Innate immune responses of epididymal epithelial cells to Staphylococcus aureus infection

The epithelium is an active participant in the host response to infection. We hypothesized that epididymal epithelia play a role in the innate immune responses by sensing the presence of pathogens, expressing and secreting inflammatory cytokines that recruit inflammatory cells in response to invading pathogens. Our results indicated that TNF-alpha and IL-1beta could be secreted by the primary cultured rat epididymal cauda epithelia infected with Staphylococcus aureus. Epididymal epithelial-induced nitric oxide synthase (iNOS) expression was up-regulated after S. aureus infection and nitric oxide (NO) was also found to be produced significantly. NF-kappaB inhibitor BAY11-7082 inhibited TNF-alpha secretion completely and p38 mitogen-activated protein kinases (MAPKs) inhibitor SB203580 decreased TNF-alpha secretion partly, indicating that NF-kappaB and p38 signal pathways were involved in this inflammation response. Toll-like receptor (TLR)-2 and -4 were shown to be expressed in primary cultured rat epididymal epithelia. After infection the level of TLR2 expression was up-regulated rather than TLR4. These results demonstrated that epididymal epithelium have an innate immune response through activation of p38 MAPK and NF-kappaB after TLR2 activation by S. aureus infection.     

Analysis of differential immune responses induced by innate and adaptive immunity following transplantation

The roles of innate and adaptive immunity in allograft rejection remain incompletely understood. Previous studies analysing lymphocyte deficient or syngeneic graft recipients have identified subsets of inflammatory chemokines and cytokines induced by antigen independent mechanisms. In the current study, we analysed a panel of 60 inflammatory parameters including serum cytokines, intragraft chemokines and cytokines, receptors, and cellular markers. Our results confirmed the up-regulation of a subset of markers by innate mechanisms and also identified a subset of parameters up-regulated only in the context of an adaptive response. Thus, we successfully differentiated markers of the innate and adaptive phases of rejection. Current paradigms emphasize that innate signals can promote a subsequent adaptive response. Interestingly, in our studies, expression of the markers induced by innate mechanisms was markedly amplified in the allogeneic, but not syngeneic or lymphocyte deficient, recipients. These results suggest that inflammatory mediators can have functional overlap between the innate and adaptive responses, and that the adaptive component of the rejection process amplifies the innate response by positive feedback regulation.     

Recognition of Staphylococcus aureus by the innate immune system

The gram-positive bacterium Staphylococcus aureus is a major pathogen responsible for a variety of diseases ranging from minor skin infections to life-threatening conditions such as sepsis. Cell wall-associated and secreted proteins (e.g., protein A, hemolysins, and phenol-soluble modulin) and cell wall components (e.g., peptidoglycan and alanylated lipoteichoic acid) have been shown to be inflammatory, and these staphylococcal components may contribute to sepsis. On the host side, many host factors have been implicated in the innate detection of staphylococcal components. One class of pattern recognition molecules, Toll-like receptor 2, has been shown to function as the transmembrane component involved in the detection of staphylococcal lipoteichoic acid and phenol-soluble modulin and is involved in the synthesis of inflammatory cytokines by monocytes/macrophages in response to these components. Nod2 (nucleotide-binding oligomerization domain 2) is the intracellular sensor for muramyl dipeptide, the minimal bioactive structure of peptidoglycan, and it may contribute to the innate immune defense against S. aureus. The staphylococcal virulence factor protein A was recently shown to interact directly with tumor necrosis factor receptor 1 in airway epithelium and to reproduce the effects of tumor necrosis factor alpha. Finally, peptidoglycan recognition protein L is an amidase that inactivates the proinflammatory activities of peptidoglycan. However, peptidoglycan recognition protein L probably plays a minor role in the innate immune response to S. aureus. Thus, several innate immunity receptors may be implicated in host defense against S. aureus.     

