Yanbao Xiong

The Asp299Gly Polymorphism Alters TLR4 Signaling by Interfering with Recruitment of MyD88 and TRIF

Asp299Gly (D299G) and, to a lesser extent, Thr399Ile (T399I) TLR4 polymorphisms have been associated with Gram-negative sepsis and other infectious diseases, but the mechanisms by which they affect TLR4 signaling are unclear. In this study, we determined the impact of the D299G and T399I polymorphisms on TLR4 expression, interactions with myeloid differentiation factor 2 (MD2), LPS binding, and LPS-mediated activation of the MyD88- and Toll/IL-1R resistance domain-containing adapter inducing IFN-β (TRIF) signaling pathways. Complementation of human embryonic kidney 293/CD14/MD2 transfectants with wild-type (WT) or mutant yellow fluorescent protein-tagged TLR4 variants revealed comparable total TLR4 expression, TLR4–MD2 interactions, and LPS binding. FACS analyses with anti-TLR4 Ab showed only minimal changes in the cell-surface levels of the D299G TLR4. Cells transfected with D299G TLR4 exhibited impaired LPS-induced phosphorylation of p38 and TANK-binding kinase 1, activation of NF-κB and IFN regulatory factor 3, and induction of IL-8 and IFN-β mRNA, whereas T399I TLR4 did not cause statistically significant inhibition. In contrast to WT TLR4, expression of the D299G mutants in TLR4−/− mouse macrophages failed to elicit LPS-mediated induction of TNF-α and IFN-β mRNA. Coimmunoprecipitation revealed diminished LPS-driven interaction of MyD88 and TRIF with the D299G TLR4 species, in contrast to robust adapter recruitment exhibited by WT TLR4. Thus, the D299G polymorphism compromises recruitment of MyD88 and TRIF to TLR4 without affecting TLR4 expression, TLR4–MD2 interaction, or LPS binding, suggesting that it interferes with TLR4 dimerization and assembly of intracellular docking platforms for adapter recruitment.

Induction of endotoxin tolerance in vivo inhibits activation of IRAK4 and increases negative regulators IRAK-M, SHIP-1, and A20.

TLRs mediate host defense against microbial pathogens by eliciting production of inflammatory mediators and activating expression of MHC, adhesion, and costimulatory molecules. Endotoxin tolerance limits excessive TLR‐driven inflammation during sepsis and reprograms macrophage responses to LPS, decreasing expression of proinflammatory cytokines without inhibiting anti‐inflammatory and antimicrobial mediators. Molecular mechanisms of reprogramming of TLR4 signaling upon in vivo induction of endotoxin tolerance are incompletely understood. We used an in vivo model of endotoxin tolerance, whereby C57BL/6 mice were i.p.‐inoculated with LPS or PBS, followed by in vitro challenge of peritoneal or splenic macrophages with LPS to examine activation of IRAK4 and expression of negative regulatory molecules. Administration of LPS in vivo‐induced endotoxin tolerance in peritoneal and splenic macrophages, as evidenced by decreased degradation of IκBα, suppressed phosphorylation of p38 and reduced expression of TNF‐α, IL‐6, and KC mRNA upon in vitro LPS challenge. Macrophages from control and endotoxin‐tolerant mice exhibited comparable TLR4 mRNA levels and similar expression of IL‐1RA and IL‐10 genes. Endotoxin tolerization in vivo blocked TLR4‐driven IRAK4 phosphorylation and activation in macrophages, while increasing expression of IRAK‐M, SHIP‐1, A20 mRNA, and A20 protein. Thus, induction of endotoxin tolerance in vivo inhibits expression of proinflammatory mediators via impaired activation of IRAK4, p38, and NF‐κB and increases expression of negative regulators of TLR4 pathways.

Endotoxin Tolerance Impairs IL-1 Receptor-Associated Kinase (IRAK) 4 and TGF-β-activated Kinase 1 Activation, K63-linked Polyubiquitination and Assembly of IRAK1, TNF Receptor-associated Factor 6, and IκB Kinase γ and Increases A20 Expression

