Staphylococcus aureus protein A induced inflammatory response in human corneal epithelial cells

In the present study, we examined the role of Staphylococcus aureus protein A (SpA) in inducing inflammatory response in human corneal epithelial cells (HCECs). Exposure of HCECs to SpA induces rapid NF-kappaB activation and secretion of proinflammatory cytokine/chemokines (TNF-alpha and IL-8) in both concentration and time-dependent manner. Challenge of HCECs with live SpA(-/-) mutant S. aureus strains resulted in significantly reduced production of the cytokines when compared to the wild-type S. aureus strain. SpA also elicited the activation of MAP Kinases P38, ERK, but not JNK, in HCECs. SpA-induced production of proinflammatory cytokine were completely blocked by the NF-kappaB and p38 inhibitors and partially inhibited by the Jnk inhibitor. Pretreatment with anti-TLR2 neutralizing antibody had no effect on SpA-induced inflammatory response in HCECs, suggesting that this response is independent of TLR2 signaling. Moreover, unlike TLR2 ligands, SpA failed to induce the expression of antimicrobial peptides (hBD2 and LL-37) in HCECs. These studies indicate that SpA is a S. aureus virulence factor that stimulates HCEC inflammatory response through a pathway distinct from TLR2 in HCECs.     

Tolerance induced by the lipopeptide Pam3Cys is due to ablation of IL-1R-associated kinase-1

Stimulation of the human monocytic cell line Mono Mac 6 with the synthetic lipopeptide (S)-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys(4)-OH, trihydrochloride (Pam(3)Cys) at 10 microg/ml induces a rapid expression of the TNF gene in a TLR2-dependent fashion. Preculture of the cells with Pam(3)Cys at 1 microg/ml leads to a reduced response after subsequent stimulation with Pam(3)Cys at 10 microg/ml, indicating that the cells have become tolerant to Pam(3)Cys. The CD14 and TLR2 expression is not decreased on the surface of the tolerant cells, but rather up-regulated. Analysis of the NF-kappaB binding in Pam(3)Cys-tolerant cells shows a failure to mobilize NF-kappaB-p50p65 heterodimers, while NF-kappaB-p50p50 homodimers remain unchanged. Pam(3)Cys-tolerant cells showed neither IkappaBalpha-Ser(32) phosphorylation nor IkappaBalpha degradation but MyD88 protein was unaltered. However, IRAK-1 protein was absent in Pam(3)Cys-induced tolerance, while IRAK-1 mRNA was still detectable at 30% compared with untreated cells. In contrast, in LPS-tolerized cells, p50p50 homodimers were induced, IRAK-1 protein level was only partially decreased, and p50p65 mobilization remained intact. It is concluded that in Mono Mac 6 monocytic cells, inhibition of IRAK-1 expression at the mRNA and protein levels is the main TLR-2-dependent mechanism responsible for Pam(3)Cys-induced tolerance, but not for TLR-4-dependent LPS-induced tolerance.     

Retinal Photoreceptor Expresses Toll-Like Receptors (TLRs) and Elicits Innate Responses Following TLR Ligand and Bacterial Challenge

Toll-like receptors (TLRs) play an important role in host defense against microbial pathogens. Our previous studies have shown that TLRs are expressed on various retinal cells (Microglia and Müller glia) and orchestrate retinal innate responses in bacterial endophthalmitis. In this study, we used a well-characterized mouse cone photoreceptor cell line (661W); and demonstrated that these cells express all known TLRs. Although the stimulation of 661W cells with TLR ligands (Pam3Cys, PolyI:C, LPS, Flagellin, Poly DT, and ODN) did not alter TLR expression, downstream TLR-signaling pathways (NF-κB, p38, and ERK) are activated. Moreover, TLR-activated 661W cells secreted significant amounts of inflammatory mediators (IL-6, IL-1β, MIP-2, and KC) in their culture supernatant, as assessed by ELISA. A similar trend was observed in 661W cells challenged with live bacteria (Staphylococcus aureus). Interestingly, the neutralization of TLR2, a major receptor for S. aureus recognition, did not significantly attenuate bacterial-induced inflammatory mediators, suggesting the existence of TLR2-independent mechanisms in photoreceptor cells. Together, these results indicate that photoreceptors constitutively express functional TLRs and possess the ability to initiate innate responses following pathogen challenge, implicating their role in retinal innate immunity.     