Escherichia coli and Staphylococcus aureus Elicit Differential Innate Immune Responses following Intramammary Infection

Staphylococcus aureus and Escherichia coli are among the most prevalent species of gram-positive and gram-negative bacteria, respectively, that induce clinical mastitis. The innate immune system comprises the immediate host defense mechanisms to protect against infection and contributes to the initial detection of and proinflammatory response to infectious pathogens. The objective of the present study was to characterize the different innate immune responses to experimental intramammary infection with E. coli and S. aureus during clinical mastitis. The cytokine response and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide-binding protein (LBP), two proteins that contribute to host recognition of bacterial cell wall products, were studied. Intramammary infection with either E. coli or S. aureus elicited systemic changes, including decreased milk output, a febrile response, and induction of the acute-phase synthesis of LBP. Infection with either bacterium resulted in increased levels of interleukin 1β (IL-1β), gamma interferon, IL-12, sCD14, and LBP in milk. High levels of the complement cleavage product C5a and the anti-inflammatory cytokine IL-10 were detected at several time points following E. coli infection, whereas S. aureus infection elicited a slight but detectable increase in these mediators at a single time point. Increases in IL-8 and tumor necrosis factor alpha were observed only in quarters infected with E. coli. Together, these data demonstrate the variability of the host innate immune response to E. coli and S. aureus and suggest that the limited cytokine response to S. aureus may contribute to the well-known ability of the bacterium to establish chronic intramammary infection.     

Staphylococcus aureus isolates from blood and anterior nares induce similar innate immune responses in endothelial cells

To evaluate the possibility to distinguish virulent from non‐virulent isolates, gene expression in human umbilical vein endothelial cells (HUVEC) induced by invasive and colonizing isolates of Staphylococcus aureus was compared. Gene expression in HUVEC was analyzed by microarray analysis after 4 h of infection with Staphylococcus aureus, isolated from healthy nasal carriers (n = 5) and from blood of septic patients (n = 5), to explore possible differences between the groups of bacteria in interaction with HUVEC. All isolates were spa‐typed to disclose strain relatedness. Moreover, the isolates were characterized with DNA microarray to determine the presence of virulence genes and to investigate the potential genes of importance in HUVEC interaction. The expression of 41 genes was up‐regulated, and four were down‐regulated in HUVEC by all isolates. Most of the up‐regulated genes encode cytokines, chemokines, interferon‐induced proteins, proteins regulating apoptosis and cell proliferation. There was no difference in the gene expression pattern between HUVEC infected with invasive or colonizing isolates. Furthermore, there was no difference in the presence of bacterial virulence genes between the two groups. In conclusion, our data indicate that S. aureus isolates induce comparable expression patterns in HUVEC, irrespective of invasiveness or presence of virulence genes.     

Differential induction of complement fragment C5a and inflammatory cytokines during intramammary infections with Escherichia coli and Staphylococcus aureus

The prompt recruitment of neutrophils to the site of infection is essential for the defense of the bovine mammary gland against invading pathogens and is determinant for the outcome of the infection. Escherichia coli is known to induce clinical mastitis, characterized by an intense neutrophil recruitment leading to the eradication of the bacteria, whereas Staphylococcus aureus induces subclinical mastitis accompanied by a moderate neutrophil recruitment and the establishment of chronic mastitis. To elicit the neutrophil recruitment into the udder, inflammatory mediators must be produced after recognition of the invading pathogen. To our knowledge, those mediators have never been studied during S. aureus mastitis, although understanding of the neutrophil recruitment mechanisms could allow a better understanding of the differences in the pathogeneses elicited by E. coli and S. aureus. Therefore, we studied, at several time points, the accumulation of neutrophils and the presence of the chemoattractant complement fragment C5a and of the cytokines interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and IL-8 in milk after inoculation of E. coli or S. aureus in lactating bovine udders. The low levels of C5a and the absence of cytokines in milk from S. aureus-infected cows, compared to the high levels found in milk from E. coli-infected animals, mirror the differences in the severities of the two inflammatory reactions. The cytokine deficit in milk after S. aureus inoculation in the lactating bovine mammary gland could contribute to the establishment of chronic mastitis. This result could help in the design of preventive or curative strategies against chronic mastitis.     

