Staphylococcus aureus and Escherichia coli elicit different innate immune responses from bovine mammary epithelial cells

Escherichia coli and Staphylococcus aureus are the most important pathogenic bacteria causing bovine clinical mastitis and subclinical mastitis, respectively. However, little is known about the molecular mechanisms underlying the different host response patterns caused by these bacteria. The aim of this study was to characterize the different innate immune responses of bovine mammary epithelium cells (MECs) to heat-inactivated E. coli and S. aureus. Gene expression of Toll-like receptor 2 (TLR2) and TLR4 was compared. The activation of nuclear factor kappa B (NF-κB) and the kinetics and levels of cytokine production were analyzed. The results show that the mRNA for TLR2 and TLR4 was up-regulated when the bovine MECs were stimulated with heat-inactivated E. coli, while only TLR2 mRNA was up-regulated when the bovine MECs were stimulated with heat-inactivated S. aureus. The expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and IL-8 increased more rapidly and higher when the bovine MECs were stimulated with heat-inactivated E. coli than when they were stimulated with heat-inactivated S. aureus. E. coli strongly activated NF-κB in the bovine MECs, while S. aureus failed to activate NF-κB. Heat-inactivated S. aureus could induce NF-κB activation when bovine MECs cultured in medium without fetal calf serum. These results were confirmed using TLR2- and TLR4/MD2-transfected HEK293 cells and suggested that differential TLR recognition and the lack of NF-κB activation account for the impaired immune response elicited by heat-inactivated S. aureus.     

Bovine natural killer cells are present in Escherichia coli infected mammary gland tissue and show antimicrobial activity in vitro

Natural killer (NK) cells are early responders in bacterial infections but their role in bovine mastitis has not been characterized. For the first time, we show the presence of NK cells (NKp46⁺/CD3⁻) in bovine mammary gland tissue after an intramammary challenge with Escherichia (E.) coli. A small number of NK cells was detected in milk from quarters before and during an E. coli challenge. In vitro cultures of primary bovine mammary gland epithelial cells stimulated with UV irradiated E. coli induced significant migration of peripheral blood NK cells (pbNK) within 2 h. Furthermore, pbNK cells significantly reduced counts of live E. coli in vitro within 2 h of culture. The results show that bovine NK cells have the capacity to migrate to the site of infection and produce antibacterial mediators. These findings introduce NK cells as a leukocyte population in the mammary gland with potential functions in the innate immune response in bovine mastitis.     

Regulation of Innate Immune Function in Bovine Oviduct Epithelial Cells in Culture: The Homeostatic Role of Epithelial Cells in Balancing Th1/Th2 Response

This study aimed to investigate the role of epithelial cells in regulating innate immunity in bovine oviduct epithelial cell (BOEC) culture. We studied the effect of Escherichia coli lipopolysaccharide (LPS) and its interaction with ovarian steroids, estradiol (E2) and progesterone (P4), and luteinizing hormone (LH) at concentrations observed during the preovulatory period on immune responses in BOEC culture. Immunohistochemistry of oviduct tissue showed intensive expression of Toll-like receptor-4 (TLR-4) and TLR-2 in epithelial cells. A dose of 10 ng/ml LPS stimulated TLR-4, cyclooxygenase-2 (COX-2), nuclear factor kappa B inhibitor A (NFKBIA), interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) expression, indicating an early pro-inflammatory response. A dose of 100 ng/ml LPS did not induce expression of these genes but stimulated TLR-2, IL-10,IL-4 and microsomal prostaglandin E synthase-1 (mPGES-1) expression and PGE2 secretion, indicating an anti-inflammatory response. Ovarian steroids and LH completely block LPS (10 ng/ml)-induced TLR-4, IL-1β and TNF-α expression as well as LPS (100 ng/ml)-induced TLR-2 expression. Taken together, this study suggests the existence of an early signaling system to respond to infection in the BOEC. In addition, ovarian steroids and LH may play a critical role in inducing homeostasis and in controlling hyperactive pro-inflammatory responses detrimental to epithelial cells, sperm and the embryo.     