Flagellin-Induced Corneal Antimicrobial Peptide Production and Wound Repair Involve a Novel NF-κB–Independent and EGFR-Dependent Pathway

Background

The bacterial protein flagellin plays a major role in stimulating mucosal surface innate immune response to bacterial infection and uniquely induces profound cytoprotection against pathogens, chemicals, and radiation. This study sought to determine signaling pathways responsible for the flagellin-induced inflammatory and cytoprotective effects on human corneal epithelial cells (HCECs).

Methodology/Principal Findings

Flagellin purified from Pseudomonas aeruginosa (strain PAK) or live bacteria were used to challenge cultured HCECs. The activation of signaling pathways was assessed with Western blot, and the secretion of cytokine/chemokine and production of antimicrobial peptides (AMPs) were measured with ELISA and dot blot, respectively. Effects of flagellin on wound healing were assessed in cultured porcine corneas. L94A (a site mutation in TLR5 binding region) flagellin and PAK expressing L94A flagellin were unable to stimulate NF-κB activation, but were potent in eliciting EGFR signaling in a TGF-α–related pathway in HCECs. Concomitant with the lack of NF-κB activation, L94A flagellin was ineffective in inducing IL-6 and IL-8 production in HCECs. Surprisingly, the secretion of two inducible AMPs, LL-37 and hBD2, was not affected by L94A mutation. Similar to wild-type flagellin, L94A induced epithelial wound closure in cultured porcine cornea through maintaining EGFR-mediated signaling.

Conclusions/Significance

Our data suggest that inflammatory response mediated by NF-κB can be uncoupled from epithelial innate defense machinery (i.e., AMP expression) and major epithelial proliferation/repair pathways mediated by EGFR, and that flagellin and its derivatives may have broad therapeutic applications in cytoprotection and in controlling infection in the cornea and other mucosal tissues.     

Role of TLR in B cell development: signaling through TLR4 promotes B cell maturation and is inhibited by TLR2

The role of TLR4 in mature B cell activation is well characterized. However, little is known about TLR4 role in B cell development. Here, we analyzed the effects of TLR4 and TLR2 agonists on B cell development using an in vitro model of B cell maturation. Highly purified B220(+)IgM(-) B cell precursors from normal C57BL/6 mouse were cultured for 72 h, and B cell maturation in the presence of the TLR agonists was evaluated by expression of IgM, IgD, CD23, and AA4. The addition of LPS or lipid A resulted in a marked increase in the percentage of CD23(+) B cells, while Pam3Cys had no effect alone, but inhibited the increase of CD23(+) B cell population induced by lipid A or LPS. The TLR4-induced expression of CD23 is not accompanied by full activation of the lymphocyte, as suggested by the absence of activation Ag CD69. Experiments with TLR2-knockout mice confirmed that the inhibitory effects of Pam3Cys depend on the expression of TLR2. We studied the effects of TLR-agonists on early steps of B cell differentiation by analyzing IL-7 responsiveness and phenotype of early B cell precursors: we found that both lipid A and Pam3Cys impaired IL-7-dependent proliferation; however, while lipid A up-regulates B220 surface marker, consistent with a more mature phenotype of the IgM(-) precursors, Pam3Cys keeps the precursors on a more immature stage. Taken together, our results suggest that TLR4 signaling favors B lymphocyte maturation, whereas TLR2 arrests/retards that process, ascribing new roles for TLRs in B cell physiology.     

