Streptococcus mutans Wall‐Associated Protein A Promotes TLR4‐Induced Dendritic Cell Maturation

Dendritic cells orchestrate innate and adaptive immune responses, which are central to establishing efficient responses to vaccination. Wall‐associated protein A (WapA) of Streptococcus mutans was previously used as a vaccine in animal studies for immunization against dental caries. However, as a cell surface protein, whether WapA activates innate immune responses and the effects of WapA on DCs remain unclear. In this study, WapA was cloned into the GST fusion vector pEBG, which can be expressed efficiently in mammalian cells. We found that when added before stimulation with LPS, purified WapA‐GST protein increased TLR4‐induced NF‐κB and MAPK signalling pathway activation. Pretreatment with WapA‐GST also increased LPS‐induced proinflammatory cytokine production by DCs, including IL‐12, IL‐6 and TNF‐α. Furthermore, expression of the DC maturation markers CD80/86, CD40 and MHC II was also increased by WapA pretreatment. These data indicate that WapA is recognized by DCs and promotes DC maturation.     

The cyclin‐dependent kinase inhibitor R‐roscovitine down‐regulates Mcl‐1 to override pro‐inflammatory signalling and drive neutrophil apoptosis

Successful resolution of inflammation requires inflammatory cells such as neutrophils to undergo apoptosis prior to non‐inflammatory phagocytosis by professional phagocytes. Recently, cyclin‐dependent kinase (CDK) inhibitors (e.g. R‐roscovitine) have been shown to induce neutrophil apoptosis and enhance the resolution of inflammation. Interestingly, NF‐κB and MAPK pathways and key endogenous survival proteins (typified by Mcl‐1) are involved in the regulation of neutrophil apoptosis and, in cancer‐cell lines, have been implicated as possible targets of CDK inhibitors. Here, we demonstrate that R‐roscovitine over‐rides TNF‐α and LPS‐induced survival (determined by morphological examination and binding of fluorescently labelled annexin‐V) of isolated peripheral blood neutrophils. This effect did not appear to be mediated via effects on early markers of neutrophil activation (e.g. surface marker expression, shape change, aggregation and superoxide anion generation), by direct inhibition of NF‐κB activation (assessed by cytoplasmic IκBα proteolysis and NF‐κB p65 subunit translocation) and ERK activation (determined by specific ERK phosphorylation) but due to down‐regulation (at protein and mRNA level) of the survival protein Mcl‐1 but not the pro‐apoptotic bcl‐2 homologue Bim. These findings suggest that key endogenous survival proteins may be the targets of CDK inhibitors and consequently may be of critical importance in the resolution of inflammation.     

Children with atopic histories exhibit impaired lipopolysaccharide‐induced Toll‐like receptor‐4 signalling in peripheral monocytes

Background The hygiene hypothesis states that early exposure to bacterial products such as lipopolysaccharide (LPS) may be protective against the development of allergic diseases. Whether atopic disease affects the ability of immune cells to respond to LPS is unclear. Our laboratory has demonstrated previously that children express high levels of Toll‐like receptor (TLR)‐4 on CD4⁺ cells in nasal mucosa. Objective To determine if children with a history of allergic disease have impaired responses to LPS on circulating CD4⁺ leucocytes. Methods Peripheral blood mononuclear cells from children (aged 2–18) and adults with or without a history of atopic conditions were cultured with/without IL‐4 or LPS for up to 24 h. Expression of surface TLR‐4, CD14, CD4, CD3, as well as of intracellular phosphorylated ^p42/p44ERK and p38 mitogen‐activated protein kinase (MAPK) were assessed by flow cytometry. Results A history of atopy in children was associated with impaired LPS‐induced TLR‐4‐dependent phosphorylation of ^p42/44ERK and p38 MAPK by CD4⁺ monocytes. Decreased LPS signalling was reproduced by pre‐incubation of control cells with recombinant IL‐4. LPS stimulation also decreased TLR‐4 expression on monocytes from children without atopic histories but not from atopic subjects. CD4⁺ T lymphocytes showed limited LPS responsiveness, regardless of atopic status. In contrast with non‐atopic children, TLR‐4 expression on monocytes of children with atopic histories decreased as a function of age. Conclusions This study provides evidence for defective LPS recognition on circulating CD4⁺ leucocytes of subjects with atopic histories compared with those from non‐atopic children. CD4⁺TLR4⁺ monocytes from children with atopic histories failed to phosphorylate MAPKs. Our results suggest that a history of atopic disease is associated with impaired TLR‐4‐mediated innate immune function compared with non‐atopic children. Cite this as: D. Préfontaine, A.‐A. Banville‐Langelier, P.‐O. Fiset, J. Guay, J. An, M. Mazer, Q. Hamid and B. D. Mazer, Clinical & Experimental Allergy, 2010 (40) 1648–1657.     