Comparison of innate immune agonists for induction of tracheal antimicrobial peptide gene expression in tracheal epithelial cells of cattle

Bovine respiratory disease is a complex of bacterial and viral infections of economic and welfare importance to the beef industry. Although tracheal antimicrobial peptide (TAP) has microbicidal activity against bacterial pathogens causing bovine respiratory disease, risk factors for bovine respiratory disease including BVDV and stress (glucocorticoids) have been shown to inhibit the induced expression of this gene. Lipopolysaccharide is known to stimulate TAP gene expression, but the maximum effect is only observed after 16 h of stimulation. The present study investigated other agonists of TAP gene expression in primary cultures of bovine tracheal epithelial cells. PCR analysis of unstimulated tracheal epithelial cells, tracheal tissue and lung tissue each showed mRNA expression for Toll-like receptors (TLRs) 1–10. Quantitative RT-PCR analysis showed that Pam3CSK4 (an agonist of TLR1/2) and interleukin (IL)-17A significantly induced TAP gene expression in tracheal epithelial cells after only 4–8 h of stimulation. Flagellin (a TLR5 agonist), lipopolysaccharide and interferon-α also had stimulatory effects, but little or no response was found with class B CpG ODN 2007 (TLR9 agonist) or lipoteichoic acid (TLR2 agonist). The use of combined agonists had little or no enhancing effect above that of single agonists. Thus, Pam3CSK4, IL-17A and lipopolysaccharide rapidly and significantly induce TAP gene expression, suggesting that these stimulatory pathways may be of value for enhancing innate immunity in feedlot cattle at times of susceptibility to disease.     

Inducible expression of enhanced green fluorescent protein by interleukin-1α, interleukin-1β and Toll-like receptor 2 promoters in goat mammary epithelial cells in response to bacterial challenges

The development of a bacteria-inducible expression system has several advantages compared with persistent expression of anti-bacterial proteins in milk to prevent and treat mastitis. The present study determined whether mastitis responsive promoters could regulate enhanced green fluorescent protein (EGFP) expression in goat mammary epithelial cells (GMECs) in response to challenges with Escherichia coli, Staphylococcus aureus or Streptococcus agalactiae. The level of expression of interleukin (IL)-1α was significantly increased in GMECs challenged with E. coli, S. aureus or S. agalactiae compared with untreated GMECs. IL-1β was induced by E. coli and S. aureus, while Toll-like receptor 2 (TLR2) was induced by E. coli only.GMECs were transfected with IL-1α, IL-1β and TLR2 promoter-EGFP reporter gene lentiviral expression vectors and the levels of expression of EGFP were measured by flow cytometry and Western blot analysis after bacterial challenge. EGFP expression driven by the IL-1α and IL-1β promoters was higher in GMECs challenged with E. coli, S. aureus or S. agalactiae than in untreated GMECs. There were no differences in EGFP expression driven by the TLR2 promoter between GMECs challenged with S. aureus or S. agalactiae and untreated GMECs, but EGFP expression was significantly increased in GMECs challenged with E. coli. Overall, these results indicate that the promoters of some bacteria-inducible genes can regulate EGFP expression in GMECs in response to bacterial challenges. This bacteria-inducible expression strategy could be used for production of mastitis resistant animals by regulating the expression of anti-bacterial proteins in the mammary gland.     

Alteration in lymphocyte responses,cytokine and chemokine profiles in chickens infected with genotype VII and VIII velogenic Newcastle disease virus

Newcastle disease (ND) is a highly contagious avian disease and one of the major causes of economic losses in the poultry industry. The emergence of virulent NDV genotypes and repeated outbreaks of NDV in vaccinated chickens have raised the need for fundamental studies on the virus–host interactions. In this study, the profiles of B and T lymphocytes and macrophages and differential expression of 26 immune-related genes in the spleen of specific-pathogen-free (SPF) chickens, infected with either the velogenic genotype VII NDV strain IBS002 or the genotype VIII NDV strain AF2240, were evaluated. A significant reduction in T lymphocyte population and an increase in the infiltration of IgM⁺ B cells and KUL01⁺ macrophages were detected in the infected spleens at 1, 3 and 4 days post-infection (dpi) (P < 0.05). The gene expression profiles showed an up-regulation of CCLi3, CXCLi1, CXCLi2 (IL-8), IFN-γ, IL-12α, IL-18, IL-1β, IL-6, iNOS, TLR7, MHCI, IL-17F and TNFSF13B (P < 0.05). However, these two genotypes showed different cytokine expression patterns and viral load. IBS002 showed higher viral load than AF2240 in spleen at 3 and 4 dpi and caused a more rapid up-regulation of CXCLi2, IFN-γ, IL-12α, IL-18, IL-1β, iNOS and IL-10 at 3 dpi. Meanwhile, the expression levels of CCLI3, CXCLi1, IFN-γ, IL-12α, IL-1β and iNOS genes were significantly higher in AF2240 at 4 dpi. In addition, the expression levels of IL-10 were significantly higher in the IBS002-infected chickens at 3 and 4 dpi. Hence, infection with velogenic genotype VII and VIII NDV induced different viral load and production of cytokines and chemokines associated with inflammatory reactions.     

