The significance of autoantibodies to DFS70/LEDGFp75 in health and disease: integrating basic science with clinical understanding

DFS70/LEDGFp75 and its short splice variant LEDGF/p52 (hereafter referred to as p52) (Fig. 2a) are derived from the same PS1P1/LEDGF gene, which consists of 15 exons and 14 introns, with exons 1–15 encoding DFS70/LEDGFp75, and exons 1–9 and a small part of intron 9 (24 nucleotides) encoding p52 [30]. Although other alternatively spliced variants of this gene have been identified [31], DFS70/LEDGFp75 and p52 are the most common based on immunoblotting analysis of cell lysates (Fig. 2b) [32‐34]. DFS70/LEDGFp75 and p52 share an amino (N)-terminal region (residues 1–325); however, p52 has an intron-derived C-terminal tail (CTT, residues 326–333) that is not present in DFS70/LEDGFp75 (Fig. 2a). These variants localize to different nuclear regions and appear to play opposing roles when ectopically overexpressed, with DFS70/LEDGFp75 acting as a stress survival protein and p52 as a pro-apoptotic protein [33, 35]. P52 has been particularly implicated in splicing regulation through binding to trimethylated histone H3K36me3 and splicing factor SRSF1, and in the regulation of neurite growth in rat retinal ganglion cells [36‐39].

The N-terminal region shared by DFS70/LEDGFp75 and p52 contains a PWWP domain (Fig. 2a), defined by a proline-tryptophan-tryptophan-proline motif (residues 19–22). PWWP domains are highly conserved in members of the hepatoma-derived growth factor (HDGF) family and in several DNA-binding proteins and have been implicated in chromatin binding, HIV-integration, protein–protein interactions, transcription, and DNA methylation [40‐43]. This domain facilitates the dynamic scanning and hopping of DFS70/LEDGFp75 along the chromatin, and the locking into chromatin of interacting proteins that are bound to its C-terminus [44]. Binding of this domain to chromatin is facilitated by its interaction with H3K36me3 [45]. Other sequences such as positively charged regions, a nuclear localization signal and AT-hook motifs (Fig. 2a), also contribute to DFS70/LEDGFp75 binding to chromatin, particularly to H3K4me3 at active transcription sites [46‐50].

Both the N- and C-terminal regions of DFS70/LEDGFp75 contribute to its transcription and stress survival functions by engaging in interactions with chromatin-binding proteins, or by binding to promoters of specific stress genes [33, 42, 48, 51‐56]. Chromosomal translocations in leukemia produce a NUP98-LEDGFp75 fusion protein lacking the N-terminal region of DFS70/LEDGFp75, resulting in a PWWP-deficient protein with deregulated transcription functions [56‐60]. The C-terminal region of DFS70/LEDGFp75 (residues 325–530), absent in p52, contains two helix-turn-helix domains (residues 421–442 and 471–492) that are capable of binding heat shock elements within promoter regions of stress genes [48]. The C-terminal region of DFS70/LEDGFp75 also encompasses the HIV integrase-binding domain (IBD, residues 347–429), which is recognized by the HIV-1 integrase (HIV-IN) [17‐20, 61].

The C-terminus of DFS70/LEDGFp75 contains the autoepitope (aa 347–429) recognized by the autoantibodies [62], which explains why these consistently recognize a single band of 70–75 kD and not the p52 variant in immunoblots of cell lysates (Fig. 1b). Intriguingly, this immunogenic region is essentially the same region comprised by the IBD [18, 61, 63] and shares significant homology with HRP-2 (HDGF-related protein 2), a member of the HDGF protein family that can also interact with HIV-IN [64].

The biological significance of these coincidental findings is not presently clear and raises interesting questions. Why would an epitope region targeted by autoantibodies correspond to the exact same region specifically recognized by the HIV-IN? What structural or functional elements within this region make it attractive for targeting by both the immune system and the HIV-1 virus? While at the present time we lack sufficient information to answer these questions, it is evident that the autoepitope/IBD region has intrinsically important cellular functions. These include, in addition to HIV-IN binding, serving as a hub for protein–protein interactions in the chromatin, regulation of gene expression, and stress survival activity. It should be noted that DFS70/LEDGFp75 is predicted to be a highly disordered protein, a feature of proteins that have multiple interacting partners [61]. One could speculate that the largely disordered nature of DFS70/LEDGFp75 and the promiscuity of its autoepitope/IBD domain in interacting with multiple proteins (both self and non-self), may influence its proteolytic processing and presentation to the immune system. This could contribute to enhanced immunogenicity under pro-inflammatory conditions, leading to autoantibody generation in susceptible individuals. The autoimmune targeting of the IBD is consistent with the notion that autoantibody-defined epitopes typically comprise highly conserved, conformational, and functional domains [1, 2].

