Recent work from various groups indicates that NET formation may be an important phenomenon in autoantigen modification and exposure to the immune system, as well as in the induction of tissue damage (9
). As such, aberrant NET formation may play an important role in the development and perpetuation of autoimmune diseases and organ damage observed in chronic inflammatory disorders. Our work now expands and reinforces this concept by reporting that a distinct subset of neutrophils found in SLE patients (LDGs) have enhanced capacity to form NETs and upregulate expression of various neutrophil proteins and enzymes implicated in NET formation and in autoimmunity induction. These NETs also expose ds-DNA, an autoantigen considered key in lupus pathogenesis. Furthermore, lupus neutrophils and, in particular, LDGs elicit enhanced EC cytotoxicity through NET formation. This phenomenon also appears to play a role in the induction of IFN-α synthesis by pDCs. We have also identified that enhanced NETosis occurs in vivo
in SLE in affected skin and kidney. Furthermore, neutrophils from blood and skin from SLE patients frequently externalize IL-17 as part of the NETosis process, which may contribute to tissue damage and immune dysregulation. Overall, these observations further support a pathogenic role for neutrophils in organ damage in SLE.
One of the findings from our study is that no differences in gene expression were found when normal density lupus neutrophils were compared to gender-matched healthy control neutrophils. Thus, previous reports examining alterations in lupus neutrophils may in part reflect responses specifically elicited in the LDG pool. In contrast, lupus LDGs obtained from the same patients from whom the normal density neutrophils were obtained showed significant differences in gene expression when compared to healthy control and lupus neutrophils. The pair-wise comparison of gene expression in LDGs and autologous neutrophils in SLE patients provides a control for many potential sources of variability such as medications, disease activity and clinical manifestations, and exposure to environmental factors. As it is expected that LDGs and autologous lupus neutrophils were exposed to a very similar cytokine milieu, these results indicate that LDGs may indeed represent a distinct subset of proinflammatory and pathogenic cells within the granulocyte spectrum.
It is unclear why LDGs upregulate mRNA of various serine proteases and bactericidal proteins present in azurophilic granules. One possibility is that these findings are indicative of a more immature phenotype of the LDGs, further supported by their immature nuclear morphology (1
). Indeed, proteins synthesized at the same time during neutrophil differentiation are co-localized in the same granules. The levels of expression of the mRNA that encode the neutrophil serine proteases are greatest at the pro-myelocytic stage of neutrophil differentiation in the marrow and are downregulated as neutrophils mature (36
). This observation could indicate that LDGs are indeed a more immature neutrophil subset, despite their apparent expression of markers of fully matured neutrophils, including CD16 and CD10. Indeed, a previous study has shown that, among the lupus bone marrow up-regulated genes (when compared to controls), the highest overexpression occurs in granulopoiesis-related genes. These genes include several of the “early granulopoiesis genes” upregulated in the LDG microarray in our study, including MPO
). This is also confirmed by a previous study that showed that the PBMC granulocyte signatures observed in pediatric SLE patients were for genes preferentially transcribed within the earliest granulocytes (myeloblast and promyelocytes) and with the presence of immature neutrophils in their peripheral blood(3
). These observations further support that LDGs could represent an aberrant immature subset originating from lupus bone marrow that may persist or expand in the blood and/or other tissues from SLE patients.
The functional consequences of high serine protease expression in LDGs may be varied. It has been proposed that all serine proteases of azurophilic granules (cathepsin G, proteinase 3 and neutrophil elastase), released after encountering immune complexes, may potentiate a positive autocrine feedback on neutrophil activation (37
). Further, these molecules have been implicated in the activation of the pro-forms of proinflammatory cytokines including TNF and IL-1β (39
). Neutrophil elastase can activate TLR4 eventually resulting in IL-8 production (40
). IL-8 levels are elevated in SLE but the exact mechanisms leading to this increase have been unclear (41
). One could propose that enhanced exposure to extracellular elastase through NET formation in SLE LDGs could promote enhanced synthesis of IL-8. This cytokine could in turn activate neutrophil recruitment and promote damage in various organs. In addition, we previously showed that LDGs synthesize enhanced levels of IL-8 and TNF upon activation, when compared to control and normal-density lupus neutrophils (1
The mechanisms by which LDGs are more primed to make NETs are unclear. To date, the exact molecular mechanisms and subcellular events leading to NETosis remain elusive. While a crucial role for elastase, reactive oxygen species and the cytoskeleton has been proposed, recent reports suggest that NETosis is quite complex. The observed higher expression of elastase and MPO in LDGs could play an important role in enhancing extracellular trap synthesis, based on what other groups have recently reported(9
). There is also evidence that type I and II IFNs can act as priming factors on mature neutrophils, allowing the formation of NETs upon subsequent stimulation with complement factor 5a (34
). One possibility may be that LDGs are more sensitive to the effects of type I IFNs and/or to IFN signaling than normal density lupus neutrophils. However, this would go against the hypothesis that LDGs represent a more immature subset of neutrophils, since previous evidence indicates that granulocyte precursors and less mature cells are fairly insensitive to type I IFN effects, when compared to fully differentiated neutrophils (34
). Further, while one possibility is that LDGs represent cells that have been exposed to elevated levels of type I IFNs in the bone marrow, we do not consider this is likely the case since these cells do not display evidence of increased type I IFN gene signature. Future studies need to explore whether the LDGs are derived from normal neutrophils or result from alterations in granulocyte development.
