TLR4 Engagement Research Articles

Autoantibodies in systemic autoimmune diseases: specificity and pathogenicity.

In this Review we focus on the initiation of autoantibody production and autoantibody pathogenicity, with a special emphasis on the targeted antigens. Release of intracellular antigens due to excessive cell death or to ineffective clearance of apoptotic debris, modification of self-antigens during inflammatory responses, and molecular mimicry contribute to the initiation of autoantibody production. We hypothesize that those autoreactive B cells that survive and produce pathogenic autoantibodies have specificity for self-antigens that are TLR ligands. Such B cells experience both B cell receptor (BCR) activation and TLR engagement, leading to an escape from tolerance. Moreover, the autoantibodies they produce form immune complexes that can activate myeloid cells and thereby establish the proinflammatory milieu that further negates tolerance mechanisms of both B and T cells.

B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses (IRM10P.608)

Abstract CSR in B cells is crucial to the maturation of antibody responses. It is induced by engagement of CD40 or dual engagement of a TLR and BCR. Here, we have addressed cross-regulation between two different TLRs or between a TLR and CD40 in CSR induction by using a B cell stimulation system involving LPS. LPS mediated long-term primary class-switched antibody responses and induced generation of class-switched B cells and plasma cells. Consistent with the requirement for dual TLR and BCR engagement in CSR induction, LPS, which engages TLR4 through its lipid A moiety, triggered cytosolic Ca2+ flux in B cells through its BCR-engaging polysaccharidic moiety. In the presence of BCR crosslinking, LPS synergized with a TLR1/2 ligand (Pam3CSK4) in CSR induction, but much less efficiently with a TLR7 (R-848) or TLR9 (CpG) ligand. In the absence of BCR crosslinking, R-848 and CpG, virtually abrogated CSR, as induced by LPS or CD154, and reduced secretion of class-switched Igs, without affecting B cell proliferation or IgM expression. The CSR inhibition by TLR9 was associated with reduced AID expression and/or IgH germline IH-S-CH transcription, and required co-stimulation of B cells by CpG with LPS or CD154. Unexpectedly, B cells also failed to undergo CSR when co-stimulated by LPS and CD154. Our study suggests the complexity of how different stimuli cross-regulate an important B cell differentiation process and an important role of TLRs in inducing T-independent antibody responses.

A role for APPL1 in TLR3/4-dependent TBK1 and IKKε activation in macrophages.

Endosomes have important roles in intracellular signal transduction as a sorting platform. Signaling cascades from TLR engagement to IRF3-dependent gene transcription rely on endosomes, yet the proteins that specifically recruit IRF3-activating molecules to them are poorly defined. We show that adaptor protein containing a pleckstrin-homology domain, a phosphotyrosine-binding domain, and a leucine zipper motif (APPL)1, an early endosomal protein, is required for both TRIF- and retinoic acid-inducible gene 1-dependent signaling cascades to induce IRF3 activation. APPL1, but not early endosome Ag 1, deficiency impairs IRF3 target gene expression upon engagement of both TLR3 and TLR4 pathways, as well as in H1N1-infected macrophages. The IRF3-phosphorylating kinases TBK1 and IKKε are recruited to APPL1 endosomes in LPS-stimulated macrophages. Interestingly, APPL1 undergoes proteasome-mediated degradation through ERK1/2 to turn off signaling. APPL1 degradation is blocked when signaling through the endosome is inhibited by chloroquine or dynasore. Therefore, APPL1 endosomes are critical for IRF3-dependent gene expression in response to some viral and bacterial infections in macrophages. Those signaling pathways involve the signal-induced degradation of APPL1 to prevent aberrant IRF3-dependent gene expression linked to immune diseases.

Transforming growth factor-β-activated kinase 1 resistance limits glucocorticoid responsiveness to Toll-like receptor 4-mediated inflammation.