Compelling evidence supports a cellular protective function for DFS70/LEDGFp75 against environmental factors that induce cellular stress, such as ultraviolet B (UVB) irradiation, hydrogen peroxide, alcohol, hyperthermia, nutrient deprivation, and certain chemotherapeutic drugs [14, 31, 32, 34, 51, 67‐73]. These stressors can lead to increased oxidative stress, which induces upregulation and activation of DFS70/LEDGFp75 [73].

DFS70/LEDGFp75 is presumed to promote cellular protection against environmental stressors by transcriptionally activating stress, antioxidant, and other protective genes [51, 74‐82]. However, to date only a few target genes of DFS70/LEDGFp75 have been identified and validated (Table 2). While global gene profiling studies on cells stably depleted of DFS70/LEDGFp75 failed to reveal a specific genetic pathway regulated by this protein [12], studies using pathway-specific gene arrays showed that ectopic overexpression or transient depletion of this protein in cancer cells under stress led to significant changes in the expression of certain stress and antioxidant genes [82]. These findings suggested that DFS70/LEDGFp75 contributes to the regulation of stress gene expression mainly when it is upregulated and activated under stress.

Both the PWWP domain and the C-terminal IBD region of DFS70/LEDGFp75 interact with various chromatin-associated proteins, likely facilitating DFS70/LEDGFp75 function in stress gene expression regulation. Interactors of the IBD in addition to HIV-IN include the pogo transposable element PogZ, the c-Myc interacting protein JPO2, the Cdc7-activator of S-phase kinase (ASK), and the leukemia-associated transcription complex Menin-MLL (mixed lineage leukemia) [52‐56, 83]. The PWWP domain has been implicated in binding to the methylation-associated protein MeCP2, transcription coactivator TOX4, and splicing cofactor NOVA1 [42, 84]. DFS70/LEDGFp75 also participates in the recruitment of polycomb group protein Bmi1 and co-repressor Ctbp1 to MLL complexes in HOX gene promoters [85].

Various groups have reported that depletion or functional inactivation of DFS70/LEDGFp75 leads to decreased cell survival [15, 32, 33, 57, 72, 86, 87]. However, these results are controversial because others have reported that this protein is not essential for cell survival based on observations that cell clones with stable depletion of the protein can survive in culture [88, 89]. In addition, a PSIP1/LEDGF ^−/− knockout mouse model revealed that disruption of this gene is not intrinsically lethal to mice [16]. While these conflicting observations are likely to be cell type- and context-dependent, it is possible that selected cell clones with stable depletion of DFS70/LEDGFp75 may have developed compensatory mechanisms to survive in the absence of this protein. This is supported by the observation that prostate cancer (PCa) cell clones with stable DFS70/LEDGFp75 depletion did not display significant changes in stress gene expression when compared to stressed cells with transient depletion [82].

The possibility that DFS70/LEDGFp75 is needed mainly in the context of stress survival was suggested by the observation that deletion mutants of the protein lacking portions of its N- and C-terminal domains did not show any effects on cell death or survival when stably overexpressed in cancer cells growing under normal conditions [32]. However, unlike the full-length protein, these mutants were unable to support cell survival under starvation stress [32].

Transactivation of protective genes by DFS70/LEDGFp75 under cellular stress is likely to contribute to preservation of the structural integrity of critical organelles that are highly susceptible to oxidative damage and that regulate cell death and survival. Consistent with this, DFS70/LEDGFp75 was shown to protect cancer cells against antitumor drugs that induce lysosomal membrane permeabilization (LMP) and cell death [34, 72]. PCa cell lines selected in culture for natural resistance to docetaxel, an antitumor drug that induces LMP and is antagonized by DFS70/LEDGFp75, express high levels of this autoantigen, consistent with the possibility that chemotherapeutic stress induces its expression [31, 34, 82].

