The MyD88-dependent, but not the MyD88-independent, pathway of TLR4 signaling is important in clearing nontypeable haemophilus influenzae from the mouse lung

TLRs are important for the recognition of conserved motifs expressed by invading bacteria. TLR4 is the signaling receptor for LPS, the major proinflammatory component of the Gram-negative cell wall, whereas CD14 serves as the ligand-binding part of the LPS receptor complex. Triggering of TLR4 results in the activation of two distinct intracellular pathways, one that relies on the common TLR adaptor MyD88 and one that is mediated by Toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF). Nontypeable Haemophilus influenzae (NTHi) is a common Gram-negative respiratory pathogen that expresses both TLR4 (LPS and lipooligosaccharide) and TLR2 (lipoproteins) ligands. To determine the roles of CD14, TLR4, and TLR2 during NTHi pneumonia, the following studies were performed: 1) Alveolar macrophages from CD14 and TLR4 knockout (KO) mice were virtually unresponsive to NTHi in vitro, whereas TLR2 KO macrophages displayed a reduced NTHi responsiveness. 2) After intranasal infection with NTHi, CD14 and TLR4 KO mice showed an attenuated early inflammatory response in their lungs, which was associated with a strongly reduced clearance of NTHi from the respiratory tract; in contrast, in TLR2 KO mice, lung inflammation was unchanged, and the number of NTHi CFU was only modestly increased at the end of the 10-day observation period. 3) MyD88 KO, but not TRIF mutant mice showed an increased bacterial load in their lungs upon infection with NTHi. These data suggest that the MyD88-dependent pathway of TLR4 is important for an effective innate immune response to respiratory tract infection caused by NTHi.     

Priming effect of lipopolysaccharide on acetyl-coenzyme A:lyso-platelet-activating factor acetyltransferase is MyD88 and TRIF independent

LPS has a priming effect on various stimuli. For instance, LPS priming enhances the production of platelet-activating factor (PAF), a proinflammatory lipid mediator that is induced by PAF itself. Among various enzymes responsible for PAF biosynthesis, acetyl-coenzyme A:1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase is one of the enzymes activated by PAF receptor stimulation. In this study we investigated the priming effect of LPS on the acetyltransferase activation by PAF in TLR4-knockout (KO) mice, MyD88-KO mice, and Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF)-KO mice. This enzyme was biphasically activated by LPS. Although the first peak occurred within 30 min in wild-type (WT), but not TLR4-KO or MyD88-KO, macrophages, the second phase reached a maximum within hours in WT, MyD88-KO, and TRIF-KO, but not in TLR4-KO, macrophages. Only in the second phase was the increase in acetyltransferase activity upon PAF receptor activation remarkably enhanced in WT, MyD88-KO, and TRIF-KO cells, but not in TLR4-KO cells. These data demonstrated that LPS exerted a priming effect on PAF receptor-mediated acetyltransferase activation through the TLR4-dependent, but MyD88- and TRIF-independent, pathway.     

Cutting edge: TLR9 and TLR2 signaling together account for MyD88-dependent control of parasitemia in Trypanosoma cruzi infection

Activation of innate immune cells by Trypanosoma cruzi-derived molecules such as GPI anchors and DNA induces proinflammatory cytokine production and host defense mechanisms. In this study, we demonstrate that DNA from T. cruzi stimulates cytokine production by APCs in a TLR9-dependent manner and synergizes with parasite-derived GPI anchor, a TLR2 agonist, in the induction of cytokines by macrophages. Compared with wild-type animals, T. cruzi-infected Tlr9(-/-) mice displayed elevated parasitemia and decreased survival. Strikingly, infected Tlr2(-/-)Tlr9(-/-) mice developed a parasitemia equivalent to animals lacking MyD88, an essential signaling molecule for most TLR, but did not show the acute mortality displayed by MyD88(-/-) animals. The enhanced susceptibility of Tlr9(-/-) and Tlr2(-/-)Tlr9(-/-) mice was associated with decreased in vivo IL-12/IFN-gamma responses. Our results reveal that TLR2 and TLR9 cooperate in the control of parasite replication and that TLR9 has a primary role in the MyD88-dependent induction of IL-12/IFN-gamma synthesis during infection with T. cruzi.     