Bacterial genotype affects the manifestation and persistence of bovine Staphylococcus aureus intramammary infection

Two-hundred seventeen Staphylococcus aureus isolates from 116 dairy cows with intramammary infections were analyzed by pulsed-field gel electrophoresis to study the association between symptom severity, persistence of infection, and bacterial genotype. Among five main genotypes infecting 90% of the cows, one was associated with severe clinical symptoms but reduced persistence.     

Cigarette smoking and innate immune responses to influenza infection

Cigarette smoking(CS) suppresses the immune system, and smoking is a well-known major risk factor for respiratory tract infections, including influenza infection. Both smoking cigarettes and passive smoking alter a wide range of immunological functions, including innate and adaptive immune responses. Past reviews on CS and innate immunity have been focused on the effects of CS on structural changes of the lung, as well as the effects on the function of alveolar macrophages, leukocytes, natural killer cells and dendritic cells. The study of innate immunity has developed rapidly in the last decade with the discovery of new receptors for virus recognition and interferon responses. This review aims to give a brief summary of recent findings on the suppressive effects of CS on the innate response to influenza virus, especially as it pertains to suppression of the function of pattern recognition receptors for influ-enza virus.     

Toll-like receptors (TLRs) in innate immune defense against Staphylococcus aureus

Toll-like receptors (TLRs) are the most important class of innate pattern recognition receptors (PRRs) by which host immune and non-immune cells are able to recognize pathogen-associated molecular patterns (PAMPs). Most mammalian species have 10 to 15 types of TLRs. TLRs are believed to function as homo- or hetero-dimers. TLR2, which plays a crucial role in recognizing PAMPs from Staphylococcus aureus, forms heterodimers with TLR1 or TLR6 and each dimer has a different ligand specificity. Staphylococcal lipoproteins, Panton-Valentine toxin and Phenol Soluble Modulins have been identified as potent TLR2 ligands. Conversely, the ligand function attributed to peptidoglycan and LTA remains controversial. TLR2 uses a MyD88-dependent signaling pathway that results in NF-kB translocation into the nucleus and activation of the expression of pro-inflammatory cytokine genes. Recognition rouses both an inflammatory response, culminating in the phagocytosis of bacteria, and an adaptive immune response, with the presentation of resulting bacterial compounds to T cells. Here, recent advances on the recognition of S. aureus by TLRs are presented and discussed, as well as the new therapeutic opportunities deriving from this new knowledge.     

Enhancement of Escherichia coli and Staphylococcus aureus antibiotic susceptibility using sesquiterpenoids

The present work examines the potential of sesquiterpenoids to sensitize Escherichia coli and Staphylococcus aureus, and modulate their susceptibility to the standard antibiotics ciprofloxacin, erythromycin, gentamicin and vancomycin. It was tested samples of three sesquiterpenoids: guaiazulene, nerolidol (racemic mixture of the cis and trans isomers) and germacrene D enriched natural extract. Experiments were conducted aiming to assess the antimicrobial effects of the antibiotic-sesquiterpenoid combination on bacterial growth inhibition, by the disc diffusion assay and the minimum inhibitory concentration (MIC) assessment, the bactericidal effects, the post-antibiotic effect (PAE) and the effect on membrane permeability. The data related with the antimicrobial activity evidenced, through the disc diffusion assay, an antibiotic S. aureus antimicrobial activity enhancement by sesquiterpenoids presence. The MIC value for E. coli decreased significantly by sesquiterpenoids combination with ciprofloxacin, erythromycin and gentamicin, and for S. aureus, with all four selected antibiotics. This combination also increased the PAE, with the exception of guaiazulene, which seemed to quench antibiotic antimicrobial action. A moderate correlation between antimicrobial action and impairment of cell membrane function was detected for germacrene D enriched extract, and nerolidol, as single treatments and in combination with antibiotic, while a poor correlation was obtained for guaiazulene. This study provides basis for the evaluation of sesquiterpenoids as alternative or possible synergistic compounds for current antimicrobial chemotherapeutics, showing the practical utility of natural derived products to increase the susceptibility of E. coli and S. aureus.     

