Tracheal Antimicrobial Peptide Research Articles

Cationic Peptides Harboring Antibiotic Capacity is Selective for Leishmania Panamensis and Leishmania Major

The fairly recent appearance of Leishmania resistance to currently-used therapy has led to the search for new therapeutic strategies. This work was thus aimed at evaluating the in vitro effect of 16 cationic synthetic antimicrobial peptides’ antileishmanial and haemolytic activity. The viability of murine (J774) and human (U937), peripheral blood monocytes, HeLa and HepG2 cells and L. (V) panamensis and L. (L) major promastigotes was then ascertained using the aforementioned peptides. All antimicrobial peptides were synthesised and each cell and parasite line was treated with different peptide concentrations. Melittin, bombinin, mastoparan 8 (MP-8), MP-X and dermaseptin-S1 reduced potential human and murine host cells’ viability at greater concentrations than pentamidine isethionate, human peripheral blood and U937 monocytes being the most sensitive to peptide action. Melittin had a toxic effect on all the cells evaluated in this study and L. (L) major was more sensitive than L. (V) panamensis to peptide effect. As MP-8, bombinin, dermaseptin-S1 and tracheal antimicrobial peptide (TAP) were active against both parasite species, and tachyplesin 1 and Polystes MA selectively so for L. (L) major, they were selected as being promising as they had a >1 selectivity index (SI) and greater than 50 μg/mL haemolytic concentration (HC50), suggesting that they should continue to be studied in in vitro and in vivo infection assays as there have been no previous reports of MP-8, bombinin, TAP and Polystes MA activity regarding L. (V) panamensis and L. (L) major.

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

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

Farrerol regulates antimicrobial peptide expression and reduces Staphylococcus aureus internalization into bovine mammary epithelial cells

Mastitis, defined as inflammation of the mammary gland, is an infectious disease with a major economic influence on dairy industry. Staphylococcus aureus is a common gram-positive pathogen that frequently causes subclinical, chronic infection of the mammary gland in dairy cows. Farrerol, a traditional Chinese medicine isolated from rhododendron, has been shown to have anti-bacterial activity. However, the effect of farrerol on S. aureus infection in mammary epithelium has not been studied in detail. The aim of this study was to investigate the effect of farrerol on the invasion of bovine mammary epithelial cells (bMEC) by S. aureus. The expression of antimicrobial peptide genes by bMEC were assessed in the presence or absence of S. aureus infection. Our results demonstrated that farrerol (4–16 μg/ml) reduced > 55% the internalization of S. aureus into bMEC. We also found that farrerol was able to down-regulate the mRNA expression of tracheal antimicrobial peptide (TAP) and bovine neutrophil β-defensin 5 (BNBD5) in bMEC infected with S. aureus. The Nitric oxide (NO) production of bMEC after S. aureus stimulation was decreased by farrerol treatment. Furthermore, farrerol treatment suppressed S. aureus-induced NF-κB activation in bMEC. These results demonstrated that farrerol modulated TAP and BNBD5 gene expression in mammary gland, enhances bMEC defense against S. aureus infection and could be useful in protection against bovine mastitis.

Stimulating innate immunity in feedlot cattle: strategies to induce tracheal antimicrobial peptide gene expression

Bovine respiratory disease (BRD) is an economically devastating complex of bacterial and viral infections that greatly affects the feedlot industry across North America. Studies have shown that viral infections such as BVDV and glucocorticoid production in stressed calves may inhibit the induction of tracheal antimicrobial peptide (TAP), a cationic β‐defensin that has direct microbicidal effects on invading microbes in the respiratory tract. Certain surface receptors of the innate immune system called the Toll‐Like‐Receptors (TLRs) recognize pathogen associated molecular patterns (PAMPs) to initiate intracellular signal transduction pathways that potentially induce TAP gene expression. PCR analyses of unstimulated tracheal epithelial cells show mRNA expression for TLRs 1,2,3,4, and 6. Therefore, stimulation of tracheal epithelial cells with agonists for these TLRs may induce TAP expression. Previous studies have shown that a TLR 4 agonist called Lipopolysaccharide (LPS) significantly induces TAP expression in epithelial cells after 16 hours of stimulation. This suggests that LPS induces TAP expression via the TLR 4 pathway. The present study examines TLR 2 agonists to determine their effect on TAP induction. Quantitative Real‐Time PCR analysis of epithelial cells stimulated by the TLR 2 agonist Lipoteichoic Acid has shown significant induction of TAP. However, the amount of induction is much less than LPS and occurs after the same amount of stimulation time. Future experiments will determine if other TLR 2 agonists can induce gene expression of TAP in a robust and rapid manner so that TAP production can be increased to ultimately reduce the incidence of BRD in feedlot calves.