MyD88-Dependent Signaling Influences Fibrosis and Alternative Macrophage Activation during Staphylococcus aureus Biofilm Infection

Bacterial biofilms represent a significant therapeutic challenge based on their ability to evade host immune and antibiotic-mediated clearance. Recent studies have implicated IL-1β in biofilm containment, whereas Toll-like receptors (TLRs) had no effect. This is intriguing, since both the IL-1 receptor (IL-1R) and most TLRs impinge on MyD88-dependent signaling pathways, yet the role of this key adaptor in modulating the host response to biofilm growth is unknown. Therefore, we examined the course of S. aureus catheter-associated biofilm infection in MyD88 knockout (KO) mice. MyD88 KO animals displayed significantly increased bacterial burdens on catheters and surrounding tissues during early infection, which coincided with enhanced dissemination to the heart and kidney compared to wild type (WT) mice. The expression of several proinflammatory mediators, including IL-6, IFN-γ, and CXCL1 was significantly reduced in MyD88 KO mice, primarily at the later stages of infection. Interestingly, immunofluorescence staining of biofilm-infected tissues revealed increased fibrosis in MyD88 KO mice concomitant with enhanced recruitment of alternatively activated M2 macrophages. Taken in the context of previous studies with IL-1β, TLR2, and TLR9 KO mice, the current report reveals that MyD88 signaling is a major effector pathway regulating fibrosis and macrophage polarization during biofilm formation. Together these findings represent a novel example of the divergence between TLR and MyD88 action in the context of S. aureus biofilm infection.     

Liposomal Lipopolysaccharide Initiates TRIF-Dependent Signaling Pathway Independent of CD14

Lipopolysaccharide (LPS) is recognized by CD14 with Toll-like receptor 4 (TLR4), and initiates 2 major pathways of TLR4 signaling, the MyD88-dependent and TRIF-dependent signaling pathways. The MyD88-dependent pathway induces inflammatory responses such as the production of TNF-α, IL-6, and IL-12 via the activation of NFκB and MAPK. The TRIF-dependent pathway induces the production of type-I IFN, and RANTES via the activation of IRF-3 and NFκB, and is also important for the induction of adaptive immune responses. CD14 plays a critical role in initiating the TRIF-dependent signaling pathway response to LPS, to support the internalization of LPS via endocytosis. Here, we clearly demonstrate that intracellular delivery of LPS by LPS-formulated liposomes (LPS-liposomes) initiate only TRIF-dependent signaling via clathrin-mediated endocytosis, independent of CD14. In fact, LPS-liposomes do not induce the production of TNF-α and IL-6 but induce RANTES production in peritoneal macrophages. Additionally, LPS-liposomes could induce adaptive immune responses effectively in CD14-deficient mice. Collectively, our results strongly suggest that LPS-liposomes are useful as a TRIF-dependent signaling-based immune adjuvant without inducing unnecessary inflammation.     

The IFN-inducible GTPase LRG47 (Irgm1) negatively regulates TLR4-triggered proinflammatory cytokine production and prevents endotoxemia

LRG47/Irgm1, a 47-kDa IFN-inducible GTPase, plays a major role in regulating host resistance as well as the hemopoietic response to intracellular pathogens. LRG47 expression in macrophages has been shown previously to be stimulated in vitro by bacterial LPS, a TLR4 ligand. In this study, we demonstrate that induction of LRG47 by LPS is not dependent on MyD88 signaling, but rather, requires STAT-1 and IFN-beta. In addition, LRG47-deficient mice are highly susceptible to LPS, but not TLR2 ligand-induced shock, an outcome that correlates with enhanced proinflammatory cytokine production in vitro and in vivo. Further analysis revealed that LPS-stimulated LRG47-deficient macrophages display enhanced phosphorylation of p38, a downstream response associated with TLR4/MyD88 rather than IFN-beta/STAT-1 signaling. In contrast, LPS-induced phosphorylation of IFN regulatory factor-3 and expression of IFN-beta or the type I IFN-regulated genes, CCL5 and CCL10, were unaltered in LRG47(-/-) cells. Together, these observations indicate that in LPS-stimulated murine macrophages LRG47 is induced by IFN-beta and negatively regulates TLR4 signaling to prevent excess proinflammatory cytokine production and shock. Thus, our findings reveal a new host-protective function for this GTPase in the response to pathogenic encounter.     

