The role of interleukin-10 receptor alpha (IL10Rα) in Mycobacterium avium subsp. paratuberculosis infection of a mammary epithelial cell line.

BACKGROUND: Johne's disease is a chronic wasting disease caused by the bacterium Mycobacterium avium subspecies paratuberculosis (MAP). Johne's disease is highly contagious and MAP infection in dairy cattle can eventually lead to death. With no available treatment for Johne's disease, genetic selection and improvements in management practices could help reduce its prevalence. In a previous study, the gene coding interleukin-10 receptor subunit alpha (IL10Rα) was associated with Johne's disease in dairy cattle. Our objective was to determine how IL10Rα affects the pathogenesis of MAP by examining the effect of a live MAP challenge on a mammary epithelial cell line (MAC-T) that had IL10Rα knocked out using CRISPR/cas9. The wild type and the IL10Rα knockout MAC-T cell lines were exposed to live MAP bacteria for 72 h. Thereafter, mRNA was extracted from infected and uninfected cells. Differentially expressed genes were compared between the wild type and the IL10Rα knockout cell lines. Gene ontology was performed based on the differentially expressed genes to determine which biological pathways were involved. RESULTS: Immune system processes pathways were targeted to determine the effect of IL10Rα on the response to MAP infection. There was a difference in immune response between the wild type and IL10Rα knockout MAC-T cell lines, and less difference in immune response between infected and not infected IL10Rα knockout MAC-T cells, indicating IL10Rα plays an important role in the progression of MAP infection. Additionally, these comparisons allowed us to identify other genes involved in inflammation-mediated chemokine and cytokine signalling, interleukin signalling and toll-like receptor pathways. CONCLUSIONS: Identifying differentially expressed genes in wild type and ILR10α knockout MAC-T cells infected with live MAP bacteria provided further evidence that IL10Rα contributes to mounting an immune response to MAP infection and allowed us to identify additional potential candidate genes involved in this process. We found there was a complex immune response during MAP infection that is controlled by many genes.

Evaluation of Immunomodulatory Effects of Fusarium Mycotoxins Using Bacterial Endotoxin-Stimulated Bovine Epithelial Cells and Macrophages in Co-Culture.

Mycotoxins are secondary metabolites produced by a variety of fungi that contaminate animal food and feeds and are capable of inducing a wide range of toxicities. Predictive in vitro models represent valuable substitutes for animal experiments to assess the toxicity of mycotoxins. The complexities of the interactions between epithelial and innate immune cells, vital for upholding barrier integrity and averting infections, remain inadequately understood. In the current study, a co-culture model of bovine epithelial cells (MAC-T) and macrophages (BoMac) was used to investigate the impact of exposure to Fusarium mycotoxins, namely deoxynivalenol (DON), zearalenone (ZEN), enniatin B (ENB), and beauvericin (BEA), on the inflammatory response elicited by the bacterial lipopolysaccharide (LPS) endotoxin. The MAC-T cells and BoMac were seeded on the apical side of a Transwell membrane and in the lower chamber, respectively, and mycotoxin exposure on the apical side of the membrane was carried out with the different mycotoxins (LC20; concentrations that elicited 20% cytotoxicity) for 48 h followed by an LPS immunity challenge for 24 h. The culture supernatants were collected from the basolateral compartment and these samples were submitted for cytokine/chemokine multiplex analysis. RNA-Seq analysis was performed using total RNA extracted from the MAC-T cells to acquire a more detailed insight into their cellular functions. The multiplex analysis indicated that IFN-γ, IL-1α, IL-8, and MCP-1 were significantly induced post-DON treatment when compared to control cells, and levels of IL-1α and IL-8 were enhanced significantly in all mycotoxin-treated groups post-LPS challenge. Analysis of the sequencing results showed that there were 341, 357, and 318 differentially expressed MAC-T cell genes that were up-regulated in the DON, ENB, and BEA groups, respectively. Gene ontology and pathway analysis revealed that these DEGs were significantly enriched in various biological processes and pathways related to inflammation, apoptosis signaling, and Wnt signaling. These results provide a comprehensive analysis of the co-culture cytokine/chemokine production and MAC-T cells' gene expression profiles elicited by Fusarium mycotoxins, which further contributes to the understanding of early endotoxemia post-mycotoxin exposure.

