Gabriel Nussbaum

T cells respond to heat shock protein 60 via TLR2: activation of adhesion and inhibition of chemokine receptors

Soluble 60 kDa heat shock protein (HSP60) activates macrophages via TLR4. We now report that soluble HSP60 activates T cells via the innate receptor TLR2. HSP60 activated T cell adhesion to fibronectin to a degree similar to other activators: IL‐2, SDF‐1α, and RANTES. T cell type and state of activation was important; nonactivated CD45RA+ and IL‐2‐activated CD45RO+ T cells responded optimally (1 h) at low concentrations (0.1–1 ng/ml), but nonactivated CD45RO+ T cells required higher concentrations (∼1 εg/ml) of HSP60. T cell HSP60 signaling was inhibited specifically by monoclonal antibodies (mAb) to TLR2 but not by a mAb to TLR4. Indeed, T cells from mice with mutated TLR4 could still respond to HSP60, whereas Chinese hamster T cells with mutated TLR2 did not respond. The human T cell response to soluble HSP60 depended on phosphatidylinositol 3‐kinase and protein kinase C signaling and involved the phosphorylation of Pyk‐2. Soluble HSP60 also inhibited actin polymerization and T cell chemotaxis through extracellular matrix‐like gels toward the chemokines SDF‐1α (CXCL12) or ELC (CCL19). Exposure to HSP60 for longer times (18 h) down‐regulated chemokine receptor expression: CXCR4 and CCR7. These results suggest that soluble HSP60, through TLR2‐dependent interactions, can regulate T cell behavior in inflammation.

Heat Shock Protein 60 Activates B Cells via the TLR4-MyD88 Pathway

We recently reported that soluble 60-kDa heat shock protein (HSP60) can directly activate T cells via TLR2 signaling to enhance their Th2 response. In this study we investigated whether HSP60 might also activate B cells by an innate signaling pathway. We found that human HSP60 (but not the Escherichia coli GroEL or the Mycobacterial HSP65 molecules) induced naive mouse B cells to proliferate and to secrete IL-10 and IL-6. In addition, the HSP60-treated B cells up-regulated their expression of MHC class II and accessory molecules CD69, CD40, and B7-2. We tested the functional ability of HSP60-treated B cells to activate an allogeneic T cell response and found enhanced secretion of both IL-10 and IFN-γ by the responding T cells. The effects of HSP60 were found to be largely dependent on TLR4 and MyD88 signaling; B cells from TLR4-mutant mice or from MyD88 knockout mice showed decreased responses to HSP60. Care was taken to rule out contamination of the HSP60 with LPS as a causative factor. These findings add B cells to the complex web of interactions by which HSP60 can regulate immune responses.

Heat Shock Protein 60 Activates Cytokine-Associated Negative Regulator Suppressor of Cytokine Signaling 3 in T Cells: Effects on Signaling, Chemotaxis, and Inflammation

Previously, we reported that treatment of T cells with the 60-kDa heat shock protein (HSP60) inhibits chemotaxis. We now report that treatment of purified human T cells with recombinant human HSP60 or its biologically active peptide p277 up-regulates suppressor of cytokine signaling (SOCS)3 expression via TLR2 and STAT3 activation. SOCS3, in turn, inhibits the downstream effects of stromal cell-derived-1α (CXCL12)-CXCR4 interaction in: 1) phosphorylation of ERK1/2, Pyk2, AKT, and myosin L chain, required for cell adhesion and migration; 2) formation of rear-front T cell polarity; and 3) migration into the bone marrow of NOD/SCID mice. HSP60 also activates SOCS3 in mouse lymphocytes and inhibits their chemotaxis toward stromal cell-derived factor-1α and their ability to adoptively transfer delayed-type hypersensitivity. These effects of HSP60 could not be attributed to LPS or LPS-associated lipoprotein contamination. Thus, HSP60 can regulate T cell-mediated inflammation via specific signal transduction and SOCS3 activation.

Macrophage Activation by Heparanase Is Mediated by TLR-2 and TLR-4 and Associates With Plaque Progression

Objective—Factors and mechanisms that activate macrophages in atherosclerotic plaques are incompletely understood. We examined the capacity of heparanase to activate macrophages. Methods and Results—Highly purified heparanase was added to mouse peritoneal macrophages and macrophage-like J774 cells, and the levels of tumor necrosis factor-&agr;, matrix metalloproteinase-9, interlukin-1, and monocyte chemotactic protein-1 were evaluated by ELISA. Gene expression was determined by RT-PCR. Cells collected from Toll-like receptor-2 and Toll-like receptor-4 knockout mice were evaluated similarly. Heparanase levels in the plasma of patients with acute myocardial infarction, stable angina, and healthy subjects were determined by ELISA. Immunohistochemistry was applied to detect the expression of heparanase in control specimens and specimens of patients with stable angina or acute myocardial infarction. Addition or overexpression of heparanase variants resulted in marked increase in tumor necrosis factor-&agr;, matrix metalloproteinase-9, interlukin-1, and monocyte chemotactic protein-1 levels. Mouse peritoneal macrophages harvested from Toll-like receptor-2 or Toll-like receptor-4 knockout mice were not activated by heparanase. Plasma heparanase level was higher in patients with acute myocardial infarction, compared with patients with stable angina and healthy subjects. Pathologic coronary specimens obtained from vulnerable plaques showed increased heparanase staining compared with specimens of stable plaque and controls. Conclusion—Heparanase activates macrophages, resulting in marked induction of cytokine expression associated with plaque progression toward vulnerability.

