Analysis of the neuroinflammatory response to TLR7 stimulation in the brain: comparison of multiple TLR7 and/or TLR8 agonists

Activation of astrocytes and microglia and the production of proinflammatory cytokines and chemokines are often associated with virus infection in the CNS as well as a number of neurological diseases of unknown etiology. These inflammatory responses may be initiated by recognition of pathogen-associated molecular patterns (PAMPs) that stimulate TLRs. TLR7 and TLR8 were identified as eliciting antiviral effects when stimulated by viral ssRNA. In the present study, we examined the potential of TLR7 and/or TLR8 agonists to induce glial activation and neuroinflammation in the CNS by intracerebroventricular inoculation of TLR7 and/or TLR8 agonists in newborn mice. The TLR7 agonist imiquimod induced astrocyte activation and up-regulation of proinflammatory cytokines and chemokines, including IFN-beta, TNF, CCL2, and CXCL10. However, these responses were only of short duration when compared with responses induced by the TLR4 agonist LPS. Interestingly, some of the TLR7 and/or TLR8 agonists differed in their ability to activate glial cells as evidenced by their ability to induce cytokine and chemokine expression both in vivo and in vitro. Thus, TLR7 stimulation can induce neuroinflammatory responses in the brain, but individual TLR7 agonists may differ in their ability to stimulate cells of the CNS.     

CX3CL1 expression induced by Candida albicans in oral fibroblasts

Oral fibroblasts as well as keratinocytes are thought to influence host inflammatory responses against Candida albicans. However, little is known about chemokine expressions in oral fibroblasts against C. albicans infection. We therefore examined whether C. albicans induced several chemokines including fractalkine/CX3CL1 (CX3CL1), a unique chemokine that has properties of both chemoattractants and adhesion molecules, in fibroblasts and keratinocytes. The addition of C. albicans live cells to human immortalized oral keratinocytes (RT7) resulted in increases in the mRNA levels of multiple chemokines, but not of CX3CL1. In contrast, live and heat-killed C. albicans caused an increase in CX3CL1 mRNA and protein expression in human immortalized oral fibroblasts (GT1). CX3CL1 mRNA expression in GT1 cells was also enhanced by stimulation with a nonalbicans species of Candida. Further, the CX3CL1 chemokine domain showed antifungal activity against C. albicans. CX3CL1 secreted by oral fibroblasts appears to play an important role in the oral immune response to C. albicans infection.     

TLR3, TLR4 and TLRs7–9 Induced Interferons Are Not Impaired in Airway and Blood Cells in Well Controlled Asthma

Defective Rhinovirus induced interferon-β and interferon-λ production has been reported in bronchial epithelial cells from asthmatics but the mechanisms of defective interferon induction in asthma are unknown. Virus infection can induce interferon through Toll like Receptors (TLR)3, TLR7 and TLR8. The role of these TLRs in interferon induction in asthma is unclear. This objective of this study was to measure the type I and III interferon response to TLR in bronchial epithelial cells and peripheral blood cells from atopic asthmatics and non-atopic non-asthmatics. Bronchial epithelial cells and peripheral blood mononuclear cells from atopic asthmatic and non-atopic non-asthmatic subjects were stimulated with agonists to TLR3, TLR4 & TLRs7–9 and type I and III interferon and pro-inflammatory cytokine, interleukin(IL)-6 and IL-8, responses assessed. mRNA expression was analysed by qPCR. Interferon proteins were analysed by ELISA. Pro-inflammatory cytokines were induced by each TLR ligand in both cell types. Ligands to TLR3 and TLR7/8, but not other TLRs, induced interferon-β and interferon-λ in bronchial epithelial cells. The ligand to TLR7/8, but not those to other TLRs, induced only type I interferons in peripheral blood mononuclear cells. No difference was observed in TLR induced interferon or pro-inflammatory cytokine production between asthmatic and non-asthmatic subjects from either cell type. TLR3 and TLR7/8,, stimulation induced interferon in bronchial epithelial cells and peripheral blood mononuclear cells. Interferon induction to TLR agonists was not observed to be different in asthmatics and non-asthmatics.     

