Systemic sclerosis (SSc) is a rare chronic autoimmune disease characterized by vasculopathy, inflammation, and fibrosis of the skin and internal organs. Depending on the distribution of skin fibrosis, SSc is clinically subdivided into limited cutaneous SSc (lcSSc) and diffuse cutaneous SSc (dcSSc) (1, 2). The pathogenesis of SSc is complex and the exact etiology of the disease is still unknown. The role of the adaptive immune system with autoreactive T and B cells producing autoantibodies in SSc pathogenesis has been well established (3, 4). Additionally, several studies have suggested the involvement of the innate immune system in early pathogenic events in SSc but this has not been explored in detail (5).

Among the innate immune cells, neutrophils are prominent contributors of inflammation in several autoimmune diseases including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), and have also been implicated in SSc pathogenesis (6–9). While neutrophils play a protective role against invading pathogens through reactive oxygen species (ROS) production, phagocytosis and formation of neutrophil extracellular traps (NETs) (10, 11), unrestrained neutrophil activation may lead to inflammation, immune dysregulation and tissue damage. Neutrophils are the most abundant immune cells in the circulation, and in SSc patient’s blood neutrophil counts are elevated and correlated with disease severity (12). Neutrophils were also increased in skin biopsies obtained from forearm lesions in SSc patients compared with controls (13). Moreover, neutrophil involvement in induction of endothelial cell apoptosis, an early event leading to fibrosis, has been reported in SSc (14). These studies indicate a critical pathogenic or modulatory role of neutrophils in SSc.

Markers of neutrophil activation, such as elevated levels of circulating calprotectin and NET formation have been previously reported in several autoimmune disorders and evolved as excellent biomarkers for inflammatory processes (6, 8, 15). Calprotectin (S100A8/A9) is a calcium-binding protein abundantly expressed in neutrophils and is essential for initiating immune responses to non-infectious inflammatory processes (16, 17). NET formation is the key feature of neutrophil cell death in which nuclear DNA is extruded together with cytoplasmic and granular contents in web-like structures that can function to entrap and eliminate extracellular pathogens (18). Elevated levels of neutrophil activation markers during an inflammatory response may be detrimental and cause bystander injury that could be perpetual or non-resolving. Among these markers, separate studies have found increased soluble levels of calprotectin, an acute phase protein expressed by neutrophils and NETs in SSc patients and associations with SSc disease manifestations were also reported in these studies (19–22). Additionally, filaments of DNA coming from elastase positive cells, such as neutrophils can be seen in SSc skin (23). Although these studies point to a role of neutrophils in SSc, a comprehensive study of neutrophil activation markers in the circulation of SSc patients and its contribution to inflammation and disease phenotype needs to be carefully addressed.

So far, factors contributing to neutrophil-mediated activation in a sterile pro-inflammatory environment like SSc remains largely unknown. During inflammation, neutrophils are activated by various molecules, including cytokines, immune complexes (ICs) and damage-associated molecular patterns (DAMPs) generated by mitochondrial components (11, 24, 25). We and others have reported mitochondrial extrusion and presence of extracellular mitochondrial components in several autoimmune disorders such as SLE and RA (26–28). Given their prokaryotic origin, mitochondria contain several pro-inflammatory components that can engage neutrophils. Among the mitochondrial protein-derived molecules, N-formyl-methionine (fMet) is a potent neutrophil chemoattractant that can trigger a variety of neutrophil functions leading to tissue damage. fMet acts through formyl peptide receptor-1 (FPR1), which is abundantly expressed on neutrophils and when bound by its cognate ligand induces inflammation (29–32). However, its role remains to be explored in SSc.

In the current study, we first assessed the levels of neutrophil activation markers in two independent SSc cohorts and investigated their association with markers of inflammation and SSc disease phenotype. Secondly, we analyzed levels of fMet in relation to neutrophil activation levels. Finally, we assessed whether fMet could contribute to neutrophil activation through FPR1-mediated signaling in SSc patients. Briefly, neutrophil activation markers were elevated in SSc patients and associated with a disease phenotype. We also made the novel observation that elevated fMet levels were associated with neutrophil activation in SSc patients. Finally, plasma samples from SSc patients induced de novo neutrophil activation through an FPR1-dependent mechanism suggesting FPR1 as a potential novel therapeutic target in these patients to reduce neutrophil-mediated inflammation.

Owning to its unknown etiology, SSc remains a poorly understood autoimmune disease and this presents one of the greatest challenges to both investigators and physicians. Despite thorough investigations, other than SSc-specific autoantibodies, few biomarkers are validated and/or widely used diagnostically or clinically (38). With respect to early immune cells in SSc pathogenesis, neutrophils are instrumental, infiltrating the lesions and participating in chronic inflammation and fibrosis (5, 39). Although neutrophils are implicated in SSc pathogenesis, neutrophil biomarkers have not been carefully investigated. In the current study we performed a comprehensive analysis on neutrophil activation biomarkers and assessed the role of extracellular mitochondrial components as potential neutrophil activating agents in SSc. Our findings indicate elevated levels of neutrophil activation markers and cell death in SSc. Neutrophil biomarkers associated with SSc disease phenotypes offering potential clinical value to these biomarkers by improving the capacity to monitor disease.

