Heat shock protein 90 is required for ex vivo neutrophil‐driven autoantibody‐induced tissue damage in experimental epidermolysis bullosa acquisita

Abstract

A broad range of immunosuppressive and immunomodulatory effects of heat shock protein 90 (Hsp90) blockade has been described in models of autoimmune bullous diseases, but the direct contribution of this chaperone to neutrophil effector pathways in the context of autoantibodydriven blistering is generally unknown. Therefore, this has been addressed in the current study on the basis of the subepidermal blistering disease epidermolysis bullosa acquisita (EBA) characterized by autoantibodies against type VII collagen, in which a crucial role of neutrophils and both their reactive oxygen species and matrix metalloproteinases in mediating tissue injury has been established. First, the Hsp90 antagonist 17-DMAG dose-dependently inhibited dermal-epidermal separation ex vivo in cryosections of human skin induced by co-incubation of EBA patient autoantibodies with neutrophils from healthy blood donors. Next, 17-DMAG dose-dependently suppressed production and release of reactive oxygen species by human neutrophils induced by both fMLP AE LPS and EBAspecific immune complexes. In addition, coimmunoprecipitation studies revealed that extracellular Hsp90 interacted with secreted matrix metalloproteinases 2 and 12 in sera of EBA patients, suggesting that these basement membranedegrading proteolytic enzymes are client proteins of Hsp90 and dependent on its chaperone function. Our findings add to the knowledge of the multimodal anti-inflammatory effects of Hsp90 blockade and implicate that Hsp90 is closely involved in the effector mechanisms of neutrophil-driven autoantibodyinduced tissue damage, thus being a relevant therapeutic target in patients with neutrophil-mediated autoimmune diseases such as inflammatory types of EBA.

Heat shock protein 90 inhibition: A potential double- or triple-edged sword in the treatment of mucous membrane pemphigoid

“…These future investigations could yield some relevant information about the role of Hsp90 in MMP and, together with already existing knowledge on the powerful anti-inflammatory and anti-scarring effects of anti-Hsp90 treatment [14][15][16][17][18][19][20][21]25,[28][29][30], possibly support the introduction of nontoxic Hsp90 inhibitors for future medical management of patients with this potentially devastating disease that may otherwise cause permanent damage when therapy is delayed or ineffective.…”

“…Based on the above, we hypothesize that pharmacological Hsp90 blockade could represent a double-edged sword in the treatment of MMP by pleiotropic targeting of the mentioned Hsp90 inhibitor-prone pathophysiological factors involved in both inflammatory subepidermal blister formation and cicatricial processes [19][20][21]25,[28][29][30]. Due to their additional anti-cancer activity, Hsp90 inhibitors could even be proclaimed as a triple-edged sword against the malignancy-associated anti-laminin 332 subtype of MMP [12].…”

“…These include that (i) Hsp90 inhibitors exhibit activity in mice with experimental epidermolysis bullosa acquisita and in a respective ex vivo human dermal-epidermal separation cryosection model by potently affecting inflammatory disease pathways (i.e., inhibition of effector T cells, B cells, and neutrophils; suppression of autoantibody, proinflammatory cytokine, and reactive oxygen species production; promotion of regulatory B cells) [19][20][21], (ii) secreted basement membrane-degrading and blister-provoking matrix metalloproteinases with partly profibrotic potential are complexed by extracellular Hsp90 in sera of patients with epidermolysis bullosa acquisita, thus representing additional treatment targets of Hsp90 antagonists [21][22][23], (iii) Hsp90 is aberrantly expressed and secreted in the skin and blood of patients with BP, and its blockade modulates anti-BP180 autoantibody-induced production of proinflammatory IL-8 by cultured keratinocytes [24,25], and (iv) circulating autoantibodies to Hsp90 are elevated and positively correlated with disease activity and serum anti-epidermal/tissue transglutaminase autoantibodies in patients with dermatitis herpetiformis [26]. With regard to MMP, it has previously been reported that, in contrast to healthy persons and those with atopic keratoconjunctivitis, Hsp90 expression is upregulated in the conjunctival stroma and vasculature of patients with ocular disease [27].…”

Clinical features and diagnosis of epidermolysis bullosa acquisita

“…To investigate the loss of tolerance to type VII collagen, mice were immunized with recombinant fragments of NC1 domain, which raised murine antitype VII collagen IgG that resulted in a long-lasting autoimmune skin disease resembling human EBA [68,74]. These animal models were subsequently used to show the pathogenic relevance of complement activation at the epidermal BMZ, neutrophils, CD18-mediated extravasation, FcγRIV, heat-shock protein 90, the glycosylation status of antitype VII collagen IgG, IL-1β, IL-6, GM-CSF, Erk 1/2, CXCL1/2, p38, Akt, RORa, HSP90, gene expression in myeloid cells, and the skin microbiome [75][76][77][78][79][80][81][82][83][84].…”

Epidermolysis Bullosa Acquisita: From Pathophysiology to Novel Therapeutic Options

“…A novel strategy to inhibit autoreactive T cell responses is derived from experiments using inhibitors of the cell stressinducible chaperone heat shock protein 90 (Hsp90) in experimental EBA (Kasperkiewicz et al, 2011). Currently tested in clinical trials for therapy of cancer patients due to its inhibitory effects on malignant cells (Solárová et al, 2015), anti-Hsp90 treatment is also increasingly becoming a research focus in autoimmune diseases, including blistering disorders, as it exerts potent immunomodulatory actions (Collins et al, 2013;de Zoeten et al, 2011;Kasperkiewicz et al, 2011;Tukaj et al, 2014aTukaj et al, , 2014bTukaj et al, , 2014cTukaj et al, , 2015aTukaj et al, , 2015b. Application of Hsp90 inhibitors before and after disease onset blocked EBA development and induced clinical recovery associated with suppressed autoantibody production compared with vehicletreated animals in immunization-induced EBA, respectively.…”