Systemic lupus erythematosus extends beyond a type I interferonopathy, as demonstrated by NET‐activated monocytes

Dear Editor, Type I interferon (IFN) signalling and neutrophil extracellular traps (NETs) formation are the primary contributors to the pathogenesis of systemic lupus erythematosus (SLE).1, 2 Anifrolumab, a recently approved monoclonal antibody prescribed for moderate to severe SLE,3 selectively inhibits type I IFN signalling but demonstrates incomplete efficacy in many patients.4 We found that NET-activated monocytes release CCL5 and promote CD4+ T cell proliferation independently of type I IFN signalling, underscoring a distinct, NET-driven mechanism in SLE that functions outside canonical type I IFN pathway. Our data highlight the need for complementary therapies targeting NETs in patients unresponsive to anifrolumab treatment. The chemokine CCL5 (RANTES), a potential biomarker elevated in SLE patients,5 may contribute to disease progression by mediating leukocytes chemotaxis6 or potentially exerting a previously unrecognized immunostimulatory function. We confirmed that plasma CCL5 concentrations were notably elevated in seven SLE patients in remission than in healthy donors (HD) (Figure 1A). In a 2-year longitudinal study, we screened nine active SLE patients, excluding patients who developed antiphospholipid syndrome.7 CCL5 levels were measured and plotted alongside Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores (Figure 1B) or flare status (Figure 1C) for a representative case (Pt. 06), with same analyses shown for the other included patients (Figures S1 and S2). Across the cohort, CCL5 levels normalized to individual baselines were elevated during periods of high SLEDAI (Figure 1D,E) and flares (Figure 1F), supporting its potential as a dynamic disease activity marker. To further explore the functional axis of CCL5, we assessed the expression of its highest-affinity receptor CCR5, on monocytes from SLE patients and healthy controls. Monocyte subsets were identified as shown in Figure 1G. While the overall distribution of monocyte subclasses was comparable between HD and SLE groups (Figure S3A), monocytes from SLE patients exhibited a notable reduction in CD16 expression (Figure 1H).8 Monocytes from SLE patients displayed higher surface expression of CCR5 compared to those from healthy donors (Figure 1I,J), particularly within the pro-inflammatory classical and intermediate subsets (Figure 1K). Within individual donors, higher plasma CCL5 levels are associated with increased CCR5 expression on monocytes (Figure 1L). To investigate the mechanisms driving CCL5/CCR5 upregulation in SLE, we mimicked the disease environment in vitro by stimulating monocytes from healthy donors with IFNα and NETs induced by LPS-activated platelet.1, 9 We found that IFNα and NETs synergistically regulate monocyte responses: CCL5 mRNA expression is upregulated by combined IFNα and NETs stimulation (Figure 2B), while CCL5 protein secretion is primarily induced by NETs in a DNA-dependent manner (Figure 2C–E). Conversely, CCR5 mRNA is increased by IFNα alone (Figure 2F), but CCR5 surface protein expression on monocytes is maximally enhanced by the combined effect of IFNα and NETs (Figure 2G). Additionally, IFNα stimulation selectively increases CD80 expression on monocytes (Figure S4A,B), suggesting a potential for increased monocyte-mediated T cell activation. To test whether CCL5 directly influenced T cell responses, we stimulated purified T cells with recombinant CCL5. Although CCL5 increased CCR5 expression on CD4+ T cells (Figure 2I), it did not promote their proliferation (Figure 2J) or activation markers expression (Figure 2K–M), indicating that additional signals are required to elicit functional T cell responses. Given that CCL5 alone does not activate T cells, we investigated whether IFNα- and/or NET-stimulated monocytes could promote CD4+ T cell activation and proliferation. To approximate physiological conditions in PBMCs culture, monocyte-derived dendritic cells (moDCs) were generated and stimulated with IFNα and/or NETs (Figure 3A). After 24 h, T cell activation markers were analysed, and