The role of toll-like receptors (TLRs) in bacteria-induced maturation of murine dendritic cells (DCS). Peptidoglycan and lipoteichoic acid are inducers of DC maturation and require TLR2

Toll-like receptors (TLRs) have been found to be key elements in pathogen recognition by the host immune system. Dendritic cells (DCs) are crucial for both innate immune responses and initiation of acquired immunity. Here we focus on the potential involvement of TLR ligand interaction in DC maturation. TLR2 knockout mice and mice carrying a TLR4 mutation (C3H/HeJ) were investigated for DC maturation induced by peptidoglycan (PGN), lipopolysaccharide (LPS), or lipoteichoic acids (LTAs). All stimuli induced maturation of murine bone marrow-derived DCs in control mice. TLR2(-)/- mice lacked maturation upon stimulation with PGN, as assessed by expression of major histocompatibility complex class II, CD86, cytokine, and chemokine production, fluorescein isothiocyanate-dextran uptake, and mixed lymphocyte reactions, while being completely responsive to LPS. A similar lack of maturation was observed in C3H/HeJ mice upon stimulation with LPS. DC maturation induced by LTAs from two different types of bacteria was severely impaired in TLR2(-)/-, whereas C3H/HeJ mice responded to LTAs in a manner similar to wild-type mice. We demonstrate that DC maturation is induced by stimuli from Gram-positive microorganisms, such as PGN and LTA, with similar efficiency as by LPS. Finally, we provide evidence that TLR2 and TLR4 interaction with the appropriate ligand is essential for bacteria-induced maturation of DCs.     

TICAM-1 and TICAM-2: toll-like receptor adapters that participate in induction of type 1 interferons

Toll-like receptor (TLR) 3 and 4 mediate the expression of many genes, including NF-kappaB- and interferon-regulatory factor (IRF)-3/interferon (IFN)-inducible genes, in macrophages and dendritic cells (DCs) in response to their ligand stimuli, polyI:C and lipopolysaccharide (LPS). Toll-IL-1 receptor homology domain (TIR)-containing adapter molecule 1 (TICAM-1) facilitates expression of IFN-inducible genes via TLR3. Although MyD88 and Mal/TIRAP adapters function downstream of TLR4, they barely induce IFN-beta. In addition, DC maturation as well as IFN-beta induction are largely independent of MyD88 and Mal/TIRAP. TICAM-1 is the functional adapter for both TLR3 and TLR4 that induces type 1 IFN and MyD88-independent DC maturation. In LPS-mediated TLR4 activation, a complex of TICAM-1 and an additional TLR4-binding adapter serves as the adapter. We named this TLR4-TICAM-1-bridging adapter TICAM-2. Our results reveal the details of MyD88-independent pathways which separately recruit the distinct adapters downstream of TLR3 and TLR4 and variations of the TLR output are in part regulated by the two additional adapters in DCs.     

Dual signaling of MyD88 and TRIF is critical for maximal TLR4-induced dendritic cell maturation

TLR4 is a unique TLR because downstream signaling occurs via two separate pathways, as follows: MyD88 and Toll IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-beta (TRIF). In this study, we compared and contrasted the interplay of these pathways between murine dendritic cells (DCs) and macrophages during LPS stimulation. During TLR4 activation, neither pathway on its own was critical for up-regulation of costimulatory molecules in DCs, whereas the up-regulation of costimulatory molecules was largely TRIF dependent in macrophages. LPS-induced secreted factors, of which type I IFNs were one of the active components, played a larger role in promoting the up-regulation of costimulatory molecules in macrophages than DCs. In both cell types, MyD88 and TRIF pathways together accounted for the inflammatory response to LPS activation. Furthermore, signaling of both adaptors allowed maximal T cell priming by LPS-matured DCs, with MyD88 playing a larger role than TRIF. In sum, in our experimental systems, TRIF signaling plays a more important role in LPS-induced macrophage activation than in DC activation.     