Assessing Larval Zebrafish Survival and Gene Expression Following Sodium Butyrate Exposure and Subsequent Lethal Bacterial Lipopolysaccharide (LPS) Endotoxin Challenge.

As aquaculture production continues to grow, producers are looking for more sustainable methods to promote growth and increase fish health and survival. Butyrate is a short-chain fatty acid (SCFA) with considerable benefits to gut health, and in recent years, butyrate has been commonly used as an alternative to antimicrobials in livestock production. In this study, we aimed to assess the protective effects of sodium butyrate (NaB) on larval zebrafish subjected to a lethal Pseudomonas aeruginosa lipopolysaccharide (LPS) endotoxin challenge and to elucidate potential protective mechanisms of action. Larval zebrafish were pre-treated with 0, 3000, or 6000 µM NaB for 24 h at 72 h post-fertilization (hpf), then immune challenged for 24 h with 60 µg/mL of LPS at 96 hpf. Our results demonstrate that larval zebrafish pre-treated with 6000 µM of NaB prior to lethal LPS challenge experienced significantly increased survival by 40%, and this same level of NaB significantly down-regulated the expression of pro-inflammatory Tumor Necrosis Factor α (TNF-alpha). Findings from this study are consistent with the beneficial effects of NaB on other vertebrate species and support the potential use of NaB in aquaculture.

N-Acetylcysteine and Its Immunomodulatory Properties in Humans and Domesticated Animals.

N-acetylcysteine (NAC), an acetylated derivative of the amino acid L-cysteine, has been widely used as a mucolytic agent and antidote for acetaminophen overdose since the 1960s and the 1980s, respectively. NAC possesses antioxidant, cytoprotective, anti-inflammatory, antimicrobial, and mucolytic properties, making it a promising therapeutic agent for a wide range of diseases in both humans and domesticated animals. Oxidative stress and inflammation play a major role in the onset and progression of all these diseases. NAC's primary role is to replenish glutathione (GSH) stores, the master antioxidant in all tissues; however, it can also reduce levels of pro-inflammatory tumor necrosis factor-alpha (TNF-∝) and interleukins (IL-6 and IL-1ß), inhibit the formation of microbial biofilms and destroy biofilms, and break down disulfide bonds between mucin molecules. Many experimental studies have been conducted on the use of NAC to address a wide range of pathological conditions; however, its effectiveness in clinical trials remains limited and studies often have conflicting results. The purpose of this review is to provide a concise overview of promising NAC usages for the treatment of different human and domestic animal disorders.

Impact of Mycobacterium avium subsp. paratuberculosis infection on bovine IL10RA knockout mammary epithelial (MAC-T) cells.

Mycobacterium avium subsp. Paratuberculosis (MAP) is an intracellular pathogen that causes Johne's disease (JD) in cattle and other ruminants. IL10RA encodes the alpha chain of the IL-10 receptor that binds the cytokine IL-10, and is one of the candidate genes that have been found to be associated with JD infection status. In this study, a previously developed IL10RA knockout (IL10RAKO) bovine mammary epithelial (MAC-T) cell line and wild-type (WT) MAC-T cells were infected with live MAP for 72 h to identify potential immunoregulatory miRNAs, inflammatory genes, and cytokines/chemokines impacted by MAP infection in the presence/absence of IL10RA. Cytokine and chemokine concentrations in culture supernatants were measured by multiplexing immunoassay. Total RNA was extracted from the MAC-T cells, and qPCR was performed to determine the expression of inflammatory genes and selected bovine miRNAs. Results showed that the levels of TNF-α, IL-6, CXCL8, CXCL10, CCL2, and CCL3 were significantly induced in WT MAC-T cells and IL-10 was significantly inhibited post-MAP infection. However, IL10RAKO MAC-T cells had greater secretion of TNF-α, IL-6, IFN-γ, CCL3, CCL4, CXCL8, and CXCL10, and lower secretion of VEGF-α. Moreover, the expression of inflammatory genes (TNF-α, IL-1α, IL-6) was also more significantly induced in IL10RAKO cells than in WT MAC-T cells post-MAP-infection, and unlike the WT cells, anti-inflammatory cytokines IL-10 and SOCS3 and chemokines CCL2 were not significantly induced. In addition, the expression of miRNAs (miR133b, miR-92a, and miR-184) was increased in WT MAC-T cells post-MAP-infection; however, there was no significant induction of these miRNAs in the IL10RAKO cells, which suggests IL10 receptor is somehow involved in regulating the miRNA response to MAP infection. Target gene function analysis further suggests that miR-92a may be involved in interleukin signaling, and miR-133b and miR-184 may be involved in other signaling pathways. These findings support the involvement of IL10RA in the regulation of innate immune response to MAP.

