anti-HSP Autoantibodies Research Articles

Interplay between the Chaperone System and Gut Microbiota Dysbiosis in Systemic Lupus Erythematosus Pathogenesis: Is Molecular Mimicry the Missing Link between Those Two Factors?

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease characterized by self-immune tolerance breakdown and the production of autoantibodies, causing the deposition of immune complexes and triggering inflammation and immune-mediated damage. SLE pathogenesis involves genetic predisposition and a combination of environmental factors. Clinical manifestations are variable, making an early diagnosis challenging. Heat shock proteins (Hsps), belonging to the chaperone system, interact with the immune system, acting as pro-inflammatory factors, autoantigens, as well as immune tolerance promoters. Increased levels of some Hsps and the production of autoantibodies against them are correlated with SLE onset and progression. The production of these autoantibodies has been attributed to molecular mimicry, occurring upon viral and bacterial infections, since they are evolutionary highly conserved. Gut microbiota dysbiosis has been associated with the occurrence and severity of SLE. Numerous findings suggest that proteins and metabolites of commensal bacteria can mimic autoantigens, inducing autoimmunity, because of molecular mimicry. Here, we propose that shared epitopes between human Hsps and those of gut commensal bacteria cause the production of anti-Hsp autoantibodies that cross-react with human molecules, contributing to SLE pathogenesis. Thus, the involvement of the chaperone system, gut microbiota dysbiosis, and molecular mimicry in SLE ought to be coordinately studied.

Detection of autoantibodies to heat shock protein 70 in the saliva and urine of normal individuals.

Cells exposed to stressors of various origin activate protective mechanisms that include the expression of heat shock proteins (Hsps)/molecular chaperones belonging to several families. Well-characterized inducible Hsp70 is present in all human cell-types and biological fluids, including blood, urine, and saliva. The presence of anti-Hsp70 autoantibodies in the serum of healthy individuals has already been confirmed, and their elevated titers positively correlated with the severity of several pathological conditions, including coeliac disease and dermatitis herpetiformis - a cutaneous manifestation of coeliac disease. Here, using an indirect enzyme-linked immunosorbent assay, we demonstrate, for the first time, that anti-Hsp70 autoantibodies are present in the saliva and urine of healthy individuals. Although the occurrence of anti-Hsp70 autoantibodies in the biological fluids of healthy individuals is intriguing, their physiological role is currently unknown. It is believed that antibodies reacting with self-molecules present in the serum of healthy individuals are part of natural autoantibody pool with multiple regulatory functions. On the other hand, some autoantibodies (e.g., typical of autoimmune bullous skin diseases or systemic lupus erythematosus) may be present before the onset of the disease and serve as specific predictive biomarkers. Therefore, we would like to initiate a discussion or future research direction on the use of anti-Hsp70 autoantibodies as a potential "biomarker" in the diagnosis or prediction of autoimmune diseases. Our findings can be considered in biomedical research to develop noninvasive, inexpensive and easy-to-use tests. Nevertheless, large-scale comparative studies should be initiated, involving the collection and analysis of biological samples such as saliva or urine from patients suffering from autoimmune diseases or other inflammatory or neoplastic diseases, to determine whether the levels of anti-Hsp70 autoantibodies are indeed elevated and whether they correlate with the clinical picture of any disease or established biomarkers.

Pathological Relevance of Anti-Hsp70 IgG Autoantibodies in Epidermolysis Bullosa Acquisita.

Stress-induced heat shock protein 70 (Hsp70) is a key intra- and extracellular molecular chaperone implicated in autoimmune processes. Highly immunogenic extracellular Hsp70 can activate innate and acquired (adaptive) immune responses driving the generation of anti-Hsp70 autoantibodies that are frequently observed in inflammatory/autoimmune disorders. We recently described the direct pathological role of extracellular Hsp70 in epidermolysis bullosa acquisita (EBA), an anti-type VII collagen autoantibody-mediated autoimmune blistering skin disease. Here, we determined the role of anti-Hsp70 autoantibodies in EBA. We observed that circulating anti-Hsp70 IgG autoantibodies were significantly elevated in EBA patients compared to healthy individuals and positively correlated with serum levels of pro-inflammatory interferon gamma (IFN-γ). The pathophysiological relevance of anti-Hsp70 IgG autoantibodies was demonstrated in an antibody transfer-induced EBA mouse model in which elevated serum levels of anti-Hsp70 IgG were found. In addition, anti-Hsp70 IgG-treated animals had a more intense clinical and histological disease activity, as well as upregulated nuclear factor kappa B (NF-κB) activation in skin biopsies compared to isotype-treated animals. Our results suggest that autoantibodies to Hsp70 may contribute to EBA development via enhanced neutrophil infiltration to the skin and activation of the NF-κB signaling pathway in an IFN-γ-associated manner.

