Abstract Background Analyses of lymphoid organs are required to further elucidate the pathogenesis of inflammatory diseases like rheumatoid arthritis (RA). Yet, invasive tissue collection methods are scarcely applied, because they are often considered burdensome, although patients do not always consider invasive methods as a high burden. We aimed to investigate the perspectives of study participants undergoing ultrasound-guided inguinal lymph node (LN) needle biopsy sampling and determine the molecular and cellular quantity and quality of LN biopsies. Methods Together with patient research partners, questionnaires were developed to evaluate the motives, expectations, and experiences of participants undergoing a LN biopsy. Healthy controls and RA(-risk) patients were asked to complete these questionnaires before and after the procedure. RNA and lymphocyte yields from obtained LN biopsies were also calculated. Results We included 50 individuals, of which 43 (86%) reported their pre- and post-procedure experiences. The median reported pain on a 5-point Likert scale (1 not to 5 very painful) was 1. Interestingly, almost all ( n = 32; 74%) study participants would undergo a second procedure and more than half ( n = 23; 54%) would encourage others to take part in the LN biopsy study. Motives for current and future participation were mostly altruistic. Inguinal hematoma occurred frequently, but no other significant or unexpected complications ensued. The LN biopsies yielded sufficient and high-quality RNA and lymphocyte numbers. Conclusions Ultrasound-guided inguinal LN biopsy sampling is well-tolerated, safe, and provides sufficient material for further molecular and cellular analyses. Our participants’ positive experiences endorse the application of this research tool to further elucidate the pathogenesis of RA and other inflammatory diseases.
The tumor necrosis factor (TNF) and IL-23/IL-17 axes are the main therapeutic targets in spondyloarthritis. Despite the clinical efficacy of blocking either pathway, monotherapy does not induce remission in all patients and its effect on new bone formation remains unclear. We aimed to study the effect of TNF and IL-17A dual inhibition on clinical disease and structural damage using the HLA-B27/human β2-microglobulin transgenic rat model of SpA. Immunized rats were randomized according to arthritis severity, 1 week after arthritis incidence reached 50%, to be treated twice weekly for a period of 5 weeks with either a dual blockade therapy of an anti-TNF antibody and an anti-IL-17A antibody, a single therapy of either antibody, or PBS as vehicle control. Treatment-blinded observers assessed inflammation and structural damage clinically, histologically and by micro-CT imaging. Both single therapies as well as TNF and IL-17A dual blockade therapy reduced clinical spondylitis and peripheral arthritis effectively and similarly. Clinical improvement was confirmed for all treatments by a reduction of histological inflammation and pannus formation (p < 0.05) at the caudal spine. All treatments showed an improvement of structural changes at the axial and peripheral joints on micro-CT imaging, with a significant decrease for roughness (p < 0.05), which reflects both erosion and new bone formation, at the level of the caudal spine. The effect of dual blockade therapy on new bone formation was more prominent at the axial than the peripheral level. Collectively, our study showed that dual blockade therapy significantly reduces inflammation and structural changes, including new bone formation. However, we could not confirm a more pronounced effect of dual inhibition compared to single inhibition.
