Anti–Heat Shock Protein 70 Autoantibodies from Patients with Idiopathic Pulmonary Fibrosis Epigenetically Enhance Lung Fibroblast Apoptosis Resistance and Bcl-2 Expression

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease of older adults (1). Currently available therapeutic agents for IPF are only partially effective, and this disease has a worse prognosis than many common malignancies. Although the inciting etiology of IPF remains enigmatic, a number of cellular abnormalities and processes are known to be involved in the pathogenesis of this disease (2, 3).

As an example, fibroblasts and related mesenchymal cells are downstream effectors of the lung fibrosis that is a sine qua non of IPF (2). Fibroblasts and their activated, differentiated progeny (e.g., myofibroblasts) traffic to and proliferate in injury foci, where they engage in complex repair processes that include production of connective tissue extracellular matrix. Under normal circumstances, these responses are limited, and homeostasis is ultimately maintained, by appropriately timed induction of fibroblast and myofibroblast apoptosis. In particular conditions, however, such as some chronic or recurrent tissue injuries, these mesenchymal cells do not readily undergo apoptosis and instead inappropriately continue to produce and remodel extracellular matrix, resulting in pathological fibrosis (4, 5). Pulmonary fibroblasts from patients with IPF have increased resistance to apoptosis, which has been attributed to their overexpression of Bcl-2, an antiapoptotic regulatory protein (4, 6). Despite the significance of these abnormalities, it remains to be determined if the fibroblast dysfunction of IPF is due to an inherent cellular defect(s) or a secondary consequence of some other process(es).

A growing body of evidence indicates that adaptive immune responses also participate in IPF pathogenesis (7–13). Numerous T cell and B cell abnormalities that parallel features of conventional connective tissue diseases are prevalent in patients with IPF (7–9, 11, 12). As examples, Abs with specificities for numerous and varied self-proteins are found in the majority of patients with IPF (7), and some of these have been shown to have pathological functions, as well as associations with clinical phenotypes and outcomes (10, 13). We reported that IgG autoantibodies to heat shock protein 70 (anti-HSP70) in patients with IPF are particularly associated with accelerated lung function deterioration and increased mortality (9). Moreover, anti-HSP70 autoantibodies are not unique to IPF and are also found in patients with several other immunological diseases and autoimmune syndromes (14–18).

In addition to its well-known function as an intracellular chaperone, HSP70 protein can be secreted by stressed cells (19). Among other consequences, extracellular HSP70 (eHSP70) acts as a paracrine messenger by cognate binding to specific surface receptors that transduce a variety of proinflammatory (20–22) and antiapoptotic effects (23–25). Treatments with divalent anti-HSP70 mAbs raised in animals can enhance eHSP70 functional effects (20), whereas monovalent anti-HSP70 Fab fragments are relatively inert, indicating that the autoantibody actions are dependent on cross-linkage (oligomerization) of eHSP70-receptor complexes (26).

To our knowledge, the human anti-HSP70 autoantibodies found in various diseases have not been tested to determine if they can modulate apoptosis. If so, this finding would illustrate a novel mechanism by which autoantibodies can deleteriously alter target cell functions. Given the widespread importance of programmed cell death in diverse homeostatic processes, such as fibroproliferation and immune regulation, this discovery could have considerable significance in patients with IPF and several other disease populations in which anti-HSP70 autoimmunity is also present (14, 16, 18, 27).

In the present study, we examined the effects of anti-HSP70 IgG autoantibodies, isolated from plasma of patients with IPF, on fibroblast apoptosis. We found that treatment with anti-HSP70 decreases the apoptosis susceptibility of normal human primary lung fibroblasts and increases Bcl-2 expression in these target cells. Moreover, these studies also discovered that this autoantibody effect is mediated via epigenetic regulation.

Human primary fibroblasts were derived from deidentified normal or IPF lung tissues obtained as surgical explants at the time of lung transplant, as detailed previously (7). IPF diagnoses were made by expert clinicians, blinded to these studies, who assessed all available information (including histological evaluations) according to American Thoracic Society/European Respiratory Society guidelines (1). IMR-90 lung fibroblasts were obtained from the Coriell Institute for Medical Research (Camden, NJ).