Involvement of Toll-Like Receptors 2 and 4 in the Innate Immune Response to Treponema denticola and Its Outer Sheath Components

ABSTRACT Treponema denticola is considered an important oral pathogen in the development and progression of periodontal diseases. In the present study, the mechanisms of recognition and activation of murine macrophages by T. denticola and its major outer sheath protein (MSP) and lipooligosaccharide (LOS or glycolipid) were investigated. T. denticola cells and the MSP induced innate immune responses through TLR2-MyD88, whereas LOS induced a macrophage response through TLR4-MyD88. The presence of gamma interferon (IFN-γ), or of high numbers of T. denticola, circumvented the requirement for TLR2 for the macrophage response to T. denticola, although the response was still dependent on MyD88. In contrast, synergy with IFN-γ did not alter the TLR dependence of the response to the T. denticola surface components LOS and MSP, despite enhanced sensitivity. These data suggest that although there is flexibility in the requirements for recognition of T. denticola cells (TLR2 dependent or independent), MyD88 is a requirement for the downstream signaling events that lead to inflammation. We also demonstrate that both outer sheath molecules LOS and MSP induce macrophage tolerance to further stimulation with enterobacterial lipopolysaccharide. Tolerance induced by T. denticola components during mixed infections may represent a general mechanism through which bacteria evade clearance.

TLR2-Dependent Inflammatory Response to Porphyromonas gingivalis Is MyD88 Independent, whereas MyD88 Is Required To Clear Infection

Porphyromonas gingivalis is a Gram-negative anaerobe considered to be a major periodontal pathogen. TLR2 plays a central role in the response to P. gingivalis infection in vivo. In its absence there is a weak inflammatory response; however, bacteria are cleared rapidly compared with wild-type mice. We examined the role of the TLR adaptor proteins MyD88 and TLR/IL-1R–domain-containing adaptor-inducing IFN-β in the inflammatory response to P. gingivalis in vivo and in the ability to clear the bacterial infection. Proinflammatory cytokine production in response to P. gingivalis infection depends on TLR2, but it does not require MyD88 or TLR/IL-1R–domain-containing adaptor-inducing IFN-β. In contrast, the generation of intracellular toxic oxygen species and the ultimate clearance of P. gingivalis infection depend critically on MyD88, independent of TLR2. Thus, robust cytokine production and bacterial clearance are independent events mediated by distinct signaling pathways following infection with P. gingivalis.

Induction of IgG3 to LPS via Toll-Like Receptor 4 Co-Stimulation

B-cells integrate antigen-specific signals transduced via the B-cell receptor (BCR) and antigen non-specific co-stimulatory signals provided by cytokines and CD40 ligation in order to produce IgG antibodies. Toll-like receptors (TLRs) also provide co-stimulation, but the requirement for TLRs to generate T-cell independent and T-cell dependent antigen specific antibody responses is debated. Little is known about the role of B-cell expressed TLRs in inducing antigen-specific antibodies to antigens that also activate TLR signaling. We found that mice lacking functional TLR4 or its adaptor molecule MyD88 harbored significantly less IgG3 natural antibodies to LPS, and required higher amounts of LPS to induce anti-LPS IgG3. In vitro, BCR and TLR4 signaling synergized, lowering the threshold for production of T-cell independent IgG3 and IL-10. Moreover, BCR and TLR4 directly associate through the transmembrane domain of TLR4. Thus, in vivo, BCR/TLR synergism could facilitate the induction of IgG3 antibodies against microbial antigens that engage both innate and adaptive B-cell receptors. Vaccines might exploit BCR/TLR synergism to rapidly induce antigen-specific antibodies before significant T-cell responses arise.