The association between serum anti-HSP27 levels with hypertriglyceridemia

BackgroundThere are little data on isolated hypertriglyceridemia and CVD risk. Heat shock protein 27 (Hsp27) is one of the heat shock proteins (Hsps) with low molecular weight. It has been shown that the atherosclerosis patients have higher concentrations of anti-Hsp auto-antibodies. The aim of the current study was to assess the relationship between hypertriglyceridemia with serum anti-Hsp27 titres. MethodsIn this cross-sectional study, fasting serum lipid profile and anthropometric parameters of 1688 subjects (1113 subjects with and 575 without hypertriglyceridemia) aged 35–65 years were analyzed as part of the data derived from the Mashhad stroke and heart atherosclerotic disorders (MASHAD: 2010–2020). A validated questionnaire was used to collect the demographic data. Combined dyslipidemia was defined according to the NCEP ATPIII criteria. ResultsThere were significant differences in waist and hip circumferences, level of fasting blood glucose, hs-CRP, systolic and diastolic blood pressure (p < 0.001 for all variables), between individuals with isolated hypertriglyceridemia (IHTG), those with combined dyslipidemia and control groups. After adjusting for confounding factors, logistic regression analysis showed that high levels of serum anti-Hsp 27 were associated with a lower odds of HTG risk (OR:0.62, 95% CI (0.39–0.97), p: 0.03). ConclusionWe found that serum anti-HSP27 antibody titers were significantly lower in the HTG group compared with individuals without HTG. However, levels of serum anti-HSP27 antibody can be affected by drug treatment. Therefore, the evaluation of drug treatment on the serum anti-HSP27 antibody in a larger population can help to confirm these findings.

Autoantibodies to heat shock proteins 60, 70, and 90 in patients with rheumatoid arthritis

Heat shock proteins (HSP) have been reported to impact immune responses and to be associated with rheumatoid arthritis (RA). Recently, we provided evidence for a role of autoantibodies to Hsp40 in patients with RA. In this study, we aimed at investigating the humoral autoimmune response to Hsp60, Hsp70, and Hsp90 in RA patients (n = 39). In comparison with healthy controls (n = 40), circulating IgG, IgM, and IgA autoantibodies against Hsp60, Hsp70, and Hsp90 were significantly increased in RA patients. Non-parametric statistical analysis, however, revealed no significant association between anti-HSP and disease activity or disease progression. On the other hand, positive correlations between serum levels of anti-Hsp60 IgG and IL-4 (Th2-like cytokine) or between serum levels of anti-Hsp90 IgG and IFN-ɣ (Th1-like cytokine) were found to be statistically significant in RA. In addition, a significant inverse correlation was found for serum levels of anti-Hsp70 IgM and TNF-α (Th1-like cytokine) in RA. Our results suggest a pronounced anti-Hsp60, anti-Hsp70, and anti-Hsp90 humoral autoimmune response in RA patients that seems not to be directly linked to RA pathophysiology, however, may have a potential modulatory impact on inflammatory status in this disease. Further investigations are needed to clarify the role of anti-HSP autoantibodies in RA.