Fibroblasts were grown to 70% confluence in DMEM supplemented with 10% FBS, 1% l-glutamine, 1% penicillin/streptomycin, and 0.5% amphotericin B. These primary lung fibroblasts were used prior to passage 5, and IMR90 cells were used before population doubling of 30, as previously detailed (6). Fibroblasts were cultured in serum-free medium overnight before treatments with autoantibodies (or normal control IgG) at 1 μg/ml (a concentration shown by pilot study to optimally balance treatment effects and reagent use) and collected after 24 h or at other specified times as indicated. HSP70 IgG autoantibodies were isolated by protein G and HSP70 affinity columns from plasma specimens of patients with IPF who were having or would soon have severe acute exacerbations of their lung disease (11) and who had been found to have anti-HSP70 autoreactivity on previous study (10). Details of these autoantibody purifications were previously detailed (9). Normal human control IgG was pooled from healthy volunteer donors, and for additional confirmation, normal IgG was also purchased from Sigma-Aldrich (St. Louis, MO). Endotoxin concentrations in the autoantibody preparations were below the detection threshold of a Pierce LAL Chromogenic Endotoxin Quantitation kit (Pierce Biotechnology, Rockford, IL). These studies were approved by the University of Alabama at Birmingham Institutional Review Board.

Fibroblasts were grown on sterile glass coverslips overnight in DMEM-10, which was then replaced with serum-free media for 24 h. The slides were fixed with 4% paraformaldehyde and then incubated with either anti-HSP70 rabbit polyclonal Abs (Enzo Life Sciences, Farmingdale, NY) at 1:100 dilution or anti-HSP70 autoantibodies isolated from patient plasma (2 µg/100 µl) for 2 h at room temperature. After washing, the coverslips were incubated with the appropriate secondary Abs [i.e., anti-rabbit IgG F(ab′)₂-PE; Cell Signaling Technology, Beverly, MA] at 1:500 dilution or mouse anti-human IgG-FITC (BD Biosciences, San Diego, CA) at 1:10 dilution, respectively. After more washes, slips were mounted on glass slides with DAPI mounting medium (Life Technologies, Carlsbad, CA) and imaged on a Nikon A1 confocal microscope (Nikon Instruments, Melville, NY).

Fibroblasts were harvested from cultures by scraping and then stained with rabbit polyclonal anti-human HSP70. After interval washes, the cells were stained with a secondary Ab consisting of anti-rabbit IgG F(ab′)₂ conjugated to PE in addition to 7-aminoactinomycin D. Successive gates were established on the basis of forward and side scatter characteristics and viability (7-aminoactinomycin D null) (Supplemental Fig. 1), followed by Ab and fluorochrome controls, as detailed previously (7, 12). A series of pilot studies had confirmed 98–99% of these fibroblasts were CD90 positive (data not shown).

To determine intracellular HSP70 expression, fibroblasts were prepared using reagents in a kit (Cytofix/Cytoperm Fixation/Permeabilization Kit, BD Biosciences), followed by staining with monoclonal anti-HSP70-PE (Miltenyi Biotec, Auburn, CA). Data from 10,000 events were collected on a flow cytometer (LSR II, BD Biosciences) and analyzed using FlowJo version 10 software (FlowJo LLC, Ashland, OR).

Fibroblasts treated for 24 h with anti-HSP70 autoantibody or normal control IgG were incubated with staurosporine at 500 nM or 750 nM for 1 h. Then the cells were collected and subjected to three different methods to measure apoptosis effects.

After the fibroblasts were collected as indicated above, apoptosis was quantified by annexin V binding (MBLI, Woburn, WA) and flow cytometry, as previously described (28).

Fibroblasts treated as above were also used in a kit that measured cleaved caspase-3 activity (Takara Bio).

The protein lysate of the treated fibroblasts as described above was also subjected to Western blot analysis to detect cleaved caspase-3 (detailed below).

Fibroblasts used in RT-PCR determinations and other functional assays (e.g., apoptosis and epigenetic studies) were treated for specified durations with 1 µg/ml of an IPF patient-derived HSP70-autoantibody or normal human control IgG.

