Diversification of memory B cells drives the continuous adaptation of secretory antibodies to gut microbiota.

Secretory immunoglobulin A (SIgA) shields the gut epithelium from luminal antigens and contributes to host-microbe symbiosis. However, how antibody responses are regulated to achieve sustained host-microbe interactions is unknown. We found that mice and humans exhibited longitudinal persistence of clonally related B cells in the IgA repertoire despite major changes in the microbiota during antibiotic treatment or infection. Memory B cells recirculated between inductive compartments and were clonally related to plasma cells in gut and mammary glands. Our findings suggest that continuous diversification of memory B cells constitutes a central process for establishing symbiotic host-microbe interactions and offer an explanation of how maternal antibodies are optimized throughout life to protect the newborn.

Metataxonomic analysis and host proteome response in dairy cows with high and low somatic cell count: a quarter level investigation.

Host response to invasive microbes in the bovine udder has an important role on the animal health and is essential to the dairy industry to ensure production of high-quality milk and reduce the mastitis incidence. To better understand the biology behind these host-microbiome interactions, we investigated the somatic cell proteomes at quarter level for four cows (collected before and after milking) using a shotgun proteomics approach. Simultaneously, we identified the quarter microbiota by amplicon sequencing to detect presence of mastitis pathogens or other commensal taxa. In total, 32 quarter milk samples were analyzed divided in two groups depending on the somatic cell count (SCC). The high SCC group (>100,000 cell/mL) included 10 samples and significant different proteome profiles were detected. Differential abundance analysis uncovers a specific expression pattern in high SCC samples revealing pathways involved in immune responses such as inflammation, activation of the complement system, migration of immune cells, and tight junctions. Interestingly, different proteome profiles were also identified in quarter samples containing one of the two mastitis pathogens, Staphylococcus aureus and Streptococcus uberis, indicating a different response of the host depending on the pathogen. Weighted correlation network analysis identified three modules of co-expressed proteins which were correlated with the SCC in the quarters. These modules contained proteins assigned to different aspects of the immune response, but also amino sugar and nucleotide sugar metabolism, and biosynthesis of amino acids. The results of this study provide deeper insights on how the proteome expression changes at quarter level in naturally infected cows and pinpoint potential interactions and important biological functions during host-microbe interaction.

Diverse myeloid cells are recruited to the developing and inflamed mammary gland.

The immune system plays fundamental roles in the mammary gland, shaping developmental processes and controlling inflammation during infection and cancer.Here, we reveal unanticipated heterogeneity in the myeloid cell compartment duringdevelopment of virgin, pregnant, lactating and involuting mouse mammary glands,and in milk. We investigate the functional consequences of individual and compoundchemokine receptor deficiency on cell recruitment. Diverse myeloid cell recruitmentwas also shown in models of sterile inflammation and bacterial infection.Strikingly, we have shown that inflammation and infection can alter the abundanceof terminal end buds, a key developmental structure, within the pubertal mammarygland. This previously unknown effect of inflammatory burden during puberty couldhave important implications for understanding pubertal development.

Protective effects of chlorogenic acid on inflammatory responses induced by Staphylococcus aureus and milk protein synthesis in bovine mammary epithelial cells.

Staphylococcus aureus (S. aureus) is a major mastitis-causing pathogen in dairy cows. Dairy cows with mastitis suffer from a decrease in milk yield and protein content. Chlorogenic acid (CGA) is a natural product with anti-inflammatory effects. In this study, we examined the function and mechanism of CGA with regard to its anti-inflammatory effects and evaluated its protective function in milk protein synthesis in bovine mammary epithelial cells (BMECs). BMECs were cultured with and without infection by S. aureus and CGA, and extracellular inflammatory cytokines and amino acids in the medium and milk proteins were determined by ELISA. The function of IL-10RA in anti-inflammatory processes and of SF-1 in milk protein synthesis was assessed by gene silencing. The activity of mTORC1, NF-κB, and STAT5 was examined by western blot. S. aureus caused intracellular infection and upregulated TNF-α, IL-1ß, IL-6, and IL-8, whereas uptake of amino acids and milk protein synthesis were suppressed. CGA mitigated the S. aureus-induced inflammatory response and milk protein synthesis in vitro and in vivo. CGA alleviated S. aureus-induced inhibition of mTORC1 and STAT5 and upregulated IL-10 and IL-10RA. In addition, SF-1 was predicted to be a transcription factor of the milk protein-encoding genes α-LA, ß-LG, and CSN2. S. aureus downregulated SF-1 and CGA reversed the decline in milk protein synthesis due to SF-1 knockdown. Thus, CGA mitigates the inflammatory response that is induced by S. aureus and protects the uptake of amino acids and milk protein synthesis in BMECs.