Bactericidal activity of tracheal antimicrobial peptide against respiratory pathogens of cattle

Tracheal antimicrobial peptide (TAP) is a β-defensin produced by mucosal epithelial cells of cattle. Although effective against several human pathogens, the activity of this bovine peptide against the bacterial pathogens that cause bovine respiratory disease have not been reported. This study compared the antibacterial effects of synthetic TAP against Mannheimia haemolytica, Histophilus somni, Pasteurella multocida, and Mycoplasma bovis. Bactericidal activity against M. bovis was not detected. In contrast, the Pasteurellaceae bacteria showed similar levels of susceptibility to that of Escherichia coli, with 0.125μg TAP inhibiting growth in a radial diffusion assay and minimum inhibitory concentrations of 1.56–6.25μg/ml in a bactericidal assay. Significant differences among isolates were not observed. Sequencing of exon 2 of the TAP gene from 23 cattle revealed a prevalent non-synonymous single nucleotide polymorphism (SNP) A137G, encoding either serine or asparagine at residue 20 of the mature peptide. The functional effect of this SNP was tested against M. haemolytica using synthetic peptides. The bactericidal effect of the asparagine-containing peptide was consistently higher than the serine-containing peptide. Bactericidal activities were similar for an acapsular mutant of M. haemolytica compared to the wild type. These findings indicate that the Pasteurellaceae bacteria that cause bovine respiratory disease are susceptible to killing by bovine TAP and appear not to have evolved resistance, whereas M. bovis appears to be resistant. A non-synonymous SNP was identified in the coding region of the TAP gene, and the corresponding peptides vary in their bactericidal activity against M. haemolytica.

Endometrial epithelial cells are potent producers of tracheal antimicrobial peptide and serum amyloid A3 gene expression in response to E. coli stimulation

Endometrial epithelial cells play a critical role in mediating inflammatory mechanisms key to bacterial clearance and tissue re-modelling postpartum. This study characterised innate immune gene expression by bovine endometrial epithelial cells from three animals in response to Escherichia coli, a common cause of bovine uterine disease. Expression of key innate immune genes, encoding Toll-like receptor 4 (TLR4), the transcription factor NFkB1, the chemokine interleukin 8 (IL8), inflammatory cytokines (interleukins IL1β, IL6; tumour necrosis factor, TNF), β-defensins (lingual antimicrobial peptides LAP, tracheal antimicrobial peptide TAP) and acute phase proteins (haptoglobin, HP; serum amyloid A, SAA3) was examined in endometrial epithelial cells stimulated with E. coli for 6 and 24h using qRT-PCR. Expression of all genes was increased significantly (P<0.05) 6h post-stimulation. Expression of IL1b, TNF and SAA3 genes was increased by 121-, 357- and 721-fold, respectively (P<0.05). Twenty four hours post-stimulation, IL1b, IL6, IL8, TNF and LAP gene expression was decreased compared to 6h, whereas TAP and SAA3 expression was further increased to 209- and 3452-fold (P<0.05). E. coli driven expression of immune effector genes demonstrates potent immune, antimicrobial and regulatory capacity of endometrial epithelial cells to respond to this pathogen.

