Anti-inflammatory effects of infliximab and methotrexate on peripheral blood and synovial fluid mononuclear cells: ex vivo study

Abstract

Objective

To investigate the effects of methotrexate (MTX) and the tumour necrosis factor inhibitor infliximab (IFX) on immune cells derived from peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) of inflammatory arthritis patients.

Method

Phytohaemagglutinin (PHA)-induced proliferation of healthy donors’ PBMCs and synovial intermediate monocytes (CD14⁺CD16⁺ cells) in SFMCs derived from psoriatic arthritis (PsA) and rheumatoid arthritis (RA) patients was determined by flow cytometry following co-culture with IFX and MTX. PHA-induced interferon-γ (IFN-γ) production in PBMCs was measured by enzyme-linked immunosorbent assay. The drugs’ effect on mRNA expression in SFMCs was determined by quantitative polymerase chain reaction.

Results

The combination of IFX 10 μg/mL + MTX 0.1 μg/mL had the strongest inhibitory effect on PBMC proliferation (91%), followed by MTX 0.1 μg/mL (86%) and IFX 10 μg/mL (49%). In PHA-stimulated PBMCs, IFN-γ production was reduced by IFX 10 μg/mL, MTX 0.1 μg/mL, and IFX 10 μg/mL + MTX 0.1 μg/mL by 68%, 90%, and 85%, respectively. In SFMCs, IFX 10 µg/mL significantly reduced CD14⁺CD16⁺ cells compared to medium (PsA 54%, p < 0.01; RA 46%, p < 0.05), while MTX had no effect on this population. IFX + MTX led to a similar suppression of CD14⁺CD16⁺ cells as achieved by IFX alone. The drugs had different impacts on SFMC gene expression.

Conclusion

Both IFX and MTX effectively inhibited PBMC proliferation and IFN-γ production, but only IFX reduced synovial monocytes and pro-inflammatory gene expression in SFMCs, suggesting a differential impact of IFX and MTX on critical inflammatory cell populations ex vivo.

Inflammatory arthritis (IA), present in rheumatoid arthritis (RA) and psoriatic arthritis (PsA), is characterized by joint pain and swelling as well as systemic inflammation (1, 2). Currently available anti-rheumatic therapies, disease-modifying anti-rheumatic drugs (DMARDs) and biological drugs, improve the quality of life of patients with IA by attenuating inflammation and delaying bone damage. Methotrexate (MTX) has been used as the first line synthetic DMARD in IA treatment. Approximately 30% of RA patients achieve remission with initial MTX monotherapy (3, 4). Tumour necrosis factor (TNF) is an inflammatory cytokine that plays a critical role in the pathogenesis of inflammatory rheumatic diseases (5). Consequently, tumour necrosis factor inhibitors (TNFis) are designed to block the biological function of TNF, with high efficacy in the treatment of RA and PsA. To date, five TNFi drugs have been approved for clinical use: infliximab, etanercept, adalimumab, golimumab, and certolizumab pegol (6, 7). In RA, MTX combined with a TNFi enhances the efficacy of therapy compared to TNFi monotherapy (8). Different immune cell types, such as lymphocytes and monocytes, are involved in the development and progression of IA (9, 10). Immune cells from the circulation are recruited to the synovium and accumulate in the inflamed joint. Intermediate monocytes, characterized by CD14 and CD16 expression, produce inflammatory cytokines and differentiate into inflammatory macrophages (11, 12). In IA, inflammatory responses take place in the blood and synovial fluid (SF). Hence, it is important to understand the impact of anti-rheumatic drugs on inflammatory cells in both compartments. Anti-rheumatic therapies target different immune cell populations and attenuate their activities. TNFi treatment induces apoptosis in monocytes and T cells (13), reducing T-cell activation (14, 15), and MTX decreases T-cell proliferation (16).