Role of Toll-Like Receptor 4 in Mycobacterium avium subsp. paratuberculosis Infection of Bovine Mammary Epithelial (MAC-T) Cells In Vitro.

Toll-like receptor 4 (TLR4) encodes an innate immune cell pattern-recognition receptor implicated in the recognition of Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease in ruminants. Polymorphisms in TLR4 have been associated with susceptibility to MAP infection. In this study, a previously developed TLR4 knockout (TLR4KO) bovine mammary epithelial (MAC-T) cell line and wild-type MAC-T cells (WT) were infected with live MAP for 72 h to identify potential immunoregulatory miRNAs, inflammatory genes, and cytokines/chemokines impacted by MAP infection in the presence/absence of TLR4. Cytokines/chemokines production in culture supernatants was measured by multiplexing immunoassay. Total RNA was extracted from the remaining MAC-T cells, and quantitative PCR was performed to determine the expression of inflammatory genes and selected bovine miRNAs. Results showed that the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), CXCL8, CXCL10, CCL4, and CCL3 were significantly induced in WT MAC-T cells during MAP infection. However, TLR4KO MAC-T cells had greater secretion of CCL3, IL-6, vascular endothelial growth factor (VEGF-α), and TNF-α and decreased secretion of CXCL10 and CCL2. Moreover, the expression of inflammatory genes was induced in TLR4KO cells. The expression of miRNAs (miR133b, miR-92a, and miR-184) was increased in WT MAC-T cells post-MAP infection; however, there was no significant induction of these miRNAs in TLR4KO cells, which suggests they are involved in regulating the innate immune response to MAP infection. Target gene function analysis further suggests that miR-92a may be involved in TLR and interleukin signaling and miR-133b and miR-184 may be involved in other signaling pathways. These findings support the involvement of TLR4 in the regulation of innate immune response to MAP. IMPORTANCE Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent for paratuberculosis or Johne's disease (JD) in ruminants, a disease clinically very similar to Crohn's disease in humans. Polymorphisms in the bovine Toll-like receptor genes (TLR1, TLR2, and TLR4) have been shown to affect MAP recognition and host innate immune response and have been associated with increased susceptibility of cattle to paratuberculosis. Our results demonstrated that knocking out the TLR4 gene in bovine MAC-T cells enhanced inflammation in response to MAP. These findings show divergent roles for TLR4 in Escherichia coli lipopolysaccharide and mycobacterial infections, and this may have important consequences for the treatment of these inflammatory diseases and for genetic selection to improve disease resistance. It advances our understanding of the role of TLR4 in the context of MAP infection.

Editing of HSF-1 and Na/K-ATPase α1 subunit by CRISPR/Cas9 reduces thermal tolerance of bovine skin fibroblasts to heat shock in vitro.

A follow-up to our previous findings, the present study was planned to evaluate the role of Na/K-ATPase alpha1-subunit (ATP1A1) gene in heat shock tolerance. The primary fibroblast culture was established using ear pinna tissue samples of Sahiwal cattle (Bos indicus). The knockout cell lines of Na/K-ATP1A1 and HSF-1 (heat shock factor-1, as a positive control) genes were developed by CRISPR/Cas9 method and the gene-editing was confirmed by the genomic cleavage detection assay. The two knockout cell lines (ATP1A1 and HSF-1) and wild-type fibroblasts were exposed to heat shock at 42 °C in vitro and different cellular parameters viz., apoptosis, proliferation, mitochondrial membrane potential (ΔΨm), oxidative stress, along with expression pattern of heat-responsive genes were studied. The results showed that in vitro heat shock given to knockout fibroblast cells of both ATP1A1 and HSF-1 genes resulted in decreased cell viability, while increasing the apoptosis rate, membrane depolarization, and ROS levels. However, the overall impact was more in HSF-1 knockout cells as compared to ATP1A1 knockout cells. Taken together, these results indicated that the ATP1A1 gene plays a critical role as HSF-1 under heat stress and helps cells to cope with heat shock.