Cholecalciferol (vitamin D) differentially regulates antimicrobial peptide expression in bovine mammary epithelial cells: Implications during Staphylococcus aureus internalization

Vitamin D has immunomodulatory functions regulating the expression of host defense genes. The aim of this study was to determine the effect of cholecalciferol (vitamin D3) on S. aureus internalization into bovine mammary epithelial cells (bMEC) and antimicrobial peptide (AP) mRNA expression. Cholecalciferol (1–200nM) did not affect S. aureus growth and bMEC viability; but it reduced bacterial internalization into bMEC (15–74%). Also, bMEC showed a basal expression of all AP genes evaluated, which were induced by S. aureus. Cholecalciferol alone or together with bacteria diminished tracheal antimicrobial peptide (TAP) and bovine neutrophil β-defensin (BNBD) 5 mRNA expression; while alone induced the expression of lingual antimicrobial peptide (LAP), bovine β-defensin 1 (DEFB1) and bovine psoriasin (S100A7), which was inhibited in the presence of S. aureus. This compound (50nM) increased BNBD10 mRNA expression coinciding with the greatest reduction in S. aureus internalization. Genes of vitamin D pathway (25-hydroxylase and 1 α-hydroxylase) show basal expression, which was induced by cholecalciferol or bacteria. S. aureus induced vitamin D receptor (VDR) mRNA expression, but not in the presence of cholecalciferol. In conclusion, cholecalciferol can reduce S. aureus internalization and differentially regulates AP expression in bMEC. Thus, vitamin D could be an effective innate immunity modulator in mammary gland, which leads to a better defense against bacterial infection.

Short chain fatty acids (propionic and hexanoic) decrease Staphylococcus aureus internalization into bovine mammary epithelial cells and modulate antimicrobial peptide expression

Short chain fatty acids (SCFAs) are critical nutrients for ruminants and are mainly obtained from bacterial fermentation of carbohydrates. In addition to their nutrimental function, SCFAs have antimicrobial and anti-inflammatory properties, as well as immunomodulatory roles. It has been reported that sodium butyrate reduces Staphylococcus aureus internalization into bovine mammary epithelial cells (bMEC) and modulates antimicrobial peptide mRNA expression. Nevertheless, it has not been evaluated if sodium propionate (NaP) and sodium hexanoate (NaH) have similar actions. Since they are present in milk, the aim of this study was to determinate the effect of both SCFAs on S. aureus internalization into bMEC and to evaluate their effects on modulation of innate immunity elements. Our data showed that both SCFAs (0.25–5mM) did not affect S. aureus growth and bMEC viability. By gentamicin protection assay (MOI 30:1) we showed that NaP and NaH reduced bacterial internalization into bMEC, which ranged 27–55% and 39–65%, respectively, in relation to non treated controls. Also, both SCFAs up-regulate tracheal antimicrobial peptide (TAP) mRNA expression; however, bovine neutrophil β-defensin 5 (BNBD5) mRNA expression was not modified or was down-regulated. In addition, TAP and BNBD5 expression was up-regulated by S. aureus. Finally, the decrease in bacterial internalization under SCFA treatments is not related to nitric oxide production. In conclusion, NaP and NaH decrease S. aureus internalization into bMEC and modulate TAP gene expression, which may be related to the reduction in bacterial internalization.

Staphylococcus aureus inhibits nuclear factor kappa B activation mediated by prolactin in bovine mammary epithelial cells

The hormone prolactin (PRL) regulates differentiation and lactation in the bovine mammary epithelium. This tissue is especially prone to contracting mastitis, a disease characterized by an inflammatory response in the mammary gland. Staphylococcus aureus is the infectious agent primarily responsible for mastitis. In a previous study, we have shown that bovine PRL (bPRL) stimulates S. aureus internalization in bovine mammary epithelial cells (bMECs) by regulating several host innate immune elements, which are often modulated by nuclear factor kappa B (NF-κB). However, it is unknown whether the activation of the NF-κB transcription factor is regulated by bPRL during S. aureus internalization. The objective of this study was to determine the role of NF-κB in bPRL-stimulated bMECs during S. aureus internalization. Our results showed that bPRL (5 ng/ml) induced NF-κB activation in bMECs; however, it was inhibited by S. aureus in presence of the hormone. When we blocked NF-κB activation with acetylsalicylic acid, we detected an inhibition in S. aureus internalization (48%) in bPRL-stimulated bMECs. The infection-induced inhibition of NF-κB activation in the presence of bPRL correlates with the downregulation in bPRL-mediated tumor necrosis factor (TNF)-α (27%) and tracheal antimicrobial peptide (TAP, 70%) mRNA expression and nitric oxide (NO) production in bMECs. We also detected an inhibition in the expression of the bPRL target gene κ-casein (50%) under these conditions. Interestingly, these effects are not achieved through increased PRL receptor expression (PRLR), as it was inhibited (48%) compared to control cells. In conclusion, NF-κB activation in bMECs is inhibited by S. aureus in the presence of bPRL, suggesting a mechanism by which the host innate immune response may be compromised during subclinical mastitis.