CD5 blockade enhances ex vivo CD8+ T cell activation and tumour cell cytotoxicity.

CD5 is expressed on T cells and a subset of B cells (B1a). It can attenuate TCR signalling and impair CTL activation and is a therapeutic targetable tumour antigen expressed on leukemic T and B cells. However, the potential therapeutic effect of functionally blocking CD5 to increase T cell anti-tumour activity against tumours (including solid tumours) has not been explored. CD5 knockout mice show increased anti-tumour immunity: reducing CD5 on CTLs may be therapeutically beneficial to enhance the anti-tumour response. Here, we show that ex vivo administration of a function-blocking anti-CD5 MAb to primary mouse CTLs of both tumour-naïve mice and mice bearing murine 4T1 breast tumour homografts enhanced their capacity to respond to activation by treatment with anti-CD3/anti-CD28 MAbs or 4T1 tumour cell lysates. Furthermore, it enhanced TCR signalling (ERK activation) and increased markers of T cell activation, including proliferation, CD69 levels, IFN-γ production, apoptosis and Fas receptor and Fas ligand levels. Finally, CD5 function-blocking MAb treatment enhanced the capacity of CD8+ T cells to kill 4T1-mouse tumour cells in an ex vivo assay. These data support the potential of blockade of CD5 function to enhance T cell-mediated anti-tumour immunity.

Species-Specific Humoral Immune Responses in Sheep and Goats upon Small Ruminant Lentivirus Infections Inversely Correlate with Protection against Virus Replication and Pathological Lesions.

Maedi-Visna-like genotype A strains and Caprine arthritis encephaltis-like genotype B strains are small ruminant lentiviruses (SRLV) which, for incompletely understood reasons, appear to be more virulent in sheep and goats, respectively. A 9-month in vivo infection experiment using Belgian genotype A and B SRLV strains showed that almost all homologous (genotype A in sheep; genotype B in goats) and heterologous (genotype A in goats; genotype B in sheep) intratracheal inoculations resulted in productive infection. No differences in viremia and time to seroconversion were observed between homologous and heterologous infections. Higher viral loads and more severe lesions in the mammary gland and lung were however detected at 9 months post homologous compared to heterologous infection which coincided with strongly increased IFN-γ mRNA expression levels upon homologous infection. Pepscan analysis revealed a strong antibody response against immune-dominant regions of the capsid and surface proteins upon homologous infection, which was absent after heterologous infection. These results inversely correlated with protection against virus replication in target organs and observed histopathological lesions, and thus require an in-depth evaluation of a potential role of antibody dependent enhancement in SRLV infection. Finally, no horizontal intra- and cross-species SRLV transmission to contact animals was detected.

Transcriptome Profiling of m6A mRNA Modification in Bovine Mammary Epithelial Cells Treated with Escherichia coli.

Mastitis is a common disease in dairy cows that is mostly caused by E. coli, and it brings massive losses to the dairy industry. N6-Methyladenosine (m6A), a methylation at the N6 position of RNA adenine, is a type of modification strongly associated with many diseases. However, the role of m6A in mastitis has not been investigated. In this study, we used MeRIP-seq to sequence the RNA of bovine mammary epithelial cells treated with inactivated E. coli for 24 h. In this in vitro infection model, there were 16,691 m6A peaks within 7066 mRNA transcripts in the Con group and 10,029 peaks within 4891 transcripts in the E. coli group. Compared with the Con group, 474 mRNAs were hypermethylated and 2101 mRNAs were hypomethylated in the E. coli group. Biological function analyses revealed differential m6A-modified genes mainly enriched in the MAPK, NF-κB, and TGF-ß signaling pathways. In order to explore the relationship between m6A and mRNA expression, combined MeRIP-seq and mRNA-seq analyses revealed 212 genes with concomitant changes in the mRNA expression and m6A modification. This study is the first to present a map of RNA m6A modification in mastitis treated with E. coli, providing a basis for future research.

Sodium butyrate attenuated iE-DAP induced inflammatory response in the mammary glands of dairy goats fed high-concentrate diet.