Fentanyl Suppresses the Survival of CD4+ T Cells Isolated from Human Umbilical Cord Blood through Inhibition of IKKs‐mediated NF‐κB Activation

The aim of this study was to investigate the effects and the underlying mechanisms of fentanyl anaesthetic on T lymphocytes isolated from human umbilical cord blood in vitro. The percentages of CD4⁺, CD8⁺ and regulatory T (Treg) cells in human umbilical cord blood mononuclear cells (UBMC) treated with fentanyl in vitro were analysed by flow cytometry. The levels of cytokines IFN‐γ, IL‐2, IL‐4 and IL‐17 secreted by activated CD4⁺ T cells were measured by ELISA assays. Expressions of MAPK and NF‐κB signalling pathway proteins were determined by Western blotting. Effects of fentanyl on IKK and p65 expression promoter activities were analysed by luciferase assay. Fentanyl decreased the percentages and amounts of CD4⁺, CD8⁺ and Foxp3⁺Treg T lymphocyte subsets in UBMCs in a dose‐dependent manner. Fentanyl inhibited the proliferation and induced apoptosis of activated CD4⁺ T cells dose dependently. Fentanyl could not reverse the increase of cell proliferation in activated groups to be equivalent with those in inactivated group. Secretions of IFN‐γ, IL‐2 and IL‐4 cytokines were significantly decreased by moderate to high dose of fentanyl compared with controls. No significant differences were observed in protein expressions of MAPK pathway. In addition, fentanyl suppressed the IKKs‐mediated activation of NF‐κB. This study demonstrates that fentanyl exerts immunosuppressive effects on T lymphocytes obtained from UBMCs. Thus, the clinical application of fentanyl would not only relieve pain caused by surgery but regulate immune responses post‐operation possibly through inhibition of IKKs‐mediated NF‐κB activation.     

The interaction between DC and Plasmodium berghei/chabaudi‐infected erythrocytes in mice involves direct cell‐to‐cell contact,internalization and TLR

Early interactions between blood‐stage Plasmodium parasites and cells of the innate immune system are very important in shaping the adaptive immune response to malaria, and a number of studies have suggested that DC are responsible for this phenomenon. Therefore, we examined the capacity of murine BM‐derived DC to internalize parasites, be activated and produce cytokines upon in vitro interaction with murine erythrocytes infected with two different strains of rodent malaria parasites (Plasmodium berghei and Plasmodium chabaudi chabaudi). We show that the increased expression of MHC class II and co‐stimulatory molecules and increased production of cytokines by DC following Plasmodium infection involves internalization of infected RBC. Such DC activation not only requires direct cell‐to‐cell contact and internalization of infected RBC by DC but also involves TLR4, TLR9, MyD88 and signaling via NF‐κB; however, TLR involvement in survival to Plasmodium infection was found to be negligible.     

Triggering of DC migration by dengue virus stimulation of COX‐2‐dependent signaling cascades in vitro highlights the significance of these cascades beyond inflammation

A term “bone‐breaking fever” is used in Chinese medicine to describe the symptoms of patients infected with dengue virus (DV). We examined the significance of the COX‐prostaglandin pathway in human DC infected by DV. We show that DV infection induced the expression of COX‐2 and the production of prostaglandin E₂ (PGE₂) in DC, and stimulated the DNA binding of NF‐κB and the kinase activity of both IκBα kinase (IKK) α and β. DV infection also activated MAPK and AP‐1 signaling. Both IκBα kinase‐NF‐κB and MAPK‐AP‐1 were upstream of COX‐2 activation. Our investigation into the significance of COX‐2‐PGE₂ pathway also revealed that DV infection enhances DC migration by inducing CC chemokine receptor 7 (CCR7) expression, and that blocking COX‐2 or MAPK activity suppresses DV‐induced DC migration. Our data also suggest that PGE₂ can induce CCR7 expression on DC and that antagonists of the PGE₂ receptors EP2 and EP4 suppress DV‐induced DC migration. We further show that the increased CCR7 expression was observed in both DV‐infected and bystander DC, suggesting the presence of secondary effects in inducing CCR7 expression. Collectively, this study reveals not only the pathways involved in COX‐2 synthesis in DV‐infected DC but also the autocrine action of PGE₂ on the migration of DV‐infected DC.     

