145) miRNAs were quantified by RT-qPCR. Results showed that incubation with 2.5% of GTB/ST8 secretome increased the viability of cells. In contrast, incubation with the GTS/ST398 secretome strongly decreased cell viability, preventing any further assays. The GTB/ST8 secretome promoted PBMC polarization towards the pro-inflammatory phenotype inducing the overexpression of IL1-β, STAT1 and miR-155-5p, while the expression of genes involved in the anti-inflammatory response was not affected. In conclusion, the challenge of PBMC to the GTS/ST398 secretome strongly impaired cell viability, while exposure to the GTB/ST8 secretome increased cell viability and enhanced a pro-inflammatory response, further highlighting the different effects exerted on host cells by S. aureus strains with epidemiologically divergent behaviors.

Introduction

Mastitis is a primary health and economic issue addressed in dairy farming. Among the numerous mastitis-causing pathogens, Staphylococcus aureus is one of the most relevant worldwide. It can establish both acute and chronic infections often leading to subclinical mastitis. Due to its ability to persist inside the mammary gland [1, 2] and to internalize within mammary epithelial cells and phagocytes such as monocytes, S. aureus can evade the host immune response [3, 4]. The immune escape strategies vary greatly in vivo and in vitro, according to the bacterial genotype [5,6,7,8]. S. aureus genotypes can have strikingly different genomic, transcriptomic, and proteomic profiles, as well as diverse pathogenic and epidemiological behaviors [9,10,11,12]. In many European countries, most S. aureus strains isolated from cows with intramammary infection (IMI) belong to genotype B (GTB), generally corresponding to Sequence Type (ST) 8 [13], which is a highly contagious bovine-adapted strain [14, 15]. Genotype S (GTS), corresponding to ST398, is more likely associated with sporadic IMI and can affect livestock animals and humans, developing antimicrobial resistance and, thus, representing a public health issue [16].

Secreted molecules are essential elements in bacterial infections. Based on the released virulence factors, they can exert different activities, killing target cells or helping the bacterial pathogen establishment in the host cell [17]. A recent comparative study provided a thorough characterization of the secreted proteins (secretome) of GTB/ST8 and GTS/ST398, connecting their secretome profiles with the respective epidemiological behaviors: GTB/ST8 preferentially released virulence factors associated with the infection development and persistence, avoiding both the innate and adaptive humoral responses, while GTS/ST398 secretomes enhanced cellular damage and inflammation [10]. The same study demonstrated that the secretome of GTB/ST8 did not exert cytotoxic activities on bovine PBMCs, further supporting the hypothesis of its ability to evade the host immune response. At the same time, GTS/ST398 reduced cell viability at high concentrations (2.5 and 10%) [10]. Although the S. aureus ability to evade the host immune response by directly interacting with the immune cells has been previously investigated [7], no data on the immunomodulatory ability of secreted molecules (secretome) has been reported on bovine immune cells so far. This study aimed to investigate whether GTB/ST8 and GTS/ST398 secretomes in vitro could modulate the bovine immune response of peripheral blood mononuclear cells at the molecular level, focusing on genes and miRNAs related to the M1/Th1 and M2/Th2 phenotypes polarization.

Materials and methods

Purification of PBMC from bovine peripheral blood

Peripheral blood from clinically healthy multiparous Holstein cows at the second parity in their second trimester (90 to 180 DIM) of lactation was collected in sterile tubes treated with K2EDTA (Vacutainer) during routine slaughtering procedures. The isolation of PBMC was performed using Ficoll-Paque Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) 1.077 g/mL density gradient centrifugation, as previously described [18]. Briefly, whole blood was centrifuged at 1260 × g for 30 min at 18 °C to collect the buffy coat. The buffy coat was diluted 1:5 with cold PBS + EDTA 2 mM without Ca2+ and Mg2+ (Sigma-Aldrich, St. Louis, MO, USA), layered on Ficoll, and centrifuged at 1700 × g for 30 min at 4 °C to isolate PBMC ring. The PBMC ring was collected and washed twice with cold PBS + EDTA 2 mM by centrifuging at 500 × g for 7 min at 4 °C to remove the platelets. The pellet was treated with Red Blood Lysis Buffer (Sigma-Aldrich) and then washed with cold PBS + EDTA 2 mM by centrifuging at 500 × g for 7 min at 4 °C to remove red blood cells. Isolated PBMC were counted using an Automatic Cell Counter (BioRad) and resuspended at the desired final concentration in RPMI 1640 medium with 25mM Hepes and l-glutamine, complemented with 1% nonessential amino acid solution (100×), 1% penicillin-streptomycin solution (100×; Euroclone, Milano, Italy), and 10% fetal bovine serum (FBS; Sigma-Aldrich).