The role of oct-1 in the regulation of tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP) expression in bovine mammary epithelial cells

Lingual antimicrobial peptide (LAP) and tracheal antimicrobial peptide (TAP) are two important β-defensins of antimicrobial peptide family, which are evolutionarily conserved effector molecules of the innate immune response. Although known to be sensitive to pathogenic challenge, the control of their expression remains unclear. Both LAP and TAP genes showed constitutive and inducible expression in bovine mammary epithelial tissues, and the aim of this study was to investigate the mechanisms underlying their expression and regulation. Reporter plasmids fused with 5' regions of the two gene promoter regions were constructed and transiently transfected into a bovine mammary epithelial (BME) cell line. Initial serial deletion of the promoter regions from both genes identified two positive regulatory elements within the 1 kb regions upstream the transcription start sites, which co-operatively contribute to LAP and TAP gene expression. Further luciferase reporter assays revealed that an enhancer and a 61-bp region proximal to both genes are important for basal expression and regulation of transcription. Electrophoretic mobility shift assays (EMSA) indicated the involvement of the Oct-1 protein-DNA complex in regulating the promoter activity, which was confirmed by super shift EMSA with Oct-1 antibody and by knockdown of Oct-1 with small interfering RNA. The Oct-1 binding motif was also shown to be responsive to phorbol 12-myristate 13-acetate but not LPS stimulation. The results from this study clearly demonstrate the involvement of the Oct-1 transcription factor in the regulation of LAP and TAP expression.

The spatial expression pattern of antimicrobial peptides across the healthy bovine udder

Antimicrobial peptides are key molecules in local host defense. With the aim to better understand the possible involvement of these peptides in the prevention of bovine mastitis, we determined, for the first time to our knowledge, the spatial pattern of constitutive expression of 5 bovine β-defensins and bovine psoriasin (S100A7) across 5 localizations of the bovine mammary gland applying a quantitative real-time PCR approach. We observed 3 different expression patterns in the healthy udder: 1) constitutive expression of the lingual and tracheal antimicrobial peptides (LAP and TAP), as well that of bovine neutrophil β-defensins 4 and 10 (BNBD4 and BNBD10), is essentially restricted to the mammary lymph node; 2) bovine β-defensin 1 (DEFB1) is mainly expressed in the cisternal epithelium and the Rosette of Fürstenberg; 3) strong constitutive mRNA expression of the calcium-binding protein S100A7, which is also known as psoriasin and which has been reported to be highly active against Escherichia coli, was detected in the streak canal. These results indicate a crucial role of S100A7 in the early-stage prevention of coliform mastitis, and the analyzed β-defensins might be regarded as inducible weapons against already invading pathogens.

Differential activity of innate defense antimicrobial peptides against Nocardia species

BackgroundMembers of the genus Nocardia are ubiquitous environmental saprophytes capable to cause human pulmonary, disseminated and cutaneous nocardiosis or bovine mastitis. Innate immunity appears to play an important role in early defense against Nocardia species. To elucidate the contribution of antimicrobial peptides (AMPs) in innate defense against Nocardia, the activity of human α-defensins human neutrophil peptides (HNPs) 1-3, human β-defensin (hBD)-3 and cathelicidin LL-37 as well as bovine β-defensins lingual and tracheal antimicrobial peptides (LAP, TAP) and bovine neutrophil-derived indolicidin against four important Nocardia species was investigated.ResultsWhereas N. farcinica ATCC 3318 and N. nova ATCC 33726 were found to be susceptible to all investigated human and bovine AMPs, N. asteroides ATCC 19247 was killed exclusively by neutrophil-derived human α-defensins HNP 1-3 and bovine indolicidin. N. brasiliensis ATCC 19296 was found to exhibit complete resistance to investigated human AMPs and to be susceptible only to bovine indolicidin.ConclusionSelected AMPs are capable to contribute to the first line of defense against Nocardia, yet, susceptibility appears to vary across different Nocardia species. Obtained results of neutrophil-derived AMPs to possess the broadest antinocardial spectrum are remarkable, since nocardiosis is characterized by a neutrophil-rich infiltrate in vivo.

