HSP60 as a Target of Anti-Ergotypic Regulatory T Cells

The 60 kDa heat shock protein (HSP60) has been reported to influence T-cell responses in two ways: as a ligand of toll-like receptor 2 signalling and as an antigen. Here we describe a new mechanism of T-cell immuno-regulation focused on HSP60: HSP60 is up-regulated and presented by activated T cells (HSP60 is an ergotope) to regulatory (anti-ergotypic) T cells. Presentation of HSP60 by activated T cells was found to be MHC-restricted and dependent on accessory molecules - CD28, CD80 and CD86. Anti-ergotypic T cells responded to T-cell HSP60 by proliferation and secreted IFNγ and TGFβ1. In vitro, the anti-ergotypic T cells inhibited IFNγ production by their activated T-cell targets. In vivo, adoptive transfer of an anti-ergotypic HSP60-specific T-cell line led to decreased secretion of IFNγ by arthritogenic T cells and ameliorated adjuvant arthritis (AA). Thus, the presentation of HSP60 by activated T cells turns them into targets for anti-ergotypic regulatory T cells specific for HSP60. However, the direct interaction between the anti-ergotypic T regulators (anti-HSP60) and the activated T cells also down-regulated the regulators. Thus, by functioning as an ergotope, HSP60 can control both the effector T cells and the regulatory HSP60-specific T cells that control them

Blocking TLR2 Activity Attenuates Pulmonary Metastases of Tumor

Background: Metastasis is the most pivotal cause of mortality in cancer patients. Immune tolerance plays a crucial role in tumor progression and metastasis. Methods and Findings: In this study, we investigated the potential roles and mechanisms of TLR2 signaling on tumor metastasis in a mouse model of intravenously injected B16 melanoma cells. Multiple subtypes of TLRs were expressed on B16 cells and several human cancer cell lines; TLR2 mediated the invasive activity of these cells. High metastatic B16 cells released more heat shock protein 60 than poor metastatic B16-F1 cells. Importantly, heat shock protein 60 released by tumor cells caused a persistent activation of TLR2 and was critical in the constitutive activation of transcription factor Stat3, leading to the release of immunosuppressive cytokines and chemokines. Moreover, targeting TLR2 markedly reduced pulmonary metastases and increased the survival of B16-bearing mice by reversing B16 cells induced immunosuppressive microenvironment and restoring tumor-killing cells such as CD8 + T cells and M1 macrophages. Combining an anti-TLR2 antibody and a cytotoxic agent, gemcitabine, provided a further improvement in the survival of tumor-bearing mice. Conclusions and Significance: Our results demonstrate that TLR2 is an attractive target against metastasis and that targeting immunosuppressive microenvironment using anti-TLR2 antibody is a novel therapeutic strategy for combating

Toll-Like Receptor Ligands Induce Human T Cell Activation and Death, a Model for HIV Pathogenesis

Background: Recently, heightened systemic translocation of microbial products was found in persons with chronic HIV infection and this was linked to immune activation and CD4 + T cell homeostasis. Methodology: We examined here the effects of microbial Toll-like receptor (TLR) ligands on T cell activation in vitro. Conclusions/Findings: We show that exposure to TLR ligands results in activation of memory and effector CD4 + and CD8 + T cells. After exposure to each of 8 different ligands that activate TLRs 2, 3, 4, 5, 7, 8, and 9, CD8 + T cells are activated and gain expression of the C type lectin CD69 that may promote their retention in lymphoid tissues. In contrast, CD4 + T cells rarely increase CD69 expression but instead enter cell cycle. Despite activation and cell cycle entry, CD4 + T cells divide poorly and instead, disproportionately undergo activation-induced cell death. Systemic exposure to TLR agonists may therefore increase immune activation, effector cell sequestration in lymphoid tissues and T cell turnover. These events may contribute to the pathogenesis of immune dysfunction and CD4+ T cell losses in chronic infection with the human immunodeficiency virus

A Mycobacterium leprae Hsp65 Mutant as a Candidate for Mitigating Lupus Aggravation in Mice