Cell-mediated immune responses in Staphylococcus aureus infections in mice.

Delayed hypersensitivity to staphylococcal antigens was shown in mice repeatedly infected with Staphylococcus aureus. It was characterized by footpad swelling at 48 hours with a mononuclear cell infiltrate and could be transferred to non-infected recipients by T lymphocytes from infected animals, but not by serum. Recipients of immune T cells produced very severe necrotic lesions when challenged with staphylococci. This was in contrast to the protection against necrosis in recipients afforded by serum from infected donors. When both serum and cells were transferred into the same mouse the humoral effects overshadowed or perhaps inhibited those mediated by cells with resultant protection against staphylococcal dermonecrosis.     

Flagellin-dependent and -independent inflammatory responses following infection by enteropathogenic Escherichia coli and Citrobacter rodentium

Enteropathogenic Escherichia coli (EPEC) and the murine pathogen Citrobacter rodentium belong to the attaching and effacing (A/E) family of bacterial pathogens. These noninvasive bacteria infect intestinal enterocytes using a type 3 secretion system (T3SS), leading to diarrheal disease and intestinal inflammation. While flagellin, the secreted product of the EPEC fliC gene, causes the release of interleukin 8 (IL-8) from epithelial cells, it is unclear whether A/E bacteria also trigger epithelial inflammatory responses that are FliC independent. The aims of this study were to characterize the FliC dependence or independence of epithelial inflammatory responses to direct infection by EPEC or C. rodentium. Following infection of Caco-2 intestinal epithelial cells by wild-type and DeltafliC EPEC, a rapid activation of several proinflammatory genes, including those encoding IL-8, monocyte chemoattractant protein 1, macrophage inflammatory protein 3alpha (MIP3alpha), and beta-defensin 2, occurred in a FliC-dependent manner. These responses were accompanied by mitogen-activated protein kinase activation, as well as the Toll-like receptor 5 (TLR5)-dependent activation of NF-kappaB. At later infection time points, a subset of these proinflammatory genes (IL-8 and MIP3alpha) was also induced in cells infected with DeltafliC EPEC. The nonmotile A/E pathogen C. rodentium also triggered similar innate responses through a TLR5-independent but partially NF-kappaB-dependent mechanism. Moreover, the EPEC FliC-independent responses were increased in the absence of the locus of enterocyte effacement-encoded T3SS, suggesting that translocated bacterial effectors suppress rather than cause the FliC-independent inflammatory response. Thus, we demonstrate that infection of intestinal epithelial cells by A/E pathogens can trigger an array of proinflammatory responses from epithelial cells through both FliC-dependent and -independent pathways, expanding our understanding of the innate epithelial response to infection by these pathogens.     

Recombinant soluble CD14 reduces severity of intramammary infection by Escherichia coli