Traditional and emerging Fusarium mycotoxins disrupt homeostasis of bovine mammary cells by altering cell permeability and innate immune function.

High incidence of traditional and emerging Fusarium mycotoxins in cereal grains and silages can be a potential threat to feed safety and ruminants. Inadequate biodegradation of Fusarium mycotoxins by rumen microflora following ingestion of mycotoxin-contaminated feeds can lead to their circulatory transport to target tissues such as mammary gland. The bovine udder plays a pivotal role in maintaining milk yield and composition, thus, human health. However, toxic effects of Fusarium mycotoxins on bovine mammary gland are rarely studied. In this study, the bovine mammary epithelial cell line was used as an in-vitro model of bovine mammary epithelium to investigate effects of deoxynivalenol (DON), enniatin B (ENB) and beauvericin (BEA) on bovine mammary gland homeostasis. Results indicated that exposure to DON, ENB and BEA for 48 h significantly decreased cell viability in a concentration-dependent manner (P < 0.001). Exposure to DON at 0.39 µmol/L and BEA at 2.5 µmol/L for 48 h also decreased paracellular flux of FITC-40 kDa dextran (P < 0.05), whereas none of the mycotoxins affected transepithelial electrical resistance after 48 h exposure. The qPCR was performed for assessment of expression of gene coding tight junction (TJ) proteins, toll-like receptor 4 (TLR4) and cytokines after 4, 24 and 48 h of exposure. DON, ENB and BEA significantly upregulated the TJ protein zonula occludens-1, whereas markedly downregulated claudin 3 (P < 0.05). Exposure to DON at 1.35 µmol/L for 4 h significantly increased expression of occludin (P < 0.01). DON, ENB and BEA significant downregulated TLR4 (P < 0.05). In contrast, ENB markedly increased expression of cytokines interleukin-6 (IL-6) (P < 0.001), tumor necrosis factor α (TNF-a) (P < 0.05) and transforming growth factor-ß (TGF-ß) (P < 0.01). BEA significantly upregulated IL- 6 (P < 0.001) and TGF-ß (P = 0.01), but downregulated TNF-α (P < 0.001). These results suggest that DON, ENB and BEA can disrupt mammary gland homeostasis by inducing cell death as well as altering its paracellular permeability and expression of genes involved in innate immune function.

Expression Profile of miRNA from High, Middle, and Low Stress-Responding Sheep during Bacterial Endotoxin Challenge.

Animals respond to stress by activating a wide array of physiological and behavioral responses that are collectively referred to as the stress response. MicroRNAs (miRNAs) are small, noncoding RNAs that play key roles in the regulation of homeostasis. There are many reports demonstrating examples of stress-induced miRNA expression profiles. The aim of this study was to determine the circulatory miRNA profile of variable stress-responding lambs (n = 112) categorized based on their cortisol levels as high (HSR, 336.2 ± 27.9 nmol/L), middle (MSR, 147.3 ±9.5 nmol/L), and low (LSR, 32.1 ± 10.4 nmol/L) stress responders post-LPS challenge (400 ng/kg iv). Blood was collected from the jugular vein at 0 (T0) and 4 h (T4) post-LPS challenge, and miRNAs were isolated from four animals from each group. An array of 84 miRNAs and 6 individual miRNAs were evaluated using qPCR. Among 90 miRNAs, there were 48 differentially expressed (DE) miRNAs (log fold change (FC) > 2 < log FC) in the HSR group, 46 in the MSR group, and 49 in the LSR group compared with T0 (control) samples. In the HSR group, three miRNAs, miR-485-5p, miR-1193-5p, and miR-3957-5p were significantly (p < 0.05) upregulated, while seven miRNAs, miR-376b-3p, miR-376c-3p, miR-411b-5p, miR-376a-3p, miR-376b-3p, miR-376c-3p, and miR-381-3p, were downregulated (p < 0.05) as compared to the LSR and MSR groups. Functional analysis of DE miRNAs revealed their roles in Ras and MAPK signaling, cytokine signaling, the adaptive immune system, and transcription pathways in the HSR phenotype, implicating a hyper-induced acute-phase response. In contrast, in the LSR group, enriched pathways included glucagon signaling metabolic regulation, the transportation of amino acids and ions, and the integration of energy metabolism. Taken together, these results indicate variation in the acute-phase response to an immune stress challenge, and these miRNAs are implicated in regulating responses within cortisol-based phenotypes.

