Role of Heat Shock Proteins in Diseases and Their Therapeutic Potential

Heat shock proteins are ubiquitously expressed intracellular proteins and act as molecular chaperones in processes like protein folding and protein trafficking between different intracellular compartments. They are induced during stress conditions like oxidative stress, nutritional deficiencies and radiation. They are released into extracellular compartment during necrosis. However, recent research findings highlights that, they are not solely present in cytoplasm, but also released into extracellular compartment during normal conditions and even in the absence of necrosis. When present in extracellular compartment, they have been shown to perform various functions like antigen presentation, intercellular signaling and induction of pro-inflammatory cytokines. Heat shock proteins represents as dominant microbial antigens during infection. The phylogenetic similarity between prokaryotic and eukaryotic heat shock proteins has led to proposition that, microbial heat shock proteins can induce self reactivity to host heat shock proteins and result in autoimmune diseases. The self-reactivity of heat shock proteins protects host against disease by controlling induction and release of pro-inflammatory cytokines. However, antibodies to self heat shock proteins haven been implicated in pathogenesis of autoimmune diseases like arthritis and atherosclerosis. Some heat shock proteins are potent inducers of innate and adaptive immunity. They activate dendritic cells and natural killer cells through toll-like receptors, CD14 and CD91. They play an important role in MHC-antigen processing and presentation. These immune effector functions of heat shock proteins are being exploited them as therapeutic agents as well as therapeutic targets for various infectious diseases and cancers.     

Oral administration of HSP-containing E.Coli extract OM-89 has suppressive effects in autoimmunity. Regulation of autoimmune processes by modulating peripheral immunity towards hsp’s?

OM-89 (Subreum) is anE. coli extract used for oral administration in the treatment of rheumatoid arthritis. It contains bacterial heat shock proteins, namely hsp60 and hsp70, which were shown to be major immunogenic constitutents of the drug. Immunity to bacterial heat-shock antigens was shown to be a means of immunomodulation of (experimental) autoimmune disease and possibly inflammation in general. This was demonstrated for mycobacterial hsp60 respectively hsp70 in autoimmune disease models for arthritis, diabetes and encephalitis. Parallel to the effects displayed by immunisation with hsp, oral administration of hsp-containing OM-89 was found to modify autoimmune disease in a number of animal models, such as for arthritis, diabetes and SLE. In rats immunisation with OM-89 was found to lead to proliferative T cell responses to hsp60 and hsp70 of bothE. coli and mycobacterial origin. Conversely, immunisation with hsp antigens could induce T cell reactivity specific for OM-89. Given this and the autoimmune disease modulating properties of both hsp and OM-89 it is argued that OM-89 acts via the same mechanism as proposed for hsp: that peripheral tolerance is induced at the level of regulatory T cells with specificity for heat-shock proteins. This may constitute one mode of action for OM-89 as an arthritis suppressive oral drug in man.     

T cell hyporesponsiveness induced by oral administration of ovalbumin is associated with impaired NFAT nuclear translocation and p27kip1 degradation

Oral tolerance is an important physiological component of the immune system whereby the organism avoids dangerous reactions such as hypersensitivity to ingested food proteins and other luminal Ags which may cause tissue damage and inflammation. In addition, it has been shown in animal models and in humans that oral tolerance can be applied to controlling undesired immune responses, including autoimmune diseases, allergies, and organ transplant rejections. However, the molecular mechanisms of oral tolerance have been poorly defined. In this study, we investigated the molecular basis underlying the hyporesponsiveness of orally tolerant CD4 T cells using a TCR transgenic mouse system in which oral tolerance was induced by long-term feeding with high dose Ag. We demonstrate that the hyporesponsive state of the CD4 T cells was maintained by a selective impairment in the TCR-induced calcium/NFAT signaling pathway and in the IL-2R-induced degradation of p27(kip1) and cell cycle progression. Thus, physiological mucosal tolerance is revealed to be associated with a unique type of T cell hyporesponsiveness which differs from previously described anergic T cells.     