BACKGROUND: Long-term high-concentrate (HC) diet feeding increased bacterial endotoxins, which translocated into the mammary glands of dairy goats and induced inflammatory response. γ-d-Glutamyl-meso-diaminopimelic acid (iE-DAP), bacterial peptidoglycan component, triggered inflammatory response through activating nucleotide oligomerization domain protein 1 (NOD1) signaling pathway. While dietary supplemented with sodium butyrate (SB) relieved inflammatory response and improved animal health and production. To investigate the effects and the mechanisms of action of SB on the inflammatory response in the mammary glands of dairy goats fed HC diet, 12 Saanen dairy goats were randomly assigned into HC group and SB regulated (BHC) group. RESULTS: The results showed that SB supplementation attenuated ruminal pH decrease caused by HC diet in dairy goats resulting in a decrease of proinflammatory cytokines and iE-DAP plasma concentration and the mRNA expression of NOD1 and other inflammation-related genes. The protein levels of NOD1, NF-κB p65 and NF-κB pp65 were decreased by the SB supplementation. The expression of histone deacetylase 3 (HDAC3) was also inhibited by the SB supplementation. Meanwhile, the chromatin compaction ratios and DNA methylation levels of NOD1 and receptor-interacting protein 2 (RIP2) of BHC group were upregulated. CONCLUSION: Collectively, the SB supplementation mitigated the inflammatory response in the mammary glands of dairy goats during HC-induced subacute ruminal acidosis (SARA) by inhibiting the activation of the NOD1/NF-κB signaling pathway through the decrease of the iE-DAP concentration in the rumen fluid and plasma and HDAC3 expression. DNA methylation and chromatin remodeling also contributed to the anti-inflammatory effect of SB. © 2020 Society of Chemical Industry.

Immunosuppression in Cows following Intramammary Infusion of Mycoplasma bovis.

Mycoplasma bovis is a destructive pathogen that causes large economic losses in rearing cattle for beef and dairy worldwide. M. bovis causes suppression of and evades the host immune response; however, the mechanisms of host immune function involved in M. bovis mastitis have not been elucidated. The purpose of this study was to elucidate the characteristics of the bovine immune response to mycoplasmal mastitis. We evaluated the responsiveness of the bovine mammary gland following infusion of M. bovis Somatic cell counts and bacterial counts in milk from the infected quarter were increased. However, the proliferation of peripheral blood mononuclear cells (blood MNCs) and mononuclear cells isolated from M. bovis-stimulated mammary lymph nodes (lymph node MNCs) did not differ from that in the unstimulated cells. Transcriptome analysis revealed that the mRNA levels of innate immune system-related genes in blood MNCs, complement factor D (CFD), ficolin 1 (FCN1), and tumor necrosis factor superfamily member 13 (TNFSF13) decreased following intramammary infusion of M. bovis The mRNA levels of immune exhaustion-related genes, programmed cell death 1 (PD-1), programmed cell death-ligand 1 (PD-L1), lymphocyte activation gene 3 (LAG3), and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) of milk mononuclear cells (milk MNCs) in the infected quarter were increased compared with those before infusion. Increase in immune exhaustion-related gene expression and decrease in innate immune response-related genes of MNCs in quarters from cows were newly characterized by M. bovis-induced mastitis. These results suggested that M. bovis-induced mastitis affected the immune function of bovine MNCs, which is associated with prolonged duration of infection with M. bovis.

In utero estrogenic endocrine disruption alters the stroma to increase extracellular matrix density and mammary gland stiffness.

BACKGROUND: In utero endocrine disruption is linked to increased risk of breast cancer later in life. Despite numerous studies establishing this linkage, the long-term molecular changes that predispose mammary cells to carcinogenic transformation are unknown. Herein, we investigated how endocrine disrupting compounds (EDCs) drive changes within the stroma that can contribute to breast cancer susceptibility. METHODS: We utilized bisphenol A (BPA) as a model of estrogenic endocrine disruption to analyze the long-term consequences in the stroma. Deregulated genes were identified by RNA-seq transcriptional profiling of adult primary fibroblasts, isolated from female mice exposed to in utero BPA. Collagen staining, collagen imaging techniques, and permeability assays were used to characterize changes to the extracellular matrix. Finally, gland stiffness tests were performed on exposed and control mammary glands. RESULTS: We identified significant transcriptional deregulation of adult fibroblasts exposed to in utero BPA. Deregulated genes were associated with cancer pathways and specifically extracellular matrix composition. Multiple collagen genes were more highly expressed in the BPA-exposed fibroblasts resulting in increased collagen deposition in the adult mammary gland. This transcriptional reprogramming of BPA-exposed fibroblasts generates a less permeable extracellular matrix and a stiffer mammary gland. These phenotypes were only observed in adult 12-week-old, but not 4-week-old, mice. Additionally, diethylstilbestrol, known to increase breast cancer risk in humans, also increases gland stiffness similar to BPA, while bisphenol S does not. CONCLUSIONS: As breast stiffness, extracellular matrix density, and collagen deposition have been directly linked to breast cancer risk, these data mechanistically connect EDC exposures to molecular alterations associated with increased disease susceptibility. These alterations develop over time and thus contribute to cancer risk in adulthood.