Divergent antimicrobial peptide (AMP) and acute phase protein (APP) responses to Trypanosoma congolense infection in trypanotolerant and trypanosusceptible cattle

African animal trypanosomiasis (AAT) is endemic across Sub-Saharan African and is a major constraint to livestock production. The ability of certain cattle breeds to remain productive despite infection is known as trypanotolerance; however, the underlying immune mechanisms contributing to this trait remain poorly understood. Antimicrobial peptides (AMPs) and acute phase proteins (APPs) are evolutionarily conserved effector molecules of the innate immune system that have important roles in the resolution of infection and activation of the adaptive immune response. Expression levels of AMP genes (TAP, LAP, BNBD4, DEFB1, DEFB5 and LEAP2) and APP genes (HP, CP, AGP, LBP, SAA3 and CRP) were investigated using real time quantitative reverse transcription PCR (qRT-PCR) in peripheral blood mononuclear cells (PBMC) isolated from two breeds of African cattle (trypanotolerant N’Dama and trypanosusceptible Boran), experimentally infected with Trypanosoma congolense. Haptoglobin and serum amyloid A (SAA) were also measured in plasma using quantitative protein assays. Results demonstrated that tracheal antimicrobial peptide (TAP) gene expression increased by 32-fold in Boran, compared to only 3-fold in N’Dama, by 14 days post-infection (dpi) and rising to 136-fold at 29dpi in Boran, compared to 47-fold in N’Dama (P<0.05). Protein expression levels of SAA are elevated in N’Dama, rising to 163μg/ml at 14dpi compared with 72μg/ml in Boran. The SAA expression profile mirrors the wave of parasitaemia detected in N’Dama. Seven single nucleotide polymorphisms (SNPs) were identified in the promoter regions of the SAA3 and SAA4 genes, which are predicted to affect transcription factor binding and thereby contributing to the differential patterns of expression detected between the breeds. Whereas elevated TAP expression is a conserved component of the innate immune response to infection in both breeds, higher SAA expression levels may contribute toward trypanotolerance in N’Dama.

Sodium butyrate inhibits Staphylococcus aureus internalization in bovine mammary epithelial cells and induces the expression of antimicrobial peptide genes

A distinctive feature of bovine milk fat is the presence of butyrate, molecule with recognized antimicrobial and antiinflammatory properties. Bovine mastitis is a pathology characterized by inflammatory and infectious processes; however, the role of sodium butyrate on Staphylococcus aureus infection in mammary epithelium has not been studied. In this work we assess the role of sodium butyrate on the invasion of bovine mammary epithelial cells (bMEC) by S. aureus responsible of mastitis and on the expression of antimicrobial peptide genes. Our data show that sodium butyrate (0.25–0.5 mM) reduces ∼50% the internalization of S. aureus (ATCC 27543) into bMEC. By RT-PCR analysis, we showed that sodium butyrate is able to up-regulate the expression of tracheal antimicrobial peptide (TAP), β-defensin and inducible nitric oxide synthase (iNOS) mRNAs, as well as nitric oxide production. Also, sodium butyrate and infection increased acetylation of histone H3 in bMEC. These results indicate that sodium butyrate could be effective to modulate innate immune gene expression in mammary gland that leads to a better defense against bacterial infection. To our knowledge, this is the first report that shows a role of sodium butyrate during the internalization of S. aureus into bMEC.