Preparation and quantification of the S. aureus secretome

The stock solution of S. aureus secretomes concentrated at 8–10 µg/µL was produced as previously reported [10]. Briefly, bacteria were revitalized in Brain Heart Infusion (BHI) broth overnight at 37 °C. Overnight culture suspensions were then diluted 1:100 in RPMI-1640 and incubated at 37 °C with agitation for 3.5 h. The bacterial culture was centrifuged at 9300 × g for 5 min, and proteins were processed with Amicon Ultra-0.5 centrifugal filter units with Ultracel-10 membrane (Millipore, Billerica, MA, USA). Protein concentration was assessed using the Pierce™ 660 nm Protein Assay Kit (Thermo Scientific, San Jose, CA, USA).

Cell viability assay

Cell viability was determined using Cell Proliferation Kit, I (MTT) from Roche, following the manufacturer’s instructions. PBMC from 6 different animals (1 × 105 cells/well) were challenged with increasing concentrations (0.5%, 1%, 2.5%) of GTB/ST8 and GTS/ST398 secretomes in 96-well plates, incubated at 37 °C and 5% CO2 for 18 and 48 h. The secretome concentrations were selected based on previously reported results [10]. Cells without secretome were included as a control. After incubation, the MTT reagent (10 µL) was added and incubated for 4 h at 37 °C. Solubilization buffer (100 µL) was then added and incubated overnight. The absorbance was measured with a Lab Systems Multiskan plate reader spectrophotometer (Lab, Midland, Canada) at 550 nm.

PBMC stimulation with S. aureus secretome

A total of 5 × 105 cells/well from 10 animals were seeded in triplicate in sterile 24-well plates (Falcon COD 351147) and incubated for 18 and 48 h at 37 °C and 5% CO2 with increasing concentrations of S. aureus secretome (0.5%, 1%, and 2.5%). Cells without secretome were included as a control. After incubation, the cells were washed with PBS and centrifuged at 500 × g for 7 min. Finally, PBMCs were lysed, adding 700 µL of Fenozol plus (A&A biotechnology COD 203-50P), and stored at −80 °C.

Long and small RNA extraction and quantification

Long and small RNAs were extracted from cells using a MicroRNA concentrator kit (A&A biotechnology COD 010AAB035), following the manufacturer’s instructions. Caenorhabditis elegans miRNA cel-miR-39 (25 fmol final concentration) was added and used as exogenous synthetic spike-in control. RNA concentration and quality were assessed using a NanoDrop ND-1000 UV–vis spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). The Minimum Information for Publication of Quantitative Real-Time PCR (MIQE) guidelines were followed [19].

mRNA quantification

The reverse transcription (RT) reaction from 190 ng mRNA was carried out using the iScript cDNA Synthesis Kit (BioRad), according to the manufacturer’s instructions. The expression of genes involved in the Th1-M1 and Th2-M2 pathways was quantified using qPCR, and the reaction was carried out in duplicate. The selected targets included pro- (IL1-β and STAT1) and anti-inflammatory (IL-10, STAT6, and TGF-β) genes, amplified using previously described primers [20]. The reaction was carried out in a scaled-down reaction volume (15 µL) in a CFX Connect Real-Time PCR Detection System (BioRad), using 7.5 µL of SsoFast™ EvaGreenSupermix (Bio-Rad, California, USA), forward and reverse primers, 1 µL of cDNA sample, and RNase- and DNase free water to make up the remaining volume. The thermal profile consisted of 50 °C for 2 min, 95 °C for 3 min, 40 cycles of 95 °C for 15 s, and 60 °C for 30 s. Two reference genes (YWHAZ and H3F3A) were selected [20], and their mean was used for normalization using the 2−ΔΔCq method.

miRNA quantification

To synthesize cDNA from the isolated small RNA, the TaqMan® Advanced miRNA Assays kit (Thermo Fisher Scientific, A25576) was used according to the manufacturer’s recommendations.

MicroRNAs were selected according to previous studies in which these miRNAs were found to exert pro- (miR-155-5p and miR-125b-5p) and anti-inflammatory (miR-146a and miR-145) activities [21,22,23]. The selected miRNAs included miR-155-5p (assay ID 477927_mir), miR-125b-5p (assay ID 480907_mir), miR-145-5p (assay ID 480938_mir), and miR-146a-5p (assay ID 478399_mir). The reaction was carried out in a scaled-down reaction volume (15 µL) in a CFX Connect Real-Time PCR Detection System (BioRad), using 7.5 µL of Advanced Master Mix 2X (Thermo Fisher Scientific, 4444557), 0.75 µL of miRNA-specific TaqMan advance assay reagents (20×), 1 µL of cDNA sample, and RNase-free water to reach the final volume. Each sample was tested in duplicate. The thermal cycling profile of the reaction was: 50 °C for 2 min, 95 °C for 3 min, 40 cycled at 95 °C for 15 s, and 60 °C for 30 s. To evaluate the stability of reference miRNAs, namely miR-320a-3p (assay ID 478594_mir) and miR-187-5p (assay ID 477941_mir), a geNorm analysis was performed using Biogazelle’s qbase+ software. Data normalization was carried out using the arithmetic mean of two reference miRNAs. Relative quantification of each miRNA was calculated using the BioRad CFX Maestro Software by the 2−ΔΔCq method.