Type 3 immunity: a perspective for the defense of the mammary gland against infections.

Type 3 immunity encompasses innate and adaptive immune responses mediated by cells that produce the signature cytokines IL-17A and IL-17F. This class of effector immunity is particularly adept at controlling infections by pyogenic extracellular bacteria at epithelial barriers. Since mastitis results from infections by bacteria such as streptococci, staphylococci and coliform bacteria that cause neutrophilic inflammation, type 3 immunity can be expected to be mobilized at the mammary gland. In effect, the main defenses of this organ are provided by epithelial cells and neutrophils, which are the main terminal effectors of type 3 immunity. In addition to theoretical grounds, there is observational and experimental evidence that supports a role for type 3 immunity in the mammary gland, such as the production of IL-17A, IL-17F, and IL-22 in milk and mammary tissue during infection, although their respective sources remain to be fully identified. Moreover, mouse mastitis models have shown a positive effect of IL-17A on the course of mastitis. A lot remains to be uncovered before we can safely harness type 3 immunity to reinforce mammary gland defenses through innate immune training or vaccination. However, this is a promising way to find new means of improving mammary gland defenses against infection.

Proteomic analysis of bovine mammary epithelial cells after in vitro incubation with S. agalactiae: potential biomarkers.

Streptococcus agalactiae is one of the causative agents of subclinical mastitis, a common disease of dairy cows that causes great economic losses in the industry worldwide. It is thought that pathology is mainly due to inflammatory damage of bovine mammary epithelial cells (bMECs); however, the mechanism by which S. agalactiae damages the bMECs is not clear. The aim of this study was to evaluate the inflammatory effects of S. agalactiae on bMECs and the resulting changes in protein profiles. The bMECs were incubated with S. agalactiae for different times and assayed for cell viability by MTT assay, apoptosis by annexin V and propidium iodide dual staining, and morphological and ultrastructural changes by scanning and transmission electron microscopy. Quantitative real-time PCR was used to determine the effect of S. agalactiae on expression of mRNA of inflammatory factors in bMECs and protein levels were quantitated by liquid chromatography/mass spectrometry. Exposure to S. agalactiae significantly decreased the cell viability and triggered apoptosis, as well as up-regulating TNF-α, IL-1ß and IL-6 mRNA, and inhibiting IL-8 expression. S. agalactiae also induced morphological and ultrastructural changes. Furthermore, we identified 325 up-regulated and 704 down-regulated proteins in the treated vs control group. All significant differentially expressed proteins (DSEPs) were classified into three major areas by function: biological processes, cellular components and molecular functions. These differentially expressed proteins included enzymes and proteins associated with various metabolic processes and cellular immunity. Pathway enrichment analysis showed that eight down-regulated signaling pathways were significantly enriched. Exposure to even subclinical levels of S. agalactiae can lead to inflammation and bMEC damage. Our data suggest some possible molecular mechanisms for the harmful effects of subclinical mastitis in dairy cows.

Immune response in nonspecific mastitis: What can it tell us?