Caspase‐11 non‐canonical inflammasome: a critical sensor of intracellular lipopolysaccharide in macrophage‐mediated inflammatory responses

Inflammatory responses mediated by macrophages are part of the innate immune system, whose role is to protect against invading pathogens. Lipopolysaccharide (LPS) found in the outer membrane of Gram‐negative bacteria stimulates an inflammatory response by macrophages. During the inflammatory response, extracellular LPS is recognized by Toll‐like receptor 4, one of the pattern recognition receptors that activates inflammatory signalling pathways and leads to the production of inflammatory mediators. The innate immune response is also triggered by intracellular inflammasomes, and inflammasome activation induces pyroptosis and the secretion of pro‐inflammatory cytokines such as interleukin‐1β (IL‐1β) and IL‐18 by macrophages. Cysteine‐aspartic protease (caspase)‐11 and the human orthologues caspase‐4/caspase‐5 were recently identified as components of the ‘non‐canonical inflammasome’ that senses intracellular LPS derived from Gram‐negative bacteria during macrophage‐mediated inflammatory responses. Direct recognition of intracellular LPS facilitates the rapid oligomerization of caspase‐11/4/5, which results in pyroptosis and the secretion of IL‐1β and IL‐18. LPS is released into the cytoplasm from Gram‐negative bacterium‐containing vacuoles by small interferon‐inducible guanylate‐binding proteins encoded on chromosome 3 (GBP^chr3)‐mediated lysis of the vacuoles. In vivo studies have clearly shown that caspase‐11^−/− mice are more resistant to endotoxic septic shock by excessive LPS challenge. Given the evidence, activation of caspase‐11 non‐canonical inflammasomes by intracellular LPS is distinct from canonical inflammasome activation and provides a new paradigm in macrophage‐mediated inflammatory responses.     

Rituximab‐mediated Raf kinase inhibitor protein induction modulates NF‐κB in Sjögren syndrome

Primary Sjögren syndrome (pSS) is an autoimmune disorder characterized by an epithelial injury surrounded by dense lymphocytic infiltrates. The conditions for the long‐term maintenance of human salivary gland epithelial cells from pSS patients and a co‐culture system with pSS lymphocytes were used to assess the effect of Rituximab (RTX) on the inflammatory condition and progression in pSS. Quantitative real‐time PCR, genes and protein array analysis, Western blot, flow cytometry, small interfering RNA transfection and nuclear factor‐κB (NF‐κB) DNA binding assays were used as methods. Supporting the benefits of RTX, this study demonstrates that RTX decreases NF‐κB activity and interrupts the NF‐κB signalling pathway through the up‐regulation of the Raf‐1 kinase inhibitor protein (RKIP). Over‐expression of RKIP down‐regulates interleukins, their receptors and the expression of genes encodes proteins that attracted lymphocytes. Silencing of the RKIP gene leads to significantly increased expression and release of pro‐inflammatory mediators supporting that RKIP expression could be involved in the suppression of NF‐κB activation in pSS salivary gland epithelial cells.     

Lipopolysaccharide‐primed heterotolerant dendritic cells suppress experimental autoimmune uveoretinitis by multiple mechanisms

Exposure of bone‐marrow‐derived dendritic cells (BMDC) to high‐dose ultrapure lipopolysaccharide for 24 hr (LPS‐primed BMDC) enhances their potency in preventing inter‐photoreceptor retinoid binding protein: complete Freund's adjuvant‐induced experimental autoimmune uveoretinitis (EAU). LPS‐primed BMDC are refractory to further exposure to LPS (= endotoxin tolerance), evidenced here by decreased phosphorylation of TANK‐binding kinase 1, interferon regulatory factor 3 (IRF3), c‐Jun N‐terminal kinase and p38 mitogen‐activated protein kinase as well as impaired nuclear translocation of nuclear factor κB (NF‐κB) and IRF3, resulting in reduced tumour necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), IL‐12 and interferon‐β secretion. LPS‐primed BMDC also show reduced surface expression of Toll‐like receptor‐4 and up‐regulation of CD14, followed by increased apoptosis, mediated via nuclear factor of activated T cells (NFATc)‐2 signalling. LPS‐primed BMDC are not only homotolerant to LPS but are heterotolerant to alternative pathogen‐associated molecular pattern ligands, such as mycobacterial protein extract (Mycobacterium tuberculosis). Specifically, while M. tuberculosis protein extract induces secretion of IL‐1β, TNF‐α and IL‐6 in unprimed BMDC, LPS‐primed BMDC fail to secrete these cytokines in response to M. tuberculosis. We propose that LPS priming of BMDC, by exposure to high doses of LPS for 24 hr, stabilizes their tolerogenicity rather than promoting immunogenicity, and does so by multiple mechanisms, namely (i) generation of tolerogenic apoptotic BMDC through CD14:NFATc signalling; (ii) reduction of NF‐κB and IRF3 signalling and downstream pro‐inflammatory cytokine production; and (iii) blockade of inflammasome activation.     