Statistical analysis

Statistical analysis was carried out using GraphPad Prism 9.0.0 (San Diego, CA, USA). For the viability assay, the normal distribution of the data was assessed by performing the Shapiro-Wilk test. Not normally distributed data were analyzed using Friedman and Dunn’s multiple comparison tests. A grouped analysis was performed using multiple comparisons 2-way ANOVA test to analyze the mRNA and miRNA expression.

Results

The S. aureus secretomes had opposite effects on cell viability

After 18 h of incubation with GTS/ST398-secreted molecules (Figure 1A), PBMC treated with 1% and 2.5% concentration decreased their viability by 0.7 and 0.48 folds compared with the control (P = 0.0052 and P = 0.0010, respectively). After 48 h of incubation (Figure 1B), 1% and 2.5% concentrations significantly decreased immune cell viability by 0.25 and 0.08 folds compared with the control (P = 0.0006 and P = 0.0086, respectively). On the other hand, no effect on viability was observed on cells incubated with GTB/ST8 secretome for 18 h (Figure 1C); indeed, after 48 h of incubation, the highest concentration of secretome (2.5%) increased the PBMC viability by 1.4-folds compared with the control (P = 0.011; Figure 1D). Since cell viability results showed that GTS/ST398-secreted molecules have a strong cytotoxic effect, further molecular assessments were carried out testing only the GTB/ST8 secretome.

Figure 1
figure 1

PBMC viability after (A–C) 18- and (B–D) 48-h incubation with increasing concentration of GTS/ST398 and GTB/ST8 secretome, respectively. The viability is expressed as fold-change compared to the control (cells incubated without secretome) in six biological replicates. Significance was accepted at P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***). The lines inside the boxes denote the median.

The GTB/ST8 secretome increased the expression level of proinflammatory genes

To evaluate the ability of the GTB/ST8 secretome to promote a pro- or anti-inflammatory gene expression at the molecular level, PBMCs were incubated for 18 and 48 h with increasing concentrations (0.5%, 1%, and 2.5%). The results are presented in Figure 2.

Figure 2
figure 2

Relative expression of mRNA related to M1/Th1 phenotype (A and B) and M2/Th2 phenotype (C, D, and E) in PBMC incubated for 18 and 48 h with increasing concentrations of GTB/ST8 secreted proteins. Cells incubated without secretome were used as control. Data are means ± SD of 10 different animals. Significance was accepted for P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***).

The expression of IL1β was upregulated after the incubation with 2.5% secretome for 18 h (IL1βFC(2.5%/control) = 6.1, P = 0.014), while no effect was observed after 48 h (Figure 2A). STAT1 abundance was significantly affected by the challenge with GTB/ST8-secreted molecules. After 18 h, cells treated with 0.5%, 1% and 2.5% secretome increased the expression of STAT1 (STAT1FC(0.5%/control) = 5.5, P = 0.001; STAT1FC(1%/control) = 4.9, P = 0.003; STAT1FC(2.5%/control) = 4.6, P = 0.009, respectively; Figure 2B). After 48 h, cells stimulated with 0.5%, 1% and 2.5% secretome upregulated the expression of STAT1 compared with the control (STAT1FC(0.5%/control), P = 0.008, STAT1FC(1%/control), P = 0.001, STAT1FC(2.5%/control), P = 0.029, respectively; Figure 2B).

The challenge with GTB/ST8 secretome did not affect the expression of anti-inflammatory genes (Figures 2C–E).

The GTB/ST8 secretome increased the expression level of proinflammatory miR-155-5p

The miRNA’s relative abundance was quantified using RT-qPCR. Analysis of the reference miRNAs expression stability by geNorm indicated that both were suitable with average M values of 0.787. Their mean was used for the normalization of the relative quantification data experiments (Figure 3).

Figure 3
figure 3

Relative expression of miRNA related to M1/Th1 phenotype (A and B) and M2/Th2 phenotype (C and D), in PBMC, incubated for 18 and 48 h with increasing concentrations of GTB/ST8 secreted proteins (0.5%, 1%, and 2.5%). Cells incubated with medium only were used as control. Data are means ± SD of 10 different animals. Significance was accepted for P < 0.05 (*), P < 0.01 (**), P < 0.0001 (****).

The relative abundance of miRNA targets involved in modulating pro- and anti-inflammatory responses was evaluated. All targets were quantifiable in the samples at different time points, and one of the four tested targets was differentially expressed. In detail, the level of miR-155-5p increased after 48 h of challenge with 1% and 2.5% GTB/ST8 compared with the control (miR-155-5pFC(1%/control) = 8.87, P = 0.014; miR-155-5p FC(2.5%/control) = 18, P < 0.0001) (Figure 3B).