Hsp60 is an abundant and highly conserved family of intracellular molecules. Increased levels of this family of proteins have been observed in the extracellular compartment in chronic inflammation. Administration of M. leprae Hsp65 [WT] in [NZBxNZW]F1 mice accelerates the Systemic Lupus Erythematosus [SLE] progression whereas the point mutated K409A Hsp65 protein delays the disease. Here, the biological effects of M. leprae Hsp65 Leader pep and K409A pep synthetic peptides, which cover residues 352–371, are presented. Peptides had immunomodulatory effects similar to that observed with their respective proteins on survival and the combined administration of K409A+Leader pep or K409A pep+WT showed that the mutant forms were able to inhibit the deleterious effect of WT on mortality, indicating the neutralizing potential of the mutant molecules in SLE progression. Molecular modeling showed that replacing Lysine by Alanine affects the electrostatic potential of the 352–371 region. The number of interactions observed for WT is much higher than for Hsp65 K409A and mouse Hsp60. The immunomodulatory effects of the point-mutated protein and peptide occurred regardless of the catalytic activity. These findings may be related to the lack of effect on survival when F1 mice were inoculated with Hsp60 or K409A pep. Our findings indicate the use of point-mutated Hsp65 molecules, such as the K409A protein and its corresponding peptide, that may minimize or delay the onset of SLE, representing a new approach to the treatment of autoimmune diseases

PKC Theta Ablation Improves Healing in a Mouse Model of Muscular Dystrophy

Inflammation is a key pathological characteristic of dystrophic muscle lesion formation, limiting muscle regeneration and resulting in fibrotic and fatty tissue replacement of muscle, which exacerbates the wasting process in dystrophic muscles. Limiting immune response is thus one of the therapeutic options to improve healing, as well as to improve the efficacy of gene- or cell-mediated strategies to restore dystrophin expression. Protein kinase C θ (PKCθ) is a member of the PKCs family highly expressed in both immune cells and skeletal muscle; given its crucial role in adaptive, but also innate, immunity, it is being proposed as a valuable pharmacological target for immune disorders. In our study we asked whether targeting PKCθ could represent a valuable approach to efficiently prevent inflammatory response and disease progression in a mouse model of muscular dystrophy. We generated the bi-genetic mouse model mdx/θ−/−, where PKCθ expression is lacking in mdx mice, the mouse model of Duchenne muscular dystrophy. We found that muscle wasting in mdx/θ−/− mice was greatly prevented, while muscle regeneration, maintenance and performance was significantly improved, as compared to mdx mice. This phenotype was associated to reduction in inflammatory infiltrate, pro-inflammatory gene expression and pro-fibrotic markers activity, as compared to mdx mice. Moreover, BM transplantation experiments demonstrated that the phenotype observed was primarily dependent on lack of PKCθ expression in hematopoietic cells

Toll-Like Receptor 3 and Suppressor of Cytokine Signaling Proteins Regulate CXCR4 and CXCR7 Expression in Bone Marrow-Derived Human Multipotent Stromal Cells

The use of bone marrow-derived human multipotent stromal cells (hMSC) in cell-based therapies has dramatically increased in recent years, as researchers have exploited the ability of these cells to migrate to sites of tissue injury, inflammation, and tumors. Our group established that hMSC respond to "danger" signals--by-products of damaged, infected or inflamed tissues--via activation of Toll-like receptors (TLRs). However, little is known regarding downstream signaling mediated by TLRs in hMSC.We demonstrate that TLR3 stimulation activates a Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 1 pathway, and increases expression of suppressor of cytokine signaling (SOCS) 1 and SOCS3 in hMSC. Our studies suggest that each of these SOCS plays a distinct role in negatively regulating TLR3 and JAK/STAT signaling. TLR3-mediated interferon regulatory factor 1 (IRF1) expression was inhibited by SOCS3 overexpression in hMSC while SOCS1 overexpression reduced STAT1 activation. Furthermore, our study is the first to demonstrate that when TLR3 is activated in hMSC, expression of CXCR4 and CXCR7 is downregulated. SOCS3 overexpression inhibited internalization of both CXCR4 and CXCR7 following TLR3 stimulation. In contrast, SOCS1 overexpression only inhibited CXCR7 internalization.These results demonstrate that SOCS1 and SOCS3 each play a functionally distinct role in modulating TLR3, JAK/STAT, and CXCR4/CXCR7 signaling in hMSC and shed further light on the way hMSC respond to danger signals

Human Regulatory T Cell Suppressive Function Is Independent of Apoptosis Induction in Activated Effector T Cells

CD4(+)CD25(+)FOXP3(+) Regulatory T cells (Treg) play a central role in the immune balance to prevent autoimmune disease. One outstanding question is how Tregs suppress effector immune responses in human. Experiments in mice demonstrated that Treg restrict effector T cell (Teff) responses by deprivation of the growth factor IL-2 through Treg consumption, resulting in apoptosis of Teff.In this study we investigated the relevance of Teff apoptosis induction to human Treg function. To this end, we studied naturally occurring Treg (nTreg) from peripheral blood of healthy donors, and, to investigate Treg function in inflammation in vivo, Treg from synovial fluid of Juvenile Idiopathic Arthritis (JIA) patients (SF-Treg). Both nTreg and SF-Treg suppress Teff proliferation and cytokine production efficiently as predicted. However, in contrast with murine Treg, neither nTreg nor SF-Treg induce apoptosis in Teff. Furthermore, exogenously supplied IL-2 and IL-7 reverse suppression, but do not influence apoptosis of Teff.Our functional data here support that Treg are excellent clinical targets to counteract autoimmune diseases. For optimal functional outcome in human clinical trials, future work should focus on the ability of Treg to suppress proliferation and cytokine production of Teff, rather than induction of Teff apoptosis

