Changes in a few key transcriptional regulators can lead to different biological states. Extracting the key gene regulators governing a biological state allows us to gain mechanistic insights. Most ...current tools perform pathway/GO enrichment analysis to identify key genes and regulators but tend to overlook the gene/protein regulatory interactions. Here we present RegEnrich, an open-source Bioconductor R package, which combines differential expression analysis, data-driven gene regulatory network inference, enrichment analysis, and gene regulator ranking to identify key regulators using gene/protein expression profiling data. By benchmarking using multiple gene expression datasets of gene silencing studies, we found that RegEnrich using the GSEA method to rank the regulators performed the best. Further, RegEnrich was applied to 21 publicly available datasets on in vitro interferon-stimulation of different cell types. Collectively, RegEnrich can accurately identify key gene regulators from the cells under different biological states, which can be valuable in mechanistically studying cell differentiation, cell response to drug stimulation, disease development, and ultimately drug development.
Antiplatelet-antibody-producing B cells play a key role in immune thrombocytopenia (ITP) pathogenesis; however, little is known about T-cell dysregulations that support B-cell differentiation. During ...the past decade, T follicular helper cells (TFHs) have been characterized as the main T-cell subset within secondary lymphoid organs that promotes B-cell differentiation leading to antibody class-switch recombination and secretion. Herein, we characterized TFHs within the spleen of 8 controls and 13 ITP patients. We show that human splenic TFHs are the main producers of interleukin (IL)-21, express CD40 ligand (CD154), and are located within the germinal center of secondary follicles. Compared with controls, splenic TFH frequency is higher in ITP patients and correlates with germinal center and plasma cell percentages that are also increased. In vitro, IL-21 stimulation combined with an anti-CD40 agonist antibody led to the differentiation of splenic B cells into plasma cells and to the secretion of antiplatelet antibodies in ITP patients. Overall, these results point out the involvement of TFH in ITP pathophysiology and the potential interest of IL-21 and CD40 as therapeutic targets in ITP.
•Human splenic TFH expansion during ITP participates in B-cell differentiation and antiplatelet-antibody production.•IL-21 and CD40 are key TFH molecules that could be promising targets in the treatment of ITP.
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.
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.
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.
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.
Interleukin (IL)‐12 and IL‐23 are pro‐inflammatory cytokines produced by dendritic cells (DCs) and associated with Psoriasis (Pso) and Psoriatic Arthritis (PsA) pathogenesis. Tofacitinib, a Janus ...kinase inhibitor, effectively suppresses inflammatory cascades downstream the IL‐12/IL‐23 axis in Pso and PsA patients. Here, we investigated whether Tofacitinib directly regulates IL‐12/IL‐23 production in DCs, and how this regulation reflects responses to Tofacitinib in Pso patients. We treated monocyte‐derived dendritic cells and myeloid dendritic cells with Tofacitinib and stimulated cells with either lipopolysaccharide (LPS) or a combination of LPS and IFN‐γ. We assessed gene expression by qPCR, obtained skin microarray and blood Olink data and clinical parameters of Pso patients treated with Tofacitinib from public data sets. Our results indicate that in DCs co‐stimulated with LPS and IFN‐γ, but not with LPS alone, Tofacitinib leads to the decreased expression of IL‐23/IL‐12 shared subunit IL12B (p40). In Tofacitinib‐treated Pso patients, IL‐12 expression and psoriasis area and severity index (PASI) are significantly reduced in patients with higher IFN‐γ at baseline. These findings demonstrate for the first time that Tofacitinib suppresses IL‐23/IL‐12 shared subunit IL12B in DCs upon active IFN‐γ signaling, and that Pso patients with higher IFN‐γ baseline levels display improved clinical response after Tofacitinib treatment.