Discussion

In this study, we aimed to assess the ability of the secretome of S. aureus strains with opposite epidemiological behavior to micromanage the bovine immune response in vitro by acting differentially on PBMC activation.

Staphylococcus aureus can express a wide array of secreted virulence factors, which can interact with innate and adaptive immune responses, influencing leukocyte activation [24, 25], and the particular virulence pattern of each strain is strongly related to the S. aureus genotype [8, 12, 14, 26]. Recently, a comparative secretome study has analyzed the secreted protein profile of GTB/ST8 and GTS/ST398 strains [10]. Specific proteins, such as immunoglobulin G binding protein A (Spa), immunoglobulin-binding protein (Sbi), and the staphylococcal complement inhibitor (Scin), were found among the differentially secreted proteins of these two genotypes and were identified as promoters of host immune evasion by acting on different pathways [10]. That study also provided the first evidence that the molecules released in culture by the two genotypes had dramatically different impacts on cell viability, showing that exposure to low concentrations of the GTS/ST398 secretomes could lead to cell death. On the other hand, following previous studies, the S. aureus virulence factors may manipulate the host’s immune response, alternately activating a pro- or anti-immune response [27].

Mononuclear cells are a heterogeneous cell population composed of monocytes and lymphocytes, characterized by remarkable plasticity and diversity. Both functions are settled in response to microenvironmental signals, driving the polarized programs. The polarization extremes are represented by the M1/Th1 and M2/Th2 dichotomy, which occurs in pathophysiological conditions [28]. This dynamic skewing leads the cells to exert opposed functions: the M1/Th1 phenotype is involved in the onset of inflammation, while the M2/Th2 phenotype is involved in its resolution. M1/Th1 phenotype can be activated in response to microbial stimuli enhancing the secretion of proinflammatory cytokines, including IL1-β, IL-6, and TNF-α [29, 30].

Conversely, M2/Th2 lineages are involved in angiogenesis and tissue remodeling pathways, expressing anti-inflammatory cytokines, such as TGF-β and IL-10, and contributing to the resolution of the inflammation [18, 29, 30]. The regulation and the balance between these phenotypes are crucial for the correct onset and resolution of inflammation. However, the functional heterogeneity is only partially reflected by different phenotypes and morphological appearances, while different transcriptional programs, specifically activated by microenvironmental signals, enhance functional polarization [31]. STAT signaling plays a key role in the modulation of the immune response, as STAT1 can be used as a marker of the M1 polarization while STAT6 of the M2 phenotype. STAT1 is crucial for the immune response against bacterial infection, and a decrease in its activity is linked to a higher bacterial infection susceptibility [32]. After 18 and 48 h, we observed that the incubation of PBMC with the GTB/ST8 secretome led to a significant increase in the expression of STAT1 compared to the control. The STAT1 mRNA was also significantly upregulated in cells stimulated with a low concentration of secretome. A similar effect was previously observed in human monocyte-derived macrophages stimulated with S. aureus [33]. This suggests the critical and multifaceted role secreted molecules played in eliciting the host immune response. The STAT1 expression can be activated by the presence of Gram-positive bacteria such as S. aureus thanks to the binding effect of lipoteichoic acid (LTA) on the bacterial surface with the monocyte Toll-like receptors (TLR2) [34, 35]. The present results are therefore consistent with what has been reported in the literature.

The innate immune response occurs after sensing DAMPs and PAMPs as the molecules released by S. aureus by immune cells promoting the transcription of pro-IL-1α/β and other cytokines via TLR/MYD88/NF-kB pathway [36, 37]. PBMCs stimulated with GTB/ST8 secretome (2.5%) for 18 h significantly increased the expression of IL-1β. This proinflammatory cytokine can increase endothelial cell permeability and stimulate the release of chemokines, summoning inflammatory cells, including neutrophils and macrophages [38]. The IL-1β exerts wide-ranging effects in modulating immune cells and plays a pivotal role in controlling S. aureus infection, promoting phagocytosis and killing by neutrophils and macrophages [35, 39]. Moreover, T cell expansion is promoted by IL-1β toward Th1, Th2, and Th17 [40,41,42]. Souza et al. [24] demonstrated that bovine PBMC stimulated with different S. aureus strains causing persistent IMI increased IL-17 A and IFN-γ release in the supernatant, while only S. aureus strain promoted the lymphocyte polarization toward CD4+ and CD8+ phenotypes. The present work demonstrated that the challenge with 2.5% secretome increased the expression of IL-1β after 18 h and the PBMC viability after 48 h, supporting the hypothesis that S. aureus GTB/ST8 secreted molecules may induce lymphocyte activation and proliferation in vitro, also triggering an adaptive immune response. The results are consistent with previously reported data, which demonstrated that the innate immune response, mediated by monocytes, macrophages, Natural Killer cells, and cytokines, including IL-1β, predominates in the early stage of mammary gland infection regulating the expression of adhesins by endothelial cells and neutrophil chemotaxis and then stimulating the acquired immune response [43].