Glucocorticoids (GC) are among the most effective anti-inflammatory drugs, but are often associated with serious adverse effects or inadequate therapeutic responses. Here, we use activation of different Toll-like receptors (TLRs) by their respective ligands to evaluate context-specific GC sensitivity in the macrophage. Recruitment and activation of transforming growth factor-β-activated kinase 1 (TAK1), downstream of TLR engagement, is crucial in activating multiple inflammatory pathways, and contributes to inflammatory disorders. We hypothesize that GC exert anti-inflammatory effects through regulation of TAK1. Both in vivo and in vitro, in comparison to other TLRs, there was limited GC potency in restricting TLR4 ligand-mediated secretion of interleukin-6, tumour necrosis factor-α and interleukin-12. Also, we found that inactivation of TAK1 both in vivo and in vitro strongly inhibits TLR4-induced inflammation-associated genes beyond the suppressive effects from GC treatment. However, there was no effect of TAK1 inactivation on GC inhibition of TLR3- or TLR9-initiated inflammatory actions. Together, our findings demonstrate that GC resistance for TAK1 activation associated with TLR4 engagement may be an important contributor to GC resistance in inflammatory disorders.

Innate immune response to Streptococcus pyogenes depends on the combined activation of TLR13 and TLR2.

Innate immune recognition of the major human-specific Gram-positive pathogen Streptococcus pyogenes is not understood. Here we show that mice employ Toll-like receptor (TLR) 2- and TLR13-mediated recognition of S. pyogenes. These TLR pathways are non-redundant in the in vivo context of animal infection, but are largely redundant in vitro, as only inactivation of both of them abolishes inflammatory cytokine production by macrophages and dendritic cells infected with S. pyogenes. Mechanistically, S. pyogenes is initially recognized in a phagocytosis-independent manner by TLR2 and subsequently by TLR13 upon internalization. We show that the TLR13 response is specifically triggered by S. pyogenes rRNA and that Tlr13 −/− cells respond to S. pyogenes infection solely by engagement of TLR2. TLR13 is absent from humans and, remarkably, we find no equivalent route for S. pyogenes RNA recognition in human macrophages. Phylogenetic analysis reveals that TLR13 occurs in all kingdoms but only in few mammals, including mice and rats, which are naturally resistant against S. pyogenes. Our study establishes that the dissimilar expression of TLR13 in mice and humans has functional consequences for recognition of S. pyogenes in these organisms.

MiR-127 modulates macrophage polarization and promotes lung inflammation and injury by activating the JNK pathway.

A polarized macrophage response is presumed to have a pivotal role in a variety of immunological pathophysiology. However, the molecular mechanism underlying macrophage functional shaping remains largely unknown. In this study, we reveal a pivotal role of miR-127 in macrophage development and thereby the pathogenesis of inflammation and lung injury. In particular, miR-127 was demonstrated to be prominently induced upon TLR engagement and repressed by the M2-prone cytokines. Enforced expression of miR-127 in macrophages resulted in significantly increased production of proinflammatory cytokines, whereas deletion of miR-127 impaired M1 gene expression and led to a M2-biased response. Accordingly, intratracheal administration of miR-127 resulted in an exaggerated pulmonary inflammation and injury. Conversely, antagonizing of miR-127 suppressed production of the proinflammatory cytokines and rendered the mice more refractory to the inflammation-associated pathology. Mechanistically, miR-127 demonstrated to target B cell lymphoma 6 (Bcl6) and remarkably downregulated its expression and subsequently dual specificity phosphatase 1 (Dusp1), which in turn enhanced the activation of JNK kinase and hence the development of proinflammatory macrophages. Thereby, reconstitution with the expression of Bcl6 or Dusp1 or inhibition of JNK activity impaired miR-127-mediated skewing of M1 proinflammatory macrophages, whereas interference of Bcl6 or Dusp1 expression abrogated the anti-inflammatory property of anti-miR-127. Together, these data establish miR-127 as a molecular switch during macrophage development and as a potential target for treatment of inflammatory diseases.

Coxiella burnetii lipopolysaccharide blocks p38α-MAPK activation through the disruption of TLR-2 and TLR-4 association.