Primary Sjögren's syndrome (pSS) is a systemic auto-immune disease typified by dryness of the mouth and eyes. A majority of patients with pSS have a type-I interferon (IFN)-signature, which is ...defined as the increased expression of IFN-induced genes in circulating immune cells and is associated with increased disease activity. As plasmacytoid dendritic cells (pDC) are the premier type-I IFN-producing cells and are present at the site of inflammation, they are thought to play a significant role in pSS pathogenesis. Considering the lack of data on pDC regulation and function in pSS patients, we here provided the first in-depth molecular characterization of pSS pDCs. In addition, a group of patients with non-Sjögren's sicca (nSS) was included; these poorly studied patients suffer from complaints similar to pSS patients, but are not diagnosed with Sjögren's syndrome. We isolated circulating pDCs from two independent cohorts of patients and controls (each
= 31) and performed RNA-sequencing, after which data-driven networks and modular analysis were used to identify robustly reproducible transcriptional "signatures" of differential and co-expressed genes. Four signatures were identified, including an IFN-induced gene signature and a ribosomal protein gene-signature, that indicated pDC activation. Comparison with a dataset of
activated pDCs showed that pSS pDCs have higher expression of many genes also upregulated upon pDC activation. Corroborating this transcriptional profile, pSS pDCs produced higher levels of pro-inflammatory cytokines, including type-I IFN, upon
stimulation with endosomal Toll-like receptor ligands. In this setting, cytokine production was associated with expression of hub-genes from the IFN-induced and ribosomal protein gene-signatures, indicating that the transcriptional profile of pSS pDCs underlies their enhanced cytokine production. In all transcriptional analyses, nSS patients formed an intermediate group in which some patients were molecularly similar to pSS patients. Furthermore, we used the identified transcriptional signatures to develop a discriminative classifier for molecular stratification of patients with sicca. Altogether, our data provide in-depth characterization of the aberrant regulation of pDCs from patients with nSS and pSS and substantiate their perceived role in the immunopathology of pSS, supporting studies that target pDCs, type-I IFNs, or IFN-signaling in pSS.
Dermal fibroblasts are strategically positioned underneath the basal epidermis layer to support keratinocyte proliferation and extracellular matrix production. In inflammatory conditions, these ...fibroblasts produce cytokines and chemokines that promote the chemoattraction of immune cells into the dermis and the hyperplasia of the epidermis, two characteristic hallmarks of psoriasis. However, how dermal fibroblasts specifically contribute to psoriasis development remains largely uncharacterized. In this study, we investigated through which cytokines and signaling pathways dermal fibroblasts contribute to the inflammatory features of psoriatic skin. We show that dermal fibroblasts from lesional psoriatic skin are important producers of inflammatory mediators, including IL-6, CXCL8, and CXCL2. This increased cytokine production was found to be regulated by ZFP36 family members ZFP36, ZFP36L1, and ZFP36L2, RNA-binding proteins with mRNA-degrading properties. In addition, the expression of ZFP36 family proteins was found to be reduced in chronic inflammatory conditions that mimic psoriatic lesional skin. Collectively, these results indicate that dermal fibroblasts are important producers of cytokines in psoriatic skin and that reduced expression of ZFP36 members in psoriasis dermal fibroblasts contributes to their inflammatory phenotype.
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Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterized by infiltration of the exocrine glands and prominent B cell hyperactivity. Considering the key role of monocytes in ...promoting B cell hyperactivity, we performed RNA-sequencing analysis of CD14
monocytes from patients with pSS, non-Sjögren's sicca (nSS), and healthy controls (HC). We demonstrated that the transcriptomic profile of pSS patients is enriched in intermediate and non-classical monocyte profiles, and confirmed the increased frequency of non-classical monocytes in pSS patients by flow-cytometry analysis. Weighted gene co-expression network analysis identified four molecular signatures in monocytes from pSS patients, functionally annotated for processes related with translation, IFN-signaling, and toll-like receptor signaling. Systemic and local inflammatory features significantly correlated with the expression of these signatures. Furthermore, genes highly associated with clinical features in pSS were identified as hub-genes for each signature. Unsupervised hierarchical cluster analysis of the hub-genes identified four clusters of nSS and pSS patients, each with distinct inflammatory and transcriptomic profiles. One cluster showed a significantly higher percentage of pSS patients with higher prevalence of anti-SSA autoantibodies, interferon-score, and erythrocyte sedimentation rate compared to the other clusters. Finally, we showed that the identified transcriptomic differences in pSS monocytes were induced in monocytes of healthy controls by exposure to serum of pSS patients. Representative hub-genes of all four signatures were partially inhibited by interferon-α/β receptor blockade, indicating that the circulating inflammatory mediators, including type I interferons have a significant contribution to the altered transcriptional profile of pSS-monocytes. Our study suggests that targeting key circulating inflammatory mediators, such as type I interferons, could offer new insights into the important pathways and mechanisms driving pSS, and holds promise for halting immunopathology in Sjögren's Syndrome.