The S. aureus secretome enhances the expression of miR-155-5p, a key transcriptional regulator for cancer and inflammation-related diseases [44, 45]. MiR-155-5p promotes the polarization of monocytes towards the M1 lineage, being in negative correlation with the suppressor of cytokine signaling 1 (SOCS1) expression [21, 22, 46]. Previous in vitro study on bovine CD14+ monocytes challenged with Staphylococcal enterotoxin B (SEB) demonstrated a decrease in miR-155-5p level, suggesting that S. aureus may induce immunosuppression to survive inside the host [47]. Conversely, this study showed the upregulation of miR-155-5p in mononuclear cells stimulated with GTB/ST8 secretome, consistently with the upregulation of the STAT1 expression. Both MiR-155-5p and STAT1 are regulated by positive feedback in response to inflammatory signals or infection [48]. MiR-155-5p modulates STAT1 expression suppressing SOCS1 expression in hepatoma cells and, thus, promoting the JAK/STAT signaling [49, 50].

The current study suffers from some limitations. Since no data have been previously reported on the ability of GTS/ST398 and GTB/ST8 to modulate bovine mononuclear cell response, the present investigation focused on the whole PBMC population. Further studies will investigate if and how the secretome produced by different S. aureus strains may promote macrophage and lymphocyte polarization toward different subpopulations. Moreover, the cells’ viability has been tested using the MTT test, while no information on death pathways, including pyroptosis, in which IL-1β plays a pivotal role [51], or necroptosis, efficiently promoted by S. aureus [52, 53], has been evaluated. Finally, in this work, we used the growth conditions previously applied for the proteomic characterization of the S. aureus secretome, carried out on the same strains, to ensure reproducibility [10]. Nevertheless, further adjustments of culture conditions might improve S. aureus growth that more closely resembles the in vivo situation during mastitis [54], and this should be considered in future research.

The present study demonstrated for the first time that the molecules secreted by S. aureus can modulate the immune response of bovine leukocytes in vitro, highlighting how secretomes from S. aureus strains with different epidemiological behaviors could elicit dramatically different responses in bovine PBMCs. The GTS/ST398 secretome led to significant losses in cell viability, while the GTB/ST8 secretome positively affected bovine PBMC viability. The immune response was also studied at a molecular level for GTB/ST8, revealing that the bacterial secretome can trigger the upregulation of genes involved in the Th1/M1 polarization. On the contrary, no effects could be observed in the expression of targets involved in the anti-inflammatory response. Further studies on S. aureus-secreted proteins will clarify whether stimulating secretomes isolated from different strains may differentially modulate the host immune cells’ response.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

References

  1. Campos B, Pickering AC, Rocha LS, Aguilar AP, Fabres-Klein MH, de Oliveira Mendes TA, Fitzgerald JR, de Oliveira Barros Ribon A (2022) Diversity and pathogenesis of Staphylococcus aureus from bovine mastitis: current understanding and future perspectives. BMC Vet Res 18:115

    Article  PubMed  PubMed Central  Google Scholar 

  2. Sordillo LM, Streicher KL (2002) Mammary gland immunity and mastitis susceptibility. J Mammary Gland Biol Neoplasia 7:135–146

    Article  PubMed  Google Scholar 

  3. Côté-Gravel J, Malouin F (2019) Symposium review: features of Staphylococcus aureus mastitis pathogenesis that guide vaccine development strategies. J Dairy Sci 102:4727–4740

    Article  PubMed  Google Scholar 

  4. Kubica M, Guzik K, Koziel J, Zarebski M, Richter W, Gajkowska B, Golda A, Maciag-Gudowska A, Brix K, Shaw L, Foster T, Potempa J (2008) A potential new pathway for Staphylococcus aureus dissemination: the silent survival of S. aureus phagocytosed by human monocyte-derived macrophages. PLoS One 3:e1409

    Article  PubMed  PubMed Central  Google Scholar 

  5. Hensen SM, Pavičić MJAMP, Lohuis JACM, Poutrel B (2000) Use of bovine primary mammary epithelial cells for the comparison of adherence and invasion ability of Staphylococcus aureus strains. J Dairy Sci 83:418–429

    Article  CAS  PubMed  Google Scholar 

  6. Pereyra EAL, Sacco SC, Duré A, Baravalle C, Renna MS, Andreotti CS, Monecke S, Calvinho LF, Dallard BE (2017) Immune response of Staphylococcus aureus strains in a mouse mastitis model is linked to adaptive capacity and genotypic profiles. Vet Microbiol 204:64–76

    Article  CAS  PubMed  Google Scholar 

  7. Murphy MP, Niedziela DA, Leonard FC, Keane OM (2019) The in vitro host cell immune response to bovine-adapted Staphylococcus aureus varies according to bacterial lineage. Sci Rep 9:6134