Neutrophils have several effector functions crucial for eliminating invading pathogens. In contrast, uncontrolled activation of neutrophils, including release of inflammatory mediators such as calprotectin can lead to chronic inflammation and immune dysregulation. Calprotectin is primarily produced by neutrophils comprising approximately 45% of its cytoplasmic content (40) and can be released into the extracellular environment upon degranulation. Once extracellular, calprotectin is an efficient damage-associated molecular pattern (DAMP) inducing inflammation by signaling through Toll-like receptor 4 and receptor for advanced glycation end products (RAGE) (41, 42). Thus, in view of its vital role in the physiology of inflammation, calprotectin is a valuable candidate as a neutrophil activation biomarker for inflammation-associated diseases.

Calprotectin (S100A8 and S100A9), either as homodimers, or heterodimer (e.g. calprotectin), have been found at elevated levels in plasma, sera (20, 43), feces (19), saliva (44), BAL fluids (21), and skin (43) of SSc patients. These studies reflect that SSc is not only a skin disease but also affects several visceral organs. Thus, it’s possible that neutrophil infiltrates might be present in skin and/or other organs affected by the disease, which needs to be further investigated. With regards to neutrophil activation markers, elevated calprotectin levels in the BAL fluid (21) and serum (20, 43) were associated with extensive lung fibrosis and anti-topoisomerase I (ATA) positivity in the SSc patients. Moreover, fecal calprotectin levels corelated with clinically important features of gastrointestinal (GI) disease and has been explored as a possible biomarker of GI disease in SSc (19). In the same study, plasma calprotectin levels were measured which corelated with systemic inflammation markers like C-reactive protein (CRP) (19). In the current study, we were able to validate elevated levels of calprotectin in SSc patients as well as add to previous findings regarding its potential clinical applications. In particular, our study showed increased calprotectin levels in patients with vasculature-related manifestations like cutaneous pitting scars. Additionally, correlation of calprotectin with proBNP was observed which has been shown to be a useful biomarker for assessing extent of skin fibrosis, degree of restricted pulmonary involvement and a diagnostic marker for pulmonary arterial hypertension (PAH) in SSc (38, 45). These observations further highlight the pathological significance of calprotectin levels in SSc and a promising non-invasive biomarker of SSc.

Effector functions of neutrophils like ROS production has been linked to pathogenesis of SSc. Generation of ROS is crucial for NET formation, a neutrophil cell death process and a well-known biomarker in diseases like RA and SLE (46). Similar to calprotectin, we found elevated levels of NETs (MPO-DNA complexes) in plasma samples of SSc patients, consistent with prior work (22). This study also observed elevated NET levels in the plasma of active SSc patients (22). Moreover, our study showed correlation of NETs with skin score, a measure of SSc disease progression over time, offering a potential clinical value to NET levels. Association of NETs with vascular manifestations (pitting scars, telangiectasia, nail fold capillary abnormalities) were also observed in our study and the above-mentioned study indicating NETs as a reliable marker to detect the presence of vascular involvement in active SSc patients (22). Elevated level of circulating NETs observed in SSc could be due to increased formation of NETs. Consistent with the hypothesis, prior findings have demonstrated increased capacity of SSc neutrophils to undergo NET formation after stimulation with autologous serum. In addition, neutrophils from SSc patients with severe vascular complications were significantly more prone to releasing NETs compared to other SSc patients (47). Other mechanisms contributing to elevated levels of circulating NETs such as decreased degradation/clearance of NETs in SSc patients are not reported yet and need further investigation. Thus, NET formation might represent a new pathophysiological as well as potential SSc biomarker.

The initial trigger(s) of neutrophil activation in SSc patients remain to be determined. In a recent study, microparticles released from activated platelets expressing the DAMP HMGB1 were abundantly found in blood of SSc patients (22). These microparticles resulted in neutrophil activation and generation of NETs and were further ablated in presence of BoxA, a competitive inhibitor of HMGB1, indicating a platelet-microparticle specific neutrophil activation via HMGB1 (22). Although not in the context of SSc, our group has recently demonstrated extracellular mitochondrial N-formyl methionine (fMet) peptides abundantly present in RA patients (28) as well as in systemic vasculitides (Michailidou et al., under revision) suggesting fMet-mediated neutrophil activation possibly being a central process in several autoimmune inflammatory conditions. Whether SSc patients have elevated levels of extracellular mitochondrial fMet and their role in neutrophil-mediated inflammation has so far not been investigated. Consistent with the RA study, elevated level of fMet peptides were found in SSc patients prompting us to investigate its role in neutrophil activation. Additionally, increased levels of fMet were observed in DcSSc as compared to LcSSc patients, suggesting fMet levels might play an important role in the pathogenesis of DcSSc and could be useful serological marker for evaluating type of SSc disease. Mitochondrial fMet peptides can be sensed by N-formyl peptide receptor FPR1, which has high affinity for fMet, and is expressed on various host cell types but most strongly on neutrophils. Activation of neutrophils by fMet via FPR1 triggers a wide variety of downstream effector functions including chemotaxis, degranulation, ROS production, and phagocytosis bridging an association between mitochondrial fMet proteins, FPR1 and neutrophils (48).