Expression and regulation of the human beta-defensins hBD-1 and hBD-2 in intestinal epithelium

The intestinal epithelium forms a physical barrier to limit access of enteric microbes to the host and contributes to innate host defense by producing effector molecules against luminal microbes. To further define the role of the intestinal epithelium in antimicrobial host defense, we analyzed the expression, regulation, and production of two antimicrobial peptides, human defensins hBD-1 and hBD-2, by human intestinal epithelial cells in vitro and in vivo. The human colon epithelial cell lines HT-29 and Caco-2 constitutively express hBD-1 mRNA and protein but not hBD-2. However, hBD-2 expression is rapidly induced by IL-1alpha stimulation or infection of those cells with enteroinvasive bacteria. Moreover, hBD-2 functions as a NF-kappaB target gene in the intestinal epithelium as blocking NF-kappaB activation inhibits the up-regulated expression of hBD-2 in response to IL-1alpha stimulation or bacterial infection. Caco-2 cells produce two hBD-1 isoforms and a hBD-2 peptide larger in size than previously described hBD-2 isoforms. Paralleling the in vitro findings, human fetal intestinal xenografts constitutively express hBD-1, but not hBD-2, and hBD-2 expression, but not hBD-1, is up-regulated in xenografts infected intraluminally with Salmonella. hBD-1 is expressed by the epithelium of normal human colon and small intestine, with a similar pattern of expression in inflamed colon. In contrast, there is little hBD-2 expression by the epithelium of normal colon, but abundant hBD-2 expression by the epithelium of inflamed colon. hBD-1 and hBD-2 may be integral components of epithelial innate immunity in the intestine, with each occupying a distinct functional niche in intestinal mucosal defense.     

Extracellular HIV-1 Tat induces human beta-defensin-2 production via NF-kappaB/AP-1 dependent pathways in human B cells

Defensins, a family of antimicrobial peptides, are one of the first lines of host defense. Human beta-defensins (hBD) such as hBD-2 and -3 have anti-HIV activity. Previous studies have shown that HIV-1 virion can induce the expression of hBD, although the exact components of HIV-1 virion that are responsible for hBD expression have not yet been elucidated. In this study, we examined the effect of HIV-1 Tat on the expression of hBD in B cells. Stimulation of B cells with HIV-1 Tat protein significantly increased the mRNA and protein levels of hBD-2. HIV-1 Tat also induced the activation of a reporter gene for hBD-2 in a dose-dependent manner in B cells. Pretreatment of B cells with a JNK inhibitor suppressed HIV-1 Tat-induced hBD-2 expression. Pretreatment of B cells with AP-1 inhibitors or NF-κB inhibitors led to a decrease in HIV-1 Tat-induced protein and mRNA expression of hBD-2. Taken together, our results indicate that HIV-1 Tat can up-regulate the expression of hBD-2 via JNK-NF-κB/AP-1-dependent pathways in human B cells.     

Novel anti-bacterial activities of β-defensin 1 in human platelets: suppression of pathogen growth and signaling of neutrophil extracellular trap formation

Human β-defensins (hBD) are antimicrobial peptides that curb microbial activity. Although hBD's are primarily expressed by epithelial cells, we show that human platelets express hBD-1 that has both predicted and novel antibacterial activities. We observed that activated platelets surround Staphylococcus aureus (S. aureus), forcing the pathogens into clusters that have a reduced growth rate compared to S. aureus alone. Given the microbicidal activity of β-defensins, we determined whether hBD family members were present in platelets and found mRNA and protein for hBD-1. We also established that hBD-1 protein resided in extragranular cytoplasmic compartments of platelets. Consistent with this localization pattern, agonists that elicit granular secretion by platelets did not readily induce hBD-1 release. Nevertheless, platelets released hBD-1 when they were stimulated by α-toxin, a S. aureus product that permeabilizes target cells. Platelet-derived hBD-1 significantly impaired the growth of clinical strains of S. aureus. hBD-1 also induced robust neutrophil extracellular trap (NET) formation by target polymorphonuclear leukocytes (PMNs), which is a novel antimicrobial function of β-defensins that was not previously identified. Taken together, these data demonstrate that hBD-1 is a previously-unrecognized component of platelets that displays classic antimicrobial activity and, in addition, signals PMNs to extrude DNA lattices that capture and kill bacteria.     