Both hemopoietic and resident cells are required for MyD88-dependent pulmonary inflammatory response to inhaled endotoxin

Inhaled endotoxin induces an inflammatory response that contributes to the development and severity of asthma and other forms of airway disease. Here, we show that inhaled endotoxin-induced acute bronchoconstriction, TNF, IL-12p40, and KC production, protein leak, and neutrophil recruitment in the lung are abrogated in mice deficient for the adaptor molecule MyD88. Bronchoconstriction, inflammation, and protein leak are normal in Toll/IL-1R domain-containing adaptor inducing IFN-beta-deficient mice. MyD88 is involved in TLR, but also in IL-1R-associated kinase 1-mediated IL-1R and -18R signaling. We exclude a role for IL-1 and IL-18 pathways in this response, as IL-1R1 and caspase-1 (ICE)-deficient mice develop lung inflammation while TLR4-deficient mice are unresponsive to inhaled LPS. Significantly, using bone marrow chimera, we demonstrate that both hemopoietic and resident cells are necessary for a full MyD88-dependent response to inhaled endotoxin; bronchoconstriction depends on resident cells while cytokine secretion is mediated by hemopoietic cells.     

MyD88-dependent and MyD88-independent pathways in synergy, priming, and tolerance between TLR agonists

TLRs sense components of microorganisms and are critical host mediators of inflammation during infection. Different TLR agonists can profoundly alter inflammatory effects of one another, and studies suggest that the sequence of exposure to TLR agonists may importantly impact on responses during infection. We tested the hypothesis that synergy, priming, and tolerance between TLR agonists follow a pattern that can be predicted based on differential engagement of the MyD88-dependent (D) and the MyD88-independent (I) intracellular signaling pathways. Inflammatory effects of combinations of D and I pathway agonists were quantified in vivo and in vitro. Experiments used several D-specific agonists, an I-specific agonist (poly(I:C)), and LPS, which acts through both the D and I pathways. D-specific agonists included: peptidoglycan-associated lipoprotein, Pam3Cys, flagellin, and CpG DNA, which act through TLR2 (peptidoglycan-associated lipoprotein and Pam3Cys), TLR5, and TLR9, respectively. D and I agonists were markedly synergistic in inducing cytokine production in vivo in mice. All of the D-specific agonists were synergistic with poly(I:C) in vitro in inducing TNF and IL-6 production by mouse bone marrow-derived macrophages. Pretreatment of bone marrow-derived macrophages with poly(I:C) led to a primed response to subsequent D-specific agonists and vice versa, as indicated by increased cytokine production, and increased NF-kappaB translocation. Pretreatment with a D-specific agonist augmented LPS-induced IFN-beta production. All D-specific agonists induced tolerance to one another. Thus, under the conditions studied here, simultaneous and sequential activation of both the D and I pathways causes synergy and priming, respectively, and tolerance is induced by agonists that act through the same pathway.     

Cigarette smoke-induced pulmonary inflammation is TLR4/MyD88 and IL-1R1/MyD88 signaling dependent

Acute cigarette smoke exposure of the airways (two cigarettes twice daily for three days) induces acute inflammation in mice. In this study, we show that airway inflammation is dependent on Toll-like receptor 4 and IL-1R1 signaling. Cigarette smoke induced a significant recruitment of neutrophils in the bronchoalveolar space and pulmonary parenchyma, which was reduced in TLR4-, MyD88-, and IL-1R1-deficient mice. Diminished neutrophil influx was associated with reduced IL-1, IL-6, and keratinocyte-derived chemokine levels and matrix metalloproteinase-9 activity in the bronchoalveolar space. Further, cigarette smoke condensate (CSC) induced a macrophage proinflammatory response in vitro, which was dependent on MyD88, IL-1R1, and TLR4 signaling, but not attributable to LPS. Heat shock protein 70, a known TLR4 agonist, was induced in the airways upon smoke exposure, which probably activates the innate immune system via TLR4/MyD88, resulting in airway inflammation. CSC-activated macrophages released mature IL-1beta only in presence of ATP, whereas CSC alone promoted the TLR4/MyD88 signaling dependent production of IL-1alpha and pro-IL-1beta implicating cooperation between TLRs and the inflammasome. In conclusion, acute cigarette exposure results in LPS-independent TLR4 activation, leading to IL-1 production and IL-1R1 signaling, which is crucial for cigarette smoke induced inflammation leading to chronic obstructive pulmonary disease with emphysema.     

