Bovine mastitis is the inflammatory reaction of the mammary gland and is commonly caused by bacterial infections in high-yielding dairy cows. The detailed investigation of the immunotranscriptomic response of bovine mammary epithelial (BME) cells to pattern recognition receptors (PRRs) activation by microbial-associated molecular patterns (MAMPs) can be of great importance for understanding the innate immune defense mechanisms, and for exploring the immunomodulatory candidate genes. In this work, we investigated the transcriptome modifications of BME cells after the in vitro stimulation with Escherichia coli derived lipopolysaccharide (LPS) and heat-killed Staphylococcus aureus JE2 and S. aureus SA003. In addition, the effect of Pam3CSK4 (a synthetic triacylated lipopeptide that activates Toll-like receptor 2 (TLR2)), and the intracellular chemotactic protein cyclophilin A (CyPA), which is secreted by BME cells during mastitis, in the expression changes of selected cytokines and chemokines were evaluated by qPCR. Microarray analysis identified 447, 465 and 520 differentially expressed genes (DEGs) in the BME cells after LPS, S. aureus JE2 and S. aureus SA003 stimulation, respectively. A major differential response in the inflammatory gene expression was noticed between the stimulation of LPS and S. aureus strains. Unlike the S. aureus strains, LPS stimulation resulted in significant upregulation of CCL2, CXCL2, CXCL3, CXCL8, IL1α and IL1β, which were confirmed by qPCR analysis. Pam3CSK4 was not able to induce significant changes in the expression of cytokines and chemokines in challenged BME cells. The exogenous CyPA administration was able to upregulate CXCL2, CXCL3, CXCL8, IL1α and IL1β expression in BME cells indicating its ability to promote inflammation. The identification of transcriptional markers of mastitis specific for individual inflammatory factors such as LPS, Pam3CSK4 or CyPA, which can be evaluated in vitro in BME cells, may enable the development of novel diagnostics and/or immunomodulatory treatments, providing new tools for the effective management of mastitis in dairy cows. The results of this work are an advance in this regard.

Bovine mastitis, the inflammation of the mammary gland, affects the quality and quantity of milk yield. Mastitis control relies on single or multiple combinations of antibiotic therapy. Due to increasing antibiotic resistance in pathogens, the intramammary infusion of lactic acid bacteria (LAB) has been considered as a potential alternative to antibiotics for treating and preventing bovine mastitis through the improvement of the host immunity. Probiotic effects are a strain-dependent characteristic; therefore, candidate LAB strains have to be evaluated efficiently to find out the ones with the best potential. Here, we investigated LAB strains originally isolated from feedlot cattle’s environment regarding their ability in inducing the Toll-like receptor (TLR)-triggered inflammatory responses in bovine mammary epithelial (BME) cells in vitro. The BME cells were pre-stimulated with the LAB strains individually for 12, 24, and 48 h and then challenged with Escherichia coli-derived lipopolysaccharide (LPS) for 12 h. The mRNA expression of selected immune genes—interleukin 1 alpha (IL-1α), IL-1β, monocyte chemotactic protein 1 (MCP-1), IL-8, chemokine (C-X-C motif) ligand 2 (CXCL2), and CXCL3 were quantified by real-time quantitative PCR (RT-qPCR). Results indicated that pretreatment with some Lactobacillus strains were able to differentially regulate the LPS inflammatory response in BME cells; however, strain-dependent differences were found. The most remarkable effects were found for Lactobacillus acidophilus CRL2074, which reduced the expression of IL-1α, IL-1β, MCP-1, IL-8, and CXCL3, whereas Lactobacillus rhamnosus CRL2084 diminished IL-1β, MCP-1, and IL-8 expression. The pre-stimulation of BME cells with the CRL2074 strain resulted in the upregulated expression of three negative regulators of the TLRs, including the ubiquitin-editing enzyme A20 (also called tumor necrosis factor alpha-induced protein 3, TNFAIP3), single immunoglobin IL-1 single receptor (SIGIRR), and Toll interacting protein (Tollip) after the LPS challenge. The CRL2084 pre-stimulation upregulated only Tollip expression. Our results demonstrated that the L. acidophilus CRL2074 strain possess remarkable immunomodulatory abilities against LPS-induced inflammation in BME cells. This Lactobacillus strain could be used as candidate for in vivo testing due to its beneficial effects in bovine mastitis through intramammary infusion. Our findings also suggest that the BME cells immunoassay system could be of value for the in vitro evaluation of the immunomodulatory abilities of LAB against the inflammation resulting from the intramammary infection with mastitis-related pathogens.

