Novel Interleukin-11 Inhibitors Attenuate Collagen Production in Patient-Derived Synovial Fibroblasts

Abstract

Purpose: Arthrofibrosis (AF), or excessive joint scarring, is a debilitating condition that causes pain and stiffness secondary to osteoarthritis and is often worsened by the surgical trauma of total knee arthroplasty (TKA). The underlying pathology involves dysregulated transforming growth factor-beta 1 (TGF-β1) signaling, which drives fibrogenesis. However, evidence from other organ systems suggests that interleukin-11 (IL-11) mediates fibrosis downstream of TGF-β1. This study examines the relationship between IL-11 and synovial fibrosis, a hallmark of AF, in patients with end-stage knee osteoarthritis (kOA) and evaluates the potential of novel small-molecule IL-11 inhibitors (NMX compounds) as a therapeutic option.

Methods: Synovial fluid and tissue were collected from 24 patients undergoing TKA for kOA, who were divided into low (n = 12) and high (n = 12) fibrosis severity groups. Enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN) and quantitative immunodetection were used to analyze IL-11 and TGF-β1 levels in synovial fluid and to relate them to histological fibrosis status. The effectiveness of NMX compounds (NM922, NM1157, and NM1332) was then tested on commercial synoviocytes and patient-derived fibroblastic synovial cells (FSCs) by measuring IL-11 and collagen type 1 (COL1) production before and during TGF-β1 stimulation in vitro. Parametric tests were employed to compare groups and evaluate associations.

Results: IL-11 and TGF-β1 levels in patient-derived synovial fluid were strongly correlated (R = 0.602; p = 0.003), and each was significantly linked to the severity of synovial fibrosis (R = 0.759; p = 0.0097 and R = 0.527; p < 0.0001, respectively). In vitro, NMX compound treatment reduced TGF-β1-induced IL-11 and COL1 by over 40% compared to untreated controls. Notably, patient-derived FSCs conserved a hyper-fibrotic phenotype, with baseline production of IL-11 and COL1 elevated by 182.86% and 139.63%, respectively, compared to commercial synoviocytes. In the pathologically primed FSCs, the lead compound NM1157 effectively countered fibrogenic stimulation, significantly reducing COL1 production by 44.5% (p = 0.0189) and IL-11 secretion by 28.4% (p = 0.0415).

Conclusions: Our findings demonstrate a strong connection between IL-11 and synovial fibrosis status in patients with advanced kOA, indicating that IL-11 is a key mediator of this process. The ability of the new NMX compounds to significantly reduce fibrotic markers in patient-derived cells offers a promising preclinical basis for developing targeted therapies to prevent or treat AF attributable to kOA or other arthropathies and traumatic joint injuries.

Keywords: interleukin 11; knee arthrofibrosis; osteoarthritis (oa); synovial membrane; transforming growth factor-β1; type 1 collagen.

Figure 1. Elevated IL-11 and TGF-β1 levels correlate with high-grade synovial fibrosis in kOA patients

(A) Patients undergoing TKA for kOA were categorized based on (B) low (n=12) and (C) high (n=12) histological measures of synovial fibrosis severity according to established guidelines using picrosirius (PS) fluorescence. Bars = 50 μm in representative confocal photomicrographs. (D) Synovial fluid concentrations of (D) IL-11 and (E) TGF-β1 collected from these patients during TKA were measured using competitive and sandwich ELISA, respectively, and compared as mean ± SEM with Student’s t-test (*p < 0.05 and ****p < 0.0001). (F) The levels of these two cytokines were analyzed through Pearson’s correlation, revealing a moderately high association (R = 0.6019; p = 0.0030). kOA: knee osteoarthritis, TKA: total knee arthroplasty, IL-11: interleukin-11, TGF-β1: transforming growth factor-beta 1, ELISA: enzyme-linked immunosorbent assay.