DFS70/LEDGFp75 has also been implicated in cellular protection against oxidative DNA damage. It enhanced the survival of retinal pigment epithelial cells challenged by oxidative stress or UVB irradiation [70], a survival effect associated with DFS70/LEDGFp75-mediated protection of DNA from single-strand breakage and upregulation of Hsp27. This is consistent with studies showing that DFS70/LEDGFp75 promotes repair of DNA double-strand breaks through the homologous recombination repair pathway [90].

Increased cellular expression of the Sp1 transcription factor leads to upregulation of DFS70/LEDGFp75 via TATA-less promoter activation, while its inhibition represses this upregulation [91, 92]. However, during LEC exposure to UVB, a histone deacetylase/histone methylase (HDAC1/SUV39H1) complex is recruited to Sp1 responsive elements in the DFS70/LEDGFp75 gene promoter, leading to attenuation of Sp1 binding, repression of DFS70/LEDGFp75 expression, and increased cellular oxidative stress and death [93]. These results suggest that certain stressors may either upregulate or repress DFS70/LEDGFp75 depending on context.

Transforming growth factor beta 1 (TGF-β1) is also known to downregulate DFS70/LEDGFp75 in LECs by binding to its promoter region [77]. This is consistent with the observations that a Prdx6 ^−/− knockout mouse cell line displayed increased TGF-β1 levels with reduced DFS70/LEDGFp75 [94] and that an inverse expression relationship between these genes exists in diabetic and galactosemic cataractous rat lenses [95].

There is also evidence that DFS70/LEDGFp75 is regulated at the transcriptional level by micro-RNAs (miRNAs). Macrophages stimulated with lipopolysaccharide induced miR-155, concomitant with downregulation of DFS70/LEDGFp75, and ectopic expression of this miRNA reduced DFS70/LEDGFp75 expression at the transcriptional level [96]. Another miRNA, miR-135b, also downregulated DFS70/LEDGFp75 both in human cell lines and in murine vestibular sensory epithelia of the inner ear, and it was suggested that this downregulation could influence inner ear cell survival, protection against stress, development, and differentiation [97].

Differential expression of DFS70/LEDGFp75 and its short splice variant p52 was observed in a panel of cancer cell lines, with DFS70/LEDGFp75 expressed at higher levels [32‐34]. Interestingly, ectopic overexpression of p52 induced decreased cell survival via caspase-dependent apoptosis associated with DFS70/LEDGFp75 cleavage [33]. During apoptosis, caspase-3 cleaves DFS70/LEDGFp75 to generate several fragments [32, 33, 98, 99]. As shown in Fig. 3, treatment of Jurkat T cells for 6 h with staurosporine (STS) induces the classical apoptosis morphology (Fig. 3a), which is associated with cleavage of DFS70/LEDGFp75 into fragments of 68, 65, and 58 kD that are recognized by the autoantibodies in immunoblots of whole-cell lysates (Fig. 3b). Consistent with this, autoantibody recognition of DFS70/LEDGFp75 in apoptotic blebs can be detected by IIF microscopy (Fig. 3c). These fragments are produced by caspase-3-mediated sequential cleavage of the protein at specific aspartic acid residues located in the N-terminal PWWP domain and the C-terminal region (Fig. 3d). This apoptotic cleavage impairs DFS70/LEDGFp75’s stress survival activity and generates fragments that enhance cell death under stress [32]. An interesting observation was that during apoptosis, p52 is also cleaved by caspase-3 to generate a p38 fragment that antagonizes the transcriptional function of DFS70/LEDGFp75 [33].

The pro-survival protein Bcl-2 was also shown to attenuate DFS70/LEDGFp75 stress survival and transcriptional activities in LECs by interfering with its binding to the promoter region of the gene encoding αB crystallin [100]. It is plausible that these pro-survival proteins antagonize different cell death pathways, prompting cells to tightly regulate their expression.

DFS70/LEDGFp75 is post-translationally sumoylated at different sites in its N-terminal and C-terminal regions [101]. While sumoylation does not affect its cellular localization and chromatin-binding ability, it provides a mechanism to regulate its transcriptional activity, as suggested by the observation that mutations of DFS70/LEDGFp75 sumoylated sites extended its half-life and increased its transcriptional activity [101]. Consistent with these observations, LECs expressing sumoylation-deficient DFS70/LEDGFp75 displayed increased transcriptional and cellular survival activities compared to wild-type protein [102]. These results suggested that sumoylation of this autoantigen is a mechanism to regulate stress responses.