We previously showed that TNFi, but not biological agents with different mechanisms of action, such as interleukin-17A (IL-17A) or IL-6 receptor inhibitors, reduced in vitro peripheral blood mononuclear cell (PBMC) proliferation to a similar extent in both patients with PsA and healthy donors (17). We also found that co-culture of synovial fluid mononuclear cells (SFMCs) with TNFi decreased CD14⁺CD16⁺ monocytes ex vivo. The effect of TNFis on the synovial monocytes was unique since other therapeutic agents, namely, glucocorticoids and biologics with different mechanisms of action, did not exhibit such activity (18). In the present study, we investigated the cellular pathways induced by infliximab (IFX) and MTX, alone and in combination, on two different cell populations: (i) phytohaemagglutinin (PHA)-induced proliferative response and interferon-γ (IFN-γ) production in healthy donors’ PBMCs; and (ii) modulation of intermediate monocytes and gene expression in SFMCs derived from patients with IA.

Method

Human subjects

Blood samples were collected from healthy donors (n = 25) at Tel Aviv Sourasky Medical Center (Tel Aviv, Israel). (PBMCs derived from one donor was assessed at two different time-points in the proliferation assay.) SF samples were collected from patients with active PsA and RA patients. PsA patients were diagnosed according the Classification Criteria for Psoriatic Arthritis (CASPAR) (19). RA patients were diagnosed according to the American College of Rheumatology (ACR)/European Alliance of Associations for Rheumatology (EULAR) 2010 classification criteria (20). SF was collected by arthrocentesis from RA and PsA patients who presented with swollen knees at the Rheumatology Department, Tel Aviv Sourasky Medical Center. The procedure was performed by an expert rheumatologist. The primary reasons for joint aspiration were diagnosis and therapeutic relief. SF was obtained from 14 PsA and six RA patients. (SFMCs derived from one PsA patient and two RA patients were assessed at two different time-points in the analysis of CD14⁺CD16⁺ monocytes.) Clinical information, including physical examination of tender and swollen joint counts (28 joints for RA and 66 swollen and 68 tender joints for PsA), was collected prospectively (Table 1).

Table 1. Baseline clinical and demographic characteristics of patients with psoriatic arthritis (PsA) and rheumatoid arthritis (RA), and healthy donors.

Characteristic	PsA (n = 14)	RA (n = 6)	Healthy donors (n = 25)
Age (years)	48.9 ± 12.8	40.5 ± 21.0	36.8 ± 12.7
Sex, female/male	4/10	8/1	3/22
Tender joint count*	1.9 ± 1.1	2.6 ± 1.9	na
Swollen joint count^†	1.4 ± 1.0	2.2 ± 1.6	na
Leucocyte count in SF (cells/µL)	31 500 ± 18 000	22 660 ± 19 500	na
Current anti-rheumatic treatment			na
Untreated	6	1
MTX	1	0
Leflunomide	0	2
TNFi	5	3
IL-17A inhibitor	1	0
IL-6R inhibitor	0	1
JAK inhibitor	1	1
Prednisone (5 mg/day)	0	2
Refractory to MTX in the past	3	2	na

Data are shown as mean ± sd or number of participants.

SF, synovial fluid; MTX, methotrexate; TNFi, tumour necrosis factor inhibitor; IL-17A, interleukin-17A; IL-6R, interleukin-6 receptor; JAK, janus kinase; na, not applicable.

*Tender joint count for RA: 0–28; PsA: 0–68.

^†Swollen joint count for RA: 0–28; PsA: 0–66.

Ethics statement

This study was performed between 2020 and 2023 and according to the recommendations of the Declaration of Helsinki, and was approved by the Institutional Review Board of Tel Aviv Sourasky Medical Center (0182-18-TLV). All of the study participants signed an informed written consent form before enrolment.

Isolation of PBMCs and SFMCs

PBMCs were derived from heparinized venous blood of healthy donors, and SFMCs were obtained from the SF of PsA and RA patients. Both the PBMCs and SFMCs were extracted by density gradient centrifugation with Lymphoprep™ (Axis-Shield, Oslo, Norway). PBMCs were used to determine the impact of the drugs for their inhibitory effects on proliferation and IFN-γ production. SFMCs were used to determine whether the drugs demonstrated alterations in synovial monocyte inhibition and the gene expression profile.

