Since their recent discovery, NETs have been the focus of considerable study examining their roles in innate immunity, and in particular, assessing several putative links to autoimmunity. Histone proteins which are a significant component of NETs, have long been the subject of intense study as they comprise a major class of autoantigens in SLE [52
] and are richly decorated with PTMs that dynamically encode epigenetic information in chromatin [53
]. However, few studies have characterized the post-translational state of histones within NETs or examined their association with autoimmunity. We tested the hypothesis that NETs and histone PTMs have the capacity to induce autoantibodies that target histones with a focus on SLE.
First, we asked whether histone PTM-specific reactivity could be identified and characterized in SLE, in order to serve as a basis of comparison for any histone PTMs identified in NETs. In comprehensive autoantibody profiling of a well-characterized cohort of patients with SLE within the ABCoN on human epigenome microarrays, we confirmed serum IgG reactivity to acetyl-histone H2B peptides in concordance with previous work [28
]. We also observed statistically significant IgM reactivity to multiple H3 and H4 PTM epitopes as well as widespread serum IgM reactivity to methyl-H3 PTM epitopes. One possible explanation is that endogenous histone PTMs may induce a low-level autoantibody response that is present in both healthy and SLE patients and that additional pro-inflammatory signals and T cells help are required to induce autoreactive B cells to affinity maturation and isotype switching to IgG [54
]. Surprisingly, serum reactivity to citrullinated epitopes was observed at only low levels for both IgM and IgG. The biological and clinical significance of these findings will require additional and ongoing studies.
To ascertain whether NETs contain SLE serum-reactive PTM antigens, we characterized human and murine-derived NETs using a broad panel of unique, commercially available antibodies recognizing specific histone PTMs. We observed that histones within NETs harbored most of the examined methylation marks, including mono-, di-, and tri-methyl H3 at K4, K9, K27, K36 and H4 at K20. Separately, we identified major trends in the pattern of other histone PTMs enriched in NETs derived from activation of neutrophils using diverse stimuli, including marks associated with transcriptional repression as well as hypercitrullination. This suggests that examining the PTM state of NET chromatin may yield insights into the underlying mechanisms responsible for the profound changes to the chromatin during the process of NETosis.
The hypercitrullination that we observed during NETosis in primary human PMNs and EPRO cells stimulated with diverse stimuli confirms an earlier report of citrullination in HL-60 cell-derived NETs [55
]. Consistent with this finding, we observed a corresponding decrease in arginine methylation during NETosis of human and mouse neutrophils, likely reflecting conversion of arginine residues to citrulline. Other, more subtle differences were observed in multiple marks across different conditions; however, given the ECL amplification approach used in most standard immunoblotting approaches, along with the performance of most commercial polyclonal antibodies, it is difficult to ascertain whether such differences are biologically significant.
However, many but not all PTMs that were recognized by serum autoantibodies from SLE patients were found in NETs. Many of the serum-reactive PTMs found in NETs were detected at only modest levels in both SLE histone positive and healthy serum samples, for both IgG and IgM isotypes. Surprisingly, histone PTMs toward which significantly more reactivity was observed in sera from SLE patients known to have anti-histone antibodies (in particular, acetyl-H2B), were absent or detected at only low levels in NETs produced from HL-60 or EPRO cells.
To account for this discordance, one possibility may be that acetylated histones are dissociated during the chromatin condensation step in NETosis-acetylated histones are thought to be in a looser conformation within the nucleosome due to loss of positive charge on acetyl lysine residues [56
]. It is also conceivable that these acetyl histones may be particularly immunogenic since they may disperse more widely, increasing their chances of uptake by a professional antigen presenting cell, generating a subsequent proinflammatory response by the adaptive immune system. A second possibility is that only the NETs from a special subpopulation of polymorphonuclear cells are responsible for their immunogenicity in SLE. A recent study described the discovery of low density granulocytes (LDGs) whose greater tendency to undergo NETosis elicited a stronger immune response than conventional neutrophils [57
Numerous studies have investigated the significance of histone acetylation in SLE, and the pattern of evidence suggests that histone acetylation within cells negatively correlates with disease activity. Splenocytes from MRL/lpr
mice have hypoacetylated histones H3 and H4 when compared to control MRL/MPJ mice [51
] and administration of trichostatin A (a histone deacetylase (HDAC) inhibitor) to MRL/lpr
mice can be used to improve disease outcome [51
]. In T cells isolated from SLE patients, global H3 and H4 hypoacetylation was observed when compared to cells obtained from healthy donors [59
]. Furthermore, mice with a conditional knock-in of the p300 acetyltransferase gene (in which the histone acetyltransferase activity was absent) develop spontaneous lupus-like disease [60
]. Collectively, these studies demonstrate that hypoacetylated histones are associated with increased disease activity and that interventions restoring acetylation in MRL/lpr
mice improved disease outcome.