Allprep (Qiagen, Valencia, CA) or EpiQuick Nuclear Extraction kits (EpiGentek, Farmingdale, NY) were used to isolate total RNA or the nuclear extract, respectively. RNA was transcribed using a cDNA synthesis kit (Clontech, Mountain View, CA). Real-time RT-PCR was performed in triplicate and normalized to β-actin using the ΔΔC_t method (29). Primers for RT-PCR were as follows: HSP70, forward (F) 5′-CGTGGAGGAGTTCAAGAGAAA-3′, reverse (R) 5′-GTAGAAGTCGATGCCCTCAAA-3′; Bcl-2, F 5′-TGAACCGGCACCTGCACACC-3′, R 5′-GTTGGAGTGCGGGTGGGCTC-3′; Mof (males absent on the first), F 5′-AATGGCACAGCTGGGACTAGAACT-3′, R 5′-GCTTGGCTATAGCAACTGCCGAAT-3′; β-actin, F 5′-TGCTATCCAG GCTGTGCTAT-3′, R 5′-AGTCCATCACGATGCCAG-3′.

Western blotting was performed as previously described (30). Abs against Bcl-2, caspase-3, cleaved caspase-3, and β-actin were purchased from Cell Signaling Technology (Beverly, MA), and anti-Mof (A300-992A) was from Bethyl Laboratories (Montgomery, TX). β-Actin was used as a loading control. The signals were detected by an ECL system and imaged with an Amersham Biosciences 600 Imager (GE Healthcare). Densitometry was analyzed with ImageJ software.

Cells were transfected with either siMOF or scrambled nontargeting siRNA control, both from Thermo Fisher Scientific (Waltham, MA), using Lipofectamine 2000 reagent, as previously reported (31). Sequences of MOF siRNA were as follows: sense: 5′-ACUUUGACGUGGAGCCGUU-3′, and antisense: 5′-CGGCUCCACGUCAAAGUUU-3′. Transfected cells were recovered in DMEM-10 and used in functional assays described in the text.

ChIP assays were performed per the manufacturer’s protocol (ChIP Kit, ab500, Abcam, Cambridge, MA), as detailed previously (32). Anti-H4K16Ac Ab was purchased from Active Motif (Carlsbad, CA). ChIP-DNA was amplified by real-time PCR with SYBR Green PCR Master Mix (Life Technologies). The primers used for Bcl-2 were as follows: F: 5′-GAGTGGGATGCGGGAGATGTG-3′; R: 5′-CGGGATGCGGCTGTATGGG-3′. Results were normalized to input DNA.

Comparisons were made with a paired Student t test if data were paired and parametrically distributed or with the Wilcoxon test if nonparametric. Intergroup comparisons were made by unpaired Student t test if independent data were parametrically distributed or Mann–Whitney test if nonparametric. Unless otherwise specified, data are depicted as the mean ± SD. A p value <0.05 was considered significant. Data were analyzed using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA) and/or StatView 5.0.1 (SAS Institute, Cary, NC).

HSP70 was found on human lung fibroblast surfaces by both confocal microscopy (Fig. 1A–1D) and flow cytometry (Fig. 1E). Quantification by flow cytometry showed fibroblasts isolated from IPF lungs more frequently expressed cell surface HSP70 and greater upregulation of this stress response molecule with serum starvation in comparison with normal lung fibroblasts (Fig. 1F).

Intracellular HSP70 was detected in all fibroblasts (both IPF and normal). The intensity of intracellular staining tended to be greater in the IPF fibroblasts, but it was not obviously increased by the serum starvation in either group (Supplemental Fig. 1). HSP70 mRNA expression was reflective of flow cytometry findings (Fig. 1G).

After confirming that HSP70 was expressed on lung fibroblasts, we were particularly interested in testing whether HSP70 autoantibodies found in patients with IPF affected the survival of these mesenchymal cells. The primary normal lung fibroblasts were subjected to staurosporine-induced apoptosis after treatment with HSP70 autoantibodies or control normal IgG. We found that treatment with IPF HSP70 autoantibodies rendered fibroblasts resistant to apoptosis induction by staurosporine at either 750 nM or 500 nM, as demonstrated by significant increases of live cells (by flow cytometry using an Annexin V-FITC staining kit; Fig. 2A), decreased intensity (by Western blots; Fig. 2B), or decreased activity (by a kit measuring cleaved caspsase-3 activity; Fig. 2C) of cleaved caspase-3.

In consideration of previous reports that Bcl-2 may be increased in IPF fibroblasts (4, 6), as well as other descriptions that HSP70 is involved in the regulation of this antiapoptotic molecule (23–25, 33), we examined the expression changes of Bcl-2 RNA and protein in these HSP70 autoantibody-treated cells.

Despite some heterogeneity with respect to time courses, all the primary lung fibroblasts that we examined had significant upregulation of cellular Bcl-2 mRNA (Fig. 3A) and protein levels (Figs. 3B, 3C) after treatment with Hsp70 autoantibodies from different patients with IPF, in contrast to IgG control treatments (additional samples in Supplemental Fig. 2A).