Toll‐like receptor‐mediated eosinophil–basophil differentiation: autocrine signalling by granulocyte–macrophage colony‐stimulating factor in cord blood haematopoietic progenitors

Eosinophils are multi‐functional leucocytes that play a role in inflammatory processes including allergy and infection. Although bone marrow (BM) inflammatory cells are the main source of eosinophil‐basophil (Eo/B) differentiation‐inducing cytokines, a recent role has been demonstrated for cytokine induction through Toll‐like receptor (TLR)‐mediated signalling in BM progenitors. Having previously demonstrated that cord blood (CB) progenitors induce Eo/B colony‐forming units (CFU) after lipopolysaccharide (LPS) stimulation, we sought to investigate the intracellular mechanisms by which LPS induces Eo/B differentiation. Freshly isolated CD34‐enriched human CB cells were stimulated with LPS (and/or pharmacological inhibitors) and assessed for alterations in haematopoietic cytokine receptor expression and signalling pathways by flow cytometry, Eo/B CFU in methylcellulose cultures, and cytokine secretion using Luminex assays. The LPS stimulation resulted in a significant increase in granulocyte–macrophage colony‐stimulating factor (GM‐CSF)‐responsive, as opposed to interleukin‐5‐responsive, Eo/B CFU, which also correlated with significant increases in CD34⁺ cell GM‐CSFRα expression. Functionally, CB CD34⁺ cells secrete abundant amounts of GM‐CSF following LPS stimulation, via a p38 mitogen‐activated protein kinase (MAPK)‐dependent mechanism; this secretion was responsible for Eo/B CFU formation ex vivo, as shown by antibody blockade. We show for the first time that LPS stimulation of CB progenitor cells results in autocrine activation of p38 MAPK‐dependent GM‐CSF secretion facilitating Eo/B differentiation ex vivo. This work provides evidence that early life exposure to products of bacterial agents can modulate Eo/B differentiation, representing a novel mechanism by which progenitor cells can respond to microbial stimuli and so affect immune and inflammatory responses.     

Inactivation of TMEM106A promotes lipopolysaccharide‐induced inflammation via the MAPK and NF‐κB signaling pathways in macrophages

Pattern recognition receptors, such as Toll‐like receptors (TLRs), play an important role in the host defense against invading microbial pathogens. Their activation must be precisely regulated, as inappropriate activation or overactivation of TLR signaling pathways may result in inflammatory disorders, such as septic shock or autoimmune diseases. TMEM106A is a type II transmembrane protein constitutively expressed in macrophages. Our current study demonstrated that TMEM106A levels were increased in macrophages upon lipopolysaccharide (LPS) stimulation, as well as in the peripheral monocytes of patients with sepsis. Tmem106a knockout mice were more sensitive to lipopolysaccharide (LPS)‐induced septic shock than wild‐type mice. Further experiments indicated that Tmem106a ablation enhanced the expression of CD80, CD86 and major histocompatibility complex (MHC)‐II in mouse macrophages upon LPS stimulation, accompanied with up‐regulation of tumor necrosis factor (TNF)‐α, interleukin (IL)‐6, interferon (IFN)‐β and inducible nitric oxide synthase (iNOS), indicating the activation of macrophages and polarization towards the M1 inflammatory phenotype. Moreover, elevated mitogen‐activated protein kinase (MAPK) and nuclear factor kappa B (NF‐κB) signaling were found to be involved in the LPS‐induced inflammatory response in Tmem106a^?/? macrophages. However, this effect was largely abrogated by macrophage deletion in Tmem106a^?/? mice. Therefore, deficiency of Tmem106a in macrophages may enhance the M1 polarization in mice, resulting in inflammation. This suggests that TMEM106A plays an important regulatory role in maintaining macrophage homeostasis.     

