Unraveling IFN-I response dynamics and TNF crosstalk in the pathophysiology of systemic lupus erythematosus.

Introduction: The innate immune system serves the crucial first line of defense against a wide variety of potential threats, during which the production of pro-inflammatory cytokines IFN-I and TNFα are key. This astonishing power to fight invaders, however, comes at the cost of risking IFN-I-related pathologies, such as observed during autoimmune diseases, during which IFN-I and TNFα response dynamics are dysregulated. Therefore, these response dynamics must be tightly regulated, and precisely matched with the potential threat. This regulation is currently far from understood. Methods: Using droplet-based microfluidics and ODE modeling, we studied the fundamentals of single-cell decision-making upon TLR signaling in human primary immune cells (n = 23). Next, using biologicals used for treating autoimmune diseases [i.e., anti-TNFα, and JAK inhibitors], we unraveled the crosstalk between IFN-I and TNFα signaling dynamics. Finally, we studied primary immune cells isolated from SLE patients (n = 8) to provide insights into SLE pathophysiology. Results: single-cell IFN-I and TNFα response dynamics display remarkable differences, yet both being highly heterogeneous. Blocking TNFα signaling increases the percentage of IFN-I-producing cells, while blocking IFN-I signaling decreases the percentage of TNFα-producing cells. Single-cell decision-making in SLE patients is dysregulated, pointing towards a dysregulated crosstalk between IFN-I and TNFα response dynamics. Discussion: We provide a solid droplet-based microfluidic platform to study inherent immune secretory behaviors, substantiated by ODE modeling, which can challenge the conceptualization within and between different immune signaling systems. These insights will build towards an improved fundamental understanding on single-cell decision-making in health and disease.

Phenotypical Diversification of Early IFNα-Producing Human Plasmacytoid Dendritic Cells Using Droplet-Based Microfluidics.

Plasmacytoid dendritic cells (pDCs) are a rare type of highly versatile immune cells that besides their specialized function of massive type I interferon (IFN-I) production are able to exert cytotoxic effector functions. However, diversification upon toll like receptor (TLR)-induced activation leads to highly heterogeneous responses that have not been fully characterized yet. Using droplet-based microfluidics, we showed that upon TLR7/8 and TLR9-induced single-cell activation only 1-3% secretes IFNα, and only small fractions upregulate cytotoxicity markers. Interestingly, this 1-3% of early IFN-producing pDCs, also known as first responders, express high levels of programmed death-ligand 1 (PD-L1) and TNF-related apoptosis-inducing ligand (TRAIL), which makes these hybrid cells similar to earlier described IFN-I producing killer pDCs (IKpDCs). IFN-I priming increases the numbers of IFNα producing cells up to 40%, but does not significantly upregulate the cytotoxicity markers. Besides, these so-called second responders do not show a cytotoxic phenotype as potent as observed for the first responders. Overall, our results indicate that the first responders are the key drivers orchestrating population wide IFN-I responses and possess high cytotoxic potential.

CXCL4 triggers monocytes and macrophages to produce PDGF-BB, culminating in fibroblast activation: Implications for systemic sclerosis.

OBJECTIVE: To analyze how monocyte and macrophage exposure to CXCL4 induces inflammatory and fibrotic processes observed in Systemic sclerosis (SSc) patients. METHODS: In six independent experiments, monocytes of healthy controls (HC) and SSc patients were stimulated with CXCL4, TLR-ligands, IFNɑ or TGFß and the secretion of cytokines in the supernatant was assessed by multiplex immunoassays. PDGF-BB production by monocyte-derived macrophages was quantified using immunoassays. The number of monocytes and PDGF-BB in circulation was quantified in HC and SSc patients with the Sysmex XT-1800i haematology counter and immunoassays. Intracellular PDGF-BB was quantified in monocytes by Western blot. PDGF-receptor inhibition was achieved using siRNA-mediated knockdown or treatment with Crenolanib. The production of inflammatory mediators and extracellular matrix (ECM) components by dermal fibroblasts was analyzed by qPCR, ELISA and ECM deposition assays. RESULTS: SSc and HC monocytes released PDGF-BB upon stimulation with CXCL4. Conversely, TLR ligands, IFNɑ or TGFß did not induce PDGF-bb release. PDGF-BB plasma levels were significantly (P = 0.009) higher in diffuse SSc patients (n = 19), compared with HC (n = 21). In healthy dermal fibroblasts, PDGF-BB enhanced TNFɑ-induced expression of inflammatory cytokines and increased ECM production. Comparable results were observed in fibroblasts cultured in supernatant taken from macrophages stimulated with CXCL4. This effect was almost completely abrogated by inhibition of the PDGF-receptor using Crenolanib. CONCLUSION: Our findings demonstrate that CXCL4 can drive fibroblast activation indirectly via PDGF-BB production by myeloid cells. Hence, targeting PDGF-BB or CXCL4-induced PDGF-BB release could be clinically beneficial for patients with SSc.

Low RUNX3 expression alters dendritic cell function in patients with systemic sclerosis and contributes to enhanced fibrosis.

OBJECTIVES: Systemic sclerosis (SSc) is an autoimmune disease with unknown pathogenesis manifested by inflammation, vasculopathy and fibrosis in skin and internal organs. Type I interferon signature found in SSc propelled us to study plasmacytoid dendritic cells (pDCs) in this disease. We aimed to identify candidate pathways underlying pDC aberrancies in SSc and to validate its function on pDC biology. METHODS: In total, 1193 patients with SSc were compared with 1387 healthy donors and 8 patients with localised scleroderma. PCR-based transcription factor profiling and methylation status analyses, single nucleotide polymorphism genotyping by sequencing and flow cytometry analysis were performed in pDCs isolated from the circulation of healthy controls or patients with SSc. pDCs were also cultured under hypoxia, inhibitors of methylation and hypoxia-inducible factors and runt-related transcription factor 3 (RUNX3) levels were determined. To study Runx3 function, Itgax-Cre:Runx3f/f mice were used in in vitro functional assay and bleomycin-induced SSc skin inflammation and fibrosis model. RESULTS: Here, we show downregulation of transcription factor RUNX3 in SSc pDCs. A higher methylation status of the RUNX3 gene, which is associated with polymorphism rs6672420, correlates with lower RUNX3 expression and SSc susceptibility. Hypoxia is another factor that decreases RUNX3 level in pDC. Mouse pDCs deficient of Runx3 show enhanced maturation markers on CpG stimulation. In vivo, deletion of Runx3 in dendritic cell leads to spontaneous induction of skin fibrosis in untreated mice and increased severity of bleomycin-induced skin fibrosis. CONCLUSIONS: We show at least two pathways potentially causing low RUNX3 level in SSc pDCs, and we demonstrate the detrimental effect of loss of Runx3 in SSc model further underscoring the role of pDCs in this disease.