Because Bcl-2 has been shown to be associated with histone H4 lysine 16 acetylation (H4K16Ac) in pulmonary fibroblasts (6) and H4K16Ac is reportedly upregulated in some IPF patient fibroblasts (31), we evaluated the possibility that this process could also be involved in the antiapoptotic effects of IPF HSP70 autoantibodies. ChIP assays confirmed the enhanced association of Bcl-2 with this active histone mark in fibroblasts treated with HSP70 autoantibodies (Fig. 4A).

MOF (also known as KAT8 or MYSTI) is the main histone acetyltransferase that acetylates H4K16 (34), and MOF knockdown has been shown to significantly decrease H4K16Ac levels in lung fibroblasts (31). To further substantiate the role of H4K16Ac in the regulation of Bcl-2 responses to HSP70 autoantibodies, we evaluated effects of these autoantibodies in cells deficient in MOF (following MOF knockdown with siRNA; Fig. 4B and Supplemental Fig. 2B, 2C). Among cells that had Mof knockdown by siRNA, Bcl-2 was significantly reduced in the MOF siRNA fibroblasts incubated with HSP70 autoantibodies compared with control cells (Fig. 4B, 4C). In addition, ChIP assays of HSP70 autoantibody-treated lung fibroblasts that had Mof knockdown siRNA transfections showed reduced associations of Bcl-2 and H4K16Ac in the cells with siRNA MOF transfection compared with nontargeting controls (Fig. 4D), which corresponds to the reduced Bcl-2 mRNA expression in these cells (Fig. 4C). These data indicate that HSP70 autoantibody-induced Bcl-2 upregulation is mediated by increased Bcl-2 promoter binding with active histone H4K16Ac.

These data show that IgG autoantibodies with specificity for HSP70, isolated directly from the plasma of patients with IPF with severe clinical manifestations (10), significantly decrease the apoptosis susceptibility of human lung fibroblasts. IPF fibroblasts have previously been demonstrated to resist apoptosis, and this dysregulation is an important contributor to the pathogenic lung fibrosis that typifies this disease (2, 4, 5). Other findings of the present study show that the antiapoptotic effects of HSP70 autoantibodies are associated with increased Bcl-2, which, in turn, is mediated by epigenetic regulation. We are unaware of previous reports that describe autoantibody-induced alterations of target cell apoptosis susceptibility, and we do not know of prior examples in which deleterious functional effects of autoantibodies have been shown to be epigenetically mediated.

The immune responses to HSP70 in patients with IPF fulfill characteristics of Ag-specific autoimmunity (10). The frequency of HSP70-reactive profibrotic CD4 T cells is far greater in these patients with lung disease than in healthy individuals (10). HSP70 autoantibody responses in subjects with IPF are HLA biased, and their presence in this disease population is associated with more rapid pulmonary function deterioration and substantially greater 1-y mortality (10). Moreover, HSP70 autoantibodies are detected in 70% of the patients with IPF who are having or will soon have an acute exacerbation (10), which is a morbid and usually mortal manifestation of this lung disease (1, 11). Autoreactivity to various HSPs, and HSP70 in particular, has also been implicated in other immunological syndromes, including chronic obstructive pulmonary disease (14), malignancies (15), atherosclerosis (16), and various autoimmune disorders (17, 18, 35).

The processes that result in autoimmunity against ubiquitous HSPs, such as HSP70, are unknown. Because HSPs are highly conserved among widely divergent species, immune responses that develop against microbial HSP epitopes during infections may be capable of cross-reacting with host orthologues (18, 27). It has also been postulated that immune responses initially directed at other antigenic peptides could generalize to HSPs because of the close physical associations of these protein chaperones with the immunogen(s) (36). Autoreactivity can also develop against otherwise inert self-proteins that are pathologically overexpressed, particularly when proximate to inflammatory foci (37, 38). HSP70 expression is upregulated by a wide range of physiological and environmental insults such as oxidative stress and inflammation (20–22) and is significantly increased in the highly abnormal lungs of patients with IPF (10).