To survive in macrophages, Coxiella burnetii hijacks the activation pathway of macrophages. Recently, we have demonstrated that C. burnetii, via its lipopolysaccharide (LPS), avoids the activation of p38α-MAPK through an antagonistic engagement of Toll-like receptor (TLR)-4. We investigated the fine-tuned mechanism leading to the absence of activation of the p38α-MAPK despite TLR-4 engagement. In macrophages challenged with LPS from the avirulent variants of C. burnetii, TLR-4 and TLR-2 co-immunoprecipitated. This association was absent in cells challenged by the LPS of pathogenic C. burnetii. The disruption makes TLRs unable to signal during the recognition of the LPS of pathogenic C. burnetii. The disruption of TLR-2 and TLR-4 was induced by the re-organization of the macrophage cytoskeleton by C. burnetii LPS. Interestingly, blocking the actin cytoskeleton re-organization relieved the disruption of the association TLR-2/TLR-4 by pathogenic C. burnetii and rescued the p38α-MAPK activation by C. burnetii. We elucidated an unexpected mechanism allowing pathogenic C. burnetii to avoid macrophage activation by the disruption of the TLR-2 and TLR-4 association.

Squalene emulsion potentiates the adjuvant activity of the TLR4 agonist, GLA, via inflammatory caspases, IL-18, and IFN-γ.

The synthetic TLR4 agonist glucopyranosyl lipid adjuvant (GLA) is a potent Th1-response-inducing adjuvant when formulated in a squalene oil-in-water emulsion (SE). While the innate signals triggered by TLR4 engagement are well studied, the contribution of SE remains unclear. To better understand the effect of SE on the adjuvant properties of GLA-SE, we compared the innate and adaptive immune responses elicited by immunization with different formulations: GLA without oil, SE alone or the combination, GLA-SE, in mice. Within the innate response to adjuvants, only GLA-SE displayed features of inflammasome activation, evidenced by early IL-18 secretion and IFN-γ production in memory CD8(+) T cells and neutrophils. Such early IFN-γ production was ablated in caspase-1/11(-/-) mice and in IL-18R1(-/-) mice. Furthermore, caspase-1/11 and IL-18 were also required for full Th1 CD4(+) T-cell induction via GLA-SE. Thus, we demonstrate that IL-18 and caspase-1/11 are components of the response to immunization with the TLR4 agonist/squalene oil-in-water based adjuvant, GLA-SE, providing implications for other adjuvants that combine oils with TLR agonists.

X-Chromosome complement and estrogen receptor signaling independently contribute to the enhanced TLR7-mediated IFN-α production of plasmacytoid dendritic cells from women.

Human plasmacytoid dendritic cells (pDCs) play a major role in innate immunity through the production of type I IFNs after TLR engagement by pathogens. Sex-based differences in the innate function of human pDCs have been established, with pDCs from women exhibiting enhanced TLR7-mediated IFN-α production as compared with pDCs from males. In mice, we recently provided evidence for a role of estrogens as a positive regulator of pDC innate functions through cell-intrinsic estrogen receptor α signaling, but did not exclude a role for other X-linked factors, particularly in human pDCs. In this study, we investigated the respective contribution of X chromosome dosage and sex hormones using a humanized mouse model in which male or female NOD-SCID-β2m(-/-) were transplanted with human progenitor cells purified from either male or female cord blood cells. We showed that, in response to TLR7 ligands, the frequency of IFN-α- and TNF-α-producing pDCs from either sex was greater in female than in male host mice, suggesting a positive role for estrogens. Indeed, blockade of estrogen receptor signaling during pDC development in vitro inhibited TLR7-mediated IFN-α production by human pDCs, which expressed both ESR1 and ESR2 genes. Interestingly, we also found that X chromosome dosage contributed to this sex bias as female pDCs have an enhanced TLR7-mediated IFN-α response as compared with male ones, irrespective of the sex of the recipient mice. Together, these results indicate that female sex hormones, estrogens, and X chromosome complement independently contribute to the enhanced TLR7-mediated IFN-α response of pDCs in women.

Neuroimmunological communication via CGRP promotes the development of a regulatory phenotype in TLR4-stimulated macrophages.