Endotoxin tolerance reprograms Toll-like receptor 4 responses by impairing LPS-elicited production of pro-inflammatory cytokines without inhibiting expression of anti-inflammatory or anti-microbial mediators. In septic patients, Toll-like receptor tolerance is thought to underlie decreased pro-inflammatory cytokine expression in response to LPS and increased incidence of microbial infections. The impact of endotoxin tolerance on recruitment, post-translational modifications and signalosome assembly of IL-1 receptor-associated kinase (IRAK) 4, IRAK1, TNF receptor-associated factor (TRAF) 6, TGF-β-activated kinase (TAK) 1, and IκB kinase (IKK) γ is largely unknown. We report that endotoxin tolerization of THP1 cells and human monocytes impairs LPS-mediated receptor recruitment and activation of IRAK4, ablates K63-linked polyubiquitination of IRAK1 and TRAF6, compromises assembly of IRAK1-TRAF6 and IRAK1-IKKγ platforms, and inhibits TAK1 activation. Deficiencies in these signaling events in LPS-tolerant cells coincided with increased expression of A20, an essential deubiquitination enzyme, and sustained A20-IRAK1 associations. Overexpression of A20 inhibited LPS-induced activation of NF-κB and ablated NF-κB reporter activation driven by ectopic expression of MyD88, IRAK1, IRAK2, TRAF6, and TAK1/TAB1, while not affecting the responses induced by IKKβ and p65. A20 shRNA knockdown abolished LPS tolerization of THP1 cells, mechanistically linking A20 and endotoxin tolerance. Thus, deficient LPS-induced activation of IRAK4 and TAK1, K63-linked polyubiquitination of IRAK1 and TRAF6, and disrupted IRAK1-TRAF6 and IRAK1-IKKγ assembly associated with increased A20 expression and A20-IRAK1 interactions are new determinants of endotoxin tolerance.

R753Q Polymorphism Inhibits Toll-like Receptor (TLR) 2 Tyrosine Phosphorylation, Dimerization with TLR6, and Recruitment of Myeloid Differentiation Primary Response Protein 88

Background: TLR2 SNPs are linked to tuberculosis, but the mechanisms by which they alter TLR signaling are unclear. Results: R753Q TLR2 showed impaired tyrosine phosphorylation, dimerization with TLR6, MyD88 recruitment, and induction of NF-κB and cytokines upon mycobacterial challenge. Conclusion: R753Q polymorphism blocks TLR2 tyrosine phosphorylation and signalosome assembly. Significance: Deciphering how SNPs alter TLR signaling advances TLR immunobiology and facilitates design of new therapeutic strategies. The R753Q polymorphism in the Toll-IL-1 receptor domain of Toll-like receptor 2 (TLR2) has been linked to increased incidence of tuberculosis and other infectious diseases, but the mechanisms by which it affects TLR2 functions are unclear. Here, we studied the impact of the R753Q polymorphism on TLR2 expression, hetero-dimerization with TLR6, tyrosine phosphorylation, and recruitment of myeloid differentiation primary response protein (MyD) 88 and MyD88 adapter-like (Mal). Complementation of HEK293 cells with transfected WT or R753Q TLR2 revealed their comparable total levels and only minimal changes in cell surface expression of the mutant species. Notably, even a 100-fold increase in amounts of transfected R753Q TLR2 versus WT variant did not overcome the compromised ability of the mutant TLR2 to activate nuclear factor κB (NF-κB), indicating that a minimal decrease in cell surface levels of the R753Q TLR2 cannot account for the signaling deficiency. Molecular modeling studies suggested that the R753Q mutation changes the electrostatic potential of the DD loop and results in a discrete movement of the residues critical for protein-protein interactions. Confirming these predictions, biochemical assays demonstrated that R753Q TLR2 exhibits deficient agonist-induced tyrosine phosphorylation, hetero-dimerization with TLR6, and recruitment of Mal and MyD88. These proximal signaling deficiencies correlated with impaired capacities of the R753Q TLR2 to mediate p38 phosphorylation, NF-κB activation, and induction of IL-8 mRNA in transfected HEK293 cells challenged with inactivated Mycobacterium tuberculosis or mycobacterial components. Thus, the R753Q polymorphism renders TLR2 signaling-incompetent by impairing its tyrosine phosphorylation, dimerization with TLR6, and recruitment of Mal and MyD88.

Involvement of TLR2 and TLR4 in cell responses to Rickettsia akari.

A better understanding of the pathogenesis of rickettsial disease requires elucidation of mechanisms governing host defense during infection. TLRs are primary sensors of microbial pathogens that activate innate immune cells, as well as initiate and orchestrate adaptive immune responses. However, the role of TLRs in rickettsia recognition and cell activation remains poorly understood. In this study, we examined the involvement of TLR2 and TLR4 in recognition of Rickettsia akari, a causative agent of rickettsialpox. Transfection‐based complementation of TLR2/4‐negative HEK293T cells with human TLR2 or TLR4 coexpressed with CD14 and MD‐2 enabled IκB‐α degradation, NF‐κB reporter activation, and IL‐8 expression in response to heat‐killed (HK) R. akari. The presence of the R753Q TLR2 or D299G TLR4 polymorphisms significantly impaired the capacities of the respective TLRs to signal HK R. akari‐mediated NF‐κB reporter activation in HEK293T transfectants. Blocking Ab against TLR2 or TLR4 markedly inhibited TNF‐α release from human monocytes stimulated with HK R. akari, and TNF‐α secretion elicited by infection with live R. akari was reduced significantly only upon blocking of TLR2 and TLR4. Live and HK R. akari exerted phosphorylation of IRAK1 and p38 MAPK in 293/TLR4/MD‐2 or 293/TLR2 stable cell lines, whereas only live bacteria elicited responses in TLR2/4‐negative HEK293T cells. These data demonstrate that HK R. akari triggers cell activation via TLR2 or TLR4 and suggest use of additional TLRs and/or NLRs by live R. akari.