Previous studies have shown that the antimicrobial peptide LL-37 is a key factor that mediates pDC activation in psoriasis (23
). LL-37 converts inert self-DNA into a potent trigger of type I IFN production. This occurs by binding the DNA to form condensed structures that are delivered to and retained within early endocytic compartments in pDCs to trigger TLR9 (23
). LL-37 also converts self-RNA into a trigger of TLR7 and TLR8 in human DCs (22
). In general, LL-37 is involved in a myriad of important immune functions including chemoattraction of immune cells and release of inflammatory mediators from epithelial cells (42
). Our findings support that the enhanced release of LL-37 through NETosis could promote enhanced inflammatory responses in SLE organs. Other molecules overexpressed in lupus LDGs, including various defensins, may also be immunostimulatory (43
). Our findings are in agreement with a recent study that reported that SLE patients have elevated serum levels of various neutrophil peptides including MPO and defensins (44
). It is possible that these are derived from LDGs. Further, increased anti-defensin and cathepsin-G ANCA antibodies are found in lupus patients (46
). Given their immunomodulatory role, overproduction of alarmins might activate the adaptive immune and promote autoimmune responses, as is manifested in SLE (47
). Another overexpressed molecule in LDGs, neutrophil-gelatinase associated lipocalin (NGAL/lipocalin2), has recently been proposed as a biomarker of and to have a pathogenic role in lupus nephritis(49
). Finally, the role of MMP-8 overexpression in LDGs remains to be determined, but this metalloproteinase may play a key role in vascular damage in other conditions (50
Lupus patients develop accelerated atherosclerosis, leading to premature cardiovascular disease. We had previously found that ECs from lupus patients undergo accelerated apoptosis in vivo
and that this phenomenon may be pathogenic, as it correlates with endothelial dysfunction development (35
). LDGs are capable of killing ECs but the mechanism involved was unclear (1
). The current study shows that, through enhanced NET formation, LDGs acquire a heightened capability to damage the endothelium, as cytotoxicity was abrogated with MNAse treatment. As such, accelerated NETosis may represent an important mechanism of premature vascular damage in SLE.
Death by NETosis may also represent an important immunostimulatory event with regards to activation of innate immunity in SLE, given the enhanced capacity of netting lupus neutrophils to activate pDCs. This could promote chronic activation of the immune system and perpetuation of type I IFN synthesis characteristic of this disease(51
). Enhanced NETosis in SLE may be one of the mechanisms explaining why infections may trigger flares in this disease(52
), with NET induction by microorganisms which in turn leads to autoantigen externalization and immune system activation. The observation that both lupus neutrophils and LDGs induced comparable induction of IFN-α synthesis by PDCs may indicate that this process cannot be fully explained by pure enhancement of NETosis in LDGs and that other variables are involved in this phenomenon. Future studies are needed to better understand how this process occurs.