Cutting edge: TLR2-mediated proinflammatory cytokine and chemokine production by microglial cells in response to herpes simplex virus

Recent studies indicate that TLRs are critical in generating innate immune responses during infection with HSV-1. In this study, we investigated the role of TLR2 signaling in regulating the production of neuroimmune mediators by examining cytokine and chemokine expression using primary microglial cells obtained from TLR2-/- as well as wild-type mice. Data presented here demonstrate that TLR2 signaling is required for the production of proinflammatory cytokines and chemokines: TNF-alpha, IL-1beta, IL-6, IL-12, CCL7, CCL8, CCL9, CXCL1, CXCL2, CXCL4, and CXCL5. CXCL9 and CXCL10 were also induced by HSV, but their production was not dependent upon TLR2 signaling. Because TLR2-/- mice display significantly reduced mortality and diminished neuroinflammation in response to brain infection with HSV, the TLR2-dependent cytokines identified here might function as key players influencing viral neuropathogenesis.     

Muramyldipeptide and diaminopimelic acid-containing desmuramylpeptides in combination with chemically synthesized Toll-like receptor agonists synergistically induced production of interleukin-8 in a NOD2- and NOD1-dependent manner, respectively, in human monocytic cells in culture

Two types of synthetic peptidoglycan fragments, diaminopimelic acid (DAP)-containing desmuramylpeptides (DMP) and muramyldipeptide (MDP), induced secretion of interleukin (IL)-8 in a dose-dependent manner in human monocytic THP-1 cells, although high concentrations of compounds are required as compared with chemically synthesized Toll-like receptor (TLR) agonists mimicking bacterial components: TLR2 agonistic lipopeptide (Pam3CSSNA), TLR4 agonistic lipid A (LA-15-PP) and TLR9 agonistic bacterial CpG DNA. We found marked synergistic IL-8 secretion induced by MDP or DAP-containing DMP in combination with synthetic TLR agonists in THP-1 cells. Suppression of the mRNA expression of nucleotide-binding oligomerization domain (NOD)1 and NOD2 by RNA interference specifically inhibited the synergistic IL-8 secretion induced by DMP and MDP with these TLR agonists respectively. In accordance with the above results, enhanced IL-8 mRNA expression and the activation of nuclear factor (NF)-kappaB induced by MDP or DMP in combination with synthetic TLR agonists were markedly suppressed in NOD2- and NOD1-silenced cells respectively. These findings indicated that NOD2 and NOD1 are specifically responsible for the synergistic effects of MDP and DMP with TLR agonists, and suggested that in host innate immune responses to invading bacteria, combinatory dual signalling through extracellular TLRs and intracellular NODs might lead to the synergistic activation of host cells.     

Effect of the antimicrobial peptide LL-37 on Toll-like receptors 2-, 3- and 4-triggered expression of IL-6, IL-8 and CXCL10 in human gingival fibroblasts

The antimicrobial peptide LL-37 is known to have a potent LPS-neutralizing activity in monocytes and macrophages. Recently, LL-37 in gingival crevicular fluids is suggested to be the major protective factor preventing infection of periodontogenic pathogens. In this study, we tried to address the effect of LL-37 on proinflammatory responses of human gingival fibroblasts (HGFs) stimulated with Toll-like receptor (TLR)-stimulant microbial compounds. LL-37 potently suppressed LPS-induced gene expression of IL6, IL8 and CXCL10 and intracellular signaling events, degradation of IRAK-1 and IκBα and phosphorylation of p38 MAPK and IRF3, indicating that the LPS-neutralizing activity is also exerted in HGFs. LL-37 also suppressed the expression of IL6, IL8 and CXCL10 induced by the TLR3 ligand poly(I:C). LL-37 modestly attenuated the expression of IL6 and IL8 induced by the TLR2/TLR1 ligand Pam₃CSK₄, but did not affect the expression induced by the TLR2/TLR6 ligand MALP-2. Interestingly, LL-37 rather upregulated the expression of IL6, IL8 and CXCL10 induced by another TLR2/TLR6 ligand FSL-1. Thus, the regulatory effect of LL-37 is differently exerted towards proinflammatory responses of HGFs induced by different microbial stimuli, which may lead to unbalanced proinflammatory responses of the gingival tissue to infection of oral microbes.     