Overexpression of DFS70/LEDGFp75 has been shown to induce the IL-6/STAT3 signaling pathway in HaCaT skin cells, whereas its knockdown reduced IL-6 levels [103, 104]. Interestingly, the intracellular localization of both DFS70/LEDGFp75 and phosphorylated STAT3 in HaCaT cells appears to be regulated by Ran-binding proteins, which suggested that a similar mechanism may be operating in psoriatic keratinocytes [105]. Additional evidence supporting a link between DFS70/LEDGFp75 and STAT3 came from studies demonstrating that switching the expression of STAT3 to STAT3β, its dominant negative truncated variant, in cancer cells led to DFS70/LEDGFp75 repression [106]. These observations suggested that DFS70/LEDGFp75 is activated by STAT3 in an autocrine/paracrine loop and raised the interesting possibility that a regulatory crosstalk between this autoantigen and the IL-6/STAT3 pathway may contribute to inflammatory processes [106]. Consistent with a link between DFS70/LEDGFp75 activation and inflammation, Takeichi et al. [104] also observed that DFS70/LEDGFp75 stimulated release of the cytokines TNF and IL-8 from keratinocytes. Interestingly, treatment of LECs with sublethal doses of TNF resulted in induction of oxidative stress and elevated expression of DFS70/LEDGFp75, which in turn transactivated the protective protein gamma glutamylcysteine synthetase [71].

Ochs et al. [3, 4] investigated the presence of ANAs in sera of IC patients, especially noting whether or not their specificities were unique or similar to SARD-related ANAs. Among the ANA patterns observed in sera from 96 patients, there was a predominance of the nuclear DFS-IIF pattern (69 % of all ANA-positive patients and 9 % of the total cohort). Immunoblotting analysis confirmed that these DFS-positive sera contained autoantibodies to DFS70/LEDGFp75 [3]. A later study found only a 5 % frequency of these antibodies in IC patients when detected by the Quanta Flash DFS70 chemiluminescence assay (DFS70-CIA) [107].

Ochs et al. [3] also observed that sera from 28 to 16 % patients with atopic dermatitis (AD) and asthma, respectively, produced the DFS-IIF pattern. Immunoblotting with recombinant DFS70/LEDGFp75 confirmed the presence of IgG and IgE autoantibodies to this protein. Other disease cohorts tested, including SARD, revealed low frequency (<5 %) of these antibodies, suggesting that this autoantibody-autoantigen system is not associated with SARD [3].

The presence of anti-DFS70/LEDGFp75 autoantibodies in AD was confirmed by the observation that some AD patients producing these autoantibodies also had cataracts [108]. In addition, these antibodies (both IgE and IgG4) were detected at a prevalence of 15 % in AD patients, and their presence correlated with elevated thymus and activation-regulated chemokine, which is associated with increased AD severity [23]. Immunohistochemical (IHC) analysis showed that DFS70/LEDGFp75 was present in epidermal cells and infiltrating monocytes in the skin of AD patients, suggesting that DFS70/LEDGFp75 upregulation or release from damaged tissue or invading cells may trigger autoantibody responses [23]. It should be noted, however, that the elevated prevalence of these autoantibodies in AD could not be confirmed using DFS70-CIA, highlighting the inter-laboratory and inter-diagnostic platform differences in the detection of these autoantibodies [107]. Differences in cohort collection and composition (age, gender, race) may also explain these discrepancies.

DFS70/LEDGFp75 is predominantly located in the nucleus of basal epidermal cells and then translocates into the cytoplasm during differentiation, where it accumulates in the granular layer of keratohyalin granules, which are important for proper keratinocyte apoptosis [109]. It was hypothesized that anti-DFS70/LEDGFp75 autoantibodies may affect its pro-survival function and contribute to the development of skin disease [109]. However, this would be plausible only in the context of extracellular release of DFS70/LEDGFp75.

Takeichi et al. [103] observed that DFS70/LEDGFp75, in addition to inducing the IL-6/STAT-3 pathway in cultured skin cells, localized to the nucleus of keratinocytes in psoriatic lesions, which suggested a pivotal role for this autoantigen in protecting psoriatic keratinocytes under a stressful microenvironment. They suggested that downregulation of DFS70/LEDGFp75 may mitigate psoriasis symptoms by attenuating keratinocyte proliferation in psoriatic lesions.