Measurement of lymphocyte proliferation in healthy donors’ PBMCs via CFSE assay

Cell proliferation was assessed by means of carboxyfluorescein succinimidyl ester (CFSE). In brief, the healthy donors’ PBMCs were pretreated with CFSE before the start of the co-culture. The cells were suspended in phosphate-buffered saline (PBS) with 5 µM CFSE (cat no: 21888; Sigma, St Louis, MO, USA), incubated for 15 min at 37°C, and then washed twice with PBS/2% foetal calf serum (FCS). The cells were cultured at 2 × 10⁵ cells/well in a volume of 200 μL, and suspended in complete RPMI 1640 medium containing 10% FCS supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), 2 mmol/L L-glutamine, and 50 μM 2β-mercaptoethanol, and cultured with PHA (cat no: L1688; Sigma) at a final concentration of 5 µg/mL. In the CFSE-based assay, following a proliferative stimulus (i.e. PHA), the labelled cells undergo cell division. The fluorescent dye, CFSE, permits the direct visualization of cell division. The assay controls consisted of PHA-stimulated PBMCs without therapeutic agents (representing maximal proliferation) and PBMCs without PHA (representing no proliferation). Experimental samples were cultured with PHA in the presence of the TNFi IFX 1 and 10 µg/mL, MTX 0.01 µg/mL and 0.1 µg/mL, IFX 10 µg/mL + MTX 0.01 µg/mL, and IFX 10 µg/mL + MTX 0.1 µg/mL. The IFX and MTX concentrations used in co-culture reflect their therapeutic concentrations in the patients’ circulation (21, 22) and RA patients’ immune cells and synoviocytes in vitro (23). The cells were harvested after 5 days of incubation and analysed by flow cytometry, and a total of 60 000 cells was acquired from each sample.

IFN-γ in cell culture supernatants

Altered activity of PBMCs and production of pro-inflammatory cytokines may affect the course of rheumatic diseases. PHA-stimulated PBMCs secrete high levels of the pro-inflammatory cytokines IFN-γ and TNF. Since in this study we tested the impact of MTX and a TNFi (IFX) on PBMCs, we determined IFN-γ levels as a measure of PBMC activity, and avoided measuring levels of TNF owing to its potential neutralization by IFX. PBMCs (2 × 10⁶ cells/mL) were cultured in RPMI medium for 5 days. Cells were unstimulated or stimulated with PHA 5 µg/mL in the absence or presence of the above-indicated drugs (see Measurement of lymphocyte proliferation in healthy donors’ PBMCs via CFSE assay). The culture supernatants were collected and stored at −80°C until use. The level of IFN-γ in the supernatant was measured using a human IFN-γ enzyme-linked immunoassay (ELISA) kit (DuoSet ELISA kit; R&D systems; Minneapolis, MN, USA) and used according to the manufacturer’s instructions.

In vitro culture of SFMCs

SFMCs at 1.5–2 × 10⁶ cells/mL were cultured with the following agents: IFX 1 and 10 μg/mL, MTX 0.01 and 0.1 µg/mL, IFX 10 µg/mL + MTX 0.01 µg/mL, and IFX 10 µg/mL + MTX 0.1 µg/mL. The cells were cultured for 48 h for gene expression analysis and for 7 days for CD14⁺CD16⁺-expressing cells, as determined by flow cytometry analysis. The selected culture periods of 48 h for gene expression and 7 days for CD14⁺CD16⁺ cell analyses were based on our previous study conducted with SFMCs (18).

Flow cytometry

SFMCs were stained with the following antibodies: FITC anti-human CD14 (cat no: BLG-325604; BioLegend) and APC anti-human CD16 (cat no: BLG-302012; BioLegend, San Diego, CA, USA) for 30 min at room temperature. The flow cytometry gating strategy used to identify the CD14⁺CD16⁺ subset from SFMCs was based on forward scatter (FSC) versus side scatter (SSC) gating. Cells were first gated to exclude debris and apoptotic cells. Next, cells were further analysed for CD14 and CD16 double staining. The quadrant lines were determined based on each single-stained and unstained sample. The results are presented as the percentage of CD14⁺CD16⁺ cells. Flow cytometry was performed with a fluorescence-activated cell sorting (FACS) Canto™ II instrument (BD Biosciences), and the data were analysed with FlowJo software (Tree Star, Ashland, OR, USA).