Interleukin‐8 induction due to diffusely adherent Escherichia coli possessing Afa/Dr genes depends on flagella and epithelial Toll‐like receptor 5

DAEC is considered potentially diarrheagenic. For diffuse adhesion, the role of the Afa, which was originally identified as a uropathogenic factor, is now understood. However, the role of DAEC in diarrheal disease remains controversial because DAEC is often isolated not only from patients but also from healthy individuals. Previously, we suggested that Afa/Dr DAEC, which can induce high levels of IL‐8 secretion in cultures of human carcinoma epithelial cells (HEp‐2, Caco‐2), is enterovirulent. In the present study, we examined whether IL‐8 secretion induced by certain Afa/Dr DAEC strains was primarily due to flagella via TLR5. All IL‐8 high‐inducing strains were highly motile in swarming tests. Partially purified flagella induced IL‐8 in a dose‐dependent manner. However, IL‐8 induction was inhibited by small‐interfering RNA against TLR5 or by treating flagella with disialoganglioside‐GD1a, a TLR5 blocker. TLR5 is reportedly located on the basolateral side of intestinal epithelia; flagella should not have reached TLR5 from the apical side beyond tight junctions. Reduction in the number of intracellular organisms by wortmannin, a PI3K inhibitor, did not reduce IL‐8 secretion. Afa/Dr DAEC seemed to loosen the tight junctions because it quickly reduced transepithelial electrical resistance after infection. Decreased resistance led to increased IL‐8 production. In conclusion, diffuse adhesion itself is insufficient to induce high levels of IL‐8, and simultaneous stimulation by flagella via TLR5 is likely required for additional induction. Clinically, high motility may be a candidate criterion for predicting the ability of Afa/Dr DAEC strains to induce higher levels of IL‐8 secretion.     

Commensal and pathogenic biofilms differently modulate peri‐implant oral mucosa in an organotypic model

The impact of oral commensal and pathogenic bacteria on peri‐implant mucosa is not well understood, despite the high prevalence of peri‐implant infections. Hence, we investigated responses of the peri‐implant mucosa to Streptococcus oralis or Aggregatibacter actinomycetemcomitans biofilms using a novel in vitro peri‐implant mucosa‐biofilm model. Our 3D model combined three components, organotypic oral mucosa, implant material, and oral biofilm, with structural assembly close to native situation. S. oralis induced a protective stress response in the peri‐implant mucosa through upregulation of heat shock protein (HSP70) genes. Attenuated inflammatory response was indicated by reduced cytokine levels of interleukin‐6 (IL‐6), interleukin‐8 (CXCL8), and monocyte chemoattractant protein‐1 (CCL2). The inflammatory balance was preserved through increased levels of tumor necrosis factor‐alpha (TNF‐α). A. actinomycetemcomitans induced downregulation of genes important for cell survival and host inflammatory response. The reduced cytokine levels of chemokine ligand 1 (CXCL1), CXCL8, and CCL2 also indicated a diminished inflammatory response. The induced immune balance by S. oralis may support oral health, whereas the reduced inflammatory response to A. actinomycetemcomitans may provide colonisation advantage and facilitate later tissue invasion. The comprehensive characterisation of peri‐implant mucosa‐biofilm interactions using our 3D model can provide new knowledge to improve strategies for prevention and therapy of peri‐implant disease.     

Tonsil-derived mesenchymal stromal cells produce CXCR2-binding chemokines and acquire follicular dendritic cell-like phenotypes under TLR3 stimulation

We previously isolated mesenchymal stromal cells from human tonsils (T-MSCs) and showed the potential of these cells to differentiate into the mesodermal lineage and acquire a follicular dendritic cell (FDC) phenotype under cytokine stimulation. Because these T-MSCs were originally isolated from inflamed tonsillar tissues, we were curious about their activation status in response to innate immune stimuli, such as Toll-like receptors (TLRs). Therefore, we analyzed the expression profile of TLRs in T-MSCs and stimulated the T-MSCs with TLR agonists. TLR3 stimuli induced C–C chemokine receptor type 6 expression in T-MSCs after 24 h. Furthermore, results from cytokine arrays showed increases in epithelial neutrophil-activating peptide-78/C-X-C motif chemokine (CXCL) 5, granulocyte chemotactic protein-2/CXCL6, growth-related oncogene-α/CXCL1, interleukin-8/CXCL8, and interferon gamma-induced protein-10/CXCL10. CD54 expression was also increased after TLR3 stimulation. However, co-culturing T-MSCs with human B cells did not induce B-cell proliferation. This suggests that TLR3 stimulates the differentiation of T-MSCs into FDC-like cells and induces chemokine secretion, possibly by recruiting C–X–C chemokine receptor 2-expressing immune cells. In addition, T-MSCs also appeared to exert immunomodulatory effects by inhibiting B-cell proliferation, possibly by down-regulating CD18.     