Environmental signals shape the phenotype and function of activated macrophages. Here, we show that the neuropeptide calcitonin gene-related peptide (CGRP), which is released from sensory nerves, modulates the phenotype of TLR4-activated murine macrophages by enhancing expression of the regulatory macrophage markers IL-10, sphingosine kinase 1 (SPHK1), and LIGHT (lymphotoxin-like, exhibits inducible expression and competes with HSV glycoprotein D for herpesvirus entry mediator, a receptor expressed by T lymphocytes). In contrast, CGRP inhibits production of cytokines characteristic of inflammatory macrophages and does not affect expression of wound-healing macrophage markers upon TLR4 engagement. In IL-4-stimulated macrophages, CGRP increased LIGHT expression, but failed to induce IL-10 and SPHK1. The stimulatory effect of CGRP on IL-10 production required activation of protein kinase A and was linked to prolonged phosphorylation of CREB and sustained nuclear accumulation of CRTC2 and CRTC3 (where CRTC is CREB-regulated transcriptional cofactor). CGRP enhanced expression of regulatory macrophage markers during the early, but not late, phase of LPS-stimulation and this effect was independent of autocrine type-I IFN activity. In contrast, autocrine type-I IFN activity and treatment of macrophages with IFN-β promoted late-phase IL-10 production, but had only minor influence on LIGHT and SPHK1 expression. Together, the results identify neuroimmunological communication through CGRP as a novel costimulatory pathway promoting the development of a regulatory phenotype of TLR4-stimulated macrophages. CGRP appears to act through a mechanism that involves sustained activation of CREB-dependent gene transcription.

GSK3β-dependent inhibition of AMPK potentiates activation of neutrophils and macrophages and enhances severity of acute lung injury.

Although AMP-activated protein kinase (AMPK) is involved in regulating carbohydrate and lipid metabolism, activated AMPK also plays an anti-inflammatory role in many cell populations. However, despite the ability of AMPK activation to diminish the severity of inflammatory responses, previous studies have found that AMPK activity is diminished in LPS-treated neutrophils and also in lungs of mice with LPS-induced acute lung injury (ALI). Since GSK3β participates in regulating AMPK activity, we examined potential roles for GSK3β in modulating LPS-induced activation of neutrophils and macrophages and in influencing severity of ALI. We found that GSK3β-dependent phosphorylation of T479-AMPK was associated with pT172 dephosphorylation and inactivation of AMPK following TLR4 engagement. GSK3β inhibitors BIO (6-bromoindirubin-3'-oxime), SB216763, or siRNA knockdown of GSK3β, but not the PI3K/AKT inhibitor LY294002, prevented Thr172-AMPK dephosphorylation. Exposure to LPS resulted in rapid binding between IKKβ and AMPKα, and phosphorylation of S485-AMPK by IKKβ. These results suggest that IKKβ-dependent phosphorylation of S485-AMPK was an essential step in subsequent phosphorylation and inactivation AMPK by GSK3β. Inhibition of GSK3β activity delayed IκBα degradation and diminished expression of the proinflammatory TNF-α in LPS-stimulated neutrophils and macrophages. In vivo, inhibition of GSK3β decreased the severity of LPS-induced lung injury as assessed by development of pulmonary edema, production of TNF-α and MIP-2, and release of the alarmins HMGB1 and histone 3 in the lungs. These results show that inhibition of AMPK by GSK3β plays an important contributory role in enhancing LPS-induced inflammatory responses, including worsening the severity of ALI.

134: TLR2 and TLR4 differentially regulate DC to stimulate T cell cytokines in response to mycoplasma superantigen

Mycoplasma arthritidis induces arthritis, toxic shock and skin necrosis in mice. M. arthritidis mitogen (MAM), a potent superantigen (SAg) secreted by M. arthritidis , contributes to some of these symptoms in certain mouse strains. We previously demonstrated that C3H mouse strains differing in expression of TLR4, differ in immune reactivity to MAM due to differential engagement of TLR2 and TLR4. Herein, we demonstrated that in TLR4-defect C3H/HeJ (TLR2+/4−) mice, following injection of MAM SAg, the splenic DCs were activated to express significantly higher B7-1 + CD8a + surface markers than expressed in wild-type mice, and produced remarkably elevated IL-1b, IL-12p40, IFNg, and TNFa. In contrast, in wild-type C3H/HeSnJ (TLR2+/4+) mice, the interaction of MAM with both TLR4 and TLR2 led to reduction of the B7-1 + CD8a + DC subset that led to the down-regulated TNFa and IFNg, but up-regulated IL-6, IL-10 and IL-17. Interestingly, in mice injected with live M. arthritidis , injections of anti-B7-1 antibody to C3H/HeSnJ but not C3H/HeJ greatly enhanced arthritis severity that is now found to be associated to the elevation of IL-6, TGFb, IL-17 and IL-23 in sera or in the culture supernatants of splenic T cells. Thus the results suggest that TLR2 and/or TLR4 differentially control distinct DC subsets to influence Th17 differentiation in response to MAM superantigen. DC function that is regulated as the consequence of the cross-talk of discrete TLRs, B7-1 and CD8a molecules in response to MAM SAg might largely influence the outcome of subsequent adaptive immune responses in murine inflammatory diseases caused by M. arthritidis .