Real-time PCR

SFMCs were cultured for 48 h with the above-mentioned drugs for analysis of the gene expression of caspase-3, IL-1β, IL-10, and matrix metalloproteinase-9 (MMP-9). After incubation, the cells were collected and RNA was isolated using an RNA extraction kit (High Pure RNA Isolation Kit, Roche Diagnostics, Mannheim, Germany). For cDNA synthesis, 300 ng total RNA was transcribed to cDNA with a High Capacity cDNA Reverse Transcription Kit (Invitrogen Carlsbad, CA, USA). Gene expression was performed with the Fast SYBR™ Green Master Mix (Applied Biosystems, Foster City, CA, USA) in the StepOnePlus™ Real-Time PCR System (Applied Biosystems). All procedures were performed according to the manufacturer’s instructions. The following primers for human genes were used: (forward and reverse, respectively): caspase-3 5′-AGAACTGGACTGTGGCATTGAG-3′ and 5′-GCTTGTCGGCATACTGTTTCAG-3′, IL-1β 5′-TGATGGCTTATTACAGTGGCAATG-3′ and 5′-GTAGTGGTGGTGGGAGATTCG-3′, IL-10 5′-TGGAGGACTTTAAGGGTTAC-3′ and 5′-GATGTCTGGGTCTTGGTT-3′, MMP-9 5′-TTGACAGCGACAAGAAGTGG-3′ and 5′-GCCATTCACGTCGTCCTTAT-3′, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 5′-ATGGGGAAGGTGAAGGTCG-3′ and 5′-GGGGTCATTGATGGCAACAATA-3′. The GAPDH levels were used to normalize gene expression levels.

Statistical analysis

Data are presented as mean ± sem. Non-parametric analyses were performed with the Mann–Whitney U test or Kruskal–Wallis test followed by Dunn’s multiple comparison test. A p value of < 0.05 was considered statistically significant. All analyses were performed with GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA).

Results

Differential influence of IFX, MTX, and IFX + MTX on inhibition of lymphocyte proliferation in healthy donors’ PBMCs

The average extent of unstimulated PBMC (no PHA) proliferation was 1.4 ± 0.2% (representing the control) (Figure 1A, upper panel). The proliferation rate of PHA-stimulated PBMCs increased to 41.8 ± 3.8%, reflecting the maximal proliferation. The PHA-stimulated PBMCs’ proliferative response in the presence of the tested drugs was then assessed. The drugs’ ability to reduce lymphocyte proliferation (when cultured with PHA) was indicated by the reduced percentage of cells showing CFSE dilutions (Figure 1A, lower panel, and Figure 1B).

Figure 1. Reduced phytohaemagglutinin (PHA)-induced proliferation in healthy donors’ peripheral blood mononuclear cells (PBMCs) by the experimental drugs infliximab (IFX) and methotrexate (MTX). Carboxyfluorescein succinimidyl ester (CFSE)-labelled PBMCs from healthy donors (n = 18; one subject was assessed at two different time-points) were incubated with or without PHA (5 µg/mL), or PHA with the drugs. The cells were harvested and analysed after 5 days. (A) Representative plots showing examples of the CFSE dilution assay with PBMCs derived from healthy subjects (upper panel): cells without PHA (no proliferation) or with PHA alone (maximal proliferation). The extent of proliferation is shown in the left side of each plot. Lower panel: PHA with the various drugs. Percentages indicate the percentage proliferation exerted by each drug. (B) Summary of data showing percentage proliferation. Comparisons were determined by the Kruskal–Wallis test followed by Dunn’s post-hoc test. Data are shown as mean ± sem. *p < 0.02, **p < 0.0001.

