Background: Activation of NLRP3 in liver macrophages contributes to alcohol-associated liver disease (ALD). Molecular chaperone heat shock protein (HSP) 90 facilitates NLRP3 inflammasome activity during infections and inflammatory diseases. We previously reported that HSP90 is induced in ALD and regulates proinflammatory cytokines, tumor necrosis factor alpha, and IL-6. Whether HSP90 affects IL-1b and IL-18 regulated by NLRP3 inflammasome in ALD is unknown. Here, we hypothesize that HSP90 modulated NLRP3 inflammasome activity and affects IL-1b and IL-18 secretion in ALD.Methods: The expression of HSP90AA1 and NLRP3 inflammasome genes was evaluated in human alcoholic livers and in mouse model of ALD. The importance of HSP90 on NLRP3 inflammasome activation in ALD was evaluated by administering HSP90 inhibitor, 17-dimethylaminoethylamino-17demethoxygeldanamycin (17-DMAG) to mice subjected to ALD, and in vitro to bone marrow-derived macrophages (BMDM) stimulated with LPS and ATP. The effect of activation of HSF1/HSPA1A axis during HSP90 inhibition or direct activation during heat shock of BMDMs on NLRP3 activity and secretion of downstream cytokines was evaluated.Results: We found positive correlation between induction of HSP90 and NLRP3 inflammasome genes in human alcoholic cirrhotic livers. Administration of 17-DMAG in mouse model of ALD significantly down-regulated NLRP3 inflammasome-mediated caspase-1 (CASP-1) activity and cytokine secretion, with reduction in ALD. 17-DMAG-mediated decrease in NLRP3 was restricted to liver macrophages. Using BMDMs, we show that inhibition of HSP90 prevented CASP-1 activity, and Gasdermin D (GSDMD) cleavage, important in release of active IL-1b and IL-18. Interestingly, activation of the heat shock factor 1 (HSF1)/HSPA1A axis, either during HSP90 inhibition or by heat shock, decreased NLRP3 inflammasome activity and reduced secretion of cytokines.Conclusion: Our studies indicate that inhibition of HSP90 and activation of HSF1/HSPA1A reduce IL-1b and IL-18 via decrease in NLRP3/CASP-1 and GSDMD activity in ALD.

BackgroundWe earlier reported a simple specific test for detection of anti-ovarian antibodies in infertile women and identified number of specific molecular and cellular targets of which human heat shock protein 90-beta (HSP90 beta) was found to be the most immunodominant. The present study focuses on prediction and validation of the immunodominant epitope/s of this protein using sera from infertile women having anti-HSP90 autoantibodies.MethodsDelineation of the immunodominant epitopes of HSP90 beta was done by using epitope prediction algorithms and 10 peptides (EP1-EP10) were custom synthesized. Their immunoreactivity was measured by ELISA using sera from patients and controls. To determine the most immunodominant epitope, the results were subjected to statistical analysis. The immunoreactivity of the immunodominant peptides were confirmed by dot blots using sera from patients. A rabbit polyclonal antibody against the immunodominant epitope was generated and its immunoreactivity to the parent protein in ovarian extracts as well in oocytes and embryos was investigated.ResultsExperimentally and statistically, peptide EP6 (380-389) seems to be the major antigenic epitope for the serum antibody binding followed by EP1 (1-12) and EP8 (488-498). Predicted 3D structures of these peptides demonstrated that they exist in the loop conformation which is the most mobile part of the protein. Also, analysis of the sequences of HSP90 beta across several species reveals that EP6 peptide forms a part of a well conserved motif. The polyclonal antibody generated to the immunodominant epitope- EP6 confirms similar biochemical and cellular immunoreactivity as seen with the patients' sera having anti-HSP90 autoantibodies.ConclusionsThe decapeptide EP6 is a major immunogenic epitope of HSP90 followed by EP1 and EP8. Knowledge of binding epitopes on the autoantigen is necessary to understand the subsequent pathologic events. The study might generate new tools for the detection of disease-inducing epitopes and a possible therapeutic intervention.

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This study underlines a critical role played by HSP90 in ovarian folliculogenesis and highlights the implications of the presence of anti-HSP90 antibodies in infertile women.

We previously established that the presence of autoantibodies to heat-shock protein 90 (HSP90) is one common causes of female infertility, and demonstrated that its presence leads to detrimental effects on ovarian and reproductive function in mice. The pathophysiological mechanism and alteration in the immune physiology, however, remain unknown. We therefore carried out detailed analysis of various immune cells in the spleen and ovary following immunization of C57BL/6 female mice to generate antibodies to HSP90 in the general circulation. We observed a significant increase in levels of CD45- cells; CD4+ T cells; Ly6G6C+ cells; and CD11b+ Ly6G+ cells in the spleen of these mice, which correlate with the increased anti-HSP90 antibody production. Ovarian- and granulosa-cell populations also showed increased infiltration of CD45+ leukocytes and neutrophil and monocyte populations, which may have led to the observed ovarian follicular degeneration that predominantly manifested as empty follicles. A decrease in the number of functional ovarian follicles was also associated with a decrease in the level of Gdf9 gene expression. Thus, changes in the immune physiology of the spleen and ovary that leads to the generation of antibodies to HSP90 can also bring about the destruction of ovarian follicles.

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Autoimmune diseases have gender bias with predominance in females, autoimmune infertility (AI) being no exception. This chapter will focus on AI in females with brief reference to the same in males. Autoimmune diseases have established protocols for detection and management of ensuing infertility, however similar protocols for unexplained infertility [tubal blockage, endometriosis, premature ovarian insufficiency (POI), undiagnosed underlying autoimmune disease (Sjögren's syndrome, IBS, celiac disease) and tubal blockage] are not established. Endometriosis and POI, in particular, have autoimmune etiology yet lack specific and sensitive biomarkers for accurate diagnosis. If autoantibodies are indeed diagnosed, then treatment regimen focuses on AI which has known adverse effects. The detection of natural antibodies as autoantibodies presents a viable alternative to organ specific biomarker panel for better management of AI.