Signaling through toll-like receptor 7/8 induces the differentiation of human bone marrow CD34+ progenitor cells along the myeloid lineage

Toll-like receptors (TLRs) play a key role in pathogen recognition and regulation of the innate and adaptive immune responses. Although TLR expression and signaling have been investigated in blood cells, it is currently unknown whether their bone marrow ancestors express TLRs and respond to their ligands. Here we found that TLRs (e.g. TLR4, TLR7 and TLR8) were expressed by freshly isolated human bone marrow (BM) hematopoietic CD34+ progenitor cells. Incubation of these primitive cells with TLR ligands such as immunostimulatory small interfering RNAs and R848, a specific ligand for TLR7/8, induced cytokine production (e.g. IL1-beta, IL6, IL8, TNF-alpha, GM-CSF). Moreover, TLR7/8 signaling induced the differentiation of BM CD34+ progenitors into cells with the morphology of macrophages and monocytic dendritic precursors characterized by the expression of CD13, CD14 and/or CD11c markers. By contrast, R848 ligand did not induce the expression of glycophorin A, an early marker for erythropoiesis. Collectively, the data indicate for the first time that human BM CD34+ progenitor cells constitutively express functional TLR7/TLR8, whose ligation can induce leukopoiesis without the addition of any exogenous cytokines. Thus, TLR signaling may regulate BM cell development in humans.     

Lipopolysaccharide directly stimulates aldosterone production via toll‐like receptor 2 and toll‐like receptor 4 related PI3K/Akt pathway in rat adrenal zona glomerulosa cells

The level of circulating endotoxin is related to the severity of cardiovascular disease. One of the indexes for the prognosis of cardiovascular disease is the plasma aldosterone level. Recently, the Toll‐like receptors (TLRs), lipopolysaccharide (LPS)‐regulated receptors, were found not only to mediate the inflammatory response but also to be important in the adrenal stress response. Whether LPS via TLRs induced aldosterone production in adrenal zona glomerulosa (ZG) cells was not clear. Our results suggest that LPS‐induced aldosterone secretion in a time‐ and dose‐dependent manner and via TLR2 and TLR4 signaling pathway. Administration of LPS can enhance steroidogenesis enzyme expression such as scavenger receptor‐B1 (SR‐B1), steroidogenic acute regulatory protein (StAR) and P450 side chain cleavage (P450scc) enzyme. LPS‐induced SR‐B1 and StAR protein expression are abolished by TLR2 blocker. Furthermore, we demonstrated that phosphorylation of Akt was elevated by LPS treatment and reduced by TLR2 blockers, TLR4 blockers, and LY294002 (PI₃K inhibitor). Those inhibitors of PI₃K/Akt pathways also abolish LPS‐induced aldosterone secretion and SR‐B1 protein level. In conclusion, LPS‐induced aldosterone production and SR‐B1 proteins expression are through the TLR2 and TLR4 related PI₃K/Akt pathways in adrenal ZG cells. J. Cell. Biochem. 111: 872–880, 2010. © 2010 Wiley‐Liss, Inc.     

Nod1/RICK and TLR signaling regulate chemokine and antimicrobial innate immune responses in mesothelial cells

Mesothelial cells that line the serous cavities and outer surface of internal organs are involved in inflammatory responses induced by microbial stimuli and bacterial infection. Upon exposure to bacterial products, mesothelial cells secrete chemokines, but the signaling pathways by which these cells recognize bacteria to mediate innate immune responses remain largely unknown. We report that stimulation of primary peritoneal mesothelial cells via nucleotide-binding oligomerization domain (Nod)1, a member of the intracytoplasmic Nod-like receptor family, induced potent secretion of the chemokines CXCL1 and CCL2 as well as expression of inducible NO synthase and such responses required the kinase RICK. Mesothelial cells also produced chemokines in response to TLR2, TLR3, TLR4, and TLR5 agonists, but unlike that induced by Nod1 stimulation, the TLR-mediated responses were independent of RICK. Yet, Nod1 stimulation of mesothelial cells via RICK enhanced chemokine secretion induced by LPS or IFN-gamma and cooperated with IFN-gamma in the production of NO. The i.p. administration of KF1B, a synthetic Nod1 agonist, elicited chemokine production in the serum and peritoneal fluid as well as the recruitment of neutrophils into the peritoneal cavity of wild-type mice, but not RICK-deficient mice. Finally, infection of mesothelial cells with Listeria monocytogenes induced production of CXCL1 and this response was significantly reduced in Nod1- or RICK-deficient cells. These results define mesothelial cells as microbial sensors through TLRs and Nod-like receptors and identify Nod1 and RICK as important mediators of chemokine and antimicrobial responses in mesothelial cells.     