Expression of tracheal antimicrobial peptide in bovine mammary epithelial cells

The production of antimicrobial peptides is an important key of innate immunity. Tracheal antimicrobial peptide (TAP) expression has been reported in bovine tracheal epithelial cells and it can be modulated by bacterial infection or bacterial components. In mammary gland TAP expression has been reported, but the cell type that produces it is unknown. The objective of this work was to evaluate if bovine mammary epithelial cells (bMEC) express TAP mRNA, and evaluate the regulation of its expression in response to Staphylococcus aureus infection, bovine prolactin (bPRL) or acetyl salicylic acid (ASA). By retrotranscription and PCR, we demonstrated that bMEC express TAP mRNA. bMEC infected with live S. aureus down-regulates TAP expression, whereas the challenge with gentamicin-killed S. aureus up-regulates it. Also, bPRL do not significantly modify TAP expression, but in the presence of 5 mM ASA it was down-regulated, suggesting that nuclear factor κB (NF-κB) pathway can be involved in its regulation.

Toll-like receptor and antimicrobial peptide expression in the bovine endometrium

BackgroundThe endometrium is commonly infected with bacteria leading to severe disease of the uterus in cattle and humans. The endometrial epithelium is the first line of defence for this mucosal surface against bacteria and Toll-like receptors (TLRs) are a critical component of the innate immune system for detection of pathogen associated molecular patterns (PAMPs). Antimicrobial peptides, acute phase proteins and Mucin-1 (MUC-1) also provide non-specific defences against microbes on mucosal surfaces. The present study examined the expression of innate immune defences in the bovine endometrium and tested the hypothesis that endometrial epithelial cells express functional receptors of the TLR family and the non-specific effector molecules for defence against bacteria.MethodsBovine endometrial tissue and purified populations of primary epithelial and stromal cells were examined using RT-PCR for gene expression of TLRs, antimicrobial peptides and MUC-1. Functional responses were tested by evaluating the secretion of prostaglandin E2 and acute phase proteins when cells were treated with bacterial PAMPs such as bacterial lipopolysaccharide (LPS) and lipoproteins.ResultsThe endometrium expressed TLRs 1 to 10, whilst purified populations of epithelial cells expressed TLRs 1 to 7 and 9, and stromal cells expressed TLRs 1 to 4, 6, 7, 9 and 10. The TLRs appear to be functional as epithelial cells secreted prostaglandin E2 in response to bacterial PAMPs. In addition, the epithelial cells expressed antimicrobial peptides, such as Tracheal and Lingual Antimicrobial Peptides (TAP and LAP) and MUC-1, which were upregulated when the cells were treated with LPS. However, the epithelial cells did not express appreciable amounts of the acute phase proteins haptoglobin or serum amyloid A.ConclusionEpithelial cells have an essential role in the orchestration of innate immune defence of the bovine endometrium and are likely to be the key to prevention of endometrial infection with bacteria.

Antimicrobial peptides as agents of mucosal immunity.

Mucosal surfaces are continually exposed to a wide range of potentially pathogenic organisms, yet the incidence of infectious disease resulting from these encounters is relatively low. This suggests the presence of highly effective defence mechanisms in these tissues. Antimicrobial peptides have recently been discovered in mucosal tissues and may play a significant role in host defence. Several mucosal peptides (andropin, magainin, tracheal antimicrobial peptide, enteric defensins and PR-39) all fulfil minimal criteria for a role in mucosal host defence, including potent in vitro antimicrobial activity and accumulation at the mucosal surface. Most of these mucosal peptides are encoded by members of large gene families that contain members found in other biological contexts more classically associated with antimicrobial defence. The abundance, activity and evolutionary history of several epithelial peptides suggest that antimicrobial peptides play a key role in host defence at mucosal surfaces.