Abstract
Objective
Class 3 semaphorins regulate diverse cellular processes relevant to the pathology of RA, including immune modulation, angiogenesis, apoptosis and invasive cell migration. ...Therefore, we analysed the potential role of class 3 semaphorins in the pathology of RA.
Methods
Protein and mRNA expression in RA synovial tissue, SF and fibroblast-like synoviocytes (FLS) were determined by immunoblotting and quantitative PCR (qPCR). RA FLS migration and invasion were determined using wound closure and transwell invasion assays, respectively. PlexinA1, neuropilin-1 and neuropilin-2 expression was knocked down using small interfering RNA (siRNA). Activation of FLS intracellular signalling pathways was assessed by immunoblotting.
Results
mRNA expression of semaphorins (Sema)3B, Sema3C, Sema3F and Sema3G was significantly lower in the synovial tissue of early arthritis patients at baseline who developed persistent disease compared with patients with self-limiting disease after 2 years follow-up. Sema3B and Sema3F expression was significantly lower in arthritis patients fulfilling classification criteria for RA compared with those who did not. FLS expression of Sema3A was induced after stimulation with TNF, IL-1β or lipopolysaccharides (LPS), while Sema3B and Sema3F expression was downregulated. Exogenously applied Sema3A induced the migration and invasive capacity of FLS, while stimulation with Sema3B or Sema3F reduced spontaneous FLS migration, and platelet-derived growth factor induced cell invasion, effects associated with differential regulation of MMP expression and mediated by the PlexinA1 and neuropilin-1 and -2 receptors.
Conclusion
Our data suggest that modulation of class 3 semaphorin signaling could be a novel therapeutic strategy for modulating the invasive behaviour of FLS in RA.
Objective:Durable blockade of tumour necrosis factor-alpha (TNF-α) in patients with rheumatoid arthritis (RA) suppresses disease activity and its progression. Cardiovascular diseases are 1.5–2-fold ...more frequent in RA patients than in the general population. Although TNF-α has well-established effects on lipid metabolism, the long-term effects of TNF-α blockade on lipid pattern are still unclear. In the present study, we investigated the effects of 1-year therapy with anti-TNF on the lipid profile of RA patients.Methods:Disease activity (DAS28) and plasma lipoproteins concentrations (total, HDL and LDL-cholesterol, triglycerides, ApoA, ApoB) were assessed in 55 RA patients and 55 controls. The whole RA group was followed up for 6 months, and 31 of the patients were followed up for 1 year.Results:In RA patients, DAS28 decreased after 2 weeks from the start of therapy (p<0.001) and remained low during the entire study duration. Short-term effects of anti-TNF on plasma lipid concentrations seemed beneficial and anti-atherogenic. However, these changes did not persist: plasma concentrations of total and LDL-cholesterol and the atherogenic index increased after 6 months and 1 year from the start of therapy. During therapy, the changes in disease activity and inflammatory status were inversely correlated with changes in plasma total and HDL cholesterol levels and positively correlated with the variation of atherogenic index.Conclusion:We conclude that one-year therapy with infliximab is likely to lead to a more pro-atherogenic pattern of the plasma lipids concentrations. However, the overall impact of these changes on the cardiovascular risk is more complex, considering the strong anti-inflammatory effects of anti-TNF drugs.
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