Comparative Assessment of Different Yeast Cell Wall-Based Mycotoxin Adsorbents Using a Model- and Bioassay-Based In Vitro Approach.

Frequently reported occurrences of deoxynivalenol (DON), beauvericin (BEA), and, to a lesser extent, ochratoxin A (OTA) and citrinin (CIT) in ruminant feed or feedstuff could represent a significant concern regarding feed safety, animal health, and productivity. Inclusion of yeast cell wall-based mycotoxin adsorbents in animal feeds has been a common strategy to mitigate adverse effects of mycotoxins. In the present study, an in vitro approach combining adsorption isotherm models and bioassays was designed to assess the efficacy of yeast cell wall (YCW), yeast cell wall extract (YCWE), and a postbiotic yeast cell wall-based blend (PYCW) products at the inclusion rate of 0.5% (w/v) (ratio of adsorbent mass to buffer solution volume). The Hill's adsorption isotherm model was found to best describe the adsorption processes of DON, BEA, and CIT. Calculated binding potential for YCW and YCWE using the Hill's model exhibited the same ranking for mycotoxin adsorption, indicating that BEA had the highest adsorption rate, followed by DON and CIT, which was the least adsorbed. PYCW had the highest binding potential for BEA compared with YCW and YCWE. In contrast, the Freundlich isotherm model presented a good fit for OTA adsorption by all adsorbents and CIT adsorption by PYCW. Results indicated that YCW was the most efficacious for sequestering OTA, whereas YCWE was the least efficacious. PYCW showed greater efficacy at adsorbing OTA than CIT. All adsorbents exhibited high adsorption efficacy for BEA, with an overall percentage average of bound mycotoxin exceeding 60%, whereas moderate efficacies for the other mycotoxins were observed (up to 37%). Differences in adsorbent efficacy of each adsorbent significantly varied according to experimental concentrations tested for each given mycotoxin (p < 0.05). The cell viability results from the bioassay using a bovine mammary epithelial cell line (MAC-T) indicated that all tested adsorbents could potentially mitigate mycotoxin-related damage to bovine mammary epithelium. Results from our studies suggested that all tested adsorbents had the capacity to adsorb selected mycotoxins in vitro, which could support their use to mitigate their effects in vivo.

Expression profile of different classes of proteases in milk derived somatic cells across different lactation stages of indigenous cows (Bos indicus) and riverine buffaloes (Bubalus bubalis).

Proteases play a significant role in milk and its products by affecting flavor, texture and longevity. The expression of endogenous proteases varies across different stages of lactation. The study was conducted to understand the transcriptional pattern of different classes of protease-pathways associated genes (CTSB, CTSD, CTSH, CTSL, CTSK, CTSS, CTSZ, PLAU, PLAT) and potential protease inhibitors (SERPIN E2 and SERPIN F2) in 40 milk somatic cells (MSC) samples isolated during early, peak, mid and late lactation stages of Sahiwal cows and Murrah buffaloes - the two most important dairy breeds of India. In Sahiwal cows, except CTSK and PLAU, the expression of other proteases class was not affected significantly (p > 0.05) across lactation stages. However, in Murrah buffaloes, the expression of different proteases increased as the lactation progressed. Most of the proteases showed lower expression during early and peak lactation stages while their expression tends to increase during mid to late lactation stages. The overall trend was somewhat similar in both the dairy species albeit the level of expression was higher in buffalo MSC as compared to cow MSC. The study has provided valuable information on expression kinetics of different proteases in milk somatic cells of two major dairy breeds of India.