The interaction among gram-negative bacteria, the innate immune system, and soluble CD14 (sCD14) has not been well documented. The effect of recombinant bovine sCD14 (rbosCD14) on milk somatic cell count (SCC), bacterial clearance, and cytokine production was investigated by using a bovine intramammary Escherichia coli infection model. We first determined whether rbosCD14 would increase the SCC during a lipopolysaccharide (LPS) challenge. Three quarters of each of six healthy lactating cows were injected with either 0.3 microg of LPS, 0.3 microg of LPS plus 100 micro g of rbosCD14, or saline. In comparison with quarters injected with LPS alone, the SCC was twofold higher (P < 0.05) in quarters injected with LPS plus rbosCD14 after the challenge. We therefore hypothesized that when E. coli bacteria invade the mammary gland, sCD14 in milk would interact with LPS and rapidly recruit neutrophils from the blood to eliminate the bacteria before establishment of infection. To test this hypothesis, two quarters of each of nine healthy cows were challenged with either 50 CFU of E. coli plus saline or 50 CFU of E. coli plus 100 microg of rbosCD14. Quarters challenged with E. coli plus rbosCD14 had a more rapid recruitment of neutrophils, which was accompanied by a faster clearance of bacteria, lower concentrations of tumor necrosis factor alpha and interleukin-8 in milk, and milder clinical symptoms, than challenged quarters injected with saline. Results indicate that increasing the concentration of sCD14 in milk may be a potential strategy with which to prevent or reduce the severity of infection by coliform bacteria.     

Staphylococcus aureus infection of epidermal keratinocytes promotes expression of innate antimicrobial peptides

Keratinocytes upregulate expression of endogenous antimicrobial peptides in response to inflammatory stimuli. We show that both viable and heat-inactivated Staphylococcus aureus and lipoteichoic acid differentially alter expression of these peptides upon contact with human keratinocytes. The findings indicate a diversity of staphylococcal factors involved in upregulation of antimicrobial peptide expression in cutaneous epithelia.     

Staphylococcus aureus enterotoxin D is secreted in milk and stimulates specific antibody responses in cows in the course of experimental intramammary infection

An enterotoxin D (SED)-producing strain of Staphylococcus aureus was used to infect one mammary gland of each of 17 lactating dairy cows. All glands became infected and shed bacteria over a sampling period of 3 weeks. Serum and milk antibodies specific for SED were monitored by an enzyme-linked immunosorbent assay for 12 weeks. Elevated anti-SED antibodies were detected in all cows after infection, and immunoglobulin of the G2 subclass comprised most of the specific serum response. SED was detected in mastitic milk samples from two cows at levels of 5 to 10 ng/ml. An in vitro lymphocyte proliferation assay showed that SED at levels below 10 pg/ml induced proliferation of bovine lymphocytes and that sheep antiserum specific for SED neutralized this proliferative response. Sera obtained from the cows pre- and postinfection inhibited lymphocyte proliferation at SED concentrations of 10 and 50 ng/ml, respectively. The addition of SED to whole blood or to isolated neutrophils had no significant effect on neutrophil function in vitro. The results show that SED is secreted during mammary gland infection, is mitogenic for bovine lymphocytes, and stimulates the production of specific antibodies.     

The association between lung innate immunity and differential airway antigen- specific immune responses

The mechanisms involved in the differential regulation of airwayimmune responses in atopic versus non-atopic individuals arepoorly understood. In this study, the association between nonspecific immunity and the differential airway antigen-specificImmune responses was examined in a murine model. The disparityIn antigen-specific IgE and IgG2a productions between the twostrains of mice was observed to be significant. C57BL/6J micewere much more efficient than BALB/cJ mice in making IgE antibodyto Inhaled ovalbumin (OVA) antigen. On the contrary, BALB/cJmice did make more IgG2a antibodies than C57BL/6J mice to InhaledOVA. These findings suggest that in C57BL/6J mouse strain apredominant T_h 2 type of Immune response develops in responseto inhaled OVA antigen. In contrast, BALB/c mice mount a T_h1 type of immune response to aerosollzed OVA antigen. Furthermore,after lipopolysaccharlde (LPS) stimulation, the IL-12 mRNA expressionof lung-derived cells from BALB/cJ mice was higher than thatfrom C57BL/6J cells. However, the lung-derived cells of C57BL/6Jmice stimulated by LPS produced higher levels of IL-b and prostaglandinE than BALB/cJ lung-derived cells did. Therefore, our studydemonstrated that the difference of lung-derived cells in theirability to produce cytokine and prostaglandln between BALBIcJand C57BL/6J mice correlates well with the type of the airwayantigen-specific immune effector functions.