Differential induction of MyD88- and TRIF-dependent pathways in equine monocytes by Toll-like receptor agonists

Our understanding of the innate immune response in the horse has been limited by a lack of definitive data concerning cell signaling in response to microbial products. Toll-like receptors (TLRs) recognize conserved molecular motifs of microbes and elicit immune responses through their coupling with intracellular adaptor molecules, particularly MyD88 and TRIF. To provide a more definitive characterization of TLR signaling in the horse, the objectives of this study were to: (1) characterize the responses of equine monocytes to TLR ligands that signal through MyD88, TRIF or both in other species, and (2) determine the profiles of gene expression initiated utilizing these adaptor molecules. Monocytes were used to establish concentration response curves for Escherichia coli lipopolysaccharide (LPS; TLR4 ligand) and N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine x 3 HCl (Pam₃CSK₄; TLR2 ligand) based on expression of procoagulant activity (PCA) and production of tumor necrosis factor-alpha (TNF-α); effects of polyinosine–polycytidylic acid (Poly I:C; TLR3 ligand) were determined by quantifying expression of mRNA for interferon-beta (IFN-ß). Expression of genes associated with the MyD88- (TNF-α, IL-1ß, IL-6 and IL-10) and TRIF-dependent pathways (IFN-ß, IP-10, RANTES and TRAF1) were measured at intervals spanning 20 h. LPS and Pam₃CSK₄ induced significantly higher expression of TNF-α, IL-1ß, and IL-10 than did Poly I:C. Poly I:C induced significantly higher expression of IFN-ß, IP-10 and RANTES than did either the TLR2 or TLR4 ligands. High concentrations of E. coli LPS did not significantly increase expression of genes associated with the TRIF-dependent pathway. The results of this study suggest that equine monocytes utilize a common intracellular pathway in response to TLR2 and TLR4 ligands, but a distinct pathway in response to TLR3 ligands.     

Toll-like receptor activation and expression in bovine alpha-herpesvirus infections

The involvement of Toll-like receptors (TLRs) in bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) infections has not been analyzed. In this study, the role of TLR signaling on virus replication was investigated. Blood leukocytes consistently express TLRs. Thus, our approach was to study in vitro the effects of agonist stimulation of TLRs expressed by peripheral blood leukocytes on BoHV-1 and BoHV-5 replication. Furthermore, the patterns of TLRs 3, 7–9 expression on virus-infected-bovine leukocytes were analyzed. Only Imiquimod (TLR7/8 agonist) showed anti-viral activity on infected MDBK cells. This is the first evidence that the timely activation of TLR7/8 signaling is effective in impairing BoHV-1 and 5 replication, thereby providing an experimental indication that Imiquimod may be a promising immune modulator. This work describes, for the first time, the expression patterns of TLRs in BoHV-1- or BoHV-5-infected-bovine leukocytes, suggesting the involvement of TLR7 and TLR9 in the recognition of these viruses.     

Differential expression of pro-inflammatory and anti-inflammatory cytokines during experimental infection with low or high virulence bovine viral diarrhea virus in beef calves