Convergent Sets of Data from In Vivo and In Vitro Methods Point to an Active Role of Hsp60 in Chronic Obstructive Pulmonary Disease Pathogenesis

BACKGROUND: It is increasingly clear that some heat shock proteins (Hsps) play a role in inflammation. Here, we report results showing participation of Hsp60 in the pathogenesis of chronic obstructive pulmonary diseases (COPD), as indicated by data from both in vivo and in vitro analyses. METHODS AND RESULTS: Bronchial biopsies from patients with stable COPD, smoker controls with normal lung function, and non-smoker controls were studied. We quantified by immunohistochemistry levels of Hsp10, Hsp27, Hsp40, Hsp60, Hsp70, Hsp90, and HSF-1, along with levels of inflammatory markers. Hsp10, Hsp40, and Hsp60 were increased during progression of disease. We found also a positive correlation between the number of neutrophils and Hsp60 levels. Double-immunostaining showed that Hsp60-positive neutrophils were significantly increased in COPD patients. We then investigated in vitro the effect on Hsp60 expression in bronchial epithelial cells (16HBE) caused by oxidative stress, a hallmark of COPD mucosa, which we induced with H\u2082O\u2082. This stressor determined increased levels of Hsp60 through a gene up-regulation mechanism involving NFkB-p65. Release of Hsp60 in the extracellular medium by the bronchial epithelial cells was also increased after H\u2082O\u2082 treatment in the absence of cell death. CONCLUSIONS: This is the first report clearly pointing to participation of Hsps, particularly Hsp60, in COPD pathogenesis. Hsp60 induction by NFkB-p65 and its release by epithelial cells after oxidative stress can have a role in maintaining inflammation, e.g., by stimulating neutrophils activity. The data open new scenarios that might help in designing efficacious anti-inflammatory therapies centered on Hsp60 and applicable to COP

Modulation of T Cell Function by Combination of Epitope Specific and Low Dose Anticytokine Therapy Controls Autoimmune Arthritis

Innate and adaptive immunity contribute to the pathogenesis of autoimmune arthritis by generating and maintaining inflammation, which leads to tissue damage. Current biological therapies target innate immunity, eminently by interfering with single pro-inflammatory cytokine pathways. This approach has shown excellent efficacy in a good proportion of patients with Rheumatoid Arthritis (RA), but is limited by cost and side effects. Adaptive immunity, particularly T cells with a regulatory function, plays a fundamental role in controlling inflammation in physiologic conditions. A growing body of evidence suggests that modulation of T cell function is impaired in autoimmunity. Restoration of such function could be of significant therapeutic value. We have recently demonstrated that epitope-specific therapy can restore modulation of T cell function in RA patients. Here, we tested the hypothesis that a combination of anti-cytokine and epitope-specific immunotherapy may facilitate the control of autoimmune inflammation by generating active T cell regulation. This novel combination of mucosal tolerization to a pathogenic T cell epitope and single low dose anti-TNFα was as therapeutically effective as full dose anti-TNFα treatment. Analysis of the underlying immunological mechanisms showed induction of T cell immune deviation

A case of mistaken identity: HSPs are no DAMPs but DAMPERs

Until recently, the immune system was seen solely as a defense system with its primary task being the elimination of unwanted microbial invaders. Currently, however, the functional significance of the immune system has obtained a much wider perspective, to include among others the maintenance and restoration of homeostasis following tissue damage. In this latter aspect, there is a growing interest in the identification of molecules involved, such as the so-called danger or damage-associated molecular patterns (DAMPs), also called alarmins. Since heat shock proteins are archetypical molecules produced under stressful conditions, such as tissue damage or inflammation, they are frequently mentioned as prime examples of DAMPs (Bianchi, J Leukoc Biol 81:1–5, 2007; Kono and Rock, Nat Rev Immunol 8:279–289, 2008; Martin-Murphy et al., Toxicol Lett 192:387–394, 2010). See for instance also a recent review (Chen and Nunez, Science 298:1395–1401, 2010). Contrary to this description, we recently presented some of the arguments against a role of heat shock protein as DAMPs (Broere et al., Nat Rev Immunol 11:565-c1, 2011). With this perspective and reflection article, we hope to elaborate on this debate and provide additional thoughts to further ignite this discussion on this critical and evolving issue