Adipocytes are dynamic cells that have critical functions to maintain body energy homeostasis. Adipocyte physiology is affected by the adipogenic differentiation, cell program, as well as by the exogenous stimulation of biochemical factors, such as serotonin and TNF-α. In this work, we investigated the global transcriptome modifications when porcine intramuscular preadipocyte (PIP) was differentiated into porcine mature adipocyte (pMA). Moreover, we studied transcriptome changes in pMA after stimulation with serotonin or TNF-α by using a microarray approach. Transcriptome analysis revealed that the expression of 270, 261, and 249 genes were modified after differentiation, or after serotonin and TNF-α stimulation, respectively. Expression changes in APP, HNF4A, ESR1, EGR1, SRC, HNF1A, FN1, ALB, STAT3, CBL, CEBPB, AR, FOS, CFTR, PAN2, PTPN6, VDR, PPARG, STAT5A and NCOA3 genes which are enriched in the ‘PPAR signaling’ and ‘insulin resistance’ pathways were found in adipocytes during the differentiation process. Dose-dependent serotonin stimulation resulted in a decreased fat accumulation in pMAs. Serotonin-induced differentially expressed genes in pMAs were found to be involved in the significant enrichment of ′GPCR ligand-binding′, ‘cell chemotaxis’, ‘blood coagulation and complement’, ‘metabolism of lipid and lipoproteins’, ‘regulation of lipid metabolism by PPARA’, and ‘lipid digestion, mobilization and transport’ pathways. TNF-α stimulation also resulted in transcriptome modifications linked with proinflammatory responses in the pMA of intramuscular origin. Our results provide a landscape of transcriptome modifications and their linked-biological pathways in response to adipogenesis, and exogenous stimulation of serotonin- and TNF-α to the pMA of intramuscular origin.

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Background: Respiratory bacterial infections represent a major health challenge in swine production, highlighting the need for novel immunomodulatory strategies that enhance host resistance. In this study, we investigated whether porcine intestinal lactobacilli could modulate the gut–lung axis and improve respiratory innate immunity in a mouse model of Streptococcus pneumoniae infection, as a surrogate of Streptococcus suis pneumonia. Methods: Three strains of Ligilactobacillus salivarius (LAFF998, LAFF1071, and LAFF1095) were orally administered to Swiss mice prior to pneumococcal challenge. The resistance to the infection, the lung damage and the respiratory innate immune response were evaluated. Results: Only strain LAFF998 significantly reduced pulmonary bacterial loads, prevented bacteremia, and attenuated lung injury. This protective effect was associated with selective modulation of respiratory immunity, characterized by reduced neutrophilic inflammation, increased lymphocyte recruitment, and enhanced activation of alveolar macrophages expressing MHC-II. LAFF998 markedly increased the production of IFN-β, IFN-γ, IL-6, IL-10, and IL-27 in the respiratory tract, without inducing excessive inflammatory damage. Ex vivo and in vitro analyses confirmed that alveolar macrophages from LAFF998-treated mice exhibited a primed phenotype with heightened cytokine responses to pneumococcal stimulation. In contrast, strains LAFF1071 and LAFF1095 failed to confer protection or significantly modulate respiratory immune responses. Conclusions: These findings demonstrate a strict strain-dependent effect among porcine L. salivarius isolates and identify LAFF998 as a potent immunobiotic capable of enhancing respiratory innate immunity through the gut–lung axis. This work supports further studies of LAFF998 as an immunobiotic strategy for the prevention of respiratory infections in pigs.

Immunomodulatory lactic acid bacteria (immunobiotics) have been proposed to prevent bovine mastitis through the enhancement of host immune responses. Furthermore, accumulating evidence suggests that non-viable immunobiotics (postimmunobiotics) can be as effective as viable bacteria in modulating immunity in the bovine host. However, the molecular mechanisms underlying the immunomodulatory effects of heat-inactivated lactic acid bacteria in bovine mammary epithelial cells remain incompletely understood. In the present study, we evaluated the effects of heat-killed Ligilactobacillus salivarius FFIG56 (hk-FFIG56) on the Toll-like receptor (TLR)-4-mediated innate immune response in bovine mammary epithelial cells (BMECs) in vitro. BMECs were pre-stimulated with hk-FFIG56 and subsequently challenged with Escherichia coli-derived lipopolysaccharide (LPS). Transcriptomic analyses revealed that, compared with cells stimulated with LPS alone, hk-FFIG56-pretreated BMECs exhibited 743 upregulated and 881 downregulated genes, indicating substantial changes in the LPS-induced transcriptional response. Among the inflammatory mediators evaluated, hk-FFIG56 pretreatment significantly reduced LPS-induced interleukin-6 (IL-6) and interleukin-8 (IL-8) expression at 12 h, as well as CCL2 expression at 6 and 12 h, compared with cells challenged with LPS alone at the corresponding time points. Differential regulation of the negative regulators of TLR signaling, IRAK-M, Tollip, and Mitogen-activated protein kinase phosphatase 1 (MKP-1), was also detected in BMECs pretreated with hk-FFIG56. In addition, hk-FFIG56 pretreatment prevented LPS-induced reduction in IκBα protein levels, whereas no significant changes were detected in the p-ERK/ERK ratio at the evaluated time points. Furthermore, hk-FFIG56 pretreatment attenuated the LPS-induced reduction in CSN3 (κ-casein) mRNA expression in BMECs. Overall, these results suggest that hk-FFIG56 modulates LPS-induced inflammatory responses and CSN3 mRNA expression in BMECs, supporting its potential as a postimmunobiotic candidate for further investigation in the context of bovine mastitis prevention.