DOI: 10.1111/exd.12476

Original Article

www.wileyonlinelibrary.com/journal/EXD

Immunomodulatory effects of heat shock protein 90 inhibition on humoral immune responses Stefan Tukaj1, Benjamin Tiburzy2, Rudolf Manz2, Andreia de Castro Marques1, Antal Orosz3, Ralf J. Ludwig1, Detlef Zillikens1 and Michael Kasperkiewicz1 1 Department of Dermatology, University of L€ ubeck, L€ ubeck, Germany; 2Institute for Systemic Inflammation Research, University of L€ ubeck, L€ ubeck, Germany; 3Tumor Cell Biology Laboratory, Anticancer Drug Research Foundation, Budapest, Hungary Correspondence: Michael Kasperkiewicz, MD, Department of Dermatology, University of L€ ubeck, Ratzeburger Allee 160, 23538 L€ ubeck, Germany, Tel.: +49-451-500-3254, Fax: +49-451-500-5162, e-mail: [email protected]

Abstract: Heat shock protein 90 (Hsp90) inhibition blocks T-celllinked inflammatory disease pathways and exhibits therapeutic activity in autoimmune disease mouse models, including the blistering disease epidermolysis bullosa acquisita. Although we previously showed that preformed autoreactive plasma cells do not seem to be directly affected by anti-Hsp90 treatment, immunomodulatory effects of Hsp90 inhibition on (auto-) antibody responses are not yet fully understood. In this study, the Hsp90 blocker 17-DMAG inhibited proliferation of activated total B cells and their IgG secretion in cultures of human peripheral B cells from healthy subjects, but IgG production was no longer affected when these activated B cells were allowed to differentiate prior to a deferred application of the inhibitor. 17-DMAG treatment was associated with induction of nuclear and cytoplasmic heat shock factor 1 and Hsp70 in stimulated human B cells, respectively. Type VII collagen (epidermolysis bullosa acquisita)-immunized mice early treated with 17-DMAG had

reduced total B cells in spleens, a relative increase in splenic regulatory B cell fractions, higher serum IL-10 concentrations, and lower levels of circulating autoantibodies (paralleled by less pronounced disease induction) compared with vehicle-treated immunized mice. Autoantibody production was blunted in isolated and autoantigen-restimulated lymph node cells from immunized mice by either 17-DMAG or purified autologous splenic regulatory B cells. Thus, in addition to the previously described T cell inhibitory effects of Hsp90 blockade, this treatment potently modulates humoral immune responses at the B cell level, further supporting the introduction of Hsp90 inhibitors into the clinical setting for treatment of autoantibodymediated disorders.

Introduction

As Hsp90 plays additional important roles in antigen presentation, activation of lymphocytes and macrophages, and activation and maturation of dendritic cells (6), its pharmacologic inhibition has also increasingly become a research focus in autoimmune diseases. In preclinical rodent studies, Hsp90 inhibitors have been shown to ameliorate autoimmune encephalomyelitis, rheumatoid arthritis, and systemic lupus erythematosus (7–11). Recently, our research group demonstrated that treatment with Hsp90 inhibitors is also clinically effective (both prophylactically before disease onset and abortively within 6 weeks of treatment once clinical disease had been established) in mice with experimental epidermolysis bullosa acquisita (EBA) (12), a chronic subepidermal blistering autoimmune disease characterized by pathogenic circulating and tissue-bound autoantibodies against the non-collagenous domain 1 of type VII collagen of the dermal– epidermal junction (13,14). Similar to what was observed in mouse models of the above mentioned autoimmune diseases (7–11), these effects were associated with an inhibitory potential of Hsp90 blockade on T cell responses (12). In another study, we observed an aberrant expression of Hsp90 in skin and blood of patients with the most common subepidermal blistering autoimmune disorder, bullous pemphigoid (BP), characterized by autoantibodies to type XVII collagen (BP180), suggesting that Hsp90 may play a pathophysiological role and represent a novel potential treatment target also in this disease (15). In fact, further