Effect of Toll-like receptor 2 and 4 of corneal fibroblasts on cytokine expression with co-cultured antigen presenting cells

Keratocytes are the first component to contact ocular pathogens when the epithelial barrier breaks down and the emerging evidences indicated keratocytes appeared to be one of the corneal cellular immune components. Little is known about the role of Toll-like receptors (TLRs) in keratocytes, although it has been well documented that keratocytes constitutively express various TLRs including TLR2 and TLR4. In this in vitro study, the authors focused on the role of keratocytes in corneal innate immune system and cross-talk of keratocytes with resident antigen presenting cells (APCs), especially through TLR2 and TLR4. Primary cultivated keratocytes (corneal fibroblasts) from C57BL/6 mice per se actively secreted pro-inflammatory cytokines, especially interleukin (IL)-6, with a dose-dependent manner in response to Pam3CSK4 or lipopolysaccharide (LPS) challenge. With co-culture of corneal fibroblasts with APCs per se, secretion of IL-6 and tumor necrosis factor (TNF)-α was markedly increased and it was counterbalanced by concurrent increase in IL-10 and tumor growth factor-β1. After Pam3CSK4 or LPS stimulation, this cytokine balance was completely broken down by overwhelming amplification of IL-6 and TNF-α secretion, especially in co-culture of corneal fibroblasts with macrophages, rather than with dendritic cells. Using corneal fibroblasts from TLR2 or TLR4 knockout mice, we could find the reversal of Pam3CSK4 or LPS-responsive dose-dependent increment in IL-6 and TNF-α. These results implied that corneal fibroblasts and their TLRs could be key components for the ocular homeostasis and pathogen-associated ocular innate immunity.     

The proinflammatory CD14+CD16+DR++ monocytes are a major source of TNF

In human blood two monocyte populations can be distinguished, i.e., the CD14(++)CD16(-)DR(+) classical monocytes and the CD14(+)CD16(+)DR(++) proinflammatory monocytes that account for only 10% of all monocytes. We have studied TNF production in these two types of cells using three-color immunofluorescence and flow cytometry on whole peripheral blood samples stimulated with either LPS or with the bacterial lipopeptide S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys(4)-OH,trihydrochloride (Pam3Cys). After stimulation with LPS the median fluorescence intensity for TNF protein was 3-fold higher in the proinflammatory monocytes when compared with the classical monocytes. After stimulation with Pam3Cys they almost exclusively responded showing 10-fold-higher levels of median fluorescence intensity for TNF protein. The median fluorescence intensity for Toll-like receptor 2 cell surface protein was found 2-fold higher on CD14(+)CD16(+)DR(++) monocytes, which may explain, in part, the higher Pam3Cys-induced TNF production by these cells. When analyzing secretion of TNF protein into the supernatant in PBMCs after depletion of CD16(+) monocytes we found a reduction of LPS-induced TNF by 28% but Pam3Cys-induced TNF was reduced by 64%. This indicates that the minor population of CD14(+)CD16(+) monocytes are major producers of TNF in human blood.     

Extremely low frequency electromagnetic field exposure does not modulate toll-like receptor signaling in human peripheral blood mononuclear cells

The effects of extremely low frequency electromagnetic fields (ELF-EMF) on human health remain unclear. It has been reported that ELF-EMF may modulate the innate immune response to microorganisms in animal models and mammalian cell-lines. With the recently gained insight in innate immune signaling and the discovery of pattern recognition, we aim to study whether ELF-EMF modulates innate inflammatory signaling pathways. We used human peripheral blood mononuclear cells (PBMCs), isolated from blood from healthy volunteers, which we stimulated with specific TLR2 and TLR4 ligands, and with several microorganisms. The cells were subsequently exposed in B(dc)=3 μT to a highly controlled and standardized ELF-EMF signal (20-5000Hz, B(ac)=5 μT, 30 min) and cytokine production was measured at different time points after stimulation. No significant difference in immune response, as reflected by IL-1β, IL-6, TNFα, IL-8 and IL-10 production, could be detected after stimulation with LPS (TLR4 ligand), Pam3Cys (TLR2 ligand) or a panel of heat killed microorganisms: Mycobacterium tuberculosis, Salmonella typhimurium, Candida albicans, Aspergillus fumigatus and Staphylococcus aureus (multiple TLR ligands). We therefore conclude that under our experimental conditions, ELF-EMF does not modulate the innate immune response of human primary cells after TLR stimulation in vitro.     