Treg engage lymphotoxin beta receptor for afferent lymphatic transendothelial migration.

Regulatory T cells (Tregs) are essential to suppress unwanted immunity or inflammation. After islet allo-transplant Tregs must migrate from blood to allograft, then via afferent lymphatics to draining LN to protect allografts. Here we show that Tregs but not non-Treg T cells use lymphotoxin (LT) during migration from allograft to draining LN, and that LT deficiency or blockade prevents normal migration and allograft protection. Treg LTαβ rapidly modulates cytoskeletal and membrane structure of lymphatic endothelial cells; dependent on VCAM-1 and non-canonical NFκB signalling via LTβR. These results demonstrate a form of T-cell migration used only by Treg in tissues that serves an important role in their suppressive function and is a unique therapeutic focus for modulating suppression.

T-bet Regulates Natural Regulatory T Cell Afferent Lymphatic Migration and Suppressive Function.

T-bet is essential for natural regulatory T cells (nTreg) to regulate Th1 inflammation, but whether T-bet controls other Treg functions after entering the inflammatory site is unknown. In an islet allograft model, T-bet−/− nTreg, but not induced Treg, failed to prolong graft survival as effectively as wild-type Treg. T-bet−/− nTreg had no functional deficiency in vitro but failed to home from the graft to draining lymph nodes (dLN) as efficiently as wild type. T-bet regulated expression of adhesion- and migration-related molecules, influencing nTreg distribution in tissues, so that T-bet−/− nTreg remained in the grafts rather than migrating to lymphatics and dLN. In contrast, both wild-type and T-bet−/− CD4+ conventional T cells and induced Treg migrated normally toward afferent lymphatics. T-bet−/− nTreg displayed instability in the graft, failing to suppress Ag-specific CD4+ T cells and prevent their infiltration into the graft and dLN. Thus, T-bet regulates nTreg migration into afferent lymphatics and dLN and consequently their suppressive stability in vivo.

Pellino-1 Positively Regulates Toll-like Receptor (TLR) 2 and TLR4 Signaling and Is Suppressed upon Induction of Endotoxin Tolerance

Background: Dysregulated Toll-like receptor (TLR) signaling is a hallmark of endotoxin-tolerized macrophages in immunosuppression stages of sepsis but Pellino-1 involvement is unknown. Results: Endotoxin tolerization suppresses LPS-induced Pellino-1, Pellino-1 potentiates TLR2/4-driven NF-κB, cytokines and K63-linked IRAK1, TBK1, TAK1, and TRAF6 polyubiquitination. Conclusion: Pellino-1 potentiates TLR2/4 signaling and is decreased by endotoxin tolerization. Significance: Uncovering mechanisms of endotoxin tolerance is critical to understand sepsis-associated immunosuppression. Endotoxin tolerance reprograms Toll-like receptor (TLR) 4-mediated macrophage responses by attenuating induction of proinflammatory cytokines while retaining expression of anti-inflammatory and antimicrobial mediators. We previously demonstrated deficient TLR4-induced activation of IL-1 receptor-associated kinase (IRAK) 4, IRAK1, and TANK-binding kinase (TBK) 1 as critical hallmarks of endotoxin tolerance, but mechanisms remain unclear. In this study, we examined the role of the E3 ubiquitin ligase Pellino-1 in endotoxin tolerance and TLR signaling. LPS stimulation increased Pellino-1 mRNA and protein expression in macrophages from mice injected with saline and in medium-pretreated human monocytes, THP-1, and MonoMac-6 cells, whereas endotoxin tolerization abrogated LPS inducibility of Pellino-1. Overexpression of Pellino-1 in 293/TLR2 and 293/TLR4/MD2 cells enhanced TLR2- and TLR4-induced nuclear factor κB (NF-κB) and expression of IL-8 mRNA, whereas Pellino-1 knockdown reduced these responses. Pellino-1 ablation in THP-1 cells impaired induction of myeloid differentiation primary response protein (MyD88), and Toll-IL-1R domain-containing adapter inducing IFN-β (TRIF)-dependent cytokine genes in response to TLR4 and TLR2 agonists and heat-killed Escherichia coli and Staphylococcus aureus, whereas only weakly affecting phagocytosis of heat-killed bacteria. Co-expressed Pellino-1 potentiated NF-κB activation driven by transfected MyD88, TRIF, IRAK1, TBK1, TGF-β-activated kinase (TAK) 1, and TNFR-associated factor 6, whereas not affecting p65-induced responses. Mechanistically, Pellino-1 increased LPS-driven K63-linked polyubiquitination of IRAK1, TBK1, TAK1, and phosphorylation of TBK1 and IFN regulatory factor 3. These results reveal a novel mechanism by which endotoxin tolerance re-programs TLR4 signaling via suppression of Pellino-1, a positive regulator of MyD88- and TRIF-dependent signaling that promotes K63-linked polyubiquitination of IRAK1, TBK1, and TAK1.