We analyzed a large number of immune response parameters from quarter milk samples with distinct bacteriological and quarter somatic cell count (qSCC) statuses. Furthermore, we sought to explore and identify displayed immune response patterns in milk samples from mammary glands with nonspecific mastitis. Thus, 92 quarter milk samples from 28 cows were stratified into 4 groups, as follows: (1) 49 culture-negative control quarters with a low qSCC (<1 × 105 cells/mL) from 19 dairy cows (so-called healthy quarters); (2) 15 culture-negative quarters with high qSCC (>2 × 105 cells/mL; so-called quarters with nonspecific mastitis) from 10 dairy cows; (3) 8 culture-positive quarters with low qSCC (noninflammatory quarters with low qSCC) from 5 dairy cows; and (4) 20 culture-positive quarters with high qSCC (so-called truly infected quarters) from 8 dairy cows. Using flow cytometry, we evaluated the percentage of milk neutrophils and their viability, intracellular reactive oxygen species production, phagocytosis, and the expression of CD62L, CD11b, and CD44 for each of the 4 quarter strata. Furthermore, the percentage of monocyte/macrophages, B cells, and T lymphocyte subsets were evaluated by flow cytometry. Milk samples from bacteriologically negative quarters (both with a low and elevated qSCC) had a lower qSCC than those with bacteriologically positive outcomes (both with a low and elevated qSCC). As expected, the healthy quarters showed the lowest percentage of neutrophils and also showed a higher percentage of milk monocytes/macrophages and lower percentage of T lymphocytes than truly infected quarters. The most prominent result of the present study is that quarters with nonspecific mastitis showed the highest percentage of milk CD4+ T lymphocytes. The healthy quarters had a lower percentage of apoptotic neutrophils than noninflammatory and truly infected quarters, although it did not differ from those from the quarters with nonspecific mastitis. Our study supports the role of differential cell counting in the diagnosis of mastitis, as the milk leukocyte populations markedly fluctuate under healthy and inflammatory conditions. Furthermore, an increase in milk CD4+ T cells was associated with nonspecific mastitis, suggesting an increase in this leukocyte subpopulation is correlated with low bacterial shedding. Our study allows us to go further in our understanding of mammary gland immunity, providing further insights on potential protective mammary gland immunity, which we hypothesize can open new avenues for the development of novel targets that can promote bovine udder health.

Sodium butyrate reduces bovine mammary epithelial cell inflammatory responses induced by exogenous lipopolysaccharide, by inactivating NF-κB signaling.

Exogenous molecules derived from catabolic states (e.g., fatty acids, ß-hydroxybutyrate) during periods of stress such as the periparturient period or pathogen challenges [e.g., lipopolysaccharide (LPS)] can trigger an inflammatory response in tissues such as the liver and the mammary gland. Butyrate is one of the major short-chain fatty acids produced in the rumen, and work with non-ruminants has demonstrated that it can alter inflammatory processes. The primary objective of this study was to explore the preventive effect of sodium butyrate (SB) on LPS-induced inflammation in bovine mammary epithelial cells along with underlying molecular mechanisms. Immortalized bovine mammary epithelial cells (MAC-T) were treated with SB (0.1, 0.25, 0.5, 1, 2, or 5 mM) or with the histone deacetylase inhibitor trichostatin A (TSA; 6.25, 12.5, 25, or 50 nM) for 18 h, followed by a challenge with 1 µg/mL LPS for an additional 6 h. Pretreatment with SB prevented increase in apoptosis of LPS-challenged MAC-T cells in a dose-dependent manner. The LPS treatment upregulated mRNA abundance of tumor necrosis factor α (TNFA), interleukin-6 (IL6), and interleukin-1B (IL1B), whereas inhibition of histone deacetylase with TSA dampened this effect. More importantly, SB had clear dose-dependent effects on the inflammatory response by preventing upregulation of TNFA, IL6, and IL1B. Furthermore, pretreatment with TSA or SB attenuated the downregulation of histone H3 acetylation protein abundance induced by LPS. The greater ratio of p-IκB α/IκB α and p-p65/p65 protein abundance and the increase in nuclear localization of NF-κB p65 protein in response to LPS were attenuated by pretreatment with SB. Overall, the data indicated that exogenous SB alleviates mammary cell pro-inflammatory responses partly through post-translational mechanisms that diminish NF-κB signaling. Thus, the cytoprotective effect of SB against an inflammatory challenge might represent a preventive tool to help the mammary gland against pathogens such as those causing mastitis.

Staphylococcal enterotoxin M induced inflammation and impairment of bovine mammary epithelial cells.