The skin is an important target organ of the immune system in SLE and a significant proportion of lupus patients have cutaneous involvement. Although mechanisms of skin damage in SLE are likely multifactorial (53
), recent evidence has identified an enhanced type I interferogenic signature and IFN-α producing pDCs in lupus skin(54
). The role of pDCs in the inflammation observed in autoimmune skin disease in animal models has recently supported the notion that IFN-α may be crucial in cutaneous involvement in SLE (55
). In a lupus murine model, tape stripping led to an influx into skin of neutrophils forming NETs which contain DNA and RNA associated with LL-37 (55
). Our data confirm a similar pattern in human lupus skin biopsies, where abundant ds-DNA+
NETs are seen in multiple layers of affected skin. Interestingly, these findings are associated with the presence of elevated anti-ds-DNA antibodies in the circulation, supporting the notion that autoantigen exposure in the NETs could promote autoantibody formation in vivo
Similar findings were seen in the kidneys of lupus patients affected by class III and IV glomerulonephritis. This supports recent findings that impaired NET degradation in SLE is associated with renal involvement in this disease (2
). Indeed, IgG deposition on NETs in tubuli and glomeruli in the kidney of an SLE patient who degraded NETs poorly was reported in that study (2
). We have now expanded this observation by studying a larger number of patients with lupus nephritis, where presence of netting neutrophils in glomeruli was observed in a majority of them. Interestingly, patients with higher proportion of glomeruli infiltrated by netting neutrophils had higher levels of circulating anti-ds-DNA antibodies and higher activity index in renal biopsies. It has been previously proposed that anti-NET antibodies and persistent NETs could form NET immune complexes that could be relevant in lupus disease severity (2
). It is possible that the presence of infiltrating netting neutrophils in lupus tissue samples represented a combination of enhanced NETosis and impaired NET degradation. We could then propose a model of profound imbalance in NET production and degradation. On one hand, NETosis would be enhanced in lupus LDGs. On the other hand, the activity of DNase1 (an enzyme important in NET degradation) is decreased in a subset of SLE patients (2
). This may provide the conditions by which NETs may persist and constitute a prolonged source of autoantigen exposure in an immunostimulatory context, leading to enhanced formation of immune complexes and induction of autoantibodies which could further contribute to tissue damage.
Whether the infiltrating neutrophils in the skin and kidney correspond primarily to LDGs is unclear at this point, as no specific cell marker has to this date been identified to distinguish these cells from normal-density neutrophils. A better understanding of the homing characteristics of LDGs versus other neutrophil subsets will be important to assess if, through enhanced NETosis or other yet unidentified mechanism, LDGs could have an increased capacity to migrate to various tissues and induce damage. It is relevant that patients with high levels of LDGs in their circulation have higher prevalence of skin involvement, and this association could also support that this cell subset is pathogenic to cutaneous tissue (1
). Future studies in a larger number of patients are required to assess the specific role that these cells play in tissue damage and progression of disease in SLE.
IL-17 has recently been linked to the pathogenesis of SLE(30
) by participation in the amplification of autoimmune responses by stimulating autoantibody production by B cells (29
). Further, it augments tissue injury and target organ damage in this disease (31
). Most studies have focused on Th17+ cells, which are elevated in SLE (56
) and infiltrate renal tissue (57
). A recent report showed IL-17+
cells infiltrating lupus affected skin, but neutrophils were not specifically studied (59
). Similar to what has been shown in other conditions (28
) and (Lin et al., manuscript under review), neutrophils expressing IL-17 are seen at significantly enhanced levels in blood and affected skin from lupus patients. This phenomenon could initiate a cycle in which IL-17- secreting cells in lupus skin lesions (including innate and adaptive immune cells) would recruit additional neutrophils. IL-17 can stimulate endothelial cells to produce chemoattractants (IL-8) that selectively drive neutrophil but not lymphocyte chemotaxis. IL-17 increases neutrophil adhesion to the endothelium which may also enhance neutrophil recruitment to organs (60
). We had previously been unable to detect IL-17A in LDG supernatants (1
). Given that we have now found evidence of IL-17A expression both at the mRNA and protein levels, it is possible that the technique previously used was not sensitive enough or that the NET-bound IL-17 had not been fully released to the supernatants to allow for quantification by ELISA. Overall, these results further support a pathogenic role for IL-17 in organ damage in SLE and that neutrophils represent an important subset of IL-17 producing cells.
Whether enhanced NETosis is a phenomenon present in other autoimmune diseases associated to autoantibody production, interferogenic signatures and/or vascular damage remains to be determined and should be the focus of future investigations. These conditions may include inflammatory myopathies, Sjögren syndrome and rheumatoid arthritis (61
In conclusion, we have identified that LDGs isolated from lupus patients have a higher capacity to synthesize NETs with immunostimulatory and cytotoxic properties. These results further expand the potential pathogenic role of aberrant lupus neutrophils through a NET-mediated effect and indicate that strategies attempting to modulate NET formation with the objective of abrogating autoimmune responses should be investigated.