Autoreactive T cells, B cells and plasma cells contribute to autoimmune processes, which have been also described to occur in healthy individuals, and thus represent treatment targets in autoimmune diseases (1,2). However, therapy of these disorders continues to remain challenging and in most cases still consists of conventional, unspecific immunosuppressive treatment with corticosteroids and cytostatic agents that are often associated with side effects and limited clinical responses (1). Heat shock proteins (Hsp) are a family of ubiquitous molecular chaperones essential for protein folding and transport within the cell, with Hsp90 being strongly involved in structural maturation and conformational regulation of a number of signalling molecules and transcription factors (3). Under normal conditions, the transcription factor heat shock factor 1 (HSF1) is a client of Hsp90 and is held in an inactive complex with this chaperone. In case of cell stress or by pharmacological blockade of Hsp90, HSF1 is activated, released from this interaction, trimerized, and translocated to the nucleus where it has been shown to repress proinflammatory and activate anti-inflammatory genes (3,4). As many oncogenes are client proteins of Hsp90, different specific inhibitors of this chaperone (i.e., geldanamycin derivatives, resorcinol derivatives, purine analogues, and other synthetic inhibitors) have been developed as anticancer agents and shown promising results in some solid tumors and haematological malignancies (5).

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 585–590

Key words: autoantibody – autoimmunity – B cell – heat shock protein

Accepted for publication 17 June 2014

585

Tukaj et al.

experiments of our group revealed that Hsp90 inhibition suppressed BP IgG-induced production of IL-8, a proinflammatory cytokine critical for BP pathology, by cultured keratinocytes (16). Regulatory B cells (Bregs), described as, among other proposed phenotypic sets of markers, CD19+CD1dhiCD5+ and CD19+IL-10+ cells, have been undoubtedly identified as a novel B cell subset in mice and humans and shown to downregulate cellular immune responses and inflammation via IL-10-mediated mechanisms (17). Although the important regulatory effects of these cells in vivo and their therapeutic potential in autoimmunity have been demonstrated in some animal models of human autoimmune diseases (18), there is currently a high need to identify drugs that could specifically expand Bregs or selectively deplete effector B cells while sparing this immunomodulatory cell type. Although pleiotropic effects of Hsp90 inhibitors on different immune cells have been described previously (7–12,19), information on the impact of Hsp90 blockers to regulate humoral immune responses especially in the context of Bregs is lacking. Although in our previous study, investigating anti-Hsp90 therapy in mice with experimental EBA, preformed autoreactive plasma cells were not directly affected by this treatment, a suppressed serum autoantibody production was found in anti-Hsp90treated mice (12). We hypothesized that this finding may not exclusively be linked to the observed inhibitory effects of Hsp90 blockade on T cells (12). Partly using the same EBA mouse model, this current experimental work therefore extends this previous study by showing that in vitro and in vivo inhibition of Hsp90 by the geldanamycin derivative 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) impacts (auto-)antibody responses at the B cell (effector and regulatory type) level.

Methods Human blood samples For the analysis of human immune cells in vitro, blood samples were obtained from 13 normal healthy adult donors aged between 28 and 35 years (mean 31.2  2.2; eight females and five males). The collection of samples was approved by the Ethics Committee of the University of L€ ubeck, and informed consent was obtained according to the Declaration of Helsinki.

Human cell cultures Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque Plus (GE Healthcare Life Sciences, Freiburg, Germany.) gradient centrifugation. Untouched human B cells with ~95% purity were obtained by depletion of magnetically labelled non-B cells (MACS Miltenyi Biotec GmbH, San Diego, CA, USA). Cells were cultured in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 lg/ml streptomycin in tissue 96-well plates (20 000 or 50 000/ml). Anti-CD40 mAb (1 lg/ml; clone 82111, R&D Systems, Minneapolis, MN, USA, Jackson ImmunoResearch, West Grove, PA, USA), anti-IgM Ab (5 lg/ml; Jackson ImmunoResearch), IL-2 (10 ng/ml; R&D Systems), IL-4 (50 ng/ml; R&D Systems) and IL-21 (100 ng/ml; Cell Sciences) were added to cultures where indicated. Stimulated cells were cultured alone or with different concentrations of 17-DMAG (0.001, 0.01, or 0.1 lM; InvivoGen, San Diego, CA, USA). This type of Hsp90 inhibitor was chosen and used throughout this study for the purpose of uniformity and therefore better comparability with our previous experiments using the same compound (12,16,19). Cell proliferation was assayed by

586

ELISA after BrdU incorporation (Roche, Mannheim, Germany) on the 6th day of treatment followed by 24 h of incubation according to manufacturer’s protocol. In addition, nuclear and cytoplasmic protein extracts were prepared from stimulated (combination of anti-IgM Ab, anti-CD40 mAb, and IL-2/4/21 cytokines) and 17-DMAG-treated or -non-treated B cells after 6 h using the NE-PER extraction kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. Nuclear HSF1 and cytoplasmic Hsp70 levels were evaluated by ELISA (Enzo Life Sciences, Lo¨rrach, Germany).