proliferation was assessed following 9 days of culture. Our data showed that IFNα exhibited anti-proliferative effects on CD4+ T cells,9 while NETs enhanced proliferation (Figure 3B) and HLA-DR expression of CD4+ T cells (Figure S5E). CD25 expression was downregulated by IFNα (Figure 3C) and CD69 was upregulated by both stimuli (Figure 3D). Subsequently, isolated monocytes and autologous CD3+ T cells were co-cultured without or with the presence of anifrolumab following monocyte pre-stimulation with IFNα and/or NETs. NETs consistently induced CD4+ T cell proliferation, which was not inhibited by anifrolumab (Figure 3G). Anifrolumab enhanced CD25 expression in the presence of IFNα (Figure 3H) and fully abrogated IFNα-induced upregulation of CD69 (Figure 3I) and IFNAR1 (Figure 3J). These data support that NET-matured monocytes/moDCs enhance CD4+ T cell proliferation and that this NET-driven activation is resistant to IFNAR blockade. To examine the direct influences of IFNα and NETs on T cells or monocytes, we stimulated each cell type separately with IFNα and/or NETs, with or without anifrolumab involved (Figure 4A). IFNα exposure attenuated proliferation of CD4+ T cells (Figure 4B)10 and reduced CD25 expression (Figure 4C), both of which were reversed by anifrolumab presence. IFNα increased CD69 and IFNAR1 surface expression, which was blocked by anifrolumab. These IFNα-driven changes corroborate our co-culture findings (Figure 3G–J). The NET-driven enhancement of CD4+ T cell proliferation is mediated indirectly via monocyte activation, substantiating the role of NET-induced monocyte phenotypic modulation in facilitating T cell proliferation. To further characterize the direct phenotypic and functional changes of monocytes in prolonged stimulation of IFNα and/or NETs, we cultured monocytes for up to 36 h. IFNα induced upregulation of maturation markers CD80 (Figure 4H), CD86 (Figure 4I), HLA-ABC (Figure S6A) and HLA-DR (Figure S6B), which were all significantly reduced by anifrolumab treatment. In contrast, NETs did not induce observable changes in these markers, suggesting that NET-activated monocytes adopt a distinct activation state. Supporting this, cytokine analysis of monocyte supernatants revealed that NETs selectively increased CCL2 (Figure S6C), IL-6 (Figure S6D), and IL-1β (Figure S6G). The CCL2 increase was abolished by anifrolumab, while IL-6 and IL-1β were unaffected, other cytokines, including IFNγ (E), TNFα (F), and IL-8 (H), were unchanged. These results suggest that NETs activate monocytes differently from IFNα, producing a cytokine profile that may enhance CD4+ T cell proliferation. Besides, NETs robustly triggered CCL5 secretion by monocytes, which was unaffected by IFNAR blockade (Figure 4J). In conclusion, our study identifies that NET-activated monocytes promote CCL5 production and CD4+ T cell proliferation in SLE via a pathway independent of type I IFN signalling. While anifrolumab effectively suppresses IFNα-mediated monocyte and T cell activation, it does not inhibit NET-induced responses. Elevated plasma CCL5 levels correlate with disease activity, suggesting that CCL5 reflects ongoing inflammatory activity within a broader cytokine network driven NET-activated monocyte. These findings suggest that persistent activation of monocytes by NETs may contribute to the limited efficacy of anifrolumab in certain SLE patients. By emphasizing the role of type I IFN-independent, monocyte-driven inflammatory pathways, our study underscores the need for complementary therapeutic strategies targeting both type I IFN signalling and NET-activated myeloid responses. These findings also encourage further investigation into the molecular mechanisms underlying NET-induced monocyte activation, including in vivo validation and extended functional studies, which may improve outcomes in patients with type I IFN-independent or anifrolumab-refractory disease. We acknowledge the patients and healthy volunteers who contributed samples for this study. We also appreciate the clinical team for their help in participant recruitment and sample collection. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.