Regulatory T cells in health and disease

Regulatory T (Treg) cells have emerged as a central control point in the modulation of various immune responses, including autoimmune responses and immunity to transplants, allergens, tumors, and infectious microbes. The immune system has evolved complex processes to ensure tolerance to autoantigens while preserving the potential to mount and maintain life-long humoral and cellular immune responses against invading pathogens. In this review, we summarize research showing that naturally occurring (nTreg) and induced Treg (iTreg) cell subsets, and in particular CD4+Foxp3+ Treg cells, are critical in the control of immune responses in rodents and humans. We also discuss the cellular and molecular factors that affect CD4+Foxp3+ Treg cell development, homeostasis, and function and consequential immunity to self and non-self antigens. Recent studies have shed light in our understanding of the development of novel methods of autoimmune disease prevention and treatment via enhancing and re-establishing Treg-mediated dominant control over self-reactive T cells in animal models and humans.     

"2-6-11" motif in heat shock protein 60 and central nervous system antigens: a preliminary study in multiple sclerosis patients

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) with unknown etiology and pathogenesis. A local autoimmune process involving activation of autoreactive T cells against CNS protein components is likely crucial in the development of MS lesions. Myelin-reactive T cells are believed to be primed in the periphery during infections by antigens of bacterial or viral origin via molecular mimicry, a postulated mechanism that might account for the trigger of an autoimmune response on the basis of sequence homology between foreign and self determinants. Immune responses to heat shock proteins (hsp) have been implicated in the initiation or progression of a number of autoimmune diseases. Hsp may function as immunodominant targets during the immune response evoked by pathogens, and theoretically a cross-reactive response to sequences shared by these immunogens and autoantigens in the CNS may contribute to the pathogenesis of MS. We examined the immune response of peripheral blood mononuclear cells (PBMNc) from MS patients and healthy subjects elicited by peptides derived from hsp60 containing a common structural motif ("2-6-11" motif) already described, which is also present in CNS putative antigens. This structural pattern consists of an apolar residue or Lys at position 2, Pro always at position 6, and Glu, Asp or Lys at residue 11. Results reported here are indicative of maturation of peripheral blood monocytes towards a differentiated CD14(+)CD16(+)DR(+) cell and release of pro-inflammatory cytokines consistent with a Th1-like pattern. These are typical features exhibited by immune cells implicated in autoimmune responses.     

The use of Mycobacterium tuberculosis HspX and GlcB proteins to identify latent tuberculosis in rheumatoid arthritis patients

Rheumatoid arthritis (RA) is an autoimmune disease characterised by the destruction of articular cartilage and bone damage. The chronic treatment of RA patients causes a higher susceptibility to infectious diseases such as tuberculosis (TB); one-third of the world’s population is latently infected (LTBI) with Mycobacterium tuberculosis (Mtb). The tuberculin skin test is used to identify individuals LTBI, but many studies have shown that this test is not suitable for RA patients. The goal of this work was to test the specific cellular immune responses to the Mtb malate synthase (GlcB) and heat shock protein X (HspX) antigens of RA patients and to correlate those responses with LTBI status. The T-helper (Th)1, Th17 and Treg-specific immune responses to the GlcB and HspX Mtb antigens were analysed in RA patients candidates for tumour necrosis factor-α blocker treatment. Our results demonstrated that LTBI RA patients had Th1-specific immune responses to GlcB and HspX. Patients were followed up over two years and 14.3% developed active TB. After the development of active TB, RA patients had increased numbers of Th17 and Treg cells, similar to TB patients. These results demonstrate that a GlcB and HspX antigen assay can be used as a diagnostic test to identify LTBI RA patients.     