Autoimmunity to heat shock proteins and vitamin D status in patients with celiac disease without associated dermatitis herpetiformis

Inflammation-induced heat shock proteins (HSPs) and hypovitaminosis D have been reported to impact immune responses and to be associated with autoimmune diseases including celiac disease (CD), a gluten-sensitive enteropathy mediated by autoantibodies against tissue transglutaminase (TG2). Recently, we provided evidence for a role of autoantibodies to HSPs in patients with dermatitis herpetiformis (DH), an inflammatory skin disease induced by underlying latent CD. In this study, we aimed at investigating the humoral autoimmune response to HSPs and vitamin D status in CD patients (n=15) presenting without the cutaneous disease manifestation. In comparison with healthy controls (n=15), circulating autoantibodies against HSP40, HSP60, and HSP90 were increased in these patients, and these autoantibodies, including anti-HSP70, correlated with serum anti-TG2 autoantibodies. Deficient and insufficient vitamin D serum levels were found in 8 out of 15 CD patients and 11 out of 15 healthy controls, and the vitamin D status showed no relationship with levels of circulating anti-TG2 or anti-HSP autoantibodies in the patients. Our results preliminarily suggest a pronounced anti-HSP autoimmune response in CD that is linked to pathophysiological processes in the gut independently of both a potential concomitant disease-related extra-intestinal inflammatory manifestation in the skin and the vitamin D status. Future studies on larger groups of patients are needed to confirm the present data and to further clarify whether autoimmunity towards HSPs is primarily related to underlying CD or skin inflammation in DH patients.

Mycobacterial heat shock protein 65 (mbHSP65)-induced atherosclerosis: Preventive oral tolerization and definition of atheroprotective and atherogenic mbHSP65 peptides.

The aim of this study was to identify atherogenic and atheroprotective peptides of bacterial HSP60 [taking mycobacterial HSP65 (mbHSP65) as a potent paradigmatic representative] that could be used as candidates for an orally applied tolerizing vaccine against atherosclerosis. ApoE(-/-) mice were immunized with mbHSP65 protein or peptides, given mbHSP65 orally and then kept either on chow or high cholesterol diet. Atherosclerosis was assessed by en face and immunohistological analysis. Anti-HSP autoantibodies were detected by ELISA. The number and in vitro suppressive function of splenic and lymph node regulatory T cells (Tregs) were analyzed by flow cytometry. Specific T cell reactivity against mbHSP65 protein or peptides was assessed by proliferation assay. Decreased lesion size was accompanied by (a) increased splenic Treg numbers; (b) increased interleukin (IL)-10 mRNA levels in the aorta; (c) increased levels of anti-mbHSP65 and anti-mouse HSP60 antibodies pointing to pro-eukaryotic HSP60 humoral crossreaction, not curtailed by oral tolerization; (d) most importantly, we identified and functionally characterized novel atherogenic and atheroprotective mbHSP65 epitopes. Atheroprotective mbHSP65 peptides may be considered as potential candidates for the development of a tolerizing vaccine to prevent and treat atherosclerosis, while keeping protective immunity to non-atherogenic domains of mbHSP65 intact.

Evidence for a role of autoantibodies to heat shock protein 60, 70, and 90 in patients with dermatitis herpetiformis

Heat shock proteins (Hsp) are highly conserved immunomodulatory molecules upregulated when cells are exposed to stressful stimuli, such as inflammation. Their involvement in various autoimmune diseases, including autoimmune bullous diseases and celiac disease, has been increasingly recognized. To further study the role of Hsp in autoimmune bullous diseases, we have investigated for the first time the humoral autoimmune response to Hsp40, Hsp60, Hsp70, and Hsp90 in patients with dermatitis herpetiformis (DH; n = 26), bullous pemphigoid (BP; n = 23), and pemphigus vulgaris (PV; n = 16), the first representing a cutaneous manifestation of celiac disease. While in patients with active BP and PV, serum levels of autoantibodies against these Hsp did not differ from the corresponding age- and gender-matched healthy controls (n = 9-14); circulating autoantibodies against Hsp60, Hsp70, and Hsp90 were found to be increased at the active disease stage of DH. Further analysis of this latter patient subgroup showed that these anti-Hsp autoantibodies decreased in parallel with serum autoantibodies against epidermal and tissue transglutaminase during remission of skin lesions following a gluten-free diet, revealing significantly positive correlations. Although further studies on larger groups of patients will be needed to confirm the present data, our results support the notion that autoantibodies against Hsp60, Hsp70, and Hsp90 deserve attention in the study of the mechanisms that promote the development and maintenance of DH and possibly also the underlying celiac disease as well as potential novel disease biomarkers.