Anti-DFS70/LEDGFp75 autoantibodies have also been found in 19.8 % of patients with alopecia areata (AA), an inflammatory skin condition that has autoimmune underpinnings, compared to 7.6 % of HI controls [21]. IHC analysis revealed that DFS70/LEDGFp75 localized to the outer root sheath cells of the hair follicle, the area that is targeted by the immune response in AA patients, suggesting that anti-DFS70/LEDGFp75 antibodies may contribute to AA pathophysiology [21].

Anti-DFS70/LEDGFp75 antibodies have been detected in diverse eye diseases [9, 22, 25, 110]. Ayaki et al. [108, 111] reported that these antibodies induce cytotoxicity in LECs, suggesting a pathogenic role. They proposed that the antibodies absorb extracellularly released DFS70/LEDGFp75, preventing its re-entry into LECs where it acts as a pro-survival factor. This is consistent with previous observations that DFS70/LEDGFp75 is secreted by LECs and that its absorption by autoantibodies added to the culture medium reduced cell survival under stress [15].

Bizzaro et al. [22] reported that over 20 % of human sera showing the DFS-IIF pattern in HEp-2 cells also reacted strongly against reticular fibers of the lens and the corneal epithelium. These DFS-positive sera, however, produced different distribution patterns, suggesting the presence of companion autoantibodies targeting interacting ligands of DFS70/LEDGFp75. These authors noted that it is not easy to recognize the DFS pattern by IIF microscopy and therefore this method should not be used alone as the preferred technology to measure anti-DFS70/LEDGFp75 antibodies [22, 112].

Autoantibodies to DFS70/LEDGFp75 have also been reported in Vogt–Koyanagi–Harada (VKH) disease, an inflammatory disorder affecting multiple organs containing melanocytes, including uvea, skin, central nervous system, and inner ears [25]. Their presence was confirmed by ELISA in 67 % of VKH patients and in other patients with panuveitis, including sympathetic opthalmia, Behcet’s disease, and sarcoidosis (Table 3). Notably, these antibodies were also detected in 22 % of HI, which suggested the influence of background reactivity or selection of a low cutoff value in the ELISA.

Chin et al. [110] identified high-titer anti-DFS70/LEDGFp75 autoantibodies in three of six patients with atypical retinal degeneration. IHC studies using these autoantibodies demonstrated the presence of DFS70/LEDGFp75 in nuclei from murine retinal ganglion and pigment epithelial cells [110]. Consistent with this, DFS70/LEDGFp75 protected retinal pigment epithelial cells from nuclear damage induced by rhodopsin, a protein that forms nuclear aggregates causing cell death and retinal degeneration [113, 114].

A low frequency of anti-DFS70/LEDGFp75 autoantibodies (3.3 %) was reported in adult patients with CFS [3]. However, other studies reported an elevated presence of these autoantibodies in children with CFS but not in children with fibromyalgia (FM) [115, 116]. These findings are intriguing and should be confirmed in large cohorts of adults and children with CFS and FM diagnoses. Children with other non-autoimmune conditions may also produce these autoantibodies, as highlighted by a recent case report of an 8-year-old patient with respiratory distress who presented high-titer anti-DFS70/LEDGFp75 autoantibodies with no evidence of SARD [117]. The authors concluded that these antibodies were a useful biomarker to rule out suspected autoimmune disease in that particular case [117]. It should be noted, however, that ANA prevalence, specificity, and titers may change during puberty, which could explain the observed differences in autoantibody frequencies between children and adults [118].

During a screening of sera from patients with PCa for the presence of autoantibodies to tumor associated antigens, Daniels et al. [99] observed that the DFS-IIF pattern was predominant in sera from PCa patients compared to matched controls. Immunoblotting analysis of these DFS-IIF-positive sera, using PCa cell lysates as substrates, revealed that the majority reacted with a 70-kD protein band, and ELISA showed that 18.4 % of PCa sera reacted with this protein, compared to 5.5 % of controls. Overall, 22.3 % of the PCa sera reacted with DFS70/LEDGFp75 either by ELISA or immunoblotting, compared to 6.7 % of matched controls. Interestingly, the authors observed an incomplete correlation in the detection of these antibodies between the different immunoassays, which was attributed to differences in sensitivity and antigen conformation in the individual assays [99]. More recently, other groups have independently confirmed the presence of DFS70/LEDGFp75 autoantibodies in PCa sera [119‐121]. These findings led to the hypothesis that DFS70/LEDGFp75 could be aberrantly expressed and functionally hyperactive in PCa and perhaps other human cancers [99]. Numerous studies have confirmed this hypothesis by showing altered DFS70/LEDGFp75 expression and function in various human cancer cell and tumor types, linking it to tumor aggressive properties [31, 34, 56‐60, 72, 80‐82, 86, 87, 90, 99, 122].