Therapy of rat autoimmune disease by a monoclonal antibody specific for T lymphoblasts

The central role of T lymphocytes in the initiation, regulation and propagation of autoimmune diseases defines them as most suitable targets for selective immunotherapy. The recent advance in culturing human and animal T cell lines allows us to select monoclonal antibodies specific for differentiation antigens expressed by activated T lymphocytes. We selected a monoclonal antibody cytotoxic for a subpopulation of activated rat T cells. In vivo, this antibody effectively blocks immune responses to foreign antigens or autoantigen and prevents development of autoimmune diseases like experimental allergic encephalomyelitis and adjuvant arthritis. Even already established disease can be blocked by a single injection of antibody. Furthermore, this monoclonal antibody can be used to monitor the course of autoimmune disease progression from peripheral blood samples.     

Animal models of autoimmune liver disease

Autoimmune liver diseases in humans are characterized by chronic active hepatitis with serum autoantibodies, hypergammaglobulinemia and liver pathology showing necroinflammatory disease and fibrosis. There are an increasing number of autoantigens believed to be associated with various autoimmune liver diseases. This review will briefly outline human autoimmune hepatitis and the immunology of the liver. Various murine models of liver inflammation will be discussed, including transgenic and non-transgenic models, with emphasis on how these models aid in our knowledge of the mechanisms of disease development and chronicity. There are limitations with all of the models, including a preponderance of T-cell-focused responses. Murine models do not easily develop fibrosis, a hallmark of autoimmune hepatitis in humans. Different experimental models may not reach the same conclusions with differences between immune responses. However, this multiplicity of responses does not necessarily imply that these models are inappropriate for the study of liver immunology and autoimmune liver diseases, as different autoantigens may induce different liver responses. Knowledge of how the liver differs from other immune organs is essential to further our understanding of liver-specific autoimmunity. The plethora of antigens implicated in autoimmune hepatitis in humans predicts that multiple mechanisms may play a role in precipitating disease in the susceptible individual.     

Heat shock proteins in health and disease: therapeutic targets or therapeutic agents?

For many years, heat shock or stress proteins have been regarded as intracellular molecules that have a range of housekeeping and cytoprotective functions, only being released into the extracellular environment in pathological situations such as necrotic cell death. However, evidence is now accumulating to indicate that, under certain circumstances, these proteins can be released from cells in the absence of cellular necrosis, and that extracellular heat shock proteins have a range of immunoregulatory activities. The capacity of heat shock proteins to induce pro-inflammatory responses, together with the phylogenetic similarity between prokaryotic and eukaryotic heat shock proteins, has led to the proposition that these proteins provide a link between infection and autoimmune disease. Indeed, both elevated levels of antibodies to heat shock proteins and an enhanced immune reactivity to heat shock proteins have been noted in a variety of pathogenic disease states. However, further evaluation of heat shock protein reactivity in autoimmune disease and after transplantation has shown that, rather than promoting disease, reactivity to self-heat shock proteins can downregulate the disease process. It might be that self-reactivity to heat shock proteins is a physiological response that regulates the development and progression of pro-inflammatory immunity to these ubiquitously expressed molecules. The evolving evidence that heat shock proteins are present in the extracellular environment, that reactivity to heat shock proteins does not necessarily reflect adverse, pro-inflammatory responses and that the promotion of reactivity to self-heat shock proteins can downregulate pathogenic processes all suggest a potential role for heat shock proteins as therapeutic agents, rather than as therapeutic targets.     

The role of FcgammaRIIa as an inflammatory mediator in rheumatoid arthritis and systemic lupus erythematosus

Despite their essential role in host protection, immunoglobulins are also involved in autoimmune processes where antibodies recognize the host's own tissue, triggering inflammatory responses that result in extensive tissue damage. A complex interaction of genetic predisposition, together with environment factors, is thought to trigger immune dysfunction. Although recent studies have dissected the essential role of Fc receptors in autoimmune antibody mediated processes, the uniquely human FcgammaRIIa has not been studied in detail. This Fc receptor is of particular interest, as it is the most abundantly expressed Fc receptor in humans and is implicated in immune complex disease. Investigation of its role has been hampered to date due to lack of suitable animal models. This review examines the evidence for the direct role of this receptor in diseases such as systemic lupus erythematosus and rheumatoid arthritis.     