Patients with Idiopathic Pulmonary Fibrosis with Antibodies to Heat Shock Protein 70 Have Poor Prognoses

Diverse autoantibodies are present in most patients with idiopathic pulmonary fibrosis (IPF). We hypothesized that specific autoantibodies may associate with IPF manifestations. To identify clinically relevant, antigen-specific immune responses in patients with IPF. Autoantibodies were detected by immunoblots and ELISA. Intrapulmonary immune processes were evaluated by immunohistochemistry. Anti-heat shock protein 70 (HSP70) IgG was isolated from plasma by immunoaffinity. Flow cytometry was used for leukocyte functional studies. HSP70 was identified as a potential IPF autoantigen in discovery assays. Anti-HSP70 IgG autoantibodies were detected by immunoblots in 3% of 60 control subjects versus 25% of a cross-sectional IPF cohort (n = 122) (P = 0.0004), one-half the patients with IPF who died (P = 0.008), and 70% of those with acute exacerbations (P = 0.0005). Anti-HSP70 autoantibodies in patients with IPF were significantly associated with HLA allele biases, greater subsequent FVC reductions (P = 0.0004), and lesser 1-year survival (40 ± 10% vs. 80 ± 5%; hazard ratio = 4.2; 95% confidence interval, 2.0-8.6; P < 0.0001). HSP70 protein, antigen-antibody complexes, and complement were prevalent in IPF lungs. HSP70 protein was an autoantigen for IPF CD4 T cells, inducing lymphocyte proliferation (P = 0.004) and IL-4 production (P = 0.01). IPF anti-HSP70 autoantibodies activated monocytes (P = 0.009) and increased monocyte IL-8 production (P = 0.049). ELISA confirmed the association between anti-HSP70 autoreactivity and IPF outcome. Anti-HSP70 autoantibodies were also found in patients with other interstitial lung diseases but were not associated with their clinical progression. Patients with IPF with anti-HSP70 autoantibodies have more near-term lung function deterioration and mortality. These findings suggest antigen-specific immunoassays could provide useful clinical information in individual patients with IPF and may have implications for understanding IPF progression.

Evidence of a role for both anti-Hsp70 antibody and endothelial surface membrane Hsp70 in atherosclerosis

Although previous studies have shown that autoantigens such as Hsps have been implicated by induction of an autoimmune process in the development of atherosclerosis, the exact role of anti-Hsp70 antibody in atherosclerosis is unknown. In the present study, the levels of anti-Hsp70 autoantibodies and oxidized low density lipoprotein (OxLDL) were all significantly increased, and they were strongly correlated in an atherosclerosis model. After the endothelial cells were incubated with 20μg/mL OxLDL for 12h at 37°C and followed by 90min recovery, Hsp70 positive staining of OxLDL-treated endothelial cells was observed on the cell surface in immunostaining and flow cytometric analysis. This membrane Hsp70 was not from culture supernatant Hsp70 and binding of extracellular Hsp70 but was defined as endothelial surface membrane Hsp70. Furthermore, only in the OxLDL-treated group, but not in the untreated group, (51)Cr-labeled endothelial cells were lysed by anti-Hsp70 antibody (BD091, Ig(AS)) in the presence of either complement or peripheral blood mononuclear cells. Control antibodies, including Ig(Nor), mAb to Hsp70 (SPA-810), and mAbs to Factor VIII, α-actin, and CD3 showed no cytotoxic effects. In conclusion, anti-Hsp70 antibodies could be reacting with the endothelial surface membrane Hsp70 induced by OxLDL and were able to mediate endothelial cytotoxicity. There is a possibility that a humoral immune reaction to endothelial surface membrane Hsp70 may play an important role in the pathogenesis of atherosclerosis.