Mice Six- to 8-week-old SJL mice were obtained from Charles River Laboratories. The experiments were approved by local authorities of the Animal Care and Use Committee and performed by certified personnel.

Production of autoantigen Recombinant forms of murine type VII collagen (mCVIIC) were prepared as described before (20–22). Recombinant tagged fragments GST-mCVIIC and His-mCVIIC were produced by use of a prokaryotic expression system and purified by glutathione and metallochelate affinity chromatography, respectively (20,22).

Immunization and treatment of mice Experimental EBA was induced in mice by active immunization, as described previously (12,22). Briefly, mice were injected subcutaneously in the hind footpads with a single injection of 100 ll of emulsion containing 60 lg of GST-mCVIIC in TiterMax (Alexis Biochemicals, San Diego, CA, USA). Mice were examined weekly for their general condition and for cutaneous symptoms (i.e., erythema, blisters, erosions, and crusts). Disease severity was calculated as percentage of body surface area affected by skin lesions. Mice were treated intra-peritoneally with 30 mg/kg of 17-DMAG (InvivoGen) or the equivalent volume of water as vehicle control. For prophylactic treatment, immunized or naive mice received a total of 18 injections with 17-DMAG or vehicle starting 1 day before immunization, each given three times a week over a 6-week treatment period. One day after the last injection, mice were sacrificed for clinical and immunologic evaluation.

Murine cell cultures Single cell suspensions were prepared from draining inguinal lymph nodes and spleens isolated 10 weeks after immunization of mice not treated by 17-DMAG or vehicle. Cells were cultured in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 lg/ml streptomycin in tissue 96-well plates (100 000/ml). Isolated lymph node cells were restimulated with the recombinant autoantigen His-mCVIIC (1 or 10 lg/ml) plus 1 lg/ml of lipopolysaccharide (LPS, E. coli 0:111 B4, c-irradiated) and cultured in absence or presence of different concentrations of 17-DMAG (0.001, 0.01, or 0.1 lM) for 7 days, as described previously with minor modifications (12). In a functional assay, isolated total splenic B cells were cultured in presence of LPS (10 lg/ml) and anti-CD40 mAb (5 lg/ml) for 24 h. A Mouse Regulatory B Cell Isolation Kit (Miltenyi Biotec) was then used to purify these cells according to the manufacturer’s instructions. Apoptotic and necrotic cells were removed from the suspension using a Dead Cell Removal Kit (Miltenyi Biotec). Either purified splenic CD19+IL-10+ cells or B cells containing only a low number of such cells (not exceed 7% of IL10+CD19+ cells) were then cocultured (1:2) with His-mCVIIC

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 585–590

Hsp90 inhibition and humoral immunity

(1 lg/ml)-restimulated draining inguinal lymph node cells in presence of LPS (10 lg/ml) for the next 72 h.

Flow cytometry immunophenotyping of murine cells Single cell suspensions from spleens of naive or immunized mice treated by 17-DMAG or vehicle over 6 weeks were stained with PE-conjugated anti-Syndecan-1 (CD138, clone 281-2; BD Biosciences, Bioscience, Heidelberg, Germany), Alexa-405 (Life Technologies GmbH)-coupled anti-B220 (clone RA3.B2; in-house production), anti-CD5 (clone 53-7.3; BD Biosciences) and antiCD1d (clone 1B1; BD Biosciences). In the case of intracellular IL-10 staining, splenocytes were cultured in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 lg/ml streptomycin in presence of LPS (10 lg/ml) PMA, and ionomycin for 5 h. Culture cells were washed, fixed, permeabilized, and stained for detection of intracellular cytokine with antiIL-10 mAb (BD Biosciences). Viable single lymphocytes based on forward and sidelight scatter properties were analysed using a LSRII flow cytometer (BD Biosciences). Data was evaluated using FlowJo software (TreeStar Inc., Ashland, USA).