IFX 1 µg/mL, IFX 10 µg/mL, and MTX 0.01 µg/mL, provided as single agents, inhibited the proliferation rate to a similar extent: proliferation in the presence of IFX 1 µg/mL was 23.3 ± 2.3%, IFX 10 µg/mL 20.7 ± 2.1% (p < 0.02), and MTX 0.01 µg/mL 24.2 ± 2.3%, and their activity was reflected as proliferation inhibition of 43%, 49%, and 42%, respectively. A high MTX concentration (0.1 µg/mL) resulted in 5.4 ± 0.7% proliferation (p < 0.0001) and inhibited the proliferation rate by up to 86%, which was significantly higher compared to each other single agent. The combination of IFX 10 µg/mL + MTX 0.01 µg/mL resulted in 13.5 ± 2.0% proliferation (p < 0.0001) and inhibited the proliferation rate by 69%, and IFX 10 µg/mL + MTX 0.1 µg/mL resulted in 3.7 ± 0.8% proliferation (p < 0.0001), which represented the highest proliferation inhibition of 91%.

Effect of IFX and MTX on IFN-γ cytokine production by healthy donors’ PBMCs

The inhibitory effect of IFX and MTX on the inflammatory response was determined by the measurement of IFN-γ cytokine secretion by PBMCs. Healthy donors’ PBMCs (n = 7) were activated with PHA in the absence or presence of IFX, MTX, and IFX + MTX, and the IFN-γ level was determined in the culture supernatants. The IFN-γ production in cultures with PHA alone achieved the highest IFN-γ level of 78.7 ± 18.4 pg/mL. The secretion of IFN-γ was reduced in cultures with the drugs compared with PHA alone. We observed that IFN-γ production was reduced by all drugs. However, among the single agents (IFX 1 µg/mL 40.6 ± 11.7 pg/mL, IFX 10 µg/mL 26.7 ± 5.8 pg/mL, MTX 0.01 µg/mL 65.0 ± 20.0 pg/mL, and MTX 0.1 µg/mL 14.3 ± 1.8 pg/mL), only MTX 0.1 µg/mL significantly reduced the IFN-γ level (p = 0.01) compared to PHA alone. The level of IFN-γ was reduced in cultures with IFX + MTX compared to the cultures with PHA alone, IFX 10 µg/mL + MTX 0.01 µg/mL 28.9 ± 10.8 pg/mL and IFX 10 µg/mL + MTX 0.1 µg/mL 15.1 ± 2.8 pg/mL (p = 0.01) (Figure 2).

Figure 2. Influence of infliximab (IFX) and methotrexate (MTX) on interferon-γ (IFN-γ) production in supernatants of healthy donors’ peripheral blood mononuclear cells (PBMCs) activated with phytohaemagglutinin (PHA). Healthy donor PBMCs (n = 7) were cultured in the absence or presence of PHA and the experimental drugs. Supernatants were collected after 5 days and analysed for IFN-γ level by enzyme-linked immunosorbent assay. Results are shown as mean ± sem. Comparisons were determined by the Kruskal–Wallis test followed by Dunn’s post-hoc test. *p = 0.01.

IFX but not MTX inhibited the proportion of CD14⁺CD16⁺ monocytes derived from SFMCs of PsA and RA patients

We investigated the long-lasting effect of the drugs on synovial cells by analysing intermediate monocytes (CD14⁺CD16⁺ cells) in SFMCs after 7 days’ culture. In PsA-derived SFMCs cultured ex vivo with medium alone (n = 11), the proportion of CD14⁺CD16⁺ cells was 10.9 ± 2.4% (control) (Figure 3). Supplementation of IFX 1 µg/mL to the culture non-significantly reduced the CD14⁺CD16⁺ cells to 6.4 ± 1.4%. A high concentration of IFX 10 µg/mL significantly reduced the CD14⁺CD16⁺ cells compared to the control (5.5 ± 1.3%, p < 0.01), resulting in a 50% reduction of the CD14⁺CD16⁺ cells. MTX at both concentrations had almost no effect on the CD14⁺CD16⁺ cells (MTX 0.01 µg/mL 9.7 ± 1.3% and MTX 0.1 µg/mL 10.2 ± 1.4%) compared to the control. IFX 10 µg/mL + MTX at a low concentration (0.01 µg/mL) had a similar inhibitory effect to IFX 10 µg/mL alone (5.6 ± 1.1%, p < 0.05). IFX 10 µg/mL + MTX at a high concentration (0.1 µg/mL) significantly reduced the CD14⁺CD16⁺ cells compared to the control (5.1 ± 1.1%, p < 0.01), and appeared to represent the highest reduction in CD14⁺CD16⁺ cells.