Modulation of Inflammatory Responses After Brain Death and Syngeneic Renal Transplantation By Systemic Overexpression of esRAGE.

Introduction: Donor brain death (BD) adversely affects organ quality. Inflammation mediated by engagement of TLR4 and RAGE may contribute to inferior outcomes. Endogenous secretory (es)RAGE is a soluble decoy receptor which sequesters HMGB1 and other RAGE ligands blocking binding to TLRs 2 and 4, as well as to RAGE. Aim: To determine whether in vivo expression of esRAGE can reduce inflammation in syngeneic KTx from BD donors. Methods: Donor and recipient C57BL/6 mice received 5×1011 VG rAAV-esRAGE or ctrl vector 7 days pre-transplant. Donor kidneys were grafted 1 hr after BD induction, and sampled at d1 and 4 post-Tx. Cellular infiltration, proliferation, and gene expression were determined. Serum and kidneys were also collected 3 hrs post-BD. Results: Serum HMGB1 levels in esRAGE-BD mice (˜10ng/ml) were significantly less than those in untreated BD mice (>25ng/ml, p=0.02). There was a trend towards reduced expression of CXCL10, MCP-1, MIP-2, CCL22, IL-10, IL-6, IL-1β and TNF-α in the kidneys of esRAGE-BD mice, but this was not consistently observed after KTx. Neutrophil and macrophage infiltrate density was substantially reduced by esRAGE (nΦ 9.1±2.0/HPF cf 23.1±6.0, p=0.03; F4/80 27.3±3.2/HPF cf 65.1±9.1, p<0.01).Figure: No Caption available.Proliferation of parenchymal cells (PCNA staining) was enhanced in esRAGE-treated mice (7.6±0.5/HPF cf 4.2±1.2, p=0.05).Figure: No Caption available.Conclusions: esRAGE significantly reduced neutrophil and macrophage infiltration following syngeneic renal Tx from BD donors. RAGE is a binding partner for the integrin MAC-1, and reduced leucocyte infiltration may reflect inhibition of this interaction. esRAGE increased proliferation of renal parenchymal cells after Tx from BD donors, similar to documented effects of soluble RAGE in liver injury. These effects may be translated into improved graft function.

TLR costimulation causes oxidative stress with unbalance of proinflammatory and anti-inflammatory cytokine production.

IL-1β acts in concert with anti-inflammatory cytokines, in particular, IL-1R antagonist (IL-1Ra), to ensure the correct development and outcome of the inflammation: imbalance in the IL-1β/IL-1Ra ratio is implicated in many human diseases and may lead to dramatic consequences. In this article, we show that single TLR engagement induces IL-1β and, with a little delay, IL-1Ra. Differently, costimulation of TLR2, TLR4, and TLR7/8 enhances IL-1β secretion but severely inhibits IL-1Ra production. The IL-1β/IL-1Ra unbalance after activation of multiple TLRs depends on the insurgence of oxidative stress, because of enhanced production of reactive oxygen species and failure of the antioxidant systems. Increased reactive oxygen species levels increase ATP externalization by monocytes, resulting in enhanced inflammasome activation and IL-1β secretion. Oxidative stress then induces cell responses to stress, including inhibition of protein synthesis, which, in turn, is responsible for the impaired production of IL-1Ra. IL-1Ra secretion is restored by exogenous antioxidants that oppose oxidative stress. Similar effects are evident also on other cytokines: TNF-α is induced, whereas IL-6 is inhibited by costimulation. Our findings provide a molecular basis to the imbalance between proinflammatory and regulatory cytokine circuits that occur in various pathologic conditions, and suggest new strategies for controlling inflammation.