Association between mitochondrial proteins and neutrophil activation was shown in our recent study of RA patients (28). Similarly, we found levels of fMet associating with neutrophil activation markers like calprotectin and NETs in SSc patients, supporting the hypothesis of fMet-mediated neutrophil activation. Given that correlations do not inform on causality, we performed neutrophil stimulation studies to identify circulating factors such as fMet present in plasma that are able to activate neutrophils in vitro. Our data demonstrate that plasma from SSc patients having high fMet levels in circulation had increased ability to induce activation of neutrophils from healthy blood donors. Blockade of FPR1 by cyclosporin H (CsH) suggested that circulating fMet activates FPR1 signaling in SSc patients and contribute significantly to the immune activation of neutrophils. These observations warrant exploring the inhibition of FPR1 signaling as a potential novel pharmaceutical intervention in SSc, a disease which is in dire need of novel therapeutics. Importance of FPR1 signaling has been previously shown in tissue fibrosis, the hallmark of SSc. FPR1 signaling has been shown to be crucial for neutrophil recruitment to the lung and support fibrosis in an in vivo mouse model of SSc (49). FPR1 is also expressed on human fibroblasts and signaling via FPR1 in SSc patients has been shown to promote transition of fibroblast-to-myofibroblasts and extracellular matrix deposition leading to tissue fibrosis (50). Thus, fMet proteins critical to FPR1 signaling and neutrophil activation might be an important mechanism contributing to SSc pathogenesis and a potential therapeutic target. There are several potential sources of extracellular mitochondria, including platelet activation, neutrophil death, and tissue damage (26, 27). Which, if any, of these mechanisms operate in SSc to promote release of extracellular mitochondria is under current investigation.

Adding to the complexity of SSc, in contrast to our hypothesis we found activation of neutrophils even upon stimulation with a subgroup of SSc patients having low fMet levels in plasma. The activation of neutrophils did not decrease upon blockage of FPR1 indicating the neutrophil activation was not driven by fMet/FPR1 interactions but due to other circulating factors present in the plasma samples. Among the circulating factors, immune complexes (ICs) containing SSc-specific autoantibodies engaging with nucleic acid or DNA/RNA binding proteins are shown to elicit proinflammatory and profibrotic effects on fibroblasts (51). The pathogenicity of the SSc-ICs has been suggested to be mediated by interaction with Toll-like receptors (TLRs) via nucleic acid fragments. Additionally, circulating ICs contributing to neutrophil activation has been shown in autoimmune diseases like SLE (52). However, in the current study, presence of SSc autoantibodies towards intracellular antigens did not associate with levels of circulating NETs (data not shown), but neutrophil activation upon stimulation with SSc plasma was abrogated upon blocking of Fcy receptor, the binding sites of ICs. Thus, fMet is an important driver of neutrophil activation and inflammation in a subgroup of SSc patients with high levels of fMet in circulation. However, other mechanisms, potentially driven through ICs, are operating in patients with low levels of fMet. These results further highlight the heterogeneity of the disease and the need for patient stratification or personalized medicine approach, as different patients might have different immunological pathways activated and might not necessarily benefit from the same treatment.

The limitation of our study includes limited clinical data for SSc Cohort I, as well as lack of longitudinal data, which would have allowed us to assess the prognostic utility of the neutrophil biomarkers. Another limitation of our study includes conducting experiments on neutrophil activation alone via fMet/FPR1 signaling. Although FPR1 is expressed strongly on neutrophils, other innate immune cells like monocytes and macrophages also express FPR1 warranting analyzing these cell types which are also implicated in early SSc pathogenesis (5).

In conclusion, our data for the first time demonstrated that levels of fMet are elevated in the circulation of SSc patients and implicate an important role for the mitochondrial component fMet in promoting neutrophil-mediated activation through FPR1 in SSc. Our data also support the clinical value of neutrophil biomarkers and fMet in monitoring SSc disease, although these observations need to be validated in larger patient cohorts as well as in appropriate animal models. We propose fMet-mediated signaling as a potential therapeutic target promoting anti-inflammatory effects in SSc by ameliorating neutrophil based inflammation.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Institutional Review Board of Fred Hutchinson Cancer Research Center (part of the University of Washington Consortium) and Lund University #2010/544. The patients/participants provided their written informed consent to participate in this study.

RK and CL conceived the study. RK, BD, and CL designed experiments, analyzed data, and interpreted results. RK, RS, BD, TW, and MF performed experiments. JN, RH, and MF provided materials and clinical cohorts. RK wrote the manuscript and RH, JN, MF, RS, BD, and CL critically reviewed the manuscript. All authors reviewed and approved the manuscript.

This work was supported by grants from the Arthritis National Research Foundation (#632002) to CL and NIH grant R01 AI-41721 and a Scleroderma Foundation grant to JN.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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