Endotoxin-induced maturation of MyD88-deficient dendritic cells

LPS, a major component of the cell wall of Gram-negative bacteria, can induce a variety of biological responses including cytokine production from macrophages, B cell proliferation, and endotoxin shock. All of them were completely abolished in MyD88-deficient mice, indicating the essential role of MyD88 in LPS signaling. However, MyD88-deficient cells still show activation of NF-kappaB and mitogen-activated protein kinase cascades, although the biological significance of this activation is not clear. In this study, we have examined the effects of LPS on dendritic cells (DCs) from wild-type and several mutant mice. LPS-induced cytokine production from DCs was dependent on MyD88. However, LPS could induce functional maturation of MyD88-deficient DCs, including up-regulation of costimulatory molecules and enhancement of APC activity. MyD88-deficient DCs could not mature in response to bacterial DNA, the ligand for Toll-like receptor (TLR)9, indicating that MyD88 is differentially required for TLR family signaling. MyD88-dependent and -independent pathways originate at the intracytoplasmic region of TLR4, because both cytokine induction and functional maturation were abolished in DCs from C3H/HeJ mice carrying the point mutation in the region. Finally, in vivo analysis revealed that MyD88-, but not TLR4-, deficient splenic CD11c(+) DCs could up-regulate their costimulatory molecule expression in response to LPS. Collectively, the present study provides the first evidence that the MyD88-independent pathway downstream of TLR4 can lead to functional DC maturation, which is critical for a link between innate and adaptive immunity.     

The proteoglycan biglycan enhances antigen-specific T cell activation potentially via MyD88 and TRIF pathways and triggers autoimmune perimyocarditis

Biglycan is a proteoglycan ubiquitously present in extracellular matrix of a variety of organs, including heart, and it was reported to be overexpressed in myocardial infarction. Myocardial infarction may be complicated by perimyocarditis through unknown mechanisms. Our aim was to investigate the capacity of TLR2/TLR4 ligand biglycan to enhance the presentation of specific Ags released upon cardiomyocyte necrosis. In vitro, OVA-pulsed bone marrow-derived dendritic cells from wild-type (WT; C57BL/6) and TLR2-, TLR4-, MyD88-, or TRIF-deficient mice were cotreated with LPS, biglycan, or vehicle and incubated with OVA-recognizing MHC I- or MHC II-restricted T cells. Biglycan enhanced OVA-specific cross-priming by >80% to MHC I-restricted T cells in both TLR2- and TLR4-pathway-dependent manners. Accordingly, biglycan-induced cross-priming by both MyD88- and TRIF-deficient dendritic cells (DCs) was strongly diminished. OVA-specific activation of MHC II-restricted T cells was predominantly TLR4 dependent. Our first in vivo correlate was a model of experimental autoimmune perimyocarditis triggered by injection of cardiac Ag-pulsed DCs (BALB/c). Biglycan-treated DCs triggered perimyocarditis to a comparable extent and intensity as LPS-treated DCs (mean scores 1.3 ± 0.3 and 1.5 ± 0.4, respectively). Substitution with TLR4-deficient DCs abolished this effect. In a second in vivo approach, WT and biglycan-deficient mice were followed 2 wk after induction of myocardial infarction. WT mice demonstrated significantly greater myocardial T lymphocyte infiltration in comparison with biglycan-deficient animals. We concluded that the TLR2/4 ligand biglycan, a component of the myocardial matrix, may enhance Ag-specific T cell priming, potentially via MyD88 and TRIF, and stimulate autoimmune perimyocarditis.     

Lipopolysaccharides from distinct pathogens induce different classes of immune responses in vivo.

The adaptive immune system has evolved distinct responses against different pathogens, but the mechanism(s) by which a particular response is initiated is poorly understood. In this study, we investigated the type of Ag-specific CD4(+) Th and CD8(+) T cell responses elicited in vivo, in response to soluble OVA, coinjected with LPS from two different pathogens. We used Escherichia coli LPS, which signals through Toll-like receptor 4 (TLR4) and LPS from the oral pathogen Porphyromonas gingivalis, which does not appear to require TLR4 for signaling. Coinjections of E. coli LPS + OVA or P. gingivalis LPS + OVA induced similar clonal expansions of OVA-specific CD4(+) and CD8(+) T cells, but strikingly different cytokine profiles. E. coli LPS induced a Th1-like response with abundant IFN-gamma, but little or no IL-4, IL-13, and IL-5. In contrast, P. gingivalis LPS induced Th and T cell responses characterized by significant levels of IL-13, IL-5, and IL-10, but lower levels of IFN-gamma. Consistent with these results, E. coli LPS induced IL-12(p70) in the CD8alpha(+) dendritic cell (DC) subset, while P. gingivalis LPS did not. Both LPS, however, activated the two DC subsets to up-regulate costimulatory molecules and produce IL-6 and TNF-alpha. Interestingly, these LPS appeared to have differences in their ability to signal through TLR4; proliferation of splenocytes and cytokine secretion by splenocytes or DCs from TLR4-deficient C3H/HeJ mice were greatly impaired in response to E. coli LPS, but not P. gingivalis LPS. Therefore, LPS from different bacteria activate DC subsets to produce different cytokines, and induce distinct types of adaptive immunity in vivo.     