ELISA. B cells were stimulated by anti-CD40 antibody mimicking B cell activation via T cell interaction or by the combination of stimuli resembling both T cell interaction (anti-CD40 antibody) and B cell receptor ligation (anti-IgM antibody). In addition, cells were treated by the mainly T cell-derived cytokines IL-2/IL-4/IL21. We observed that 17-DMAG was able to inhibit proliferation of activated B cells in a dose-dependent way (Fig. 1a). B cells stimulated by either anti-IgM, anti-CD40, or the mixture of soluble cytokines IL-2/IL-4/IL-21 alone showed a limited proliferation capacity, and the inhibitory effects of 17-DMAG were not observed (data not shown). Suppression of B cell proliferation by 17-DMAG was observed upon non-apoptotic and non-necrotic concentrations (0.001–0.1 lM) (Fig. 1b). Higher concentrations of 17-DMAG than 0.1 lM induced cell death in stimulated B cell cultures and were not considered for further analysis (Fig. 1b). To determine whether 17-DMAG had an influence on IgG production, activated human B cells were cultured and stimulated as described above. 17-DMAG inhibited IgG secretion to medium of the cultures as determined by ELISA (Fig. 1c). Notably, when

Cytokine analysis IL-4, IL-5, IL-10, IL-17A, IFN-c, and TNF-a were measured by a Mouse Th1/Th2/Th17 Cytokines Multi-Analyte ELISArray Kit (Qiagen Biosciences, Hilden, Germany) in serum of immunized mice after the 6-week treatment with 17-DMAG or vehicle.

(a)

(c)

Detection of (auto-)antibodies by ELISA Serum or culture supernatant autoantibodies against type VII collagen were detected as described before with minor modifications (23). Briefly, 96-well ELISA plates were coated with 0.5 or 5 lg of recombinant His-mCVIIC in 0.1 M bicarbonate buffer (pH 9.6) at 4°C overnight. After blocking with 1% BSA in PBS, wells were incubated with mouse sera or cell culture supernatants for 60 min. In the case of serum autoantibodies, biotinylated goat anti-mouse IgG secondary antibodies (1:500; SouthernBiotech), followed by the addition of streptavidin-coupled alkaline phosphatase (1:3000; Roche Diagnostics GmbH, Mannheim, Germany) and ALP (Roche Diagnostics GmbH) were used. Culture supernatant autoantibodies were detected using HRP-conjugated goat anti-mouse IgG secondary antibodies (1:1000; SouthernBiotech, Birmingham, AL, USA). For detection of total IgG in serum or culture supernatants, a mouse IgG ELISA quantititation set (Bethyl Laboratories, Inc., Montgomery, Texas, USA) was used. A human immunoglobulin quantification kit (RD Biotech, Besancßon, France) was used for evaluation of total IgG levels in culture.

(b) (d)

Cell viability assay Cell viability of cultured cells in response to different 17-DMAG concentrations (0.001, 0.01, 0.1, and 1 lM) was evaluated by Annexin V/propidium iodide staining and analysed by flow cytometry (FACScalibur, BD Biosciences).

Statistical analysis All analyses were performed using the Student’s t test or ANOVA (Bonferroni procedure for multiple comparisons). Data is presented as means  SEM; P < 0.05 was considered statistically significant.

Results 17-DMAG inhibits proliferation and IgG secretion of activated human peripheral blood B cells in vitro 17-DMAG was used in different concentrations to assay proliferation response of activated human B cells in culture by BrdU

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 585–590

Figure 1. 17-DMAG inhibits proliferation and IgG secretion of activated human peripheral blood B cells in vitro. (a) Human B cells were isolated from PBMCs by MACS-negative selection and cultured alone or stimulated by anti-CD40 (1 lg/ml) or both anti-CD40 (1 lg/ml) and anti-IgM antibody (5 lg/ml). In addition, all stimulated cells were treated by IL-2 (10 ng/ml), IL-4 (50 ng/ml), and IL-21 (100 ng/ ml). Incubations were performed in the absence or presence of different amounts of 17-DMAG for 7 days, and B-cell proliferation was determined by BrdU ELISA. (b) Possible toxic effects of 17-DMAG on B cells were evaluated after a 7-day incubation period using Annexin V and propidium iodide staining and flow cytometry measurements. (c) B cells were isolated, cultured, and treated as described above. IgG secretion was assayed after 7 days of treatment without or with 0.1 lM of 17-DMAG. (d) 17-DMAG (0.1 lM) was added at day 5 of culture, and IgG production was assayed over the next 3 days. IgG secretion was analysed by ELISA. Values are means  SEM of ≥3–6 healthy blood donors. *P < 0.05 different from the respective 17-DMAG-untreated control set of data.

587

Tukaj et al.

17-DMAG was added at the 5th day of culture, no effect on IgG production was noted over the next 3 days (Fig. 1d).

17-DMAG induces HSF1 and Hsp70 expression in activated human peripheral blood B cells To assess the impact of Hsp90 inhibition on nuclear HSF1 and cytoplasmic Hsp70 protein expression, anti-IgM-, anti-CD40, and IL-2/IL-4/IL-21-activated B cells were treated with 17-DMAG for 6 h. An increased level of HSF1 was found in the nucleus of these cells compared with 17-DMAG-untreated controls (Fig. 2a), which corresponded with an upregulation of cytoplasmic Hsp70 (Fig. 2b).