    Article  PubMed  PubMed Central  Google Scholar 

  8. Penadés M, Viana D, García-Quirós A, Muñoz-Silvestre A, Moreno-Grua E, Pérez-Fuentes S, Pascual JJ, Corpa JM, Selva L (2020) Differences in virulence between the two more prevalent Staphylococcus aureus clonal complexes in rabbitries (CC121 and CC96) using an experimental model of mammary gland infection. Vet Res 51:11

    Article  PubMed  PubMed Central  Google Scholar 

  9. Woudstra S, Wente N, Zhang Y, Leimbach S, Gussmann MK, Kirkeby C, Krömker V (2023) Strain diversity and infection durations of Staphylococcus spp. and Streptococcus spp. causing intramammary infections in dairy cows. J Dairy Sci 106:4214–4231

    Article  CAS  PubMed  Google Scholar 

  10. Addis MF, Pisanu S, Monistero V, Gazzola A, Penati M, Filipe J, Di Mauro S, Cremonesi P, Castiglioni B, Moroni P, Pagnozzi D, Tola S, Piccinini R (2022) Comparative secretome analysis of Staphylococcus aureus strains with different within-herd intramammary infection prevalence. Virulence 13:174–190

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Capra E, Cremonesi P, Pietrelli A, Puccio S, Luini M, Stella A, Castiglioni B (2017) Genomic and transcriptomic comparison between Staphylococcus aureus strains associated with high and low within herd prevalence of intra-mammary infection. BMC Microbiol 17:21

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Graber HU, Naskova J, Studer E, Kaufmann T, Kirchhofer M, Brechbühl M, Schaeren W, Steiner A, Fournier C (2009) Mastitis-related subtypes of bovine Staphylococcus aureus are characterized by different clinical properties. J Dairy Sci 92:1442–1451

    Article  CAS  PubMed  Google Scholar 

  13. Cosandey A, Boss R, Luini M, Artursson K, Bardiau M, Breitenwieser F, Hehenberger E, Lam T, Mansfeld M, Michel A, Mösslacher G, Naskova J, Nelson S, Podpečan O, Raemy A, Ryan E, Salat O, Zangerl P, Steiner A, Graber HU (2016) Staphylococcus aureus genotype B and other genotypes isolated from cow milk in European countries. J Dairy Sci 99:529–540

    Article  CAS  PubMed  Google Scholar 

  14. Fournier C, Kuhnert P, Frey J, Miserez R, Kirchhofer M, Kaufmann T, Steiner A, Graber HU (2008) Bovine Staphylococcus aureus: association of virulence genes, genotypes and clinical outcome. Res Vet Sci 85:439–448

    Article  CAS  PubMed  Google Scholar 

  15. Hoekstra J, Zomer AL, Rutten VPMG, Benedictus L, Stegeman A, Spaninks MP, Bennedsgaard TW, Biggs A, De Vliegher S, Mateo DH, Huber-Schlenstedt R, Katholm J, Kovács P, Krömker V, Lequeux G, Moroni P, Pinho L, Smulski S, Supré K, Swinkels JM, Holmes MA, Lam TJGM, Koop G (2020) Genomic analysis of European bovine Staphylococcus aureus from clinical versus subclinical mastitis. Sci Rep 10:18172

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Cremonesi P, Pozzi F, Raschetti M, Bignoli G, Capra E, Graber HU, Vezzoli F, Piccinini R, Bertasi B, Biffani S, Castiglioni B, Luini M (2015) Genomic characteristics of Staphylococcus aureus strains associated with high within-herd prevalence of intramammary infections in dairy cows. J Dairy Sci 98:6828–6838

    Article  CAS  PubMed  Google Scholar 

  17. Green ER, Mecsas J (2016) Bacterial secretion systems: an overview. Microbiol Spect 4:10

    Article  Google Scholar 

  18. Ceciliani F, Ávila Morales G, De Matteis G, Grandoni F, Furioso Ferreira R, Roccabianca P, Lecchi C (2020) Methods in isolation and characterization of bovine monocytes and macrophages. Methods 186:22–41

    Article  PubMed  Google Scholar 

  19. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622

    Article  CAS  PubMed  Google Scholar 

  20. Catozzi C, Ávila G, Zamarian V, Pravettoni D, Sala G, Ceciliani F, Lacetera N, Lecchi C (2020) In-vitro effect of heat stress on bovine monocytes lifespan and polarization. Immunobiology 225:151888

    Article  CAS  PubMed  Google Scholar 

  21. O’Connell RM, Taganov KD, Boldin MP, Cheng G, Baltimore D (2007) MicroRNA-155 is induced during the macrophage inflammatory response. Proc Natl Acad Sci USA 104:1604–1609

    Article  PubMed  PubMed Central  Google Scholar 

  22. Ghafouri-Fard S, Abak A, Tavakkoli Avval S, Shoorei H, Taheri M, Samadian M (2021) The impact of non-coding RNAs on macrophage polarization. Biomed Pharmacother 142:112112

    Article  CAS  PubMed  Google Scholar 

  23. Essandoh K, Li Y, Huo J, Fan GC (2016) MiRNA-mediated macrophage polarization and its potential role in the regulation of inflammatory response. Shock 46:122–131