Plant cell-made protein antigens for induction of Oral tolerance

The gut associated lymphoid tissue has effective mechanisms in place to maintain tolerance to food antigens. These can be exploited to induce antigen-specific tolerance for the prevention and treatment of autoimmune diseases and severe allergies and to prevent serious immune responses in protein replacement therapies for genetic diseases. An oral tolerance approach for the prevention of peanut allergy in infants proved highly efficacious and advances in treatment of peanut allergy have brought forth an oral immunotherapy drug that is currently awaiting FDA approval. Several other protein antigens made in plant cells are in clinical development. Plant cell-made proteins are protected in the stomach from acids and enzymes after their oral delivery because of bioencapsulation within plant cell wall, but are released to the immune system upon digestion by gut microbes. Utilization of fusion protein technologies facilitates their delivery to the immune system, oral tolerance induction at low antigen doses, resulting in efficient induction of FoxP3⁺ and latency-associated peptide (LAP)⁺ regulatory T cells that express immune suppressive cytokines such as IL-10. LAP and IL-10 expression represent potential biomarkers for plant-based oral tolerance. Efficacy studies in hemophilia dogs support clinical development of oral delivery of bioencapsulated antigens to prevent anti-drug antibody formation. Production of clinical grade materials in cGMP facilities, stability of antigens in lyophilized plant cells for several years when stored at ambient temperature, efficacy of oral delivery of human doses in large animal models and lack of toxicity augur well for clinical advancement of this novel drug delivery concept.     

Autoimmunity, immunodeficiency and mucosal infections: Chronic intestinal inflammation as a sensitive indicator of immunoregulatory defects in response to normal luminal microflora

Despite the fact that target antigens and the genetic basis of several autoimmune diseases are now better understood, the initial events leading to a loss of tolerance towards self-components remain unknown. One of the most attractive explanations for autoimmune phenomena involves various infections as possible natural events capable of initiating the process in genetically predisposed individuals. The most accepted explanation of how infection causes autoimmunity is based on the concept of “molecular mimicry” (similarity between the epitopes of an autoantigen and the epitopes in the environmental antigen). Infectious stimuli may also participate in the development of autoimmunity by inducing an increased expression of stress proteins (hsp), chaperones and transplantation antigens, which leads to abnormal processing and presentation of self antigens. Superantigens are considered to be one of the most effective bacterial components to induce inflammatory reactions and to take part in the development and course of autoimmune mechanisms. It has long been known that defects in the host defense mechanism render the individual susceptible to infections caused by certain microorganisms. Impaired exclusion of microbial antigens can lead to chronic immunological activation which can affect the tolerance to self components. Defects in certain components of the immune system are associated with a higher risk of a development of autoimmune disease. The use of animal models for the studies of human diseases with immunological pathogenesis has provided new insights into the influence of immunoregulatory factors and the lymphocyte subsets involved in the development of disease. One of the most striking conclusion arising from work with, genetically engineered immunodeficient mouse models is the existence of a high level of redundancy of the components of the immune system. However, when genes encoding molecules involved in T cell immunoregulatory functions are deleted, spontaneous chronic inflammation of the gut mucosa (similar to human inflammatory bowel disease) develops. Surprisingly, when such immunocompromised animals were placed into germfree environment, intestinal inflammation did not develop. Impairment of the mucosal immune response to the normal bacterial flora has been proposed to play a crucial role in the pathogenesis of chronic intestinal inflammation. The use of immunodeficient models colonized with defined microflora for the analysis of immune reactivity will shed light on the mode of action of different immunologically important molecules responsible for the delicate balance between luminal commensals, nonspecific and specific components of the mucosal immune system.     