The objective was to compare the mRNA expression of pro-inflammatory (TNF-α, IL-1β, IFN-γ, IL-2, IL-12, IL-15) and anti-inflammatory (IL-4, IL-10, TGF-β) cytokines, after experimental infection with low or high virulence noncytopathic (ncp) bovine viral diarrhea virus (BVDV). Thirty BVDV-naïve, beef calves were intranasally inoculated with low (LV; n = 10, SD-1) or high (HV; n = 10, 1373) virulence ncp BVDV or with BVDV-free cell culture medium (Control, n = 10). Calves were euthanized on day 5 post-inoculation, and tracheo-bronchial lymph node and spleen samples were collected for mRNA expression through quantitative-RT-PCR. mRNA levels of pro-inflammatory (TNF-α, IL-1β, IL-2, IFN-γ) and anti-inflammatory (IL-4 and IL-10) cytokines were up-regulated in tracheo-bronchial lymph nodes of HV, but not in LV, compared to the control group (P < 0.05). IL-12 mRNA level was up-regulated in tracheo-bronchial lymph nodes of both LV and HV groups (P ≤ 0.05). A significant up-regulation of IL-15 mRNA was observed in tracheo-bronchial lymph nodes for LV calves (P < 0.002), but not for HV calves. Experimental inoculation with BVDV-2 1373 stimulated significant mRNA expression of pro-inflammatory and anti-inflammatory cytokines. In contrast, inoculation with BVDV-1a SD-1 only resulted in up-regulation of IL-12 and IL-15 mRNA, which is associated with activation of macrophages and NK cells during innate immune response.     

Plaque assay of bovine rotavirus

Incorporation of trypsin and diethylaminoethyl-dextran in the overlay was found to be necessary for infectivity assay of the UK strain of bovine rotavirus by plaque assays. Small plaques of about 1 mm in radius were formed in BGM cells. Large plaques of about 3–4 mm in radius were consistently produced in monolayers of secondary calf kidney cultures.Key words: bovine rotavirus, plaque assay, trypsin, diethyl-aminoethy1-dextran     

Cell culture propagation of porcine rotavirus (reovirus-like agent).

Two isolates of porcine rotavirus (reovirus-like agent) were isolated and passaged in primary procine kidney cell cultures. Viral infectivity for cells was monitored by immunofluorescence because viral cytopathic effect was moderate. Successful passage of virus in cell culture required that viral suspensions obtained from infected cell cultures be treated with pancreatin prior to inoculation onto cell monolayers. Porcine rotavirus passage in cell culture also was accomplished, using trypsin treatments in lieu of pancreatin treatments. Porcine rotavirus passaged 10 times in cell culture infected gnotobiotic pigs and caused diarrhea. Gnotobiotic pigs that recovered from this infection were resistant to challenge exposure with porcine rotavirus but were susceptible to challenge exposure with transmissible gastroenteritis virus. As determined by immunofluorescent cross reactions, porcine rotavirus was found to be antigenically related to the human and bovine rotaviruses but not to reovirus type 3 or to transmissible gastroenteritis virus.     

Essential role of neutrophils but not mammary alveolar macrophages in a murine model of acute Escherichia coli mastitis

Mastitis, the inflammation of the mammary gland, is an important disease affecting dairy animals worldwide. The disease is caused by mammary pathogenic bacteria and Escherichia coli are frequently implicated. Virulence factors of mammary pathogenic E. coli are only partially known and intramammary challenge with LPS elicits neutrophil recruitment in experimental bovine and murine mastitis models. We have previously shown that neutrophil recruitment in LPS-induced murine mastitis is strictly dependent on mammary alveolar macrophages. However, the relative role of alveolar macrophages and blood neutrophils in E. coli mastitis is not well defined. To this end, we selectively depleted mammary alveolar macrophages or blood neutrophils before intramammary challenge with E. coli strain P4 (ECP4). Mice depleted of alveolar macrophages prior to intramammary challenge recruited neutrophils normally and restricted bacterial growth and interstitial invasion. Importantly however, upon depletion of alveolar macrophages, ECP4 invaded the mammary alveolar epithelial cells and formed intracellular bacterial communities. In contrast, neutrophil depletion prior to intramammary infection with ECP4 was associated with unrestricted bacterial growth, tissue damage, severe sepsis and mortality. This study suggests that neutrophils but not alveolar macrophages provide essential antimicrobial defense against mammary pathogenic E. coli. Furthermore, we show here similar invasion after depletion of alveolar macrophages as in our previous studies showing that LPS/TLR4 signaling on alveolar macrophages abrogates ECP4 invasion of the mammary epithelium. Interestingly, similar ECP4 invasion and formation of intracellular communities were also observed following intramammary infection of either iNOS gene-deficient or IL-1 receptor type 1 gene-deficient mice.