Correction: Karrow et al. Maternal COVID-19 Vaccination and Its Potential Impact on Fetal and Neonatal Development. Vaccines 2021, 9, 1351.

In the original publication [...].

Ochratoxin A and Citrinin Differentially Modulate Bovine Mammary Epithelial Cell Permeability and Innate Immune Function.

Frequent detection of mycotoxins ochratoxin A (OTA) and citrinin (CIT) in ruminant feed and feedstuff can be a potential threat to feed safety, animal performance and health. Ineffective biodegradation of these mycotoxins by rumen microflora following ingestion of contaminated feeds can lead to their circulatory transport to tissues such as mammary gland as the result of their biodistribution throughout the body. The bovine mammary epithelium plays a pivotal role in maintaining milk yield and composition and contributes to innate immune defense of the udder. The present study is the first to investigate individual effects of OTA and CIT on barrier and innate immune functions of the bovine mammary epithelium using a bovine mammary epithelial cell line (MAC-T). Results indicated that OTA and CIT exposure for 48 h significantly decreased cell viability in a concentration-dependent manner (p < 0.05). A decrease in transepithelial electrical resistance and increase in paracellular flux of FITC-40 kDa dextran was significantly induced by OTA treatment (p < 0.05), but not by CIT after 48 h exposure. qPCR was performed for assessment of expression of tight-junction proteins, Toll-like receptor 4 (TLR4) and cytokines after 4, 24 and 48 h of exposure. Both OTA and CIT markedly downregulated expression of claudin 3 and occludin (p < 0.05), whereas CIT did not affect zonula occludens-1 expression. Expression of TLR4 was significantly upregulated by OTA (p < 0.001) but downregulated by CIT (p < 0.05) at 48 h. Expression of IL-6, TNF-a and TGF-ß was significantly upregulated by OTA (p < 0.05), whereas IL-6 and TGF-ß expression was downregulated by CIT (p < 0.01). These results suggest that OTA and CIT could potentially differentially modulate barrier and innate immune functions of mammary epithelium. The present study not only throws light on the individual toxicity of each mycotoxin on bovine mammary epithelium but also lays the foundation for future studies on the combined effects of the two mycotoxins.

Maternal COVID-19 Vaccination and Its Potential Impact on Fetal and Neonatal Development.

Vaccines have been developed at "warp speed" to combat the COVID-19 pandemic caused by the SARS-CoV-2 coronavirus. Although they are considered the best approach for preventing mortality, when assessing the safety of these vaccines, pregnant women have not been included in clinical trials. Thus, vaccine safety for this demographic, as well as for the developing fetus and neonate, remains to be determined. A global effort has been underway to encourage pregnant women to get vaccinated despite the uncertain risk posed to them and their offspring. Given this, post-hoc data collection, potentially for years, will be required to determine the outcomes of COVID-19 and vaccination on the next generation. Most COVID-19 vaccine reactions include injection site erythema, pain, swelling, fatigue, headache, fever and lymphadenopathy, which may be sufficient to affect fetal/neonatal development. In this review, we have explored components of the first-generation viral vector and mRNA COVID-19 vaccines that are believed to contribute to adverse reactions and which may negatively impact fetal and neonatal development. We have followed this with a discussion of the potential for using an ovine model to explore the long-term outcomes of COVID-19 vaccination during the prenatal and neonatal periods.

CRISPR-Cas9-mediated knockout of TLR4 modulates Mycobacterium avium ssp. paratuberculosis cell lysate-induced inflammation in bovine mammary epithelial cells.