17-DMAG (n = 6) or vehicle (n = 6) over 6 weeks. 17-DMAGtreated mice showed significantly higher serum concentrations of IL-10 (1.12-fold; P < 0.05) and lower serum levels of IFN-c (1.13-fold; P < 0.05) at the end of treatment, whereas the remaining cytokines were not affected by the inhibitor. (a)

17-DMAG reduces total murine splenic B cells and slightly but significantly increases Bregs under normal and autoimmune conditions in vivo We have previously shown that Hsp90 inhibitors applied either in a prophylactic or therapeutic setting ameliorated clinical disease in mice with experimental EBA (12). Here, we tested the effects of early administration of 17-DMAG on total splenic B cells and Bregs in SJL mice. The analysis was performed after treatment with 17-DMAG or vehicle given to naive or murine type VII collagen autoantigen (GST-mCVIIC)-immunized mice over 6 weeks starting 1 day before immunization. We observed that both the percentage and the absolute numbers of total splenic B cells in either naive or immunized mice were significantly inhibited by 17-DMAG treatment compared with vehicle controls (Fig. 3a). In addition to total B cells in the spleens, the frequencies of Bregs were analysed after the 6-week treatment period with 17-DMAG or vehicle. We found that the percentages of CD19+CD1dhiCD5+ (Fig. 3b) and CD19+IL-10+ (Fig. 3c) cell populations in the spleens were significantly increased in both naive and immunized mice treated with 17-DMAG compared with vehicle-treated animals. Absolute numbers of Bregs were not increased with 17-DMAG treatment (data not shown), however, because of the concomitant treatment-related decline in total B cell numbers. Notably, CD19+CD1dhiCD5+ cell frequencies were found to be significantly higher in immunized mice compared with non-immunized animals independent of 17-DMAG treatment (Fig. 3b).

(b)

(c)

IL-10 levels are increased and IFN-c levels are reduced in serum of mice treated with 17-DMAG Next, we measured the cytokine levels of IL-4, IL-5, IL-10, IL-17A, IFN-c, and TNF-a in serum of immunized mice treated with

(a)

(b)

Figure 2. 17-DMAG increases HSF1 and Hsp70 expression in activated human peripheral blood B cells. (a) HSF1 and (b) Hsp70 protein levels were assayed by ELISA in nuclear (HSF1) and cytoplasmic extracts (Hsp70) of anti-IgM-, anti-CD40-, and IL-2/4/21-stimulated B cells treated for 6 h without or with 0.1 lM of 17-DMAG. Values are means  SEM of n = 4. *P < 0.05.

588

Figure 3. 17-DMAG depletes total murine B cells in spleens while slightly but significantly increasing splenic Bregs under normal and autoimmune conditions in vivo. 17-DMAG (30 mg/kg 3 times a week) or vehicle was given to naive or type VII collagen autoantigen (GST-mCVIIC)-immunized mice over 6 weeks starting 1 day before immunization. After this treatment, splenocytes were isolated from mice and analysed for frequency of total B cells and Bregs by flow cytometry. (a) Percentages and absolute numbers of splenic B220+ cells. Values are means of 8–10 mice per group. *P < 0.05. (b) Frequencies of splenic CD19+CD1dhiCD5+ cells. The numbers in the gates of the representative results are the percentages of CD19+CD1dhiCD5+ cells with respect to total cell numbers. Values are means of 6–10 mice per group. *P < 0.05. (c) Frequencies of splenic CD19+IL-10+ cells. The numbers below the gates of the representative results are the percentages of CD19+IL-10+ cells with respect to total cell numbers. Values are means of 6–10 mice per group. *P < 0.05.