Activation of acid sphingomyelinase and its inhibition by the nitric oxide/cyclic guanosine 3',5'-monophosphate pathway: key events in Escherichia coli-elicited apoptosis of dendritic cells

Depletion of dendritic cells (DCs) via apoptosis contributes to sepsis-induced immune suppression. The mechanisms leading to DC apoptosis during sepsis are not known. In this study we report that immature DCs undergo apoptosis when treated with high numbers of Escherichia coli. This effect was mimicked by high concentrations of LPS. Apoptosis was accompanied by generation of ceramide through activation of acid sphingomyelinase (A-SMase), was prevented by inhibitors of this enzyme, and was restored by exogenous ceramide. Compared with immature DCs, mature DCs expressed significantly reduced levels of A-SMase, did not generate ceramide in response to E. coli or LPS, and were insensitive to E. coli- and LPS-triggered apoptosis. However, sensitivity to apoptosis was restored by addition of exogenous A-SMase or ceramide. Furthermore, inhibition of A-SMase activation and ceramide generation was found to be the mechanism through which the immune-modulating messenger NO protects immature DCs from the apoptogenic effects of E. coli and LPS. NO acted through formation of cGMP and stimulation of the cGMP-dependent protein kinase. The relevance of A-SMase and its inhibition by NO/cGMP were confirmed in a mouse model of LPS-induced sepsis. DC apoptosis was significantly higher in inducible NO synthase-deficient mice than in wild-type animals and was significantly reduced by treatment ex vivo with NO, cGMP, or the A-SMase inhibitor imipramine. Thus, A-SMase plays a central role in E. coli/LPS-induced DC apoptosis and its inhibition by NO, and it might be a target of new therapeutic approaches to sepsis.     

Normal proportion and expression of maturation markers in migratory dendritic cells in the absence of germs or Toll-like receptor signaling

Dendritic cells (DCs) play major roles in immunosurveillance. In peripheral tissues, 'immature' DCs are dedicated to capturing antigens. Detection of pathogens through Toll-like receptors (TLRs) triggers DC migration to the lymph nodes (LNs), where they acquire a 'mature' phenotype specialized at presenting antigens. However, DCs migrate from tissues and mature even in the absence of overt infections. This has been attributed to detection of commensal flora in the skin, the gut or other peripheral tissues in the steady state. To test this assumption, we have analyzed the DCs contained in the lymphoid organs of germ-free mice and of mice lacking the TLR adapter molecules, MyD88 and TRIF. We show that the proportion and expression of maturation markers in DC immigrants in the LNs of these mice are similar to those in normal mice. These results suggest that DC migration from tissues, followed by their phenotypic maturation, is regulated in the steady state by an inherent program of DC differentiation or by the release of low levels of inflammatory signals from normal tissues.     

Deficit of CD47 results in a defect of marginal zone dendritic cells, blunted immune response to particulate antigen and impairment of skin dendritic cell migration

CD47 is a ubiquitously expressed cell surface glycoprotein that associates with integrins and regulates chemotaxis, migration, and activation of leukocytes. CD47 is also a ligand for signal regulatory protein alpha, a cell surface receptor expressed on monocytes, macrophages, granulocytes, and dendritic cell (DC) subsets that regulates cell activation, adhesion, and migration. Although the function of CD47 in macrophages and granulocytes has been studied in detail, little is known about the role of CD47 in DC biology in vivo. In this study we demonstrate that CD47(-/-) mice exhibit a selective reduction of splenic CD11c(high)CD11b(high)CD8alpha(-)CD4(+) DCs. These DCs correspond to marginal zone DCs and express signal regulatory protein alpha, possibly explaining their selective deficiency in CD47(-/-) mice. Deficiency of marginal zone DCs resulted in impairment of IgG responses to corpusculate T cell-independent Ags. Although epidermal DCs were present in normal numbers in CD47(-/-) mice, their migration to draining lymph nodes in response to contact sensitization was impaired, while their maturation was intact. In vitro, CD47(-/-) mature DCs showed normal CCR7 expression but impaired migration to CCL-19, whereas immature DC response to CCL-5 was only slightly impaired. These results demonstrate a fundamental role of CD47 in DC migration in vivo and in vitro and in the function of marginal zone DCs.     