Despite the presence of HSP70 autoantibody in many human disease populations (14, 16, 17, 27, 35), the functional effects of these Igs have not been studied extensively. eHSP70 acts like a cytokine, binding to and stimulating a number of distinct cell surface receptors, many of which transduce signals via MAPK pathways (19–21, 33, 39–41). Multivalent anti-HSP70 Abs raised in animals are capable of cross-linking (oligomerizing) these eHSP70-receptor complexes and can thus augment many of the proinflammatory functions attributable to eHSP70 per se (20, 26).

In contrast, effects of the human anti-HSP70 autoantibodies found in numerous disease populations have been comparatively little studied. We previously showed that anti-HSP70 autoantibodies isolated from patients with IPF activated monocytes and increased their production of IL-8, a chemokine implicated in IPF progression (9). HSP70 autoantibodies in the cerebrospinal fluid of patients with multiple sclerosis can similarly augment monocyte proinflammatory cytokine production (35). Previously, little was known about direct effects of HSP70 autoantibodies on lung fibroblasts, and the mechanisms by which HSP70 autoantibodies could exert effects were even more cryptic. The present findings show that the HSP70 autoantibodies present in the circulation of patients with IPF can increase antiapoptotic Bcl-2 levels in pulmonary fibroblasts. Notably, Bcl-2 may not be the only apoptosis-related gene altered in response to HSP70 autoantibodies, but it is the most consistent alteration that we have observed in this study.

Previously, we have reported that Bcl-2 can be upregulated in response to oxidative stress by a process involving an association between Bcl-2 and the active histone mark H4K16Ac (6). Acetylation of lysine residues in the tail of nucleosome core histones is a major epigenetic mechanism involved in the regulation of responses to environmental stimuli (42). These modifications result in heritable changes of gene expression, although these changes are also potentially reversible. Acetylation of H4K16 most often promotes transcriptional activation (43).

We hypothesized that functional effects of the IPF HSP70 autoantibodies might be similarly mediated after observing increased Bcl-2 levels and decreased apoptosis susceptibility of autoantibody-treated fibroblasts. ChIP assays confirmed that treatment with IPF HSP70 autoantibodies enriched the association between Bcl-2 and H4K16Ac. Moreover, the critical involvement of H4K16Ac in this process was further substantiated by experiments that showed Bcl-2 upregulation was attenuated in HSP70 autoantibody-treated fibroblasts in which this histone mark had been decreased by siRNA MOF. These findings not only demonstrate a previously unknown functional effect of a disease-associated autoantibody but also add to nascent insights regarding the roles of epigenetic regulation in IPF (44).

This is the initial report describing a series of novel observations, to our knowledge, and additional studies will be necessary to elucidate details of other upstream and downstream mechanisms, as well as the possible effects of HSP70 autoantibodies on other tissues. As an example, other experiments not described in the present study indicate that anti-HSP70 autoantibodies may alter transcription of other apoptosis-related molecules, and some of those could also affect the survival of target cells. Abnormally high concentrations of anti-HSP70 autoantibodies are seen in a minority of stable patients with IPF (10), so it is implausible that these autoantibodies account for all of the fibroblast apoptosis dysregulation in this population. Nonetheless, these autoantibodies are associated with pulmonary function deterioration among individual patients with IPF and are present in the majority of those patients with fulminant disease manifestations (10). Accordingly, it seems possible that the development of anti-HSP70 humoral autoreactivity could increase fibroblast apoptosis resistance and could contribute to or even provoke other pathogenic processes (10, 20, 45) that result in IPF progression (11). Furthermore, we do not yet know all the steps that occur between autoantibody binding and histone acetylation, although analogous studies of eHSP70 mechanisms (21, 23, 33, 39, 41, 46) will likely serve as models for those experiments. Given the ubiquitous distribution of HSP70, several other cell types, in addition to fibroblasts and monocytes (10, 26, 35), may also undergo important functional alterations with anti-HSP70 autoantibody exposure, and these studies are ongoing, too.

The findings in the present study illustrate a previously unrecognized mechanism of autoimmune pathogenesis with particular relevance for IPF that may also have broader implications to other cell types and patient populations (14, 16, 18). Better understanding of how disease-associated autoantibodies, such as anti-HSP70 autoantibodies, promote pathogenesis could ultimately lead to better, more relevant diagnostic assays, as well as provide rationales for novel interventions that could include autoantibody reduction treatments (11), inhibition of Bcl-2 or other antiapoptotic mediators, or reversal of acquired histone modifications (6, 43, 47).

The authors have no financial conflicts of interest.

We thank Dr. Thi K. Tran-Nguyen for her technical support for this project.