Evolutionarily conserved proteins as prominent immunogens during Leishmania infections

Many Leishmania antigens have been identified as members of conserved protein families, such as the acidic ribosomal proteins, the histones and the heat-shock proteins; despite this, they elicit specific immune responses. Furthermore, homologues of many of these antigens are immune targets in other infectious diseases and systemic autoimmune diseases. Here, Jose Mar a Requena, Carlos Alonso and Manuel Soto review this class of widely distributed antigens, which they call 'panantigens'. They also propose a model to explain the prominent immunogenicity of these antigens during Leishmania infection, on the basis of the fact that many panantigens are constituents of multicomponent complexes in the cell. The elucidation of the pathways by which Leishmania antigens are processed and presented to effector cells from the host immune system will shed light on the immunopathology of leishmaniasis and help in the development of protective immunotherapies.     

T-cell glucocorticoid receptor is required to suppress COX-2-mediated lethal immune activation

Glucocorticoids, acting through the glucocorticoid receptor, potently modulate immune function and are a mainstay of therapy for treatment of inflammatory conditions, autoimmune diseases, leukemias and lymphomas. Moreover, removal of systemic glucocorticoids, by adrenalectomy in animal models or adrenal insufficiency in humans, has shown that endogenous glucocorticoid production is required for regulation of physiologic immune responses. These effects have been attributed to suppression of cytokines, although the crucial cellular and molecular targets remain unknown. In addition, considerable controversy remains as to whether glucocorticoids are required for thymocyte development. To assess the role of the glucocorticoid receptor in immune system development and function, we generated T-cell-specific glucocorticoid receptor knockout mice. Here we show that the T-cell is a critical cellular target of glucocorticoid receptor signaling, as immune activation in these mice resulted in significant mortality. This lethal activation is rescued by cyclooxygenase-2 (COX-2) inhibition but not steroid administration or cytokine neutralization. These studies indicate that glucocorticoid receptor suppression of COX-2 is crucial for curtailing lethal immune activation, and suggest new therapeutic approaches for regulation of T-cell-mediated inflammatory diseases.     

Antibodies Specific for Carbamylated Proteins Precede the Onset of Clinical Symptoms in Mice with Collagen Induced Arthritis

Objective

The immune response to post-translationally modified antigens is a key characteristic of rheumatoid arthritis. Carbamylation is such a posttranslational modification. Recently, we demonstrated that autoantibodies recognizing carbamylated proteins are present in sera of rheumatoid arthritis. The molecular mechanisms underlying the break of tolerance and hence the induction of anti-CarP antibody responses are unknown as well as their appearance in mouse models for systemic arthritis. Therefore we analyzed their appearance in the mouse collagen-induced arthritis model.

Methods

collagen induced arthritis was induced by immunization with type II collagen in complete Freund''s adjuvant. Arthritis severity was monitored by clinical scoring and anti-CarP antibody levels were determined by ELISA.

Results

Anti-CarP antibodies were detectable in mice with collagen induced arthritis. We did not detect ACPA in mice with collagen induced arthritis. The specificity of the antibodies for carbamylated proteins was confirmed by inhibition assays and immunoblotting. Injection with complete Freund''s adjuvant without type II collagen could also induce anti-CarP antibodies, however, in mice with arthritis, the anti-CarP antibody response was stronger and developed more rapidly. The onset of collagen induced arthritis was preceded by an increase of anti-CarP IgG2a levels in the serum.

Conclusion

In mice with collagen induced arthritis we did not observe an immune response against citrullinated antigens, but we did observe an immune response against carbamylated antigens. This anti-CarP response already appeared before disease onset, indicating that collagen induced arthritis can be used as an in vivo model to study anti-CarP antibodies. Our data also indicate that the tolerance to carbamylated proteins, in contrast to the response to citrullinated proteins, is easily broken and that arthritis boosts the immune response against these proteins. The anti-CarP response in mice with CIA can be used as a model for immune responses to post-translationally modified proteins.     

Vitamin D and autoimmunity: is vitamin D status an environmental factor affecting autoimmune disease prevalence?