Chronic smoke exposure induces rheumatoid factor and anti‐heat shock protein 70 autoantibodies in susceptible mice and humans with lung disease

The impact of cigarette smoke (CS), a risk factor for rheumatoid arthritis (RA), on sauto-antibody production was studied in humans and mice with and without chronic lung disease (LD). Rheumatoid factor (RF), anti-cyclic citrullinated peptides (CCPs), and anti-HSP70 autoantibodies were measured in several mouse strains and in cohorts of smokers and nonsmokers with and without autoimmune disease. Chronic smoking-induced RFs in AKR/J mice, which are most susceptible to LD. RFs were identified in human smokers, preferentially in those with LD. Anti-HSP70 auto-antibodies were identified in CS-exposed AKR/J mice but not in ambient air exposed AKR/J controls. Whereas inflammation could induce anti-HSP70 IgM, smoke exposure promoted the switch to anti-HSP70 IgG autoantibodies. Elevated anti-CCP autoantibodies were not detected in CS-exposed mice or smokers. AKR/J splenocytes stimulated in vitro by immune complexes (ICs) of HSP70/anti-HSP70 antibodies produced RFs. The CD91 scavenger pathway was required as anti-CD91 blocked the HSP70-IC-induced RF response. Blocking Toll-like receptors did not influence the HSP70-IC-induced RFs. These studies identify both anti-HSP70 and RFs as serological markers of smoke-related LD in humans and mice. Identification of these autoantibodies could suggest a common environmental insult, namely CS, in a number of different disease settings.

Anti-heat shock protein 70 autoantibody epitope changes and BD091 promotes atherosclerosis in rats

It has been previously reported that the plasma levels of autoantibodies against heat shock protein 70 (HSP70) are elevated in atherosclerosis. The aim of the present study was to elucidate whether anti-HSP70 antibodies are involved in the pathogenesis of atherosclerosis. To determine this, we chose rats as an atherosclerosis model. Titers of plasma anti-HSP70 autoantibody were determined by ELISA. After the intravenous administration of antibody into the tail, the damaged areas of aorta were stained with Evans Blue, atheromatous plaque were stained by Oil Red O, and then they were measured and quantified with AxioVision computer software. The number of macrophages ([Formula: see text]), smooth muscle cells (SMCs), and T cells were determined by immunocytochemistry. The level of anti-HSP70 IgG1 antibody was apparently increased in the AS group at the tenth week, and one hybridoma of HSP70 antibody (BD091, IgG1, recognizing C-terminal) had the same binding epitope as plasma anti-HSP70 autoantibodies. After intravenous administration, the lesion area of aorta with BD091 was significantly larger than those of IgG(mouse) and SPA-810. Moreover, injection of BD091 resulted in significant endothelium damage, followed by a greater accumulation of [Formula: see text], T cells, and SMCs in lesions than in the control. In conclusion, BD091 reaction with HSP70 expressed on arterial endothelial cells inducing endothelium damage triggers the inflammatory response in the vessel wall that accelerates atherosclerosis in rats. BD091 shares the same binding epitope with HSP70 autoantibodies. These data indicated that a specific epitope of anti-HSP70 autoantibody participated in the pathogenesis of atherosclerosis.

Immunomodulatory activity of extracellular heat shock proteins and their autoantibodies

HSP are groups of stress-inducible proteins which contribute to quality control by assisting the correct folding of both nascent and denatured proteins, and promoting the degradation of unrecoverable denatured proteins. HSP also help to maintain cellular homeostasis and protect from cell death through a mechanism called thermotolerance. Cells subjected to mild stress induce HSP which then protect them against subsequent stress. However, in cells subjected to severe stress, HSP promote apoptosis. Besides these intracellular events, HSP also exist in extracellular fluids, and have been shown to contribute to immunomodulation. In innate immunity extracellular HSP, like various microbial substances, induce various proinflammatory cytokines. In acquired immunity they interact with antigenic polypeptides and assist in antigen presentation. The extracellular HSP are so-called adjuvant. Release of HSP from cells is triggered by stress and trauma, and is thus regarded as an immunological "danger signal". In addition, anti-HSP autoantibodies are frequently found in patients with autoimmune diseases and inflammatory disorders, and these autoantibodies can modulate the "danger signal" triggered by extracellular HSP.