CpG DNA-mediated induction of acute liver injury in D-galactosamine-sensitized mice: the mitochondrial apoptotic pathway-dependent death of hepatocytes

Unmethylated CpG motifs present in bacterial DNA (CpG DNA) induce innate inflammatory responses, including rapid induction of proinflammatory cytokines. Although innate inflammatory responses induced by CpG DNA and other pathogen-associated molecular patterns are essential for the eradication of infectious microorganisms, excessive activation of innate immunity is detrimental to the host. In this study, we demonstrate that CpG DNA, but not control non-CpG DNA, induces a fulminant liver failure with subsequent shock-mediated death by promoting massive apoptotic death of hepatocytes in D-galactosamine (D-GalN)-sensitized mice. Inhibition of mitochondrial membrane permeability transition pore opening or caspase 9 activity in vivo protects D-GalN-sensitized mice from the CpG DNA-mediated liver injury and death. CpG DNA enhanced production of proinflammatory cytokines in D-GalN-sensitized mice via a TLR9/MyD88-dependent pathway. In addition, CpG DNA failed to induce massive hepatocyte apoptosis and subsequent fulminant liver failure and death in D-GalN-sensitized mice that lack TLR9, MyD88, tumor necrosis factor (TNF)-alpha, or TNF receptor I but not interleukin-6 or -12p40. Taken together, our results provide direct evidence that CpG DNA induces a severe acute liver injury and shock-mediated death through the mitochondrial apoptotic pathway-dependent death of hepatocytes caused by an enhanced production of TNF-alpha through a TLR9/MyD88 signaling pathway in D-GalN-sensitized mice.     

Absence of TRIF signaling in lipopolysaccharide-stimulated murine mast cells

In macrophages, two signaling pathways, dependent on MyD88 or TIR domain-containing adaptor-inducing IFN-β (TRIF) signaling, emanate from the LPS receptor TLR4/MD-2. In this study, we show that in murine bone marrow-derived mast cells (BMMCs), only the MyD88-dependent pathway is activated by LPS. The TRIF signaling branch leading both to NF-κB activation and enhanced proinflammatory cytokine production, as well as to IRF3 activation and subsequent IFN-β production, is absent in LPS-stimulated BMMCs. IRF3 activation is also absent in peritoneal mast cells from LPS-injected mice. We observed strongly diminished TRAM expression in BMMCs, but overexpression of TRAM only moderately enhanced IL-6 and did not boost IFN-β responses to LPS in these cells. A combination of very low levels of TRAM and TLR4/MD-2 with the known absence of membrane-bound CD14 are expected to contribute to the defective TRIF signaling in mast cells. We also show that, unlike in macrophages, in BMMCs the TRIF-dependent and -independent IFN-αβ responses to other recognized IFN inducers (dsRNA, adenovirus, and B-DNA) are absent. These results show how the response to the same microbial ligand using the same receptor can be regulated in different cell types of the innate immune system.     

Cross talk between MyD88 and focal adhesion kinase pathways

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in signaling downstream of integrins, linking bacterial detection, cell entry, and initiation of proinflammatory response through MAPKs and NF-kappaB activation. In this study, using protein I/II from Streptococcus mutans as a model activator of FAK, we investigated the potential link between FAK and TLR pathways. Using macrophages from TLR- or MyD88-deficient mice, we report that MyD88 plays a major role in FAK-dependent protein I/II-induced cytokine release. However, response to protein I/II stimulation was independent of TLR4, TLR2, and TLR6. The data suggest that there is a cross talk between FAK and MyD88 signaling pathways. Moreover, MyD88-dependent, LPS-induced IL-6 secretion by human and murine fibroblasts required the presence of FAK, confirming that MyD88 and FAK pathways are interlinked.     

Mice lacking myeloid differentiation factor 88 display profound defects in host resistance and immune responses to Mycobacterium avium infection not exhibited by Toll-like receptor 2 (TLR2)- and TLR4-deficient animals