Background The cellular and molecular mechanisms driving inflammation and structural remodelling in spondyloarthritis (SpA) remain largely unknown, though the IL-23/IL-17 pathway can contribute to synovial inflammation and radiographic progression. Objectives To investigate the molecular pathways affected by IL-17A blockade (IL-17Ai) with secukinumab in SpA synovitis, and assess if this response is tissue- and/or treatment-specific. Methods Synovial biopsies were obtained from peripheral SpA patients by needle arthroscopy before and after 12 weeks of IL-17Ai with secukinumab (n=12), and analyzed by RNA-sequencing and qPCR. We performed pathway enrichment analysis of the differentially expressed genes (DEGs) to identify pathways modulated after treatment. We compared the synovial tissue response in patients with psoriatic arthritis (PsA) in our cohort (n=7) with open source gene expression data of skin biopsies of psoriasis patients receiving secukinumab (n=22)[1] and of synovial biopsies of PsA patients receiving IL-12p40/IL-23p40 blockade (n=7)[2]. Results IL-17Ai significantly modulated the expression of 1255 genes (549 up- and 706 down-regulated, FDR 0.1) in the synovium at week 12 compared to baseline (Figure 1). Genes downregulated upon IL-17Ai were significantly enriched in GO terms and KEGG pathways related to immune and inflammatory responses, including neutrophil and monocyte chemotaxis, TNF-mediated, NF-κB-, Wnt-, and JAK-STAT signalling pathways, and, importantly, bone-remodelling responses, such as osteoblast and osteoclast differentiation. Upregulated genes are enriched in JNK-, MAPK-, Wnt-, and PI3K-Akt signalling and negative regulation of osteoblast differentiation. We validated differential expression of selected genes from several pathways by qPCR, including: IL1B , p=0.027; CXCL6 , p=0.020; ADAMTS4 , p=0.002; MMP3 , p=0.020; and CHRDL2 , p= 0.039. Figure 1. Heatmap of differentially expressed genes (DEGs) and pathway enrichment analyses of changes in peripheral SpA synovium 12 weeks after anti-IL-17A treatment (IL-17Ai). A. Hierarchical cluster analysis of the top 100 most significant DEGs (FDR < 0.1) modulated by IL-17Ai separates pre- and post-treated groups. Normalized and scaled log2 gene expression levels are shown. B. Pathway enrichment analysis through DAVID. Enriched (p < 0.05) gene ontology terms from IL-17Ai-induced up- and down-regulated DEGs in peripheral SpA synovium are shown. To assess if this response is tissue- and/or treatment-specific, we compared changes in gene expression by IL-17Ai in PsA synovium versus psoriatic skin, and in the PsA synovium after IL-17Ai versus IL-12p40/IL-23p40 blockade. While many inflammation-related GO terms and KEGG pathways were over-represented in both tissues and treatments, NF-κB-, JAK-STAT-, and PI3K-Akt-signaling were enriched in DEGs in both skin and synovium after IL-17Ai, whereas JNK cascade, IL-17 signalling pathway and Th17 cell differentiation were overrepresented in DEGs after IL-17Ai in the synovium specifically. Remarkably, IL-17Ai, but not IL-12p40/IL-23p40 blockade, modulated multiple bone-remodelling related pathways. Also, IL-17Ai modulated ossification and collagen catabolic process terms in PsA synovium and psoriatic skin in the opposite direction: these terms were over-represented in downregulated genes in synovium, but in upregulated genes in skin. Accordingly, genes upregulated after IL-17Ai were enriched in negative regulation of osteoblast differentiation in the synovium, but in positive regulation of osteoblast differentiation in the skin. Conclusion These first in vivo human data provide molecular confirmation of previously reported animal data[3] that demonstrated down-modulation of disease-relevant immune and stromal pathways in the synovium in response to IL-17Ai. References [1]Krueger JG et al . J Allergy Clin Immunol. 2019. Sep;144(3):750-763 [2]Fiechter RH et al . Front Immunol. 2021. Mar 4;12:611656 [3]Van Tok MN et al . Arthritis Rheumatol. 2019. Apr;71(4):612-625 Disclosure of Interests Renée Fiechter: None declared, Leonieke van Mens: None declared, Ihsan Hammoura: None declared, Henriëtte de Jong: None declared, Desire Pots: None declared, Inka Fluri: None declared, Sander Tas: None declared, Dominique Baeten Employee of: Current employee of UCB Pharma, Marleen G.H. van de Sande Consultant of: Novartis and AbbVie, Grant/research support from: Janssen, Novartis and Eli Lilly, Nataliya Yeremenko: None declared.