A Novel Mechanism Underlying the Basic Defensive Response of Macrophages against Mycobacterium Infection

Following inhalation of Mycobacterium tuberculosis, including bacillus Calmette-Guérin (BCG), pathogens enter and grow inside macrophages by taking advantage of their phagocytic mechanisms. Macrophages often fail to eliminate intracellular M. tuberculosis, leading to the induction of host macrophage death. Despite accumulating evidence, the molecular mechanisms underlying M. tuberculosis infection-induced cell death remain controversial. In this study, we show the involvement of two distinct pathways triggered by TLR2 and β2 integrin in BCG infection-induced macrophage apoptosis. First, BCG infection induced activation of ERK1/2, which in turn caused phosphorylation/activation of the proapoptotic protein Bim in mouse macrophage-like Raw 264.7 cells. BCG-infected Raw cells treated with U0126, an MEK/ERK inhibitor, led to the suppression of Bim phosphorylation alongside a remarkable increase in the number of viable macrophages. Small interfering RNA-mediated knockdown of Bim rescued the macrophages from the apoptotic cell death induced by BCG infection. Stimulation with Pam3CSK, a TLR2 agonist, induced macrophage apoptosis with a concomitant increase in the phosphorylation/activation of MEK/ERK and Bim. These observations indicate the important role of the TLR2/MEK/ERK/Bim pathway in BCG infection-induced macrophage apoptosis. Second, we used the β2 integrin agonists C3bi and fibronectin to show that the β2 integrin-derived signal was involved in BCG infection-induced apoptosis, independent of MEK/ERK activation. Interestingly, latex beads coated with Pam3CSK and C3bi were able to induce apoptosis in macrophages to the same extent and specificity as that induced by BCG. Taken together, two distinct pattern-recognition membrane receptors, TLR2 and β2 integrin, acted as triggers in BCG infection-induced macrophage apoptosis, in which MEK/ERK activation played a crucial role following the engagement of TLR2.

Toll‐like receptors hit calcium

Mitochondrial Ca(2+) uptake is a multifarious signal that controls both the activity of matrix dehydrogenases and the sensitivity to apoptotic and necrotic challenges. Recent evidence indicates that mitochondria also play a role in triggering inflammation, as mitochondrial DNA, when released by the cell, is an important damage-associated molecular pattern (DAMP). Now, Toll-like receptors (TLRs) are shown to close the loop, by affecting in turn mitochondrial activity. Two studies by Shintani and colleagues, one in this issue of EMBO reports, identify a new TLR transduction mechanism that impinges directly on mitochondrial function. Upon binding of CpG oligodeoxynucleotides, TLR9--which in non-immune cells is retained in the ER--inhibits SERCA2, thus reducing Ca(2+) transfer to the mitochondria and aerobic metabolism.

TLR activation of tumor‐associated macrophages from ovarian cancer patients triggers cytolytic activity of NK cells

We analyzed the functional outcome of the interaction between tumor-associated macrophages (TAMs) and natural killer (NK) cells. TAMs from ascites of ovarian cancer patients displayed an alternatively activated functional phenotype (M2) characterized by a remarkably high frequency and surface density of membrane-bound IL-18. Upon TLR engagement, TAMs acquired a classically activated functional phenotype (M1), released immunostimulatory cytokines (IL-12, soluble IL-18), and efficiently triggered the cytolytic activity of NK cells. TAMs also induced the release of IFN-γ from NK cells, which however was significantly lower compared with that induced by in vitro-polarized M2 cells. Most tumor-associated NK cells displayed a CD56(bright) , CD16(neg) or CD56(bright) , CD16(dim) phenotype, and very poor cytolytic activities, despite an increased expression of the activation marker CD69. They also showed downregulation of DNAM-1, 2B4, and NTB-A activating receptors, and an altered chemokine receptor repertoire. Importantly however, when appropriately stimulated, NK cells from the patients, including those cells isolated from ascites, efficiently killed autologous TAMs that expressed low, "nonprotective" levels of HLA class I molecules. Overall, our data show the existence of a complex tumor microenvironment in which poorly cytolytic/immature NK cells deal with immunosuppressive tumor-educated macrophages.