A comparative analysis of cytokine responses, cell surface marker expression and MAPKs in DCs matured with LPS compared with a panel of TLR ligands

Dendritic cells (DCs) are professional antigen-presenting cells that play a vital role in shaping adaptive immunity. DC maturation begins when exogenous danger signals bind to the appropriate toll-like receptor (TLR) and initiate expression of cell surface markers and the secretion of cytokines. This process occurs through defined mitogen-activated protein kinase (MAPK) signalling pathways. Of the 13 known mammalian TLRs, lipopolysaccharide (LPS), which activates TLR4, is the most commonly used ligand for the maturation of DCs in vitro. This comprehensive study measures cytokine secretion and cell surface marker expression in murine bone-marrow-derived DCs following maturation with LPS compared to DCs matured with a panel of other TLR-ligands (zymosan A (TLR2/6), PGN (TLR2), poly(I:C) (TLR3), flagellin (TLR5) and CpG-ODN1826 (TLR9)). The role of MAPK signalling pathways in the maturation process was also examined. Results demonstrate that zymosan A and CpG induce comparable cytokine and cell surface marker profiles to LPS. The remaining ligands differed significantly for cytokine and CD40 expression, but not for CD80 and CD86 expression. While there were differences for MAPK signalling pathways for all ligands, the effect of the inhibitors were broadly similar. These findings broaden our knowledge of TLR ligand-matured DCs.     

Outer membrane protein A expression in Escherichia coli K1 is required to prevent the maturation of myeloid dendritic cells and the induction of IL-10 and TGF-beta

Dendritic cells (DCs) are professional APCs that direct both cellular and humoral immune responses. Escherichia coli K1 causes meningitis in neonates; however, the interactions between this pathogen and DCs have not been previously explored. In the present study, we observed that E. coli K1, expressing outer membrane protein A (OmpA), was able to enter, survive, and replicate inside DCs, whereas OmpA(-) E. coli was killed within a short period. Opsonization of OmpA(+) E. coli either with adult or cord serum did not affect its survival inside DCs. Exposure of DCs to live OmpA(+) E. coli K1 prevented DCs from progressing in their maturation process as indicated by failure to up-regulate costimulatory molecules, CD40, HLA-DR, and CD86. The distinct DC phenotype requires direct contact between live bacteria and DCs. The expression of costimulatory molecules was suppressed even after pretreatment of DCs with LPS or peptidoglycan. Furthermore, the suppressive effects of OmpA(+) E. coli on DCs were abrogated when the bacteria were incubated with anti-OmpA Ab. The inhibitory effect on DC maturation was associated with increased production of IL-10 as well as TGF-beta and decreased production of IL-6, TNF-alpha, IL-1beta, and IL-12p70 by DCs, a phenotype associated with tolerogenic DCs. These results suggest that the subversion of DC functions may be a novel strategy deployed by this pathogen to escape immune defense and persist in the infected host to reach a high degree of bacteremia, which is crucial for E. coli to cross the blood-brain barrier.     

IL-6 and maturation govern TLR2 and TLR4 induced TLR agonist tolerance and cross-tolerance in dendritic cells

Stimulation of naive mouse dendritic cells (DC) with LPS or Pam(3)CSK(4) (P3C) induces production of TNF-alpha via TLR4- or TLR2-signaling. Although tolerance in macrophages has been studied in detail, we investigated the role of TLR agonist concentration and IL-6 for tolerance in DC. P3C- or LPS-primed DC were nonresponsive to P3C or LPS restimulation in terms of TNF-alpha but not IL-6 production. The mechanisms involved in tolerance were dependent on the concentration of the TLR ligand used for DC priming. DC primed with LPS or P3C at high concentrations developed a maturation dependent, IL-6 independent tolerance associated with inhibition of TLR signaling upstream of IkappaB as indicated by decreased IkappaB degradation. In contrast, priming of DC with LPS or P3C at low concentrations resulted in IL-6-dependent tolerance, which was abolished in IL-6 deficient DC, and was not accompanied by maturation of DC or by down-regulation of TLR2 or TLR4. In homotolerogenic DC primed with LPS or P3C at high concentrations, degradation of IkappaB upon restimulation with LPS or P3C was inhibited suggesting tolerance mechanism(s) upstream of IkappaB; in contrast, cross-tolerance in DC primed with LPS or P3C at low concentrations was not associated with reduced IkappaB degradation suggesting tolerance mechanisms downstream of IkappaB. Our data indicate that in naive DC TLR4- and TLR2-stimulation results in homo- and cross-tolerance; the mechanisms involved in tolerance depend on the concentration of the TLR agonist used for DC priming and are governed by IL-6 and maturation.     