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Souza FN, Santos KR, Ferronatto JA, Ramos Sanchez EM, Toledo-Silva B, Heinemann MB, De Vliegher S, Della Libera AMMP (2023) Bovine-associated staphylococci and mammaliicocci trigger T-lymphocyte proliferative response and cytokine production differently. J Dairy Sci 106:2772–2783

    Article  CAS  PubMed  Google Scholar 

  25. Krishna S, Miller LS (2012) Innate and adaptive immune responses against Staphylococcus aureus skin infections. Semin Immunopathol 34:261–280

    Article  CAS  PubMed  Google Scholar 

  26. Monistero V, Graber HU, Pollera C, Cremonesi P, Castiglioni B, Bottini E, Ceballos-Marquez A, Lasso-Rojas L, Kroemker V, Wente N, Petzer IM, Santisteban C, Runyan J, Veiga Dos Santos M, Alves BG, Piccinini R, Bronzo V, Abbassi MS, Said MB, Moroni P (2018) Staphylococcus aureus isolates from bovine mastitis in eight countries: genotypes, detection of genes encoding different toxins and other virulence genes. Toxins 10:247

    Article  PubMed  PubMed Central  Google Scholar 

  27. Nahrendorf M, Swirski FK (2016) Abandoning M1/M2 for a network model of macrophage function. Circ Res 119:414–417

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Sica A, Erreni M, Allavena P, Porta C (2015) Macrophage polarization in pathology. Cell Mol Life Sci 72:4111–4126

    Article  CAS  PubMed  Google Scholar 

  29. Kansler ER, Li MO (2019) Innate lymphocytes-lineage, localization and timing of differentiation. Cell Mol Immunol 16:627–633

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Chávez-Galán L, Olleros ML, Vesin D, Garcia I (2015) Much more than M1 and M2 macrophages, there are also CD169(+) and TCR(+) macrophages. Front Immunol 6:263

    PubMed  PubMed Central  Google Scholar 

  31. Ostuni R, Piccolo V, Barozzi I, Polletti S, Termanini A, Bonifacio S, Curina A, Prosperini E, Ghisletti S, Natoli G (2013) Latent enhancers activated by stimulation in differentiated cells. Cell 152:157–171

    Article  CAS  PubMed  Google Scholar 

  32. Najjar I, Fagard R (2010) STAT1 and pathogens, not a friendly relationship. Biochimie 92:425–444

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Sun A, Zhang H, Pang F, Niu G, Chen J, Chen F, Zhang J (2018) Essential genes of the macrophage response to Staphylococcus aureus exposure. Cell Mol Biol Lett 23:25

    Article  PubMed  PubMed Central  Google Scholar 

  34. Liljeroos M, Vuolteenaho R, Rounioja S, Henriques-Normark B, Hallman M, Ojaniemi M (2008) Bacterial ligand of TLR2 signals Stat activation via induction of IRF1/2 and interferon-alpha production. Cell Signal 20:1873–1881

    Article  CAS  PubMed  Google Scholar 

  35. Pidwill GR, Gibson JF, Cole J, Renshaw SA, Foster SJ (2020) The role of macrophages in Staphylococcus aureus infection. Front Immunol 11:620339

    Article  CAS  PubMed  Google Scholar 

  36. Hashimoto M, Tawaratsumida K, Kariya H, Aoyama K, Tamura T, Suda Y (2006) Lipoprotein is a predominant toll-like receptor 2 ligand in Staphylococcus aureus cell wall components. Int Immunol 18:355–362

    Article  CAS  PubMed  Google Scholar 

  37. Takeuchi O, Takeda K, Hoshino K, Adachi O, Ogawa T, Akira S (2000) Cellular responses to bacterial cell wall components are mediated through MyD88-dependent signaling cascades. Int Immunol 12:113–117

    Article  CAS  PubMed  Google Scholar 

  38. Swirski FK, Nahrendorf M (2013) Leukocyte behavior in atherosclerosis, myocardial infarction, and heart failure. Science 339:161–166

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Ali RA, Wuescher LM, Dona KR, Worth RG (2017) Platelets mediate host defense against Staphylococcus aureus through direct bactericidal activity and by enhancing macrophage activities. J Immunol 198:344–351

    Article  CAS  PubMed  Google Scholar 

  40. Ben-Sasson SZ, Hu-Li J, Quiel J, Cauchetaux S, Ratner M, Shapira I, Dinarello CA, Paul WE (2009) IL-1 acts directly on CD4 T cells to enhance their antigen-driven expansion and differentiation. Proc Natl Acad Sci 106:7119–7124

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Ben-Sasson SZ, Hogg A, Hu-Li J, Wingfield P, Chen X, Crank M, Caucheteux S, Ratner-Hurevich M, Berzofsky JA, Nir-Paz R, Paul WE (2013) IL-1 enhances expansion, effector function, tissue localization, and memory response of antigen-specific CD8 T cells. J Exp Med 210:491–502