It should be noted, however, that other studies have reported low frequency of autoantibodies to DFS70/LEDGFp75 in cancer patients [22, 107]. It is not clear whether autoantibodies to DFS70/LEDGFp75 are more prevalent in PCa patients than in patients with other cancers. Therefore, it would be important to determine the frequency of these autoantibodies, using several detection platforms, in large cohorts of ethnically diverse patients with different cancer types as well as individuals at high risk of developing cancer.

The initial study on the clinical significance of these autoantibodies revealed a relatively low frequency (2–4 %) of these antibodies in patients with SARD [3]. This observation was later reproduced in more comprehensive studies performed by several other groups. For instance, Dellavance et al. [7] reported that 30 % of ANA-positive sera in a cohort of over 13,000 patients presented the DFS-IIF pattern, with IgG titers ranging from 1:80 to over 1:640. This was by far the overwhelming type of ANA-IIF pattern detected in this large unbiased sample cohort. Clinical information obtained for 81 of the DFS-IIF-positive serum donors indicated a diverse spectrum of disease conditions that included organ-based autoimmune diseases and inflammatory conditions. A key conclusion of this study was that anti-DFS70/LEDGFp75 autoantibodies are a relatively common finding among ANA-positive individuals with no evidence of SARD [7].

Muro and colleagues examined 500 SARD sera for the presence of anti-DFS70/LEDGFp75 antibodies, as well as for SARD-associated marker autoantibodies [28]. They found low frequencies of these autoantibodies and observed that 86 % of the SARD patients positive for anti-DFS70/LEDGFp75 autoantibodies also had at least one SARD-marker autoantibody. These authors concluded that patients with SARD producing anti-DFS70/LEDGFp75 antibodies as the only serum ANA-IIF pattern are rare and that such antibodies could be used as exclusion biomarkers of SARD in ANA-positive individuals.

Low prevalence (6.4 %) of these antibodies, detected by ELISA and immunoblotting, were also reported in a cohort of 103 Japanese patients with dermatomyositis (DM) [123]. Most patients producing these antibodies also produced DM-specific autoantibodies, including antibodies to MDA5, which are used as serological markers for aggressive disease, particularly complications with interstitial lung disease (ILD) [123, 124]. An interesting observation was that three DM-ILD patients producing both anti-DFS70/LEDGFp75 and anti-MDA5 antibodies who went into remission after therapy had decreased levels of anti-MDA5 autoantibodies concomitant with increased levels of anti-DFS70/LEDGFp75 antibodies [123]. However, a fourth patient with DM-ILD who produced both antibodies and succumbed to the disease showed unchanged levels of anti-MDA5 autoantibodies concomitant with decreased levels of anti-DFS70/LEDGFp75 antibodies. These observations raised the intriguing hypothesis, which needs to be further investigated in a larger patient cohort, that anti-DFS70/LEDGFp75 antibodies may serve a protective role [123]. However, it cannot be ruled out that these autoantibodies might be acting as sensors of DFS70/LEDGFp75 upregulation in response to the systemic stress produced by therapy in the surviving patients. To explore this possibility, it would be important to compare the circulating levels or diseased tissue expression of DFS70/LEDGFp75 in large cohorts of therapy-responding versus non-responding DM-ILD patients producing these antibodies.

Watanabe et al. [125] screened sera from 597 self-reported healthy hospital workers for the presence of ANAs and observed that 54 % of all ANA-positive individuals had anti-DFS70/LEDGFp75 antibodies. This led to the speculation that these antibodies may be naturally occurring in both apparently HI and diseased individuals. It should be cautioned, however, that hospital personnel tends to present higher ANA levels than blood donors or relatives of SARD patients [126].