Contributions of Unique Intracellular Domains to Switchlike Biosensing by Toll-like Receptor 4

Toll-like receptors (TLRs) mediate immune recognition of both microbial infections and tissue damage. Aberrant TLR signaling promotes disease; thus, understanding the regulation of TLR signaling is of medical relevance. Although downstream mediators of TLR signaling have been identified, the detailed mechanism by which ligand binding-mediated dimerization induces downstream signaling remains poorly understood. Here, we investigate this question for TLR4, which mediates responsiveness to bacterial LPS and drives inflammatory disease. TLR4 exhibits structural and functional features that are unique among TLRs, including responsiveness to a wide variety of ligands. However, the connection between these structural features and the regulation of signaling is not clear. Here, we investigated how the unique intracellular structures of TLR4 contribute to receptor signaling. Key conclusions include the following. 1) The unique intracellular linker of TLR4 is important for achieving LPS-inducible signaling via Toll/IL-1 receptor (TIR) domain-containing adapter-inducing interferon-β (TRIF) but less so for signaling via myeloid differentiation primary response 88 (MyD88). 2) Membrane-bound TLR4 TIR domains were sufficient to induce signaling. However, introducing long, flexible intracellular linkers neither induced constitutive signaling nor ablated LPS-inducible signaling. Thus, the initiation of TLR4 signaling is regulated by a mechanism that does not require tight geometric constraints. Together, these observations necessitate refining the model of TLR4 signal initiation. We hypothesize that TLR4 may interact with an inhibitory partner in the absence of ligand, via both TIR and extracellular domains of TLR4. In this speculative model, ligand binding induces dissociation of the inhibitory partner, triggering spontaneous, switchlike TIR domain homodimerization to initiate downstream signaling.     

Lipopolysaccharide induces cell volume increase and migration of dendritic cells

Migration of dendritic cells (DCs) plays an important role in T‐cell‐mediated adaptive immune responses. Lipopolysaccharide (LPS) sensed by Toll‐like receptor 4 (TLR4) serves as a signal for DC migration. We analyzed LPS‐induced DC volume changes preceding the directed movement towards chemoattractants. Treatment with LPS resulted in rapid, prolonged cell swelling in wild‐type (WT), but not in TLR4^?/? bone marrow‐derived (BM) DCs indicating that TLR4 signaling is essential for LPS‐induced swelling. As a consequence, LPS‐treatment enhanced the migratory activity along a chemokine (CCL21)‐gradient in WT, but not in TLR4‐deficient BMDCs suggesting that the LPS/TLR4‐induced swelling response facilitates DC migration. Moreover, the role of calcium‐activated potassium channels (K_Ca3.1) as putative regulators of immune cell volume regulation and migration was analyzed in LPS‐challenged BMDCs. We found that the LPS‐induced swelling of K_Ca3.1‐deficient DCs was impaired when compared to WT DCs. Accordingly, the LPS‐induced increase in [Ca²⁺]_i detected in WT DCs was reduced in K_Ca3.1‐deficient DCs. Finally, directed migration of LPS‐challenged K_Ca3.1‐deficient DCs was low compared to WT DCs indicating that activation of K_Ca3.1 is involved in LPS‐induced DC migration. These findings suggest that both TLR4 and K_Ca3.1 contribute to the migration of LPS‐activated DCs as an important feature of the adaptive immune response.

     

LPS response and endotoxin tolerance in Flt-3L-induced bone marrow-derived dendritic cells