To assess the role of Toll-like receptor (TLR) signaling in host resistance to Mycobacterium avium infection, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88), as well as TLR2(-/-) and TLR4(-/-) animals, were infected with a virulent strain of M. avium, and bacterial burdens and immune responses were compared with those in wild-type (WT) animals. MyD88(-/-) mice failed to control acute and chronic M. avium growth and succumbed 9-14 wk postinfection. Infected TLR2(-/-) mice also showed increased susceptibility, but displayed longer survival and lower bacterial burdens than MyD88(-/-) animals, while TLR4(-/-) mice were indistinguishable from their WT counterparts. Histopathological examination of MyD88(-/-) mice revealed massive destruction of lung tissue not present in WT, TLR2(-/-), or TLR4(-/-) mice. In addition, MyD88(-/-) and TLR2(-/-), but not TLR4(-/-), mice displayed marked reductions in hepatic neutrophil infiltration during the first 2 h of infection. Although both MyD88(-/-) and TLR2(-/-) macrophages showed profound defects in IL-6, TNF, and IL-12p40 responses to M. avium stimulation in vitro, in vivo TNF and IL-12p40 mRNA induction was impaired only in infected MyD88(-/-) mice. Similarly, MyD88(-/-) mice displayed a profound defect in IFN-gamma response that was not evident in TLR2(-/-) or TLR4(-/-) mice or in animals deficient in IL-18. These findings indicate that resistance to mycobacterial infection is regulated by multiple MyD88-dependent signals in addition to those previously attributed to TLR2 or TLR4, and that these undefined elements play a major role in determining bacterial induced proinflammatory as well as IFN-gamma responses.     

Mycobacterial infection in MyD88-deficient mice

MyD88 is an adaptor protein that plays a major role in TLR/IL-1 receptor family signaling. To understand the role of MyD88 in the development of murine tuberculosis in vivo, MyD88 knockout (KO) mice aerially were infected with Mycobacterium tuberculosis. Infected MyD88 mice were not highly susceptible to M. tuberculosis infection, but they developed granulomatous pulmonary lesions with neutrophil infiltration which were larger than those in wild-type (WT) mice (P < 0.01). The pulmonary tissue levels of mRNA for iNOS and IL-18 were slightly lower, but levels of mRNA for IL-1 beta, IL-2, IL-4, IL-6, IL-10, IFN-gamma, and TGF-beta were higher in MyD88 KO mice. IFN-gamma, TNF-alpha, IL-1 beta, and IL-12 also were high in the sera of MyD88 KO mice. There were no statistically significant differences in the expression of TNF-alpha, IL-12, and ICAM-1 mRNA between MyD88 KO and WT mice. Thus, MyD88 deficiency did not influence the development of murine tuberculosis. NF-kappa B activity was similar in the alveolar macrophages from the lung tissues of MyD88 KO and WT mice. Also, there may be a TLR2-specific, MyD88-independent IL-1 receptor/TLR-mediated pathway to activate NF-kappa B in the host defense against mycobacterial infection.     

Anti-CD180 (RP105) activates B cells to rapidly produce polyclonal Ig via a T cell and MyD88-independent pathway

CD180 is homologous to TLR4 and regulates TLR4 signaling, yet its function is unclear. We report that injection of anti-CD180 mAb into mice induced rapid Ig production of all classes and subclasses, with the exception of IgA and IgG2b, with up to 50-fold increases in serum IgG1 and IgG3. IgG production after anti-CD180 injection was not due to reactivation of memory B cells and was retained in T cell-deficient (TCR knockout [KO]), CD40 KO, IL-4 KO, and MyD88 KO mice. Anti-CD180 rapidly increased both transitional and mature B cells, with especially robust increases in transitional B cell number, marginal zone B cell proliferation, and CD86, but not CD80, expression. In contrast, anti-CD40 induced primarily follicular B cell and myeloid expansion, with increases in expression of CD80 and CD95 but not CD86. The expansion of splenic B cells was due, in part, to proliferation and occurred in wild-type and TCR KO mice, whereas T cell expansion occurred in wild-type, but not in B cell-deficient, mice, indicating a direct role for B cells in CD180 stimulation in vivo. Combination of anti-CD180 with various MyD88-dependent TLR ligands biased B cell fate because coinjection diminished Ig production, but purified B cells exhibited synergistic proliferation. Anti-CD180 had no effect on cytokine production from B cells, but it increased IL-6, IL-10, and TNF-α production in combination with LPS or CpG. Thus, CD180 stimulation induces intrinsic B cell proliferation and differentiation, causing rapid increases in IgG, and integrates MyD88-dependent TLR signals to regulate proliferation, cytokine production, and differentiation.     