Impairment of innate immune responses of airway epithelium by infection with bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) infection is an important risk factor for development of shipping fever pneumonia in feedlot cattle, and infects but does not cause morphologic evidence of damage to airway epithelial cells. We hypothesized that BVDV predisposes to bacterial pneumonia by impairing innate immune responses in airway epithelial cells. Primary cultures of bovine tracheal epithelial cells were infected with BVDV for 48 h, then stimulated with LPS for 16 h. Expression of tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP) mRNA was measured by quantitative RT-PCR, and lactoferrin concentrations were measured in culture supernatant by ELISA. BVDV infection had no detectable effect on the constitutive expression of TAP and LAP mRNA or lactoferrin concentration in culture supernatant. LPS treatment provoked a significant increase in TAP mRNA expression and lactoferrin concentration in the culture supernatant ( p < 0.01), and these effects were significantly ( p < 0.02, p < 0.01) abrogated by prior infection of the tracheal epithelial cells with the type 2 ncp-BVDV isolate. In contrast, infection with the type 1 ncp-BVDV isolate had no effect on TAP mRNA expression or lactoferrin secretion. LPS treatment induced a significant ( p < 0.001) upregulation of LAP mRNA expression, which was not significantly affected by prior infection with BVDV. These data indicate that infection with a type 2 BVDV isolate inhibits the LPS-induced upregulation of TAP mRNA expression and lactoferrin secretion by tracheal epithelial cells, suggesting a novel mechanism by which this virus abrogates respiratory innate immune responses and predisposes to bacterial pneumonia in cattle.

Effect of Corticosteroids and Neuropeptides on the Expression of Defensins in Bovine Tracheal Epithelial Cells

Susceptibility to bacterial pneumonia in cattle is enhanced by stressors such as transportation, weaning, and commingling, which trigger a physiologic stress response resulting in elevated levels of endogenous corticosteroids and catecholamines. To determine the effect of neuroendocrine mediators on the expression of innate defense peptides in the lung, bovine tracheal epithelial cells were exposed to dexamethasone, catecholamines, acetylcholine, or substance P, and then beta-defensin expression was quantified using real-time reverse transcription-PCR. Basal expression of tracheal antimicrobial peptide (TAP) mRNA was not affected by any of the mediators tested. However, induction of TAP expression by lipopolysaccharide was significantly inhibited by pretreatment with dexamethasone. Bronchial biopsy specimens from dexamethasone-treated calves had significantly lower expression of TAP and lingual antimicrobial peptide (LAP) mRNA than saline-treated controls following 48 h of treatment. Lipopolysaccharide-elicited neutrophil recruitment was enhanced in the lungs of dexamethasone-treated calves compared to saline-treated controls. These findings indicate that modulation of epithelial antimicrobial peptide expression is one mechanism through which corticosteroids and stress may impair innate pulmonary defenses.

Inhibition of β-Defensin Gene Expression in Airway Epithelial Cells by Low Doses of Residual Oil Fly Ash is Mediated by Vanadium

Poor ambient air quality is associated with increased morbidity and mortality, including respiratory infections. However, its effects on various host-defense mechanisms are poorly understood. This study utilized an in vitro model to study the effect of particulate matter (PM(2.5)) on one antimicrobial mechanism of host defense in the airway, beta-defensin-2 and its bovine homologue, tracheal antimicrobial peptide (TAP) induction in response to lipopolysaccharide (LPS) and IL-1beta. Our model utilized cultured primary bovine tracheal epithelial (BTE) cells and the human alveolar type II epithelial cell line, A549, treated with 0-20 microg/cm(2) residual oil fly ash (ROFA) for 6 h. The cells were then washed and stimulated for 18 h with 100 ng/ml LPS or for 6 h with 100 ng/ml IL-1beta. ROFA inhibited the LPS-induced increase in TAP mRNA and protein without inducing significant cytotoxicity. As little as 2.5 microg/cm(2) of ROFA inhibited LPS-induced TAP gene expression by 30%. The inhibitory activity was associated with the soluble fraction and not the washed particle. The activity in the leachate was attributed to vanadium, but not nickel or iron. SiO(2) and TiO(2) were utilized as controls and did not inhibit LPS induction of TAP gene expression in BTE. ROFA also inhibited the increase of IL-1beta-induced human beta-defensin-2, a homologue of TAP, in A549 cells. The results show that ROFA, V(2)O(5), and VOSO(4) inhibit the ability of airway epithelial cells to respond to inflammatory stimuli at low, physiologically relevant doses and suggest that exposure to these agents could result in an impairment of defense against airborne pathogens.