Fms-like tyrosine kinase-3 ligand (Flt-3L) stimulates the differentiation of bone marrow cells into dendritic cells (DCs) and was used as an adjuvant therapy in the experimental model of burn wound sepsis. In this study, we describe the phenotypical characteristics of an Flt-3L-dependent DC culture (FLDC) system following LPS stimulation, which induces an inflammatory response, and after a second LPS stimulation, which induces tolerance. Priming of FLDCs with LPS via TLR4 has been shown to induce the activation of all three mitogen-activated protein kinase (MAPK) families and enhance NF-κB complex translocation into the nucleus. Stimulated FLDCs express all maturation markers and exhibit an increase in IL-12p40 production and to a lesser extent, IL-10 production. In contrast, LPS stimulation of tolerized FLDCs was not associated with TLR4 up-regulation and led to MAPK inhibition. The decrease in p38 and JNK activation was correlated with an impairment of IL-12p40 production. Endotoxin tolerance in FLDCs was associated with enhanced ERK1/2 activation, an increase in MKP-1 phosphatase expression, a decrease in NF-κB translocation to the nucleus and an increase in IL-10 production. Overall, DCs generated from bone marrow with Flt-3 ligand have similar characteristics to DC subtypes found in the steady state in vivo, which can acquire endotoxin tolerance in some circumstances.     

T cell-dependent maturation of dendritic cells in response to bacterial superantigens

Dendritic cells (DC) express a set of germline-encoded transmembrane Toll-like receptors that recognize shared microbial products, such as Escherichia coli LPS, termed pathogen-associated molecular patterns. Analysis of the in vivo response to pathogen-associated molecular patterns has uncovered their ability to induce the migration and the maturation of DC, favoring thus the delivery of Ag and costimulatory signals to naive T cells in vivo. Bacterial superantigens constitute a particular class of pathogen-derived molecules known to induce a potent inflammatory response in vivo, secondary to the activation of a large repertoire of T cells. We demonstrate in this work that Staphylococcal superantigens induce migration and maturation of DC populations in vivo. However, in contrast to LPS, superantigens failed to induce DC maturation in RAG or MHC class II-deficient mice, suggesting that T cell activation was a prerequisite for DC maturation. This conclusion was further supported by the finding that T cell activation induced by 1) mitogenic anti-CD3 mAbs, 2) allo-MHC determinants, or 3) nominal Ag in a TCR-transgenic model induces DC maturation in vivo. These studies also revealed that DC that matured in response to T cell mitogens display, comparatively to LPS, a distinctive phenotype characterized by high expression of the MHC class II, CD40, and CD205 markers, but only moderate (CD86) to minimal (CD80) expression of CD28/CTLA4 ligands. This work demonstrates that activation of a sufficient number of naive T cells in vivo constitutes a novel form of immune danger, functionally linked to DC maturation.     

High mobility group box protein 1 (HMGB1)-partner molecule complexes enhance cytokine production by signaling through the partner molecule receptor

The nuclear protein high mobility group box protein 1 (HMGB1) promotes inflammation upon extracellular release. HMGB1 induces proinflammatory cytokine production in macrophages via Toll-like receptor (TLR)-4 signaling in a redox-dependent fashion. Independent of its redox state and endogenous cytokine-inducing ability, HMGB1 can form highly immunostimulatory complexes by interaction with certain proinflammatory mediators. Such complexes have the ability to enhance the induced immune response up to 100-fold, compared with induction by the ligand alone. To clarify the mechanisms for these strong synergistic effects, we studied receptor requirements. Interleukin (IL)-6 production was assessed in supernatants from cultured peritoneal macrophages from mice each deficient in one of the HMGB1 receptors (receptor for advanced glycation end products [RAGE], TLR2 or TLR4) or from wild-type controls. The cultures were stimulated with the TLR4 ligand lipopolysaccaride (LPS), the TLR2 ligand Pam₃CysSerLys₄ (Pam₃CSK₄), noninflammatory HMGB1 or each TLR ligand in complex with noninflammatory HMGB1. The activity of the HMGB1-TLR ligand complexes relied on engagement of the same receptor as for the noncomplexed TLR ligand, since HMGB1-LPS complexes used TLR4 and HMGB1-Pam₃CSK₄ complexes used TLR2. Deletion of any of the intracellular adaptor molecules used by TLR2 (myeloid differentiation factor-88 [MyD88], TIR domain–containing adaptor protein [TIRAP]) or TLR4 (MyD88, TIRAP, TIR domain–containing adaptor-inducing interferon-β [TRIF], TRIF-related adaptor molecule [TRAM]) had similar effects on HMGB1 complex activation compared with noncomplexed LPS or Pam₃CSK₄. This result implies that the enhancing effects of HMGB1-partner molecule complexes are not regulated by the induction of additional signaling cascades. Elucidating HMGB1 receptor usage in processes where HMGB1 acts alone or in complex with other molecules is essential for the understanding of basic HMGB1 biology and for designing HMGB1-targeted therapies.