Cerebrospinal fluids containing anti-HSP70 autoantibodies from multiple sclerosis patients augment HSP70-induced proinflammatory cytokine production in monocytic cells

Anti-heat shock protein 70 (HSP70) autoantibodies are found in cerebrospinal fluids (CSF) from patients with multiple sclerosis. These autoantibodies cross-reacted not only with HSP70 and heat shock cognate protein 70 (HSC70), but also with a bacterial homologue, DnaK. CSF with a high anti-HSP70 autoantibody titer enhanced HSP70-induced proinflammatory cytokine and chemokine production in the human monocytic cell line, THP-1. Thus, anti-HSP70 autoantibodies in the CSF of multiple sclerosis patients may play a pathophysiological role in enhancing inflammation.

Anti-HSP auto-antibodies enhance HSP-induced pro-inflammatory cytokine production in human monocytic cells via Toll-like receptors

Auto-antibodies against heat shock proteins (HSPs) are frequently found in the sera of patients with rheumatic and other autoimmune diseases. However, it is unclear whether these auto-antibodies play a role in the pathophysiology and etiology of these diseases. We found that a murine anti-HSP60 mAb enhanced the production of IL-8 and tumor necrosis factor-alpha induced by human HSP60 in the human monocytic cell lines THP-1 and U937, and human peripheral blood monocytes. Similar enhancement was observed with the combination of human HSP70 protein and a murine anti-HSP70 mAb. The enhancing effects were also observed for F(ab')2 fragment, but not for monovalent Fab fragment. This suggests that the enhancement is due to cross-linking of HSP by the anti-HSP antibodies. The induction of IL-8 was dramatically suppressed by the transfection of a dominant-negative mutant of Toll-like receptor 4. We also found that sera from patients with rheumatic autoimmune diseases, which contained higher anti-HSP60 auto-antibody titers than sera from healthy donors, significantly enhanced the IL-8 production induced by human HSP60 in THP-1 cells. We propose that auto-antibodies against HSPs have the potential to play a pathogenic role in rheumatic autoimmune diseases by enhancing inflammatory reactions.

An ELISA serum assay for autoantibodies to HSP70 in immune-mediated hearing loss

A Western blot to detect anti-HSP70 autoantibodies has been reported to be of diagnostic value for immune-mediated hearing loss patients. While setting up this Western blot in our lab, we detected two main problems. First, some patients were positive for antibodies to a 70-kDa protein when tested against a whole cell lysate, but negative if the antigen used was purified HSP70. Second, if high amounts of purified HSP70 were loaded on the gel, both patients and healthy controls were positive. We have developed and optimized an ELISA as an alternative to the Western blot. This assay is more appropriate to identify positive and negative individuals because it is semi-quantitative. The ELISA is also more sensitive, requiring very low concentrations of the antigen and thus minimizing false positives. Finally, we demonstrated that immune-mediated hearing loss patients recognize mainly the native form of HSP70, a fact that potentially leads to false negatives when a denaturing Western blot assay is used for diagnosis. To test the diagnostic value of the ELISA, we performed a blind test with 70 hearing loss patients, as well as 30 healthy controls. A sensitivity of 84% and a specificity of 93% were obtained, superior to what has been reported so far for the Western blot.

During canine viscero-cutaneous leishmaniasis the anti-Hsp70 antibodies are specifically elicited by the parasite protein

A Leishmania infantum cDNA library was screened with sera from dogs with viscero-cutaneous leishmaniasis. Sequence analysis of a positive clone isolated from the library revealed that it coded for the carboxyl-terminal region of a member of the 70-kDa heat-shock protein family. The full-length sequence of the L. infantum hsp70 gene was determined after isolation of genomic clones. This protein shows a high degree of sequence conservation with the homologous protein from other organisms. To test its antigenicity a recombinant Hsp70 protein fused to the maltose-binding protein was produced in Escherichia coli using the expression vector pMAL-cRI. By FAST-ELISA assays it was observed that while the complete recombinant protein was recognized by 100% of the sera, the 20 carboxyl-terminal amino acids of the protein were only recognized by 30% of those sera. Thus, although a B-cell epitope must be present within the carboxyl terminal end of the protein other antigenic determinant(s) must reside out of this region. The analysis of the cross-reactivity with mouse Hsp70 by Western blotting strongly suggests that the anti-Hsp70 antibodies generated by infection with L. infantum are directed at specific determinants of the L. infantum Hsp70. Thus, our results indicate that anti-Hsp70 autoantibodies are not induced during Leishmania infection.