Clearly, the acetylation state of histones in disease-relevant cells has a significant bearing on disease outcome; the discordance between observed anti-acetyl histone antibodies in SLE sera and the decrease in acetyl histones in NETs suggest the following three possibilities: (i) NETs are depleted of acetylated histones during NETosis and that these free histones may contribute to the autoimmune response in SLE; (ii) NETs are not the in vivo immunogens responsible for the observed patterns in reactivity to histone PTMs; and (iii) NETs derived from LDGs are enriched for acetyl histones and may account for their increased immunogenicity.
In our own mouse studies, we immunized BALB/c mice with NETs or with NETs combined with CRAMP and expected the mice to develop autoantibodies that recognize specific PTMs present in NETs. Here, we tested the hypothesis that NET PTMs are capable of breaking tolerance to self-antigens, including but not limited to histone PTMs, as observed in human SLE. We observed a moderately strong IgM and IgG response to DNA and a modest response to other NET self-antigens, including myeloperoxidase, elastase, and histones. This result is similar to mouse studies performed by Mevorach et al
. in which mice were immunized with material derived from apoptotic cells, leading to a modest, transient response including the production of antinuclear antibodies, anticardiolipin and anti-dsDNA antibodies, along with increased glomerular IgG deposition [22
] and slightly accelerated disease kinetics in MRL/lpr
and other autoimmune backgrounds [61
]. However, while we observed modest but significant and reproducible IgG and IgM reactivity to self-antigens, these mice did not exhibit other features of human SLE nor of autoimmune-prone SLE mouse models. One possible explanation for this observation is that NET chromatin purified in vitro
is not equivalent to NETs generated in vivo
, since the latter includes microbial determinants associated with TLR ligands and could thus act as superior adjuvants to break tolerance to self-antigens.
Anti-histone antibodies have also been observed in other diseases. For instance, NETs have been observed in the glomeruli of patients with antineutrophil cytoplasmic antibodies (ANCA) associated vasculitis, perhaps reflecting a common etiology [62
]. In drug-induced lupus (DIL), the vast majority of patients exhibit significant positive anti-histone antibody titers primarily targeting histone H2A/H2B [63
], concordant with our observed significant reactivity to acetyl and unmodified histone H2B. Extracellular histones can induce septic shock in mice and concurrent infusion of anti-histone H4 antibodies was protective against LPS-induced shock [64
]. This finding is consistent with speculation in the field that IgM autoantibodies may play a protective role against an excessive immune response [50
]. We evaluated the role of distinctive NET PTMs as a source of self-antigens with a qualitative relationship to autoimmunity. However, a clearance deficiency of NETs has also been described in patients with SLE [4
]. Therefore, persistence of NETs, through insufficient clearance by endonucleases or phagocytes, may serve as a complementary quantitative perturbation leading to SLE pathogenesis.
Nucleoprotein complexes are also candidates to be involved in both the etiology and pathogenesis of SLE and murine lupus, wherein they might complement NETs. Many dsDNA autoantibody-producing B cells from SLE patients are thought to be affinity selected by uncleared apoptotic lymphocytes in germinal centers. Therefore, it may not be surprising that NET derived modifications are not the dominant epitopes recognized by anti-histone autoantibodies. Further, in the presence of anti-dsDNA, NETs generated in several tissues may serve as targets for the anti-dsDNA. The binary complex of nucleoprotein and autoantibody may be prone to shift the clearance of dead cell remnants (apoptotic bodies and NET-structures) to inflammation.
Many of the specific post-translational histone modifications enriched in NETs overlapped those to which significant autoreactivity is seen in a subset of patients with SLE. Nonetheless, this overlap was partial, and many PTMs distinguished NETs from SLE-autoreactivity profiles. Further, while NETs were observed to be modestly immunogenic in vivo, the induced serological autoimmune responses were distinct from those observed in patients with lupus, as well as autoimmune-prone MRL/lpr mice.