Staphylococcus aureus is one of the major etiological pathogens of bovine mastitis. Its invasion into mammary epithelial cells has been proven to be a key event in the pathogenesis of mastitis. However, the specific pathogenic factors have not been clearly identified. Staphylococcus aureus often triggers infections by releasing virulence factor. Recent several studies reported that staphylococcal enterotoxin M was one of the most frequently found enterotoxin genes associated with bovine mastitis. Thus, the effect of staphylococcal enterotoxin M on inflammation and damage of the bovine mammary epithelial bovine mammary gland epithelial cell line (MAC-T) cells with 48 h treatment was explored in the present study. First, staphylococcal enterotoxin M protein was purified by a Ni-NTA spin column (GE Life Science, Westborough, MA). The levels of tumor necrosis factor-α, IL-6, and monocyte chemoattractant protein 1 (MCP-1) secretion were measured with the corresponding ELISA kits (R&D Systems, Abingdon, UK). Second, cell viability was assessed with a Cell Counting Kit-8 (Bioswamp, Wuhan, China) and the apoptotic percentage of cells was determined by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI; Beyotime, Nanjing, China) staining. Third, ATP concentration, reactive oxygen species (ROS) generation and lactate dehydrogenase (LDH) release were assayed with commercial kits, then mitochondrial membrane potential (ΔΨm) was estimated using fluorescent probe JC-1 (Beyotime). Finally, the production intercellular cell adhesion molecule-1 (ICAM-1), microtubule-associated protein 1A/1B-light chain 3 I/II (LC3 I/II), p62 (Proteintech, Rosemont, IL), and phosphorylation of IκBα, caspase 3, and mammalian target of rapamycin were detected by Western blot. The results showed that staphylococcal enterotoxin M induced inflammation of epithelial cells (upregulating tumor necrosis factor-α, IL-6, MCP-1, and ICAM-1 production) and activated NF-κB (promoting phosphorylation of IκBα). Furthermore, staphylococcal enterotoxin M impaired MAC-T cells via cell necrosis (enhancing LDH release), apoptosis (annexin V-FITC/PI stain, exacerbating oxidative stress, decreasing ΔΨm and intracellular ATP concentration, and activating caspase 3), but independent of autophagy (nonsignificantly increasing LC3-II, decreasing p62 expression, and activating mammalian target of rapamycin). Thereby, staphylococcal enterotoxin M induced the inflammatory property of bovine mammary epithelial cells by boosting cytokine, chemokine, and adhesion molecule production. Furthermore, it caused epithelial cell dysfunction via depressing cell viability and initiating cell necrosis and apoptosis. Because epithelial cells played important roles in orchestrating the inflammatory response and protecting bovine mammary tissue from mastitis, our results indicated that staphylococcal enterotoxin M may be associated with mastitis.

Correlation of the Levels of microRNA with Cell Composition of Axillary Lymph Node in Wistar Rats with Breast Cancer.

We studied the levels microRNA (miR-21, miR-221, miR-222, and miR-429) in blood serum, thoracic duct lymph, and breast cancer tissue, as well as the cell composition of the axillary lymph node in Wistar female rats with chemically induced breast cancer. The levels of miR-221 and miR-429 in the tumor tissue and in the lymph correlated with the decrease in lymphocyte number in the medullary cords of the axillary lymph nodes.

Correlation Analysis of Morphological Changes of Structural and Functional Areas of Mesenteric Lymph Nodes with Cytokines of Lymph of Thoracic Lymphatic Duct in Experimental Breast Cancer.

We performed a correlation analysis of the morphometric parameters of mesenteric lymph nodes and cytokine content in the lymph of thoracic duct in rats with chemically induced breast cancer. The study showed that activity of the local immune response in the lymph nodes in breast cancer is aimed at antitumor protection. In breast cancer, the area of the paracortical zone remained at the level of the intact group, while the area of lymphoid nodules with germinative centers and the area of medullary substance increased; the number of macrophages in the thymus-dependent zone and zone responsible for humoral immunity also increased. The following positive correlations were revealed: in germinative centers and medullary substance, number of mitotic cells correlated with cytokine IL-5 content and the number of medium lymphocytes correlated with the content of chemokine MIP-1α; in the germinative centers, the number of immunoblasts correlated with the level of cytokine GRO/KC, in the paracortical zone, the number of macrophages correlated with the level of chemokine MCP-1, the number of reticular cells correlated with IL-6 and M-CSF content; in medullary substance, the number of small lymphocytes and mature cells plasma cells (their content was reduced) correlated with the level of chemokine GRO/KC, which can be caused by their migration from the lymph node.

Heat stress negatively affects the transcriptome related to overall metabolism and milk protein synthesis in mammary tissue of midlactating dairy cows.