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Sahoo M, Ceballos-Olvera I, Del Barrio L, Re F (2011) Role of the inflammasome, IL-1β, and IL-18 in bacterial infections. Sci World J 49:1998–2011

    Google Scholar 

  43. Oviedo-Boyso J, Valdez-Alarcón JJ, Cajero-Juárez M, Ochoa-Zarzosa A, López-Meza JE, Bravo-Patiño A, Baizabal-Aguirre VM (2007) Innate immune response of bovine mammary gland to pathogenic bacteria responsible for mastitis. J Infect 54:399–409

    Article  PubMed  Google Scholar 

  44. Tian K, Xu W (2021) MiR-155 regulates Th9 differentiation in children with methicillin-resistant Staphylococcus aureus pneumonia by targeting SIRT1. Hum Immunol 82:775–781

    Article  CAS  PubMed  Google Scholar 

  45. Willerslev-Olsen A, Gjerdrum LMR, Lindahl LM, Buus TB, Pallesen EMH, Gluud M, Bzorek M, Nielsen BS, Kamstrup MR, Rittig AH, Bonefeld CM, Krejsgaard T, Geisler C, Koralov SB, Litman T, Becker JC, Woetmann A, Iversen L, Odum N (2021) Staphylococcus aureus induces signal transducer and activator of transcription 5-dependent miR-155 expression in cutaneous T-cell lymphoma. J Invest Dermatol 141:2449–2458

    Article  CAS  PubMed  Google Scholar 

  46. Xu F, Kang Y, Zhang H, Piao Z, Yin H, Diao R, Xia J, Shi L (2013) Akt1-mediated regulation of macrophage polarization in a murine model of Staphylococcus aureus pulmonary infection. J Infect Dis 208:528–538

    Article  CAS  PubMed  Google Scholar 

  47. Dilda F, Gioia G, Pisani L, Restelli L, Lecchi C, Albonico F, Bronzo V, Mortarino M, Ceciliani F (2012) Escherichia coli lipopolysaccharides and Staphylococcus aureus enterotoxin B differentially modulate inflammatory microRNAs in bovine monocytes. Vet J 192:514–516

    Article  CAS  PubMed  Google Scholar 

  48. Kohanbash G, Okada H (2012) MicroRNAs and STAT interplay. Semin Cancer Biol 22:70–75

    Article  CAS  PubMed  Google Scholar 

  49. Su C, Hou Z, Zhang C, Tian Z, Zhang J (2011) Ectopic expression of microRNA-155 enhances innate antiviral immunity against HBV infection in human hepatoma cells. Virol J 8:354

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Zhang Y, Mei H, Chang X, Chen F, Zhu Y, Han X (2016) Adipocyte-derived microvesicles from obese mice induce M1 macrophage phenotype through secreted miR-155. J Mol Cell Biol 8:505–517

    Article  CAS  PubMed  Google Scholar 

  51. Bergsbaken T, Fink SL, Cookson BT (2009) Pyroptosis: host cell death and inflammation. Nat Rev Microbiol 7:99–109

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Greenlee-Wacker MC, Rigby KM, Kobayashi SD, Porter AR, DeLeo FR, Nauseef WM (2014) Phagocytosis of Staphylococcus aureus by human neutrophils prevents macrophage efferocytosis and induces programmed necrosis. J Immunol 192:4709–4717

    Article  CAS  PubMed  Google Scholar 

  53. Kitur K, Parker D, Nieto P, Ahn DS, Cohen TS, Chung S, Wachtel S, Bueno S, Prince A (2015) Toxin-induced necroptosis is a major mechanism of Staphylococcus aureus lung damage. PLoS Pathog 11:e1004820

    Article  PubMed  PubMed Central  Google Scholar 

  54. Le Maréchal C, Jan G, Even S, McCulloch JA, Azevedo V, Thiéry R, Vautor E, Le Loir Y (2009) Development of serological proteome analysis of mastitis by Staphylococcus aureus in ewes. J Microbiol Methods 79:131–136

    Article  PubMed  Google Scholar 

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Funding

This work was funded by the University of Milan, Grant Piano di Sostegno per la Ricerca, Anno 2019—Linea 2, “Caratteri fenotipici di Staphylococcus aureus da mastite bovina”.

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CL, FC, MFA, and RP designed the research, provided funding, and analyzed the data. SDM, JF, AF, LR, VM, GS, CZ, and DP conducted the experiments, collected the data, and performed laboratory experiments. SDM and CL performed the statistical analysis and wrote the paper. All authors read and approved the final manuscript.

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Correspondence to Cristina Lecchi.

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Di Mauro, S., Filipe, J., Facchin, A. et al. The secretome of Staphylococcus aureus strains with opposite within-herd epidemiological behavior affects bovine mononuclear cell response. Vet Res 54, 120 (2023). https://doi.org/10.1186/s13567-023-01247-w

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