Figure 2. IL-11 and TGF-β1 levels in synovial fluid correlate with fibrosis severity and their distribution within synovial tissue

(A) IL-11 and TGF-β1 concentrations found in synovial fluid show a high (R = 0.759; p = 0.0097) to moderate (R = 0.527; p < 0.0001) Pearson’s correlation with histological severity scores, indicating fibrosis levels. To examine these trends in tissue, IL-11 and TGF-β1 were labeled in synovial tissues collected from all patients in the (B) low and (C) high cohorts using quantitative immunohistochemistry (qIHC). Bars = 50 μm in representative confocal photomicrographs. Spatial co-distribution of (D) both cytokines was compared using Student’s t-test (***p < 0.001 and ****p < 0.0001). IL-11: interleukin-11, TGF-β1: transforming growth factor-beta 1.

Figure 3. Impact of each NMX compound on HFLS metabolic activity

The metabolic activity of HFLS was assessed using the MTT assay after treatment with increasing concentrations (0.5-20 µM) of NM922, NM1157, and NM1332. Data are presented as the mean percent relative absorbance compared to the untreated control (0 µM), which is set to 100%. Significance was determined using a one-way ANOVA with Dunnett's multiple comparisons test. Asterisks denote significance compared to the untreated control: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. HFLS: human fibroblast-like synoviocytes, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 4. NMX compounds minimize IL-11 and collagen production in TGF-β1-stimulated, commercial synoviocytes

(A) To simulate a severe diseased state, a commercial line of otherwise healthy human fibroblastic synovial cells (HFLS) was incubated with 4 ng/mL of recombinant TGF-β1 for 48 hours to induce a fibrotic phenotype while being treated with NMX compounds diluted to 0.5 and 2.0 μM. Graphic created in BioRender: Reproduced from Marrero (2025) https://BioRender.com/89a2snn [29], licensed under the Creative Commons Attribution 4.0 International License (CC-BY 4.0). To evaluate the prophylactic effect, the same protocol was applied to HFLS treated for 24 hours with NMX before 48-hour stimulation with subsequent treatment (methodology diagram not shown). At the endpoints of the (B) 48-hour and (C) 72-hour experiments (24-hour pre-treatment plus 48-hour treatment and fibrogenic stimulation), secreted IL-11 and produced COL1 were measured using competitive ELISA (with an equivalent kit to that used with the kOA patient synovial fluid) and sandwich ELISA, respectively. Data are presented as mean ± SEM, compared by two-way ANOVA. For the data shown in B and C, statistical significance was assessed using a two-way ANOVA followed by a Dunnett's multiple comparisons test, comparing each treatment group to the TGF-β1-stimulated control. In the graphs, treatment groups that do not share a letter are statistically significantly different from one another. IL-11: interleukin-11, TGF-β1: transforming growth factor-beta 1, ELISA: enzyme-linked immunosorbent assay, kOA: knee osteoarthritis, SGM: synoviocyte growth media, COL1: collagen type 1, tx: treatment, US/NT: unstimulated cells cultured in SGM alone.

Figure 5. NM1157 suppresses collagen production in patient-derived fibroblastic synovial cells

(A) Fibroblastic synovial cells (FSCs) derived from kOA patients with severe fibrosis were isolated from banked synovial tissue collected at the time of TKA. Graphic created in BioRender: Reproduced from Marrero (2025) https://BioRender.com/wy5d4jp [30], licensed under the Creative Commons Attribution 4.0 International License (CC-BY 4.0). Banked tissues were retrieved in batches and processed through rapid warming, dissociation, and cell purification for growth under standard culture conditions, up to the third passage. High fibrosis patient-derived FSCs (n = 12) were then plated for 48-hour NM1157 treatment at an effective dose of 0.5 µM under TGF-β1 stimulation. Similar to experimental HFLS under the same conditions, (B) IL-11 and (C) COL1 concentrations were compared among US/NT, TGF-β1 stimulated, and NM1157-treated groups under TGF-β1 stimulation. Data are presented as mean ± SEM with p-values shown and calculated by one-way ANOVA with Tukey's multiple comparisons test. kOA: knee osteoarthritis, TKA: total knee arthroplasty, TGF-β1: transforming growth factor-beta 1, HFLS: human fibroblast-like synoviocytes, IL-11: interleukin-11, COL1: collagen type 1, US/NT: unstimulated cells cultured in SGM alone, FBS: fetal bovine serum, DMSO: dimethyl sulfoxide.