Legionella pneumophila induces human beta Defensin-3 in pulmonary cells

Background

Legionella pneumophila is an important causative agent of severe pneumonia in humans. Human alveolar epithelium and macrophages are effective barriers for inhaled microorganisms and actively participate in the initiation of innate host defense. The beta defensin-3 (hBD-3), an antimicrobial peptide is an important component of the innate immune response of the human lung. Therefore we hypothesize that hBD-3 might be important for immune defense towards L. pneumophila.

Methods

We investigated the effects of L. pneumophila and different TLR agonists on pulmonary cells in regard to hBD-3 expression by ELISA. Furthermore, siRNA-mediated inhibition of TLRs as well as chemical inhibition of potential downstream signaling molecules was used for functional analysis.

Results

L. pneumophila induced release of hBD-3 in pulmonary epithelium and alveolar macrophages. A similar response was observed when epithelial cells were treated with different TLR agonists. Inhibition of TLR2, TLR5, and TLR9 expression led to a decreased hBD-3 expression. Furthermore expression of hBD-3 was mediated through a JNK dependent activation of AP-1 (c-Jun) but appeared to be independent of NF-κB. Additionally, we demonstrate that hBD-3 elicited a strong antimicrobial effect on L. pneumophila replication.

Conclusions

Taken together, human pulmonary cells produce hBD-3 upon L. pneumophila infection via a TLR-JNK-AP-1-dependent pathway which may contribute to an efficient innate immune defense.     

T and B cells are not required for clearing Staphylococcus aureus in systemic infection despite a strong TLR2-MyD88-dependent T cell activation

Staphylococcus aureus infection elicits through its mature lipoproteins an innate immune response by TLR2-MyD88 signaling, which improves bacterial clearing and disease outcome. The role of dendritic cells (DCs) and T cells in this immune activation and the function of T and B cells in defense against S. aureus infection remain unclear. Therefore, we first evaluated DC and T cell activation after infection with S. aureus wild type (WT) and its isogenic mutant, which is deficient in lipoprotein maturation, in vitro. Lipoproteins in viable S. aureus contributed via TLR2-MyD88 to activation of DCs, which promoted the release of IFN-γ and IL-17 in CD4(+) T cells. This strong effect was independent of superantigens and MHC class II. We next evaluated the function of T cells and their cytokines IFN-γ and IL-17 in infection in vivo. Six days after systemic murine infection IFN-γ, IL-17, and IL-10 production in total spleen cells were MyD88-dependent and their levels increased until day 21. The comparison of CD3(-/-), Rag2(-/-), and C57BL/6 mice after infection revealed that IFN-γ and IL-17 originated from T cells and IL-10 originated from innate immune cells. Furthermore, vaccination of mice to activate T and B cells did not improve eradication of S. aureus from organs. In conclusion, S. aureus enhances DC activation via TLR2-MyD88 and thereby promotes T(H)1 and T(H)17 cell differentiation. However, neither T cells and their MyD88-regulated products, IFN-γ and IL-17, nor B cells affected bacterial clearing from organs and disease outcome.     