IRAK4 kinase activity is not required for induction of endotoxin tolerance but contributes to TLR2-mediated tolerance

Prior exposure to LPS induces “endotoxin tolerance” that reprograms TLR4 responses to subsequent LPS challenge by altering expression of inflammatory mediators. Endotoxin tolerance is thought to limit the excessive cytokine storm and prevent tissue damage during sepsis but renders the host immunocompromised and susceptible to secondary infections. Tolerance initiated via one TLR can affect cellular responses to challenge via the same TLR (“homotolerance”) or through different TLRs (“heterotolerance”). IRAK4, an essential component of the MyD88‐dependent pathway, functions as a kinase and an adapter, activating subsets of divergent signaling pathways. In this study, we addressed mechanistically the role of IRAK4 kinase activity in TLR4‐ and TLR2‐induced tolerance using macrophages from WT versus IRAK4KDKI mice. Whereas IRAK4 kinase deficiency decreased LPS signaling, it did not prevent endotoxin tolerance, as endotoxin pretreatment of WT and IRAK4KDKI macrophages inhibited LPS‐induced MAPK phosphorylation, degradation of IκB‐α and recruitment of p65 to the TNF‐α promoter, expression of proinflammatory cytokines, and increased levels of A20 and IRAK‐M. Pretreatment of WT macrophages with Pam3Cys, a TLR2–TLR1 agonist, ablated p‐p38 and p‐JNK in response to challenge with Pam3Cys and LPS, whereas IRAK4KDKI macrophages exhibited attenuated TLR2‐elicited homo‐ and heterotolerance at the level of MAPK activation. Thus, IRAK4 kinase activity is not required for the induction of endotoxin tolerance but contributes significantly to TLR2‐elicited homo‐ and heterotolerance.

Pellino-3 promotes endotoxin tolerance and acts as a negative regulator of TLR2 and TLR4 signaling

Development of endotoxin tolerance in macrophages during sepsis reprograms Toll‐like receptor 4 signaling to inhibit proinflammatory cytokines without suppressing anti‐inflammatory and antimicrobial mediators and protects the host from excessive inflammation and tissue damage. However, endotoxin tolerance renders septic patients immunocompromised and unable to control secondary infections. Although previous studies have revealed the importance of several negative regulators of Toll‐like receptor signaling in endotoxin tolerance, the role of Pellino proteins has not been addressed. The present report shows that the induction of endotoxin tolerance in vivo in mice and in vitro in human monocytes and THP‐1 and MonoMac‐6 macrophages increases the expression of Pellino‐3. Overexpression of Pellino‐3 in human embryonic kidney 293/Toll‐like receptor 2 or 293/Toll‐like receptor 4/myeloid differentiation factor‐2 cells inhibited Toll‐like receptor 2/4‐mediated activation of nuclear factor‐κB and induction of CXCL‐8 mRNA, and Pellino‐3 ablation increased these responses. Pellino‐3‐deficient THP‐1 cells had elevated Toll‐like receptor 2/4‐driven tumor necrosis factor‐α, interleukin‐6 mRNA, and Toll‐like receptor 4‐driven CCL5 gene expression in response to Toll‐like receptor agonists and heat‐killed Escherichia coli and Staphylococcus aureus, cytokines controlled by the MyD88 and Toll‐interleukin‐1R domain‐containing protein inducing interferon‐β‐mediated pathways, respectively. In addition, deficiency in Pellino‐3 slightly increased phagocytosis of heat‐killed bacteria. Transfected Pellino‐3 inhibited nuclear factor‐κB activation driven by overexpression of MyD88, TIR domain‐containing adapter inducing interferon‐β, interleukin‐1R‐associated kinase‐1, and tumor necrosis factor receptor activator of nuclear factor‐κB‐binding kinase‐1, TGF‐β‐activated kinase 1, and tumor necrosis factor receptor‐associated factor‐6, and inhibited interleukin‐1R‐associated kinase 1 modifications and tumor necrosis factor receptor activator of nuclear factor‐κB‐binding kinase 1 phosphorylation. Finally, Pellino‐3 ablation in THP‐1 decreased the extent of endotoxin tolerization. Thus, Pellino‐3 is involved in endotoxin tolerance and functions as a negative regulator of Toll‐like receptor 2/4 signaling.