Toll-like receptor 4 (TLR4) is a pattern-recognition receptor involved in the recognition of microbial pathogens and host alarmins. Ligation to TLR4 initiates a signaling cascade that leads to inflammation. Polymorphisms in bovine TLR4 have been associated with Mycobacterium avium ssp. paratuberculosis (MAP) susceptibility and resistance, the cause of Johne's disease, and milk somatic cell score, a biomarker of mastitis. Although the contribution of TLR4 to recognition of bacterial lipopolysaccharide (LPS) has been well characterized, its role in MAP recognition is less certain. Clustered regularly interspaced short palindromic repeats-Cas9 mediated gene editing was performed to generate TLR4 knockout (KO) mammary epithelial cells to determine if TLR4 expression is involved in the initiation of the host inflammatory response to MAP cell lysate (5 and 10 µg/mL) and Escherichia coli LPS (5 µg/mL). The absence of TLR4 in KO cells resulted in enhanced expression of key inflammatory genes (TNFA and IL6), anti-inflammatory genes (IL10 and SOCS3), and supernatant cytokine and chemokine levels (TNF-α, IL-6, IL-10, CCL3) in response to the MAP cell lysate (10 µg/mL). However, in response to LPS, the KO cells showed reduced expression of key inflammatory genes (TNFA, IL1A, IL1B, and IL6) and supernatant cytokine levels (TNF-α, IL-6, CCL2, IL-8) as compared with unedited cells. Overall, these results confirm that TLR4 is essential for eliciting inflammation in response to LPS; however, exacerbated gene and protein expression in TLR4 KO cells in response to MAP cell lysate suggests a different mechanism of infection and host response for MAP, at least in terms of how it interacts with TLR4. These novel findings show potential divergent roles for TLR4 in mycobacterial infections, and this may have important consequences for the therapeutic control of inflammation in cattle.

Functional analysis of bovine interleukin-10 receptor alpha in response to Mycobacterium avium subsp. paratuberculosis lysate using CRISPR/Cas9.

BACKGROUND: The interleukin-10 receptor alpha (IL10RA) gene codes for the alpha chain of the IL-10 receptor which binds the cytokine IL-10. IL-10 is an anti-inflammatory cytokine with immunoregulatory function during the pathogenesis of many inflammatory disorders in livestock, including Johne's disease (JD). JD is a chronic enteritis in cattle caused by Mycobacterium avium subsp. paratuberculosis (MAP) and is responsible for significant economic losses to the dairy industry. Several candidate genes including IL10RA have been found to be associated with JD. The aim of this study was to better understand the functional significance of IL10RA in the context of immune stimulation with MAP cell wall lysate. RESULTS: An IL10RA knock out (KO) bovine mammary epithelial cell (MAC-T) line was generated using the CRISPR/cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) gene editing system. These IL10RA KO cells were stimulated with the immune stimulant MAP lysate +/- IL-10, or with LPS as a positive control. In comparison to unedited cells, relative quantification of immune-related genes after stimulation revealed that knocking out IL10RA resulted in upregulation of pro-inflammatory cytokine gene expression (TNFA, IL1A, IL1B and IL6) and downregulation of suppressor of cytokine signaling 3 (SOCS3), a negative regulator of pro-inflammatory cytokine signaling. At the protein level knocking out IL10RA also resulted in upregulation of inflammatory cytokines - TNF-α and IL-6 and chemokines - IL-8, CCL2 and CCL4, relative to unedited cells. CONCLUSIONS: The findings of this study illustrate the broad and significant effects of knocking out the IL10RA gene in enhancing pro-inflammatory cytokine expression and further support the immunoregulatory role of IL10RA in eliciting an anti-inflammatory response as well as its potential functional involvement during the immune response associated with JD.

Identification of Ovine Serum miRNAs Following Bacterial Lipopolysaccharide Challenge.