Surfactant protein D inhibits mite-induced alveolar macrophage and dendritic cell activations through TLR signalling and DC-SIGN expression

Background Surfactant protein D (SP‐D), a secreted pattern recognition molecule associated with pulmonary innate immunity, has been shown to mediate the clearance of pathogens in multiple ways. However, how SP‐D interacts with alveolar macrophages (AMs) and dendritic cells (DCs) during allergen exposure remains unclear. Objective This study was performed to characterize the immunomodulatory effects of SP‐D on mite allergen (Dermatophagoides pteronyssinus, Der p)‐induced inflammatory signalling in AMs and DCs. Methods Murine AM, alveolar macrophage cell line derived from BALB/c mice (MH‐S cells), and human monocyte‐derived dendritic cells (MDDC) were used as model systems. The production of nitric oxide (NO) and TNF‐α, expression of surface Toll‐like receptors (TLRs), and expression of the C‐type lectin receptor known as dendritic cell (DC)‐specific ICAM‐grabbing non‐integrin (DC‐SIGN) were measured as a function of pretreatment with SP‐D and subsequent exposure to Der p. Der p‐dependent cellular activations that were modified by SP‐D in these model systems were then identified. Results Pretreatment of MH‐S cells with SP‐D reduced Der p‐dependent production of NO, TNF‐α, and the downstream activations of IL‐1 receptor‐associated kinase, mitogen activated protein kinase (MAPK) kinase, and nuclear factor‐κB. SP‐D interacted with CD14 such that CD14 binding to Der p was inhibited and Der p‐induced signalling via TLRs was blocked. DC‐SIGN expression was suppressed by Der p in MH‐S and MDDC; this down‐regulation of DC‐SIGN expression was prevented by pretreatment with SP‐D. Conclusions These results indicated that the inhibition of Der p‐induced activation of MH‐S and MDDC by SP‐D is mediated through suppression of the CD14/TLR signalling pathway and maintenance of DC‐SIGN expression, which may protect allergen‐induced airway inflammation. Cite this as: C‐F Liu, M. Rivere, H‐J Huang, G. Puzo and J‐Y Wang, Clinical & Experimental Allergy, 2010 (40) 111–122.     

5,7‐dihydroxy‐3,4,6‐trimethoxyflavone inhibits the inflammatory effects induced by Bacteroides fragilis enterotoxin via dissociating the complex of heat shock protein 90 and IκBα and IκB kinase‐γ in intestinal epithelial cell culture

Enterotoxin produced by enterotoxigenic Bacteroides fragilis (BFT) has been associated with mucosal inflammation and diarrhoeal diseases. In this study, the anti‐inflammatory molecular mechanism of 5,7‐dihydroxy‐3,4,6‐trimethoxyflavone (eupatilin) was characterized in an HT‐29 intestinal epithelial cell line stimulated with BFT. Pre‐treatment of HT‐29 cells with eupatilin decreased the production significantly of both interleukin (IL)‐8 and prostaglandin E₂ induced by BFT in a dose‐dependent manner. BFT‐activated nuclear factor‐kappaB (NF‐κB) signals in HT‐29 cells and pretreatment with eupatilin suppressed NF‐κB activation that resulted in the significant inhibition of IL‐8 and cyclo‐oxygenase‐2 expression. BFT‐induced phosphorylation of both IκBα and IκB kinase (IKK) signals was prevented in eupatilin‐pretreated HT‐29 cells. Transfection of siRNA for IKK‐α and IKK‐β decreased the production of IL‐8 and prostaglandin E₂; however, the transfection of IKK‐β siRNA showed a more significant reduction of BFT‐induced IκBα phosphorylation compared with that of IKK‐α siRNA. In addition, herbimycin A, a specific inhibitor of heat shock protein 90 (Hsp90), decreased the BFT‐induced activation of IKK and NF‐κB, suggesting that Hsp90 is associated with a pathway of IKK‐NF‐κB‐IL‐8/cyclo‐oxygenase‐2 gene signalling. Furthermore, eupatilin dissociated the complex between Hsp90 and IKK‐γ in BFT‐stimulated HT‐29 cells. These results suggest that eupatilin can suppress the NF‐κB signalling pathway by targeting the Hsp90‐IKK‐γ complex in intestinal epithelial cells and may attenuate BFT‐induced inflammatory responses.     