Heat-killed Brucella abortus induces TNF and IL-12p40 by distinct MyD88-dependent pathways: TNF,unlike IL-12p40 secretion,is Toll-like receptor 2 dependent

Cattle and humans are susceptible to infection with the Gram-negative intracellular bacterium Brucella abortus. Heat-killed B. abortus (HKBA) is a strong Th1 adjuvant and carrier. Previously, we have demonstrated that dendritic cells produce IL-12 in response to HKBA stimulation. In the present study, we use knockout mice and in vitro reconstitution assays to examine the contribution of signaling by Toll-like receptors (TLRs) and their immediate downstream signaling initiator, myeloid differentiation protein MyD88, in the activation following stimulation by HKBA. Our results show that HKBA-mediated induction of IL-12p40 and TNF is dependent on the adapter molecule MyD88. To identify the TLR involved in HKBA recognition, we examined HKBA responses in TLR2- and TLR4-deficient animals. TNF responses to HKBA were TLR4 independent; however, the response in TLR2-deficient mice was significantly delayed and reduced, although not completely abolished. Studies using Chinese hamster ovary/CD14 reporter cell lines stably transfected with either human TLR2 or human TLR4 confirmed the results seen with knockout mice, namely TLR2, but not TLR4, can mediate cellular activation by HKBA. In addition, human embryonic kidney 293 cells, which do not respond to HKBA, were made responsive by transfecting TLR2, but not TLR4 or TLR9. Taken together, our data demonstrate that MyD88-dependent pathways are crucial for activation by HKBA and that TLR2 plays a role in TNF, but not IL-12p40 pathways activated by this microbial product.     

Critical roles of myeloid differentiation factor 88-dependent proinflammatory cytokine release in early phase clearance of Listeria monocytogenes in mice

Listeria monocytogenes (LM), a facultative intracellular Gram-positive bacterium, often causes lethal infection of the host. In this study we investigated the molecular mechanism underlying LM eradication in the early phase of infection. Upon infection with LM, both IL-12 and IL-18 were produced, and then they synergistically induced IFN-gamma production, leading to normal LM clearance in the host. IFN-gamma knockout (KO) mice were highly susceptible to LM infection. IL-12/IL-18 double knockout mice were also highly susceptible. Their susceptibility was less than that of IFN-gamma KO mice, but more than that of single IL-12 or IL-18 KO mice. Mice deficient in myeloid differentiation factor 88 (MyD88), an essential adaptor molecule used by signal transduction pathways of all members of the Toll-like receptor (TLR) family, showed an inability to produce IL-12 and IFN-gamma following LM infection and were most susceptible to LM. Furthermore, MyD88-deficient, but not IFN-gamma-deficient, Kupffer cells could not produce TNF-alpha in response to LM in vitro, indicating the importance of MyD88-dependent TNF-alpha production for host defense. As TLR2 KO, but not TLR4 KO, mice showed partial impairment in their capacity to produce IL-12, IFN-gamma, and TNF-alpha, TLR2 activation partly contributed to the induction of IL-12-mediated IFN-gamma production. These results indicated a critical role for TLRs/MyD88-dependent IL-12/TNF-alpha production and for IL-12- and IL-18-mediated IFN-gamma production in early phase clearance of LM.     

Etk/BMX, a Btk family tyrosine kinase, and Mal contribute to the cross-talk between MyD88 and FAK pathways

MyD88 and focal adhesion kinase (FAK) are key adaptors involved in signaling downstream of TLR2, TLR4, and integrin alpha5beta1, linking pathogen-associated molecule detection to the initiation of proinflammatory response. The MyD88 and integrin pathways are interlinked, but the mechanism of this cross-talk is not yet understood. In this study we addressed the involvement of Etk, which belongs to the Tec family of tyrosine kinases, in the cross-talk between the integrin/FAK and the MyD88 pathways in fibroblast-like synoviocytes (FLS) and in IL-6 synthesis. Using small interfering RNA blockade, we report that Etk plays a major role in LPS- and protein I/II (a model activator of FAK)-dependent IL-6 release by activated FLS. Etk is associated with MyD88, FAK, and Mal as shown by coimmunoprecipitation. Interestingly, knockdown of Mal appreciably inhibited IL-6 synthesis in response to LPS and protein I/II. Our results also indicate that LPS and protein I/II induced phosphorylation of Etk and Mal in rheumatoid arthritis FLS via a FAK-dependent pathway. In conclusion, our data provide support that, in FLS, Etk and Mal are implicated in the cross-talk between FAK and MyD88 and that their being brought into play is clearly dependent on FAK.