The environment in which the encounter of antigen with the immune system occurs determines whether tolerance, infectious immunity, or autoimmunity results. Geographical areas with low supplies of vitamin D (for example Scandinavia) correlate with regions with high incidences of multiple sclerosis, arthritis, and diabetes. The active form of vitamin D has been shown to suppress the development of autoimmunity in experimental animal models. Furthermore, vitamin D deficiency increases the severity of at least experimental autoimmune encephalomyelitis (mouse multiple sclerosis). Targets for vitamin D in the immune system have been identified, and the mechanisms of vitamin D-mediated immunoregulation are beginning to be understood. This review discusses the possibility that vitamin D status is an environmental factor, which by shaping the immune system affects the prevalence rate for autoimmune diseases such as multiple sclerosis, arthritis, and juvenile diabetes.     

The application of Toll like receptors for cancer therapy

Toll-like receptor (TLR) proteins play key roles in immune responses against infection. Using TLR proteins, host can recognize the conserved molecular structures found in pathogens called pathogen-associated molecular patterns (PAMPs). At the same time, some TLRs are able to detect specific host molecules, such as high-mobility group box protein 1 (HMGB1) and heat shock proteins (hsp), and lead to inflammatory responses. Thus, it has been suggested that TLRs are involved in the development of many pathogenic conditions. Recent advances in TLR-related research not only provide us with scientific information, but also show the therapeutic potential against diseases, such as autoimmune disease and cancer. In this mini review, we demonstrate how TLRs pathways could be involved in cancer development and their therapeutic application, and discuss recent patentable subjects, in particular, that are targeting this unique pathway.     

Heat shock proteins HSP70 and GP96: structural insights

Several heat shock proteins (HSPs) act as potent adjuvants for eliciting anti-tumor immunity. HSP-based tumor vaccine strategies have been highly successful in animal models and are undergoing testing in clinical trials. It is generally accepted that HSPs, functioning as chaperones for tumor antigens, elicit tumor-specific adaptive immune responses. HSPs also appear to induce innate immune responses in an antigen-independent fashion. Innate responses generated by HSPs may contribute to anti-tumor immunity. Immunologically active chaperones with anti-tumor activity are referred to as “immunochaperones”. Here, we review the studies that address the role of structural domains or regions of the immunochaperones HSP70 and GP96 that may be involved in the induction of adaptive or innate immune responses. This article forms part of the Symposium in Writing “Thermal stress-related modulation of tumor cell physiology and immune responses”, edited by Elfriede Noessner.     

Heat shock proteins in autoimmune diseases

Heat shock proteins (hsp's) are among the most conserved proteins in evolution. They have been identified as important pathogen-related antigens as well as autoantigens suitable for construction of novel vaccines. The high evolutionary homology of hsp's has raised the question about the safety of such vaccines. Experimental and clinical observations have confirmed that hsp proteins are involved in the regulation of some autoimmune disease such as autoimmune arthritis, type 1 diabetes mellitus, atherosclerosis, multiple sclerosis, and other autoimmune reactions. It has been shown in experimental animals that some hsp proteins (especially hsp60, hsp70, and hsp10) can either induce or prevent autoimmune reactions depending on the circumstances. This article discusses the involvement of hsp proteins in the etiology of autoimmune diseases and it presents promising experimental data on the effects of immunization with hsp proteins in the prevention and therapy of autoimmune diseases.     

E3 ubiquitin ligase Cbl-b in innate and adaptive immunity

Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b), a RING finger E3 ubiquitin-protein ligase, has been demonstrated to play a crucial role in establishing the threshold for T-cell activation and controlling peripheral T-cell tolerance via multiple mechanisms. Accumulating evidence suggests that Cbl-b also regulates innate immune responses and plays an important role in host defense to pathogens. Understanding the signaling pathways regulated by Cbl-b in innate and adaptive immune cells is therefore essential for efficient manipulation of Cbl-b in emerging immunotherapies for human disorders such as autoimmune diseases, allergic inflammation, infections, and cancer. In this article, we review the latest developments in the molecular structural basis of Cbl-b function, the regulation of Cbl-b expression, the signaling mechanisms of Cbl-b in immune cells, as well as the biological function of Cbl-b in physiological and pathological immune responses in animal models and human diseases.