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 585–590

Hsp90 inhibition and humoral immunity

17-DMAG and Bregs dampen the (auto-)antibody response in vivo and ex vivo To determine whether 17-DMAG affected circulating antibodies in vivo, anti-type VII collagen IgG autoantibodies and total IgG levels were analysed in serum of mice after the 6-week treatment with 17-DMAG or vehicle. The generation of autoantibodies in immunized mice was sufficiently suppressed by 17-DMAG compared with control mice (Fig. 4a), which was paralleled by a significantly weaker induction of EBA clinical phenotype in the inhibitor-treated group at the time of treatment completion [2.5-fold lower disease score, P < 0.05; disease incidence of 56% and 90% in verum (n = 9) and control (n = 10) group, respectively]. In contrast, 17-DMAG-treated naive and immunized mice showed unchanged total IgG serum levels (Fig. 4b) as well as no reduction of overall plasma cell numbers in lymph nodes and spleens (data not shown). To confirm the in vivo observed inhibitory impact of 17-DMAG on autoantibody production in an ex vivo setting, we isolated draining lymph node cells from untreated mice 10 weeks after immunization with GST-mCVIIC and restimulated them with autoantigen. The addition of 17-DMAG significantly inhibited the

(a)

(c)

(b)

(d)

Figure 4. 17-DMAG and Bregs suppress the (auto-)antibody response in vivo and ex vivo. (a) Type VII collagen (mCVIIC)-specific IgG autoantibody or (b) total IgG levels were evaluated in sera of immunized and naive control mice after the 6week treatment period with 17-DMAG and vehicle by ELISA. Values are means of 8–9 mice per group. *P < 0.05. (c) Draining inguinal lymph node cells were isolated from untreated, GST-mCVIIC-immunized mice 10 weeks after immunization, restimulated with the recombinant autoantigen His-mCVIIC (1 or 10 lg/ml) plus 1 lg/ml of LPS, and cultured in absence or presence of different concentrations of 17-DMAG for 7 days. mCVIIC-specific IgG autoantibody levels were analysed in culture supernatants by ELISA. Values are means  SEM of 5 mice per group. *P < 0.05 different from the respective 17-DMAG-untreated control set of data. (d) In addition to draining inguinal lymph node cells, splenic cells were isolated from untreated, GST-mCVIIC-immunized mice 10 weeks after immunization. A regulatory B cell isolation kit (MACS) was used to purify viable IL10-producing splenic B cells (CD19+IL-10+; purities of approximately 65–70%). Autoantigen (His-mCVIIC; 1 lg/ml)-restimulated lymph node cells in presence of LPS were cultured (1:2) (i) alone, (ii) with B cells containing only a sparse number of Bregs (not exceed 7% of IL-10+CD19+ cells), or (iii) with purified CD19+IL-10+ cells for 72 h. mCVIIC-specific IgG autoantibody levels were analysed in culture supernatants by ELISA. Values are means  SEM of 5 mice per group. *P < 0.05.

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 585–590

secretion of anti-type VII collagen IgG autoantibodies in a dosedependent manner (Fig. 4c). To determine whether Bregs were able to directly regulate the secretion of autoantibodies, a regulatory B cell isolation kit (Miltenyi Biotec) was used to purify viable IL-10-producing B cells (with purities of approximately 65–70%) from spleens of untreated, immunized mice. In this functional assay, either purified splenic CD19+IL-10+ cells or B cells containing only a low number of Bregs (not exceed 7% of IL-10+CD19+ cells) were cocultured (1:2) with autoantigen-restimulated draining lymph node cells from immunized mice. The presence of enriched CD19+IL-10+ cells, but not the sparse Breg population, considerably inhibited the secretion of anti-type VII collagen IgG autoantibodies in culture (Fig. 4d).

Discussion The results of the present study show that 17-DMAG has potent direct effects on B cell responses, inhibiting their total proliferation, immunoglobulin production, and favouring a relative shift towards regulatory subtypes. In a first set of in vitro experiments, we could show that 17-DMAG in non-toxic doses effectively suppressed proliferation of activated human B cells, which resulted in significant reduction of IgG production. This is in line with previous studies reporting the capacity of Hsp90 inhibitors to inhibit proliferation in lymphocytes and in a variety of human cancer cell lines (5,7–12,19). Interestingly, inhibition of IgG production was not evident when these isolated B cells were treated with 17-DMAG after 5 days in culture, indicating that Hsp90 blockade inhibits the generation of plasma cells but not their subsequent persistence. This is in agreement with our previous and current in vivo data from the EBA mouse model, in which already preformed plasma cells as well as total IgG serum levels were not directly affected by anti-Hsp90 treatment (12). Treatment of activated B cells by 17-DMAG was associated with induction of nuclear expression of HSF1 as well as upregulation of the HSF1 response protein Hsp70 in the cytoplasm of these cells, an effect that was recently also obtained by pharmacological inhibition of Hsp90 in stimulated regulatory T cells (Tregs) (4). Hsp70 is generally accepted as both a good marker for effective Hsp90 inhibition and a potent negative regulator of inflammatory responses through, but not limited to, its negative feedback effect on NFjB signalling activity (24–26). Therefore, it is likely that the B cell inhibitory effects of 17-DMAG are, at least, partly mediated via the HSF1–Hsp70 pathway. We could confirm the above mentioned findings from human cell culture studies using naive mice or mice immunized with the EBA autoantigen type VII collagen, mimicking a normal and autoimmune in vivo situation, respectively. Both naive mice injected with 17-DMAG as well as immunized mice treated by this Hsp90 inhibitor that was administered before the appearance of EBA clinical phenotype showed reduced total B220+ cell numbers in spleens. As the impact of anti-Hsp90 treatment on Bregs has not yet been studied at all, we further investigated this novel immunoregulatory cell subtype. We observed significantly higher percentages of splenic CD19+CD1dhiCD5+ and CD19+IL-10+ cells compared with vehicle-treated control animals. In addition, serum IL-10 levels were found to be significantly higher in 17-DMAG-treated immunized mice compared with vehicle-injected immunized mice. The increase of IL-10-producing Bregs after 17-DMAG treatment