Figure 3. Synovial fluid mononuclear cell (SFMC) CD14⁺CD16⁺ monocytes derived from patients with psoriatic arthritis (PsA) are inhibited by infliximab (IFX) but not by methotrexate (MTX) ex vivo. SFMCs derived from PsA patients (n = 11; one patient was assessed at two different time-points) were co-cultured in vitro for 7 days either with the experimental drugs or with medium alone. (A) Representative flow cytometry plots of PsA SFMCs labelled with anti-CD14 and anti-CD16 monoclonal antibodies, after 7 days in culture with medium alone (control) or with the experimental drugs. Values in the upper right quadrant of each plot represent the percentage of CD14⁺CD16⁺ cells. (B) The graph shows the percentage of CD14⁺CD16⁺ monocytes. Significance was assessed by the Kruskal–Wallis test followed by Dunn’s post-hoc test. Data are shown as mean ± sem. *p < 0.05, **p < 0.01.

The drugs exhibited a similar inhibitory activity on SFMCs derived from RA patients, as shown in Figure 4. In SFMCs derived from RA patients (n = 6; two patients were assessed at two different time-points), cultured ex vivo with medium alone (control), the CD14⁺CD16⁺ cell population was 11.0 ± 1.8%. Supplementation of IFX 1 µg/mL (Figure 4) resulted in a non-significant reduction in the CD14⁺CD16⁺ cells to 6.7 ± 1.9%. IFX 10 µg/mL significantly reduced this population to 5.9 ± 1.5% compared to the control (p < 0.05). MTX barely reduced the CD14⁺CD16⁺ cells compared to the control (MTX 0.01 µg/mL 10.8 ± 2.0% and MTX 0.1 µg/mL 11.4 ± 2.2%). Both IFX 10 µg/mL + MTX 0.01 µg/mL and IFX 10 µg/mL + MTX 0.1 µg/mL reduced the CD14⁺CD16⁺ cells compared to control (5.8 ± 1.5% and 6.1 ± 1.6%), but the effect was significant only for IFX 10 µg/mL + MTX 0.01 µg/mL (p < 0.05).

Figure 4. Synovial fluid mononuclear cell (SFMC) CD14⁺CD16⁺ monocytes derived from patients with rheumatoid arthritis (RA) are inhibited by infliximab (IFX) but not by methotrexate (MTX) ex vivo. SFMCs derived from RA patients (n = 6) were co-cultured in vitro for 7 days either with the experimental drugs or with medium alone. (A) Representative flow cytometry plots of SFMCs derived from RA patients labelled with anti-CD14 and anti-CD16 monoclonal antibodies after 7 days in culture with medium alone or with the experimental drugs as indicated. Values in the upper right quadrant of each plot represent the CD14⁺CD16⁺ cells. (B) The graph shows the percentage of CD14⁺CD16⁺ monocytes. Groups were compared using a two-tailed unpaired Mann–Whitney U test. Data are shown as values ± sem. *p < 0.05.

IFX and MTX differentially regulated caspase-3, IL-1β, MMP-9, and IL-10 expression in SFMCs derived from PsA patients

Since synovial monocytes were differently inhibited by TNFi and MTX, we aimed to determine how these drugs affected apoptotic and inflammatory-related gene expression. For this purpose, we examined the potency of IFX 10 µg/mL, MTX 0.1 µg/mL, and IFX (10 µg/mL) + MTX (0.01 and 0.1 µg/mL) to modulate caspase-3, IL-1β, MMP-9, and IL-10 gene expression. The analysis was performed following 48 h co-culture of PsA patients’ SFMCs (n = 6) with the experimental drugs and compared to samples containing medium.