Poly-thymidine oligonucleotides mediate activation of murine glial cells primarily through TLR7, not TLR8

The functional role of murine TLR8 in the inflammatory response of the central nervous system (CNS) remains unclear. Murine TLR8 does not appear to respond to human TLR7/8 agonists, due to a five amino acid deletion in the ectodomain. However, recent studies have suggested that murine TLR8 may be stimulated by alternate ligands, which include vaccinia virus DNA, phosphothioate oligodeoxynucleotides (ODNs) or the combination of phosphothioate poly-thymidine oligonucleotides (pT-ODNs) with TLR7/8 agonists. In the current study, we analyzed the ability of pT-ODNs to induce activation of murine glial cells in the presence or absence of TLR7/8 agonists. We found that TLR7/8 agonists induced the expression of glial cell activation markers and induced the production of multiple proinflammatory cytokines and chemokines in mixed glial cultures. In contrast, pT-ODNs alone induced only low level expression of two cytokines, CCL2 and CXCL10. The combination of pT-ODNs along with TLR7/8 agonists induced a synergistic response with substantially higher levels of proinflammatory cytokines and chemokines compared to CL075. This enhancement was not due to cellular uptake of the agonist, indicating that the pT-ODN enhancement of cytokine responses was due to effects on an intracellular process. Interestingly, this response was also not due to synergistic stimulation of both TLR7 and TLR8, as the loss of TLR7 abolished the activation of glial cells and cytokine production. Thus, pT-ODNs act in synergy with TLR7/8 agonists to induce strong TLR7-dependent cytokine production in glial cells, suggesting that the combination of pT-ODNs with TLR7 agonists may be a useful mechanism to induce pronounced glial activation in the CNS.     

Innate immune responses to Mycobacterium ulcerans via toll-like receptors and dectin-1 in human keratinocytes

Mycobacterium ulcerans (MU), an environmental pathogen, causes Buruli ulcer, a severe skin disease. We hypothesized that epidermal keratinocytes might not be a simple barrier, but play a role during MU infection through pattern-recognition receptors expressed in keratinocytes. We found that keratinocyte Toll-like receptors (TLRs) 2 and 4 and Dectin-1 actively participate in the innate immune response to MU, which includes the internalization of bacteria, the production of reactive oxygen species (ROS), and the expression of chemokines and LL-37. Human keratinocytes constitutively expressed TLRs 2 and 4 and induced Dectin-1 in response to MU. Exposing keratinocytes to MU resulted in rapid ROS production, which in turn contributed to the mRNA and protein expression of LL-37. In addition, TLR2, Dectin-1 and, to an extent, TLR4 are essential for the MU-mediated expression of CXCL8, CCL2 and LL-37 in keratinocytes. Furthermore, confocal analysis showed that the Dectin-1 is necessary for keratinocytes to internalize bacilli. Importantly, blockade of ROS and LL-37 significantly increased the intracellular MU growth in keratinocytes, suggesting an important role of these mediators for cutaneous innate immune responses. Our results demonstrate that TLR2, TLR4 and Dectin-1 actively sense, internalize and respond in an innate way to MU in human epidermal keratinocytes.     