Dengue virus up‐regulates expression of notch ligands Dll1 and Dll4 through interferon‐β signalling pathway

The Notch signalling pathway is involved in multiple cellular processes and has been recently indicated to modulate the host immune response. However, the role of the Notch pathway in dengue virus (DENV) infection remains unknown. Our study has screened the expression profile of Notch receptors, ligands and target genes in human monocytes, macrophages and dendritic cells in response to DENV infection. The real‐time PCR data showed that Notch ligand Dll1 was significantly induced in DENV‐infected monocytes; and receptor Notch4, ligands Dll1 and Dll4, and target Hes1 were dramatically enhanced in DENV‐infected macrophages and dendritic cells. In macrophages, induction of Dll1 and Dll4 mediated by DENV2 was increased by treatment with interferon‐β (IFN‐β), and was impaired by neutralization of IFN‐β. The DENV‐induced Dll1 and Dll4 expression level was decreased by silencing key innate immune molecules, including Toll‐like receptor 3 (TLR3), MyD88, RIG‐I and IPS‐I. In IFN‐receptor‐depleted macrophages, the Dll1 and Dll4 induction was significantly alleviated. Functionally, activation of Notch signalling by Dll1 in CD4⁺ T cells enhanced the expression of a T helper type 1 (Th1) cytokine IFN‐γ, while Notch activation in macrophages had no direct effect on replication of DENV. Our data revealed that the expressions of Notch ligands in antigen‐presenting cells were differentially induced by DENV via innate immune signalling, which is important for Th1/Th2 differentiation during adaptive immune response.     

Increased interleukin (IL)‐20 and IL‐24 target osteoblasts and synovial monocytes in spondyloarthritis

The pathogenesis of spondyloarthritis (SpA) involves activation of the innate immune system, inflammation and new bone formation. The two cytokines interleukin (IL)‐20 and IL‐24 have been shown to link innate immune activation and tissue homeostasis. We hypothesized that these two cytokines are secreted as part of activation of the innate immune system and affect bone homeostasis in SpA. IL‐20 and IL‐24 were measured in plasma from axial SpA patients (n = 83). Peripheral SpA patients (n = 16) were included for in‐vitro cell culture studies. The plasma IL‐20 and IL‐24 levels were increased in SpA patients compared with healthy controls (HCs) by 57 and 83%, respectively (both P < 0·0001). The Toll‐like receptor 4‐induced secretion of the two cytokines was greater in SpA peripheral blood mononuclear cells (PBMCs) compared with HC PBMCs. IL‐20 and IL‐24 increased the production of monocyte chemoattractant protein‐1 by activated SpA synovial fluid monocytes, decreased the production of Dickkopf‐1 by SpA fibroblast‐like synovial cells and induced mineralization in human osteoblasts. Taken together, our findings indicate disease‐aggravating functions of IL‐20 and IL‐24 in SpA.     

Molecular mechanisms of the secretion of cytokines and chemokines from human monocytes activated by pneumococcal surface protein A (PspA): Roles of mitogen-activated protein kinases and NF-kappaB

Pneumococcal surface protein A (PspA) plays a key role in the pathogenesis of invasive pneumococcal infection. PspA might modulate specific immune responses in human population. Circulating monocytes are essential for the innate responses and subsequent acquired immune responses to Streptococcus pneumoniae. In this study, we investigated the effects of PspA on cytokine and chemokine secretion from human peripheral blood monocytes and the underlying intracellular signaling mechanisms. Stimulation of monocytes with purified PspA protein induced the significant release of inflammatory cytokine IL-6 and chemokines including CXCL8, CCL2, CCL4 and CCL5. Products from PspA-deficient mutant pneumococcus that did not express PspA induced significantly less secretion of these mediators than those from wild type pneumococcus. Further investigations showed that PspA activated the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen activated protein kinase (MAPK) and nuclear factor (NF)-κB signaling pathways in human monocytes. Moreover, inhibition of these pathways using selective inhibitors could significantly reduce the cytokine and chemokine secretion induced by PspA. Taken together, our findings provide insight for PspA-mediated activation of human monocytes via NF-κB and MAPKs signaling cascades in the pathogenesis of invasive pneumococcal infection.