Later studies confirmed that anti-DFS70/LEDGFp75 antibodies are more prevalent in apparently HI than in patients with SARD. For instance, Mariz et al. [26] screened 918 HI (negative history of SARD, infections, and inflammatory conditions) and 153 SARD patients for the prevalence of ANAs, as detected by HEp-2 IIF. The DFS-IIF and the nuclear fine speckled (unrelated to DFS70/LEDGFp75) patterns were the most frequent (approximately 33 and 46 %, respectively) in ANA-positive HI. Another similar HEp-2-IIF pattern characterized by fine grainy nuclear staining with staining of metaphase chromosomes, designated as quasi-homogeneous pattern, was observed in 4 % of the ANA-positive HI [26]. Confirmation that the DFS-IIF pattern was associated with antibodies to DFS70/LEDGFp75 was obtained by immunoblotting, whereas sera positive for the nuclear fine speckled and quasi-homogeneous patterns did not react with the protein. Interestingly, antibody titers reached 1:640 and 1:1280 in 50 % of the DFS-IIF-positive sera, with titers >1:5,120 in three individuals. Follow-up studies revealed that the presence and titers of anti-DFS70/LEDGFp75 antibodies were stable over the years and that the positive HI did not subsequently develop SARD or any evident disease [26]. This is in contrast to the known predictive value of disease marker ANAs for SARD diagnosis [127]. Mariz et al. [26] pointed to the difficulty of distinguishing the nuclear fine speckled and the quasi-homogeneous nuclear patterns from the DFS-IIF pattern in HEp-2 substrates, even by trained laboratory personnel, and recommended expanding efforts to address the reproducibility of ANA-HEp-2 test interpretations among different experts and commercial brands.

Mahler et al. [107] reported a prevalence of anti-DFS70/LEDGFp75 antibodies of 8.9 % (determined by DFS70-CIA) in a cohort of 124 serum samples from clinically defined HI with no history of SARD. This prevalence was significantly higher than in patients with SARD and non-SARD diseases, which exhibited prevalences below 6 %. These authors noted that in an SLE cohort there were no clinical differences between the few patients with anti-DFS70/LEDGFp75 antibodies and the patients without these antibodies, suggesting that the antibodies are not protective and do not correlate with disease activity. They also observed that in the SLE cohort all but one of the patients with anti-DFS70/LEDGFp75 antibodies also had other classical SLE-associated autoantibodies [107]. These findings reinforced the notion that anti-DFS70/LEDGFp75 antibodies are more prevalent in HI than in SARD patients. However, given that a small proportion (2–3 %) of SARD patients in this and other studies also produced these antibodies [3, 28], it cannot be asserted conclusively that these antibodies are highly accurate biomarkers for SARD exclusion, unless they are the only ANA specificity detected in the sera.

Studies with large cohorts of well-defined SARD patients are necessary to determine whether the presence of anti-DFS70/LEDGFp75 autoantibodies in these patients is coincidental or associated with a specific clinical phenotype or therapy. Along these lines, a novel immunoadsorption technology has been developed to increase the specificity of the ANA-HEp-2 cell assay [5]. Using recombinant DFS70/LEDGFp75 in the dilution buffer, anti-DFS70/LEDGFp75 antibodies are prevented from binding their target in HEp-2 cells [5, 6]. This then reveals the clinically relevant IIF pattern in sera with concomitant anti-DFS70/LEDGFp75 and other SARD-marker autoantibodies.

Mahler et al. [107] screened 3,263 serum samples submitted for ANA testing for the presence of anti-DFS70/LEDGFp75 autoantibodies and observed that 1.62 % presented the DFS-IIF pattern, which was confirmed to correspond to anti-DFS70/LEDGFp75 autoantibodies when evaluated by DFS70-specific ELISA and CIA. Bizzaro et al. [22] also observed low frequency (0.8 %) of sera displaying the DFS-IIF pattern in HEp-2 cells in 21,512 samples screened for ANA in the clinical laboratory. Two additional studies also reported low frequencies (<4 %) of this pattern in thousands of sera screened for ANAs [128, 129].

Miyara et al. [24] evaluated the clinical value of anti-DFS70/LEDGFp75 autoantibodies in patients undergoing routine ANA testing. Analysis of sera from 100 consecutive patients with DFS-IIF pattern and 100 patients with other patterns, using the ANA-HEp-2 test, DFS70 CIA, and QUANTA Lite ANA Screen ELISA (which simultaneously detects serum autoantibodies to common SARD-related autoantigens), revealed that only 5.5 % of patients with anti-DFS70/LEDGFp75 antibodies had SARD. Most of the anti-DFS70/LEDGFp75 antibody-positive samples were negative on the ANA Screen ELISA. When combining a negative ANA ELISA result with a positive anti-DFS70/LEDGFp75 antibody test result, good discrimination between SARD and non-SARD patients was obtained [24], strengthening the notion that when found as the only ANA-IIF specificity in patient serum this antibody could serve as a reliable exclusion marker of SARD.