Glucosamine inhibits IL‐1β‐mediated IL‐8 production in prostate cancer cells by MAPK attenuation

Inflammation is a complex process involving cytokine production to regulate host defense cascades. In contrast to the therapeutic significance of acute inflammation, a pathogenic impact of chronic inflammation on cancer development has been proposed. Upregulation of inflammatory cytokines, such as IL‐1β and IL‐8, has been noted in prostate cancer patients and IL‐8 has been shown to promote prostate cancer cell proliferation and migration; however, it is not clear whether IL‐1β regulates IL‐8 expression in prostate cancer cells. Glucosamine is widely regarded as an anti‐inflammatory agent and thus we hypothesized that if IL‐1β activated IL‐8 production in prostate cancer cells, then glucosamine ought to blunt such an effect. Three prostate cancer cell lines, DU‐145, PC‐3, and LNCaP, were used to evaluate the effects of IL‐1β and glucosamine on IL‐8 expression using ELISA and RT‐PCR analyses. IL‐1β elevated IL‐8 mRNA expression and subsequent IL‐8 secretion. Glucosamine significantly inhibited IL‐1β‐induced IL‐8 secretion. IL‐8 appeared to induce LNCaP cell proliferation by MTT assay; involvement of IL‐8 in IL‐1β‐dependent PC‐3 cell migration was demonstrated by wound‐healing and transwell migration assays. Inhibitors of MAPKs and NFκB were used to pinpoint MAPKs but not NFκB being involved in IL‐1β‐mediated IL‐8 production. IL‐1β‐provoked phosphorylation of all MAPKs was notably suppressed by glucosamine. We suggest that IL‐1β can activate the MAPK pathways resulting in an induction of IL‐8 production, which promotes prostate cancer cell proliferation and migration. In this context, glucosamine appears to inhibit IL‐1β‐mediated activation of MAPKs and therefore reduces IL‐8 production; this, in turn, attenuates cell proliferation/migration. J. Cell. Biochem. 108: 489–498, 2009. © 2009 Wiley‐Liss, Inc.     

The functional effects of physical interactions among Toll-like receptors 7, 8, and 9

Toll-like receptors (TLRs) TLR1, TLR2, TLR4, and TLR6 are evolutionarily conserved, highly homologous, and localized to plasma membranes of host cells and recognize pathogen-associated molecular patterns (PAMPs) derived from bacterial membranes. These receptors cooperate in a pairwise combination to elicit or inhibit the inflammatory signals in response to certain PAMPs. The other TLRs that are evolutionarily closely related and highly homologous are TLR7, TLR8, and TLR9. They are all confined to the membranes of endosomes and recognize similar molecular structures, the oligonucleotide-based PAMPs. However, the cooperative interactions among these receptors that may modulate the inflammatory signaling in response to their cognate agonists are not reported. We report here for the first time the functional effects of one TLR on the other among TLR7, TLR8, and TLR9. The results indicate that TLR8 inhibits TLR7 and TLR9, and TLR9 inhibits TLR7 but not vice versa in HEK293 cells transfected with TLRs in a pairwise combination. This is concluded by selectively activating one TLR over the other by using small molecule TLR agonists. We also show that these inhibitory interactions are the result of direct or indirect physical interactions between the TLRs. The murine TLR8 that does not respond to any known human TLR8 agonists also inhibits both murine and human TLR7. The implications of the inhibitory interactions among these TLRs in host-pathogen recognition and subsequent inflammatory responses are not obvious. However, given the complexity in expression pattern in a particular cell type and the variation in distribution and response to different pathogens and stress signals in different cell types, the inhibitory physical interactions among these TLRs may play a role in balancing the inflammatory outcome from a given cell type to a specific challenge.     

Pam3CSK4 and LTA-TLRs ligands associated with microdomains induce IL8 production in human adrenocortical cancer cells.

Bacterially derived ligands, Pam3CSK4 and LPS, can directly impact adrenal glands steroidogenesis through microdomain-related TLR1/2 and 4, respectively, and indirectly via immune cell-derived cytokines. The bilateral immunoadrenal relationship plays an important role in the proper functioning of both systems. CXC chemokine-dependent immune cell infiltration into adrenocortical carcinomas (ACC), which correlates with poor prognosis, is a common phenomenon. Recently, IL8 was identified in ACC and NCI-H295R cells, and was found to contribute to ACC tumour growth. The aim of this study was to clarify the role of different TLR ligands in IL8 production in NCI-H295R cells. This is the first study to demonstrate the expression of several TLRs including TLR1, 3, 6, 7 and 9 in human adrenocortical cells by using the RT-PCR approach. Only stimulation with TLR1/6 together with TLR2 ligands resulted in IL8 peptide and mRNA induction in a dose and time-dependent manner. Our data suggest that gram-positive bacteria-related TLR1/2/6 ligands might contribute to adrenal gland tumorigenesis via IL8 production.