Bruton tyrosine kinase mediates TLR9-dependent human dendritic cell activation

Bruton tyrosine kinase (BTK) plays an essential role in various biologic functions of different cell types. Mutations in BTK lead to X-linked agammaglobulinemia (XLA) in humans. BTK was recently linked to the innate immune system, in particular, the Toll-like receptor (TLR) pathway; however, the TLR9 pathway has never been studied in dendritic cells (DCs) of patients with XLA. The aim of this study was to investigate the role of BTK in human DC activation upon TLR9 stimulation. DCs of patients with XLA and healthy donors were stimulated via TLR4/9 and evaluated for cell activation and cytokine production. We showed that BTK plays an essential role in DC responses to unmethylated CpG oligodeoxynucleotide: although responses to lipopolysaccaride/TLR4 induce normal DC activation in terms of upregulation of specific markers (CD86, CD83, CD80, HLA-DR), the CpG/TLR9 pathway is completely impaired in patients with XLA. Furthermore, cytokine production upon TLR9 activation in patients with XLA is radically impaired in terms of IL-6, IL-12, TNF-α, and IL-10 production. Interestingly, BTK mediated STAT1/3 upregulation in a TLR9-dependent manner. The important role of BTK in human DC activation was confirmed after incubation of healthy DCs with ibrutinib, the specific BTK inhibitor, which resulted in impairment of TLR9 responses as seen in patients with XLA. Analysis of these data suggests that BTK regulates human DC responses upon TLR9 engagement in terms of activation, cytokine production, and STAT1/3 upregulation. These findings may be of important significance for better understanding and managing different clinical conditions, such as agammaglobulinemia and lymphoid malignancies.

Toll‐like receptors expressed on embryonic macrophages couple inflammatory signals to iron metabolism during early ontogenesis

Mammalian TLRs in adult animals serve indispensable functions in establishing innate and adaptive immunity and contributing to the homeostasis of surrounding tissues. However, the expression and function of TLRs during mammalian embryonic development has not been studied so far. Here, we show that CD45(+) CD11b(+) F4/80(+) macrophages from 10.5-day embryo (E10.5) co-express TLRs and CD14. These macrophages, which have the capability to engulf both apoptotic cells and bacteria, secrete a broad spectrum of proinflammatory cytokines and chemokines upon TLR stimulation, demonstrating that their TLRs are functional. Comparative microarray analysis revealed an additional set of genes that were significantly upregulated in E10.5 TLR2(+) CD11b(+) macrophages. This analysis, together with our genetic, microscopic, and biochemical evidence, showed that embryonic phagocytes express protein machinery that is essential for the recycling of cellular iron and that this expression can be regulated by TLR engagement in a MyD88-dependent manner, leading to typical inflammatory M1 responses. These results characterize the utility of TLRs as suitable markers for early embryonic phagocytes as well as molecular triggers of cellular responses, the latter being demonstrated by the involvement of TLRs in an inflammation-mediated regulation of embryonic homeostasis via iron metabolism.

TLR-induced PAI-2 expression suppresses IL-1β processing via increasing autophagy and NLRP3 degradation

The NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, a multiprotein complex, triggers caspase-1 activation and maturation of the proinflammatory cytokines IL-1β and IL-18 upon sensing a wide range of pathogen- and damage-associated molecules. Dysregulation of NLRP3 inflammasome activity contributes to the pathogenesis of many diseases, but its regulation remains poorly defined. Here we show that depletion of plasminogen activator inhibitor type 2 (PAI-2), a serine protease inhibitor, resulted in NLRP3- and ASC (apoptosis-associated Speck-like protein containing a C-terminal caspase recruitment domain)-dependent caspase-1 activation and IL-1β secretion in macrophages upon Toll-like receptor 2 (TLR2) and TLR4 engagement. TLR2 or TLR4 agonist induced PAI-2 expression, which subsequently stabilized the autophagic protein Beclin 1 to promote autophagy, resulting in decreases in mitochondrial reactive oxygen species, NLRP3 protein level, and pro-IL-1β processing. Likewise, overexpressing Beclin 1 in PAI-2-deficient cells rescued the suppression of NLRP3 activation in response to LPS. Together, our data identify a tier of TLR signaling in controlling NLRP3 inflammasome activation and reveal a cell-autonomous mechanism which inversely regulates TLR- or Escherichia coli-induced mitochondrial dysfunction, oxidative stress, and IL-1β-driven inflammation.