Fitch-Rogalsky et al. [130] analyzed the clinical and serological features of patients referred through a rheumatology central triage system because of a positive ANA test. Of 15,357 referred patients, 4.1 % had positive ANA. The frequency of the anti-DFS70/LEDGFp75 autoantibody in 225 archived sera from the patients evaluated by a rheumatologist was 15.1 %, and this was the sole autoantibody in 70.6 % of the anti-DFS70/LEDGFp75-positive patient sub-cohort. Among the anti-DFS70/LEDGFp75-positive patients, 6 % had SARD with other autoantibodies. This reinforced the notion that when these autoantibodies are present in patients with SARD they usually coexist with other disease marker autoantibodies and tend to exclude a SARD diagnosis when they are the sole ANA-IIF specificity in human sera. The detection of anti-DFS70/LEDGFp75 antibodies is now used in this triage system to help prioritize patients for referral and thereby reducing waiting times for urgent cases.

Natural autoantibodies, both IgM and IgG, play a critical, protective role by assisting in the clearance or neutralization of apoptotic cell debris, which is essential to prevent the release of intracellular self-antigens and danger signals that could induce inflammatory and autoimmune responses [137‐140]. To date there is little objective and formal evidence that anti-DFS70/LEDGFp75 autoantibodies are natural antibodies playing protective roles. While their low frequency in SARD, presence in 5–10 % of HI who do not develop autoimmune conditions after years of follow-up, and increased levels in DM-ILD patients who went into remission after therapy, suggest the possibility that they could play a protective role, further studies are warranted to support this role. These autoantibodies might function in the removal of DFS70/LEDGFp75 cleavage fragments from debris generated during cell death associated with tissue damage. This would not only attenuate local inflammatory responses, but also prevent these fragments from enhancing cell death [32].

The only evidence that these autoantibodies could play pathogenic roles comes from the studies by Ayaki et al. [108, 111] reporting their cytotoxicity in vitro against LEC and cultured lens organs. In this context, when upregulated and activated by stress, DFS70/LEDGFp75 could be released into the extracellular milieu and uptaken by cells in the local tissue microenvironment where it may transcriptionally activate stress response and pro-inflammatory pathways. Ayaki et al. [108, 111] suggested that binding of the autoantibodies to released DFS70/LEDGFp75 exerts a pathogenic role by preventing its uptake by neighboring cells.

The broad spectrum of diseases and conditions associated with the presence of anti-DFS70/LEDGFp75 autoantibodies (Table 3) points to an augmented state of cellular oxidative stress, local inflammation, and tissue damage (i.e., bladder, eye, skin, prostate), as potential common denominators. Dying cells, which in vivo can be derived from tissue damage, are a source of intracellular autoantigens that are clustered in apoptotic blebs or post-translationally modified [141‐143]. Defects in the clearance of dying cells in certain autoimmune diseases or inflammatory conditions, associated with inflammatory necrosis or progression of apoptosis to secondary necrosis, could lead to a pro-inflammatory environment, thus facilitating autoantibody responses to aberrantly modified autoantigens [144]. Primary and secondary necrosis, and necroptosis, also yield unique autoantigen cleavage fragments, generated by lysosomal cathepsins that are recognized by autoantibodies [98, 145, 146].

As mentioned previously, DFS70/LEDGFp75 is cleaved during apoptosis into fragments that are recognized by human autoantibodies and that persist during secondary necrosis [32, 33, 98, 99]. Its overexpression in disease-affected tissues, combined with its proteolytic cleavage or involvement in stress-induced protein complexes that influence its processing by the immune system, may alter its immunogenicity in a pro-inflammatory microenvironment, making it a target of autoantibodies (Fig. 4). There is evidence that tissue overexpression, mutation, or posttranslational modification of intracellular autoantigens in a pro-inflammatory context may trigger the elicitation of autoantibodies [143, 145‐147]. It is then plausible that autoantibodies to DFS70/LEDGFp75 could then be considered as “sensors” of microenvironmental stressors associated with inflammation, tissue damage, and altered expression of this protein.