589

Tukaj et al.

in these immunized mice may have at least in part accounted for the concomitantly observed lower serum concentrations of IFN-c compared with vehicle-treated immunized controls, as it was previously reported that Bregs are capable of suppressing proinflammatory IFN-c responses in vivo (27). It has to be stressed, however, that only percentages but not the absolute numbers of Bregs were increased in 17-DMAG-treated animals, suggesting that this relative and modest increase of the Breg fractions is rather related to a selective survival of this immunomodulatory cell type during effective effector B cell depletion by the Hsp90 inhibitor than to their true induction. Interestingly, CD19+CD1dhiCD5+ cell frequencies were significantly higher in immunized mice compared with non-immunized mice irrespective of 17-DMAG treatment. This observation is in good agreement with recently reported data showing that blood Bregs from patients with autoimmune diseases, including those with autoimmune bullous disorders, were expanded in comparison with healthy controls (28). Pharmacologic targeting of Hsp90 has been previously shown to be associated with reduced serum anti-dsDNA and anti-basal membrane zone autoantibody production in mouse models of systemic lupus erythematosus and EBA (10–12), respectively. In addition to our analogous current finding of reduced circulating anti-type VII collagen-specific autoantibodies (along with less pronounced disease induction) in 17-DMAG-treated immunized-mice compared with vehicle-injected immunized animals, this observation was further confirmed ex vivo by showing that autoantibody secretion to culture medium was dose dependently suppressed by 17-DMAG in isolated draining lymph node cells from immunized mice restimulated with recombinant autoantigen. Using a similar experimental approach, we were previously able to demonstrate a reduced proliferative response of isolated lymph node cells from immunized mice to in vitro restimulation with the T cell activating anti-CD3/CD28 antibody or autoantigen (12). Moreover, we recently provided evidence for inhibitory effects of Hsp90 blockade on proinflammatory human Th1 and Th17 cell subpopulations (19). Therefore, in addition to direct inhibitory effects of 17-DMAG on autoreactive B cells, suppression of autoantibody production may also be the result of an inhibitory capacity of Hsp90 inhibitors on T cells, which are known to provide help to

References 1 Davidson A, Diamond B. N Engl J Med 2001: 345: 340–350. €ßmann J, Pru €ßmann W, Recke A et al. Exp 2 Pru Dermatol 2014: doi:10.1111/exd.12445. [Epub ahead of print]. 3 Taipale M, Jarosz D F, Lindquist S. Nat Rev Mol Cell Biol 2010: 11: 515–528. 4 Collins C B, Aherne C M, Yeckes A et al. Mucosal Immunol 2013: 6: 960–971. 5 Garcia-Carbonero R, Carnero A, Paz-Ares L. Lancet Oncol 2013: 14: e358–e369. 6 Srivastava P. Nat Rev Immunol 2002: 2: 185– 194. 7 Dello Russo C, Polak P E, Mercado P R et al. J Neurochem 2006: 99: 1351–1362. 8 Rice J W, Veal J M, Fadden R P et al. Arthritis Rheum 2008: 58: 3765–3775. 9 Yun T J, Harning E K, Giza K et al. J Immunol 2011: 186: 563–575. 10 Han J M, Kwon N H, Lee J Y et al. PLoS ONE 2010: 5: e9792.