Pneumococcal Capsular Polysaccharide Is Immunogenic When Present on the Surface of Macrophages and Dendritic Cells: TLR4 Signaling Induced by a Conjugate Vaccine or by Lipopolysaccharide Is Conducive

Previously, we reported that a peptide, p458, from the sequence of the mammalian 60-kDa heat shock protein (hsp60) molecule can serve as a carrier in conjugate vaccines with capsular polysaccharide (CPS) molecules of various bacteria. These conjugate vaccines were effective injected in PBS without added adjuvants. We now report that p458 conjugated to pneumococcal CPS type 4 (PS4) manifests innate adjuvant effects: it stimulated mouse macrophages to secrete IL-12 and induced the late appearance of PS4 on the macrophage surface in a TLR4-dependent manner; PS4 alone or conjugated to other carriers did not stimulate macrophages in vitro. The injection of macrophages manifesting PS4 on the surface into mice induced long-term resistance to lethal Streptococcus pneumoniae challenge. The TLR4 ligand LPS could also induce the late appearance on the surface of unconjugated PS4 and resistance to challenge in injected mice. Resistance was not induced by macrophages containing only internalized PS4 or by pulsed macrophages that had been lysed. Glutaraldehyde-fixed macrophages pulsed with PS4 did induce resistance to lethal challenge. Moreover, bone marrow-derived dendritic cells activated by LPS and pulsed with unconjugated CPS were also effective in inducing resistance to lethal challenge. Resistance induced by the PS4-pulsed bone marrow-derived dendritic cell was specific for pneumococcal CPS serotypes (type 3 or type 4) and was associated with the induction of CPS-specific IgG and IgM Abs.

Proteins and Their Derived Peptides as Carriers in a Conjugate Vaccine for Streptococcus pneumoniae: Self-Heat Shock Protein 60 and Tetanus Toxoid

We induced T cell help for vaccination against Streptococcus pneumoniae (Pn) using self and foreign peptides and their source proteins conjugated to the capsular polysaccharide (CPS) of type 4 Pn; the carriers were self-heat shock protein 60 (HSP60) and tetanus toxoid (TT). We measured the production of IgG Abs to the CPS and the carriers, and tested resistance to challenge with highly lethal amounts of Pn injected i.p. (LD50 × 103–106). We now report that vaccination protects old and young mice from bacterial challenge; however, there were significant differences in vaccine efficacy based on the carrier. Self-HSP60 peptide p458m was more effective than the whole HSP60 molecule and was equally effective compared with TT. Both p458m and TT were more protective than the TT-derived peptide p30 after a single vaccination. However, peptide p30 was effective in more MHC genotypes than was p458m. Unlike other vaccines, protection conferred by p458m was not related to the amount of anti-CPS Ab: mice that produced very little Ab were still protected from highly lethal doses of bacteria (LD50 × 105–106). Furthermore, unlike the other carriers, there was no Ab response to the p458m carrier. Thus, peptides, self as well as foreign, can provide T cell help that differs functionally from that provided by the whole parent protein.

GAS6 is a key homeostatic immunological regulator of host–commensal interactions in the oral mucosa

Significance Understanding the mechanisms by which the immune system and local microorganisms coexist in the oral cavity is important, as disruption of this delicate balance could cause oral and systemic diseases. We revealed that growth arrest specific 6 (GAS6), a ligand of the TYRO3–AXL–MERTK signaling system, plays a critical role in this process. Upon birth, microorganisms residing in the oral cavity induce expression of GAS6 in oral tissues; GAS6 in turn regulates antibacterial function in these tissues. We also found that GAS6 expressed by cells of the immune system further contributes to its regulatory role in oral tissues. Collectively, this work proposes that GAS6 restrains the immune response in the oral cavity to maintain coexistence with favorable microorganisms residing within the oral cavity. The oral epithelium contributes to innate immunity and oral mucosal homeostasis, which is critical for preventing local inflammation and the associated adverse systemic conditions. Nevertheless, the mechanisms by which the oral epithelium maintains homeostasis are poorly understood. Here, we studied the role of growth arrest specific 6 (GAS6), a ligand of the TYRO3–AXL–MERTK (TAM) receptor family, in regulating oral mucosal homeostasis. Expression of GAS6 was restricted to the outer layers of the oral epithelium. In contrast to protein S, the other TAM ligand, which was constitutively expressed postnatally, expression of GAS6 initiated only 3–4 wk after birth. Further analysis revealed that GAS6 expression was induced by the oral microbiota in a myeloid differentiation primary response gene 88 (MyD88)-dependent fashion. Mice lacking GAS6 presented higher levels of inflammatory cytokines, elevated frequencies of neutrophils, and up-regulated activity of enzymes, generating reactive nitrogen species. We also found an imbalance in Th17/Treg ratio known to control tissue homeostasis, as Gas6-deficient dendritic cells preferentially secreted IL-6 and induced Th17 cells. As a result of this immunological shift, a significant microbial dysbiosis was observed in Gas6−/− mice, because anaerobic bacteria largely expanded by using inflammatory byproducts for anaerobic respiration. Using chimeric mice, we found a critical role for GAS6 in epithelial cells in maintaining oral homeostasis, whereas its absence in hematopoietic cells synergized the level of dysbiosis. We thus propose GAS6 as a key immunological regulator of host–commensal interactions in the oral epithelium.