Host-pathogen interactions are complex and influenced by host genetic and epigenetic modifications. Recently, the significance of microRNAs (miRNAs) in pathogenic infection and the regulation of immune response has been highlighted. However, information on miRNAs' role in the course of inflammation is still very limited in small ruminants. The present study was intended to identify changes in the expression of circulatory miRNAs post-lipopolysaccharide (LPS)-challenge. In this study, young ewes (n = 18) were challenged with Escherichia coli LPS (400 ng/kg i.v.) and blood samples were collected for serum miRNA isolation at two-time points; prior to challenge (T0), and 4 h (T4) post-challenge, reflecting the peak cortisol response. A total of 91 miRNAs were profiled, including 84 miRNAs on a commercial ovine miRNA-PCR array, and seven individual miRNAs. Forty five miRNAs were differentially expressed (DE) with 35 being up-regulated (Fold regulation, FR > 2) and 10 being down-regulated (FR < 1, p < 0.05) at T4. Among the up-regulated miRNAs, 14 were significantly (p < 0.05) induced, including oar-miRs: 369-3p, 495-3p, 376a-3p, 543-3p, 668-3p, 329a-3p, 655-3p, 411a-5p, and 154a-3p, which were located on ovine chromosome 18 forming four miRNA clusters within 10 kb. The elevated miRNAs belonged to different functional classes, playing roles in activating the hypothalamic-pituitary-adrenal axis; increasing cell survival and differentiation; and inducing inflammatory responses and targeted PI3K-Akt and MAPK signaling and chemokine signaling pathways. In summary, these results reveal the dynamic nature of ovine serum miRNAs during LPS-induced stress and highlight the potential role of identified miRNA-clusters on chromosome 18 to understand the regulation of the acute-phase response. Some of these identified circulating miRNAs may also serve as stress biomarkers for livestock in the future.

In-Vitro Cell Culture for Efficient Assessment of Mycotoxin Exposure, Toxicity and Risk Mitigation.

Mycotoxins are toxic secondary fungal metabolites that commonly contaminate crops and food by-products and thus, animal feed. Ingestion of mycotoxins can lead to mycotoxicosis in both animals and humans, and at subclinical concentrations may affect animal production and adulterate feed and animal by-products. Mycotoxicity mechanisms of action (MOA) are largely unknown, and co-contamination, which is often the case, raises the likelihood of mycotoxin interactions. Mitigation strategies for reducing the risk of mycotoxicity are diverse and may not necessarily provide protection against all mycotoxins. These factors, as well as the species-specific risk of toxicity, collectively make an assessment of exposure, toxicity, and risk mitigation very challenging and costly; thus, in-vitro cell culture models provide a useful tool for their initial assessment. Since ingestion is the most common route of mycotoxin exposure, the intestinal epithelial barrier comprised of epithelial cells (IECs) and immune cells such as macrophages, represents ground zero where mycotoxins are absorbed, biotransformed, and elicit toxicity. This article aims to review different in-vitro IEC or co-culture models that can be used for assessing mycotoxin exposure, toxicity, and risk mitigation, and their suitability and limitations for the safety assessment of animal foods and food by-products.

Heat stress modulates differential response in skin fibroblast cells of native cattle (Bos indicus) and riverine buffaloes (Bubalus bubalis).

Heat stress in hot climates is a major cause that negatively affects dairy animals, leading to substantial economic loss. The present study was aimed to analyze the effect of heat stress on cellular and molecular levels in dermal fibroblast of cattle and buffaloes. Primary fibroblast culture was established using ear pinna tissue samples of cattle (Bos indicus) and riverine buffaloes (Bubalus Bubalis). The cells were exposed to thermal stress at 42°C for 1 h and subsequently allowed to recover and harvest at 37°C at different time points (0, 2, 4, 8, 16, and 24 h) along with control samples. Different cellular parameters viz., apoptosis, proliferation, mitochondrial membrane potential (ΔΨm), oxidative stress, along with expression pattern of heat responsive genes and miRNAs were determined. Cell viability and proliferation rate of heat-stressed fibroblasts decreased significantly (P < 0.05) albeit to a different extent in both species. The cell cytotoxicity, apoptosis, production of reactive oxygen species, and ΔΨm increased more significantly (P < 0.01) in heat stressed fibroblasts of buffalo than cattle. The pattern of heat shock proteins, inflammation/immune genes, and heat responsive miRNA showed differences in induction of their expression level in buffalo and native cattle fibroblasts. Conclusively, finding indicates that heat stress induces more profound impact on buffalo fibroblasts than native cattle fibroblasts. The differential response of cellular parameters, HSP genes, and miRNA expression could be due to better adaptive capacity of skin fibroblast of Bos indicus cattle in comparison with riverine buffaloes.