590

B cells. Accumulating evidence has recently shown that Bregs themselves may control antibody production (29). In fact, our coculture experiments revealed that purified CD19+IL-10+ cells, but not B cells containing only a sparse number of Bregs, were able to efficiently blunt anti-type VII collagen autoantibody production in activated lymph node cells. As Tregs also have the capacity to control antibody production and were recently shown to develop enhanced functions through Hsp90 inhibition in experimental colitis (4,29,30), it is tempting to speculate that an additional Treg-mediated mechanism might have accounted for this suppressed autoantibody response after Hsp90 treatment in our study. Thus, pleiotropic effects of Hsp90 inhibitors on different effector as well as regulatory cell subsets may be mechanistically involved in the control of autoantibody production, making them an attractive candidate for future clinical application in patients with autoantibody-mediated diseases. In conclusion, our results contribute to the understanding of the anti-inflammatory mechanism of action underlying the known beneficial clinical effects of pharmacological Hsp90 blockade in experimental models of autoimmune diseases. In addition to the previously described T cell inhibitory effects of this novel therapeutic approach (7–12,19), it potently modulates humoral immune responses at the B cell (effector and regulatory type) level. This study therefore further supports the introduction of Hsp90 inhibitors into the clinical setting for the treatment of autoantibodymediated disorders.

Acknowledgements The authors would like to thank Susen M€ uller (Department of Dermatology, University of L€ ubeck) for technical assistance. This work was supported by Deutsche Forschungsgemeinschaft (DFG) Excellence Cluster “Inflammation at Interfaces” (EXC 306/2), DFG KA 3438/1-1, Medical Faculty of the University of L€ ubeck (E22-2013), and Focus Program “Autoimmunity” at the University of L€ ubeck.

Author contribution ST, RM, and MK designed the research study; ST, BT, ACM, and MK performed the research; RM and DZ contributed essential reagents or tools; ST and MK wrote the paper. All authors contributed to data analyses and interpretation as well as critical review of the manuscript.

Conflict of interests The authors have declared no conflicting interests.

11 Shimp S K 3rd, Chafin C B, Regna N L et al. Cell Mol Immunol 2012: 9: 255–266. €ller R, Manz R et al. Blood 12 Kasperkiewicz M, Mu 2011: 117: 6135–6142. 13 Woodley D T, Briggaman R A, O’Keefe E J et al. N Engl J Med 1984: 310: 1007–1013. 14 Woodley D T, Burgeson R E, Lunstrum G et al. J Clin Invest 1988: 81: 683–687.  ski K, Vafia K et al. PLoS ONE 15 Tukaj S, Kleszczyn 2013: 8: e70496. €ner D, Zillikens D et al. Cell Stress 16 Tukaj S, Gru Chaperones 2014: PMID: 24796797. [Epub ahead of print]. 17 Mauri C, Bosma A. Annu Rev Immunol 2012: 30: 221–241. 18 Kalampokis I, Yoshizaki A, Tedder T F. Arthritis Res Ther 2013: 15(Suppl 1): S1. 19 Tukaj S, Zillikens D, Kasperkiewicz M. J Inflamm (Lond) 2014: 11: 10. 20 Sitaru C, Mihai S, Otto C et al. J Clin Invest 2005: 115: 870–878.

21 Kasperkiewicz M, Hirose M, Recke A et al. Br J Dermatol 2010: 162: 1064–1070. 22 Ludwig R J, Recke A, Bieber K et al. J Invest Dermatol 2011: 131: 167–176. 23 Sitaru C, Chiriac M T, Mihai S et al. J Immunol 2006: 177: 3461–3468. 24 Dakappagari N, Neely L, Tangri S et al. Biomarkers 2010: 5: 31–38. 25 de Jong P R, Schadenberg A W, Jansen N J et al. Cell Stress Chaperones 2009: 14: 117– 131. 26 Stocki P, Dickinson A M. Autoimmune Dis 2012: 2012: 617213. 27 Yoshizaki A, Miyagaki T, DiLillo D J et al. Nature 2012: 491: 264–268. 28 Iwata Y, Matsushita T, Horikawa M et al. Blood 2011: 117: 530–541. 29 Fujio K, Okamura T, Sumitomo S et al. Ann Rheum Dis 2013; 72 Suppl 2: ii85–ii89. 30 de Zoeten E F, Wang L, Butler K U et al. Mol Cell Biol 2011: 31: 2066–2078.

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 585–590

Immunomodulatory effects of heat shock protein 90 inhibition on humoral immune responses.

Heat shock protein 90 (Hsp90) inhibition blocks T-cell-linked inflammatory disease pathways and exhibits therapeutic activity in autoimmune disease mo...
564KB Sizes 0 Downloads 6 Views