Cutting edge: activation of Toll-like receptor 2 induces a Th2 immune response and promotes experimental asthma

Recognition of microbial components by APCs and their activation through Toll-like receptors (TLR) leads to the induction of adaptive immune responses. In this study, we show that activation of TLR2 by its synthetic ligand Pam3Cys, in contrast to activation of TLR9 by immunostimulatory DNA (ISS-ODN), induces a prominent Th2-biased immune response. Activation of APCs by Pam3Cys resulted in the induction of Th2-associated effector molecules like IL-13, and IL-1beta, GM-CSF and up-regulation of B7RP-1, but low levels of Th1-associated cytokines (IL-12, IFNalpha, IL-18, IL-27). Accordingly, TLR2 ligands aggravated experimental asthma. These data indicate that the type of TLR stimulation during the initial phase of immune activation determines the polarization of the adaptive immune response and may play a role in the initiation of Th2-mediated immune disorders, such as asthma.     

The innate defense antimicrobial peptides hBD3 and RNase7 are induced in human umbilical vein endothelial cells by classical inflammatory cytokines but not Th17 cytokines

Antimicrobial peptides are multifunctional effector molecules of innate immunity. In this study we investigated whether endothelial cells actively contribute to innate defense mechanisms by expression of antimicrobial peptides. We therefore stimulated human umbilical vein endothelial cells (HUVEC) with inflammatory cytokines, Th17 cytokines, heat-inactivated bacteria, bacterial conditioned medium (BCM) of Staphylococcus aureus and Streptococcus sanguinis, and lipoteichoic acid (LTA). Stimulation with single cytokines induced discrete expression of human β-defensin 3 (hBD3) by IFN-γ or IL-1β and of ribonuclease 7 (RNase7) by TNF-α without any effects on LL-37 gene expression. Stronger hBD3 and RNase7 induction was observed after combined stimulation with IL-1β, TNF-α and IFN-γ and was confirmed by high hBD3 and RNase7 peptide levels in cell culture supernatants. In contrast, Th17 cytokines or stimulation with LTA did not result in AMP production. Moreover, only BCM of an invasive S. aureus bacteremia isolate induced hBD3 in HUVEC. We conclude that endothelial cells actively contribute to prevent dissemination of pathogens at the blood-tissue-barrier by production of AMPs that exhibit microbicidal and immunomodulatory functions. Further investigations should focus on tissue-specific AMP induction in different endothelial cell types, on pathogen-specific induction patterns and potentially involved pattern-recognition receptors of endothelial cells.     

The human beta-defensins (-1, -2, -3, -4) and cathelicidin LL-37 induce IL-18 secretion through p38 and ERK MAPK activation in primary human keratinocytes

In addition to its physical barrier against invading microorganisms, the skin produces antimicrobial peptides, human beta-defensins (hBDs) and cathelicidin LL-37, that participate in the innate host defense. Because IL-18 is produced by keratinocytes and involved in skin diseases in which hBDs and LL-37 are highly expressed, we hypothesized that these peptides would activate keratinocytes to secrete IL-18. We found that hBD-2, -3, and -4 and LL-37, but not hBD-1, activated normal human keratinocytes to secrete IL-18; this secretion reached peak strength at 3 h. In addition, the combination of peptides resulted in a synergistic effect on IL-18 secretion. We also revealed that hBD-2, -3, and -4 and LL-37 increased IL-18 mRNA expression, and that IL-18 secretion was more enhanced in keratinocytes differentiated in vitro with high Ca2+-containing medium. Furthermore, because IL-18 secretion induced by hBDs and LL-37 could not be suppressed by caspase-1 or caspase family inhibitors, and because these peptides failed to increase caspase-1 activity, we suggest that hBD- and LL-37-induced IL-18 secretion is probably via a caspase-1-independent pathway. To determine the molecular mechanism involved, we demonstrated that IL-18 secretion was through p38 and ERK1/2 MAPK pathways, because the inhibitors of p38 and ERK1/2, but not JNK, almost completely nullified IL-18 secretion. Moreover, hBD-2, -3, and -4 and LL-37 could induce the phosphorylation of p38 and ERK1/2, but not JNK. Thus, the ability of hBDs and LL-37 to induce IL-18 secretion by keratinocytes provides a new mechanism for these peptides in innate immunity and an understanding of their role in the pathogenesis of skin disorders.