Inadequate dry matter intake only partially accounts for the decrease in milk protein synthesis during heat stress (HS) in dairy cows. Our hypothesis is that reduced milk protein synthesis during HS in dairy cows is also caused by biological changes within the mammary gland. The objective of this study was to assess the hypothesis via RNA-Seq analysis of mammary tissue. Herein, four dairy cows were used in a crossover design where HS was induced for 9 days in environmental chambers. There was a 30-day washout between periods. Mammary tissue was collected via biopsy at the end of each environmental period (HS or pair-fed and thermal neutral) for transcriptomic analysis. RNA-Seq analysis revealed HS affected >2,777 genes (false discovery rate-adjusted P value < 0.05) in mammary tissue. Expression of main milk protein-encoding genes and several key genes related to regulation of protein synthesis and amino acid and glucose transport were downregulated by HS. Bioinformatics analysis revealed an overall decrease of mammary tissue metabolic activity by HS (especially carbohydrate and lipid metabolism) and an increase in immune activation and inflammation. Network analysis revealed a major role of TNF, IFNG, S100A8, S100A9, and IGF-1 in inducing/controlling the inflammatory response, with a central role of NF-κB in the process of immunoactivation. The same analysis indicated an overall inhibition of PPARγ. Collectively, these data suggest HS directly controls milk protein synthesis via reducing the transcription of metabolic-related genes and increasing inflammation-related genes.

DNA methylation analysis of porcine mammary epithelial cells reveals differentially methylated loci associated with immune response against Escherichia coli challenge.

BACKGROUND: Epigenetic changes such as cytosine (CpG) DNA methylations regulate gene expression patterns in response to environmental cues including infections. Microbial infections induce DNA methylations that play a potential role in modulating host-immune response. In the present study, we sought to determine DNA methylation changes induced by the mastitis causing Escherichia coli (E. coli) in porcine mammary epithelial cells (PMEC). Two time points (3 h and 24 h) were selected based on specific transcriptomic changes during the early and late immune responses, respectively. RESULTS: DNA methylation analysis revealed 561 and 898 significant (P < 0.01) differentially methylated CpG sites at 3 h and 24 h after E. coli challenge in PMEC respectively. These CpG sites mapped to genes that have functional roles in innate and adaptive immune responses. Significantly, hypomethylated CpG sites were found in the promoter regions of immune response genes such as SDF4, SRXN1, CSF1 and CXCL14. The quantitative transcript estimation indicated higher expression associated with the DNA CpG methylation observed in these immune response genes. Further, E. coli challenge significantly reduced the expression levels of DNMT3a, a subtype of de novo DNA methylation enzyme, in PMEC indicating the probable reason for the hypomethylation observed in the immune response genes. CONCLUSIONS: Our study revealed E. coli infection induced DNA methylation loci in the porcine genome. The differentially methylated CpGs were identified in the regulatory regions of genes that play important role in immune response. These results will help to understand epigenetic mechanisms for immune regulation during coliform mastitis in pigs.

Effects of chronic Staphylococcus aureus infection on immunological parameters and functionality of macrophages isolated from bovine mammary secretions.

The aim of this study was to characterize the effects of chronic S. aureus intramammary infection (IMI) on local innate and adaptive immune response during active involution. Cows in late lactation that were either uninfected or with chronic naturally acquired S. aureus IMI were included in this study. The levels of interleukin (IL)-1ß, IL-6 and IL-4 were significantly higher in mammary secretions of S. aureus-infected quarters compared with uninfected at d 7, 14 and 21 of involution. Lactoferrin (Lf), total IgG and S. aureus specific IgG1 levels were significantly lower in mammary secretions of infected quarters compared with uninfected during the first three weeks of involution. The amount of intracellular reactive oxygen species (ROS) produced per macrophage, was significantly higher in mammary secretions of infected quarters compared with uninfected at d 14 post drying off. Nitrite production was significantly higher in phagocytes from infected mammary secretions compared with uninfected at d 7 and 14 post drying off. Chronic S. aureus IMI altered normal secretion composition during bovine mammary gland involution. The high IL-1ß and IL-6 levels and increased functionality of macrophages in mammary secretions of infected quarters could be a result of the chronic inflammatory environment triggered by the presence of viable bacteria in mammary tissue. The lower levels of total and S. aureus specific antibodies and other immune factors in mammary secretion during this period may reduce the natural defense potential of the gland contributing to S. aureus persistence.