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Background: Psoriatic arthritis (PsA) is a chronic inflammatory joint disease within the spondyloarthritis (SpA) spectrum. TNF and IL17/IL23 pathways play a key role in SpA pathogenesis. Blocking of IL12p40/IL23p40 has been shown to effectively reduce disease activity in PsA [1,2]. It is however incompletely understood how IL12p40/IL23p40 blockade affects local inflammatory processes. Objectives: To investigate the cellular and molecular pathways affected by IL12p40/IL23p40 blockade with ustekinumab in PsA patients (pts). Methods: Eleven male PsA pts with at least 1 inflamed knee or ankle joint, who were scheduled to start ustekinumab treatment, were included in a 24-week single-center open-label study. All pts received ustekinumab 45 mg/sc according to standard care at week (W) 0, 4 and 16. Besides clinical outcomes, needle arthroscopic synovial tissue (ST) biopsy samples were obtained from an inflamed knee or ankle joint at baseline (BL), W12 and W24. ST samples were analyzed by immunohistochemistry (IHC), RNA sequencing and real-time quantitative polymerase chain reaction (qPCR) analysis. Results: Paired BL and W12, and paired BL, W12 and W24 ST samples were available of 9 and 6 pts, respectively. Two pts only underwent BL ST sampling (pt refusal; withdrawal after the W12 clinical visit). Two pts were excluded after W12 because of treatment adjustments. Of 1 pt no ST was obtained at W24 due to technical difficulties. Eight pts finished 24 weeks of clinical follow-up. No serious adverse events were observed. At W12 6/11 pts met ACR20, 2/11 met ACR50 and 1/11 met ACR70 improvement criteria, at W24 this was 3/8, 2/8 and 1/8 pts, respectively. Significant improvements between BL and W12 and/or W24 were seen in clinical (TJC, PASI, BASDAI) and serological markers (CRP and ESR), Table 1. IHC showed a significant decrease in sublining macrophages, a sensitive biomarker of an inflammatory response in peripheral SpA, of BL 2[1-3] vs W12 1.5[0-2]p=0.020, but not W24 1[0.5-2.5]ns. Other synovial infiltrating cells were not significantly decreased. Significant downregulation of MMP3 (p=0.047) and IL-23p19 (p=0.046), but not IL6, TNF or IL12p40 were seen with qPCR analysis at W12. RNA seq analysis showed 178 significantly differentially expressed genes between BL and W12 (FDR 0.1). Gene ontology and KEGG terms enrichment analyses identified overrepresentation of MAPK and PI3K-Akt signalling pathways among the down-regulated genes and WNT signalling pathway among the up-regulated genes. Gene expression was confirmed by qPCR analysis. Table 1. Baseline (n=11) Week 12 (n=11) Week 24 (n=8) p-value* p-value** TJC 1 (0-5) 1 (1-2) 0 (0-0.75) 0.168 0.034 SJC 2 (1-3) 2 (1-3) 1.5 (1-2) 0.394 0.715 VAS patient global 28 (16-55) 22 (12-46) 45 (2.8-50.5) 0.423 0.779 PASI 3.2 (1.2-7.4) 0.8 (0-1.8) 0 (0-2.4) 0.028 0.046 BASDAI 3.2 (2.4-4.7) 1.9 (1-3.8) 2.1 (0.4-4.2) 0.008 0.161 CRP (mg/L) 8.5 (1.7-16.4) 1.6 (0.6-3) 1.0 (0.6-7.0) 0.008 0.012 ESR (mm/hr) 20 (8-35) 6 (2-17) 8 (2-20.5) 0.008 0.017 TJC/SJC tender/swollen joint count; VAS visual analogue scale; PASI psoriasis area severity index; BASDAI Bath Ankylosing Spondylitis Disease Activity Index; CRP C-reactive protein; ESR erythrocyte sedimentation rate; All values are presented as median (IQR) p-value for the comparison of baseline and week 12 (*) or week 24 (**) Conclusion: Ustekinumab treatment reduced synovial inflammation and modulated specific molecular pathways, however inflammation was not completely resolved. Future studies comparing histological and gene expression data between different treatments targeting IL17/IL23 axis will show which changes are treatment-specific and which reflect downregulation of local inflammation. References: [1]McInnes et al. Lancet. 2013;382:780–789. [2]Kavanaugh et al. Ann Rheum Dis. 2014;73:990–999. Work was financially supported by an unrestricted grand of Janssen Pharmaceutica Disclosure of Interests: Renée Fiechter: None declared, Henriëtte de Jong: None declared, Leonieke van Mens: None declared, Inka Fluri: None declared, Sander Tas: None declared, Dominique Baeten Employee of: UCB Pharma, Nataliya Yeremenko: None declared, Marleen G.H. van de Sande Grant/research support from: Novartis, Eli lily, UCB, Jansen, Consultant of: Abbvie, Novartis, Eli lily, MSD
Abstract C-reactive protein (CRP) is an acute-phase protein produced in high quantities by the liver in response to infection and during chronic inflammatory disorders. Although CRP is known to facilitate the clearance of cell debris and bacteria by phagocytic cells, the role of CRP in additional immunological functions is less clear. This study shows that complexed CRP (phosphocholine [PC]:CRP) (formed by binding of CRP to PC moieties), but not soluble CRP, synergized with specific TLRs to posttranscriptionally amplify TNF, IL-1β, and IL-23 production by human inflammatory macrophages. We identified FcγRI and IIa as the main receptors responsible for initiating PC:CRP–induced inflammation. In addition, we identified the underlying mechanism, which depended on signaling through kinases Syk, PI3K, and AKT2, as well as glycolytic reprogramming. These data indicate that in humans, CRP is not only a marker but also a driver of inflammation by human macrophages. Therefore, although providing host defense against bacteria, PC:CRP–induced inflammation may also exacerbate pathology in the context of disorders such as atherosclerosis.
Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown etiology. One of the key factors associated with SLE pathogenesis is excessive production of type I interferons (IFNs). This could result from increased activation of type I IFN-stimulating pathways, but also from decreased activation of type I IFN-inhibitory pathways. Recently, we have identified that immunoglobulin (Ig)G immune complexes strongly inhibit type I IFN production in healthy individuals by inhibitory signaling through Fcγ receptor IIa (FcγRIIa) on dendritic cells (DCs). Because, in SLE patients, immune complexes are characteristically present, we assessed whether FcγR-induced suppression of type I IFN is functional in DCs of SLE patients. We divided the SLE patients into one group without, and one group with, previous major organ involvement, for which we chose nephritis as a prototypical example. We show that DCs of lupus nephritis patients displayed impaired FcγR-mediated type I IFN inhibition compared to SLE patients without major organ involvement or healthy controls. We verified that this impaired type I IFN inhibition was not related to differences in disease activity, medication, FcγRIIa expression or expression of IFN regulatory transcription factors (IRF)1 and IRF5. In addition, we identified that DCs of lupus nephritis patients show increased FcγR-induced interleukin (IL)-1β production, which is another important cytokine that promotes kidney inflammation. Taken together, these data indicate that DCs of lupus nephritis patients display altered FcγR-mediated regulation of cytokine production, resulting in elevated levels of type I IFN and IL-1β. This dysregulation may contribute to the development of nephritis in SLE patients.
Background: Psoriatic arthritis (PsA) is a chronic inflammatory joint disease within the spondyloarthritis spectrum. IL-12p40/IL-23p40 blockade reduces PsA disease activity, but its impact on synovial inflammation remains unclear. Objectives: To investigate the cellular and molecular pathways affected by IL-12p40/IL-23p40 blockade with ustekinumab in the synovium of PsA patients. Methods: Eleven PsA patients with at least one inflamed knee or ankle joint were included in a 24-week single-center open-label study and received ustekinumab 45 mg/sc according to standard care at week 0, 4, and 16. Besides clinical outcomes, synovial tissue (ST) samples were obtained by needle arthroscopy from an inflamed knee or ankle joint at baseline, week 12 and 24 and analyzed by immunohistochemistry, RNA-sequencing and real-time quantitative polymerase chain reaction (qPCR). Results: We obtained paired baseline and week 12, and paired baseline, week 12 and 24 ST samples from nine and six patients, respectively. Eight patients completed 24 weeks of clinical follow-up. At 12 weeks 6/11 patients met ACR20, 2/11 met ACR50 and 1/11 met ACR70 improvement criteria, at 24 weeks this was 3/8, 2/8 and 1/8 patients, respectively. Clinical and serological markers improved significantly. No serious adverse events occurred. We observed numerical decreases of all infiltrating cell subtypes at week 12, reaching statistical significance for CD68+ sublining macrophages. For some cell types this was even more pronounced at week 24, but clearly synovial inflammation was incompletely resolved. IL-17A and F, TNF, IL-6, IL-8, and IL-12p40 were not significantly downregulated in qPCR analysis of W12 total biopsies, only MMP3 and IL-23p19 were significantly decreased. RNA-seq analysis revealed 178 significantly differentially expressed genes between baseline and 12 weeks (FDR 0.1). Gene Ontology and KEGG terms enrichment analyses identified overrepresentation of biological processes as response to reactive oxygen species, chemotaxis, migration and angiogenesis as well as MAPK-ERK and PI3K-Akt signaling pathways among the downregulated genes and of Wnt signaling pathway among the upregulated genes. Furthermore, ACR20 responders and non-responders differed strikingly in gene expression profiles in a post-hoc exploratory analysis. Conclusions: Ustekinumab suppresses PsA synovial inflammation through modulation of multiple signal transduction pathways, including MAPK-ERK, Wnt and potentially PI3K-Akt signaling rather than by directly impacting the IL-17 pathway.