The caspase-3 level was significantly elevated by IFX 10 µg/mL and IFX 10 µg/mL + MTX 0.1 µg/mL compared to medium (1.47 ± 0.07, p < 0.003, and 1.38 ± 0.1, p < 0.03 vs 1.02 ± 0.06, respectively), while MTX at both concentrations did not change caspase-3 gene expression compared to medium (MTX 0.01 µg/mL 0.9 ± 0.07 and MTX 0.1 µg/mL 1.2 ± 0.07) (Figure 5). The opposite trend was shown for IL-1β expression, which was significantly reduced by IFX 10 µg/mL compared to medium (0.3 ± 0.04 vs 1.0 ± 0.04, p < 0.01). MTX at both concentrations did not change IL-1β expression compared to medium (MTX 0.01 µg/mL 1.0 ± 0.07 and MTX 0.1 µg/mL 0.8 ± 0.07). As seen with IFX alone, IFX + MTX significantly reduced IL-1β expression compared to medium (IFX 10 µg/mL + MTX 0.01 µg/mL 0.4 ± 0.07 and IFX 10 µg/mL + MTX 0.1 µg/mL 0.3 ± 0.04, p < 0.03). Similarly, MMP-9 was significantly reduced by IFX 10 µg/mL (0.3 ± 0.04, p < 0.0001) as well as by IFX + MTX at both MTX concentrations (IFX 10 µg/mL + MTX 0.01 µg/mL 0.4 ± 0.07 and IFX 10 µg/mL + MTX 0.1 µg/mL 0.3 ± 0.04, p < 0.0001) compared to the medium (1.1 ± 0.07). Consistent with the effect on IL-1β, MTX did not significantly modulate MMP-9 expression compared to medium (MTX 0.01 µg/mL 1.0 ± 0.1 and MTX 0.1 µg/mL 1.0 ± 0.09) (Figure 5). IL-10 was elevated by both IFX 10 µg/mL (2.4 ± 0.4) and IFX 10 µg/mL + MTX 0.01 µg/mL (2.5 ± 0.5). The strongest increase in IL-10 expression was exhibited by IFX 10 µg/mL + MTX 0.1 µg/mL compared to medium (2.7 ± 0.6 vs 1.0 ± 0.4, p < 0.03). MTX alone at both concentrations did not modulate IL-10 expression compared to medium (MTX 0.01 µg/mL 0.9 ± 0.6 and MTX 0.1 µg/mL 1.0 ± 0.07).

Figure 5. Infliximab (IFX) modulates gene expression in psoriatic arthritis (PsA) patients’ synovial fluid mononuclear cells (SFMCs) differently from methotrexate (MTX). SFMCs derived from PsA patients (n = 6) were cultured (1.5 × 10⁶ cells/well) for 48 h in the presence of IFX 10 µg/mL, MTX 0.01 µg/mL, or MTX 0.1 µg/mL, as well as combinations of IFX 10 µg/mL + MTX 0.01 µg/mL and IFX 10 µg/mL + MTX 0.1 µg/mL. Caspase-3, interleukin-1β (IL-1β), matrix metalloproteinase-9 (MMP-9), and interleukin-10 (IL-10) mRNA expression was determined by real-time polymerase chain reaction. Data are shown as relative expression, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), mean ± sem. Data were analysed with the Kruskal–Wallis test followed by Dunn’s post-hoc comparisons. *p < 0.03, **p < 0.003, ***p < 0.0001.

Discussion

This exploratory study aimed to test ex vivo effects of two principal anti-rheumatic drugs, IFX and MTX, at the cellular level of two different immune cell populations ex vivo: healthy donors’ PBMCs, and SFMCs from RA and PsA patients with active knee synovitis.

Our findings demonstrated that both MTX and IFX inhibited the PHA-induced proliferation of healthy donors’ PBMCs. MTX alone exhibited a more pronounced inhibitory effect than IFX, whereas the greatest inhibition of proliferation was achieved by a combination of IFX 10 µg/mL + MTX 0.1 µg/mL. We further found that MTX inhibited IFN-γ secretion from PHA-stimulated PBMCs more effectively than IFX. There was also a different effect of the drugs on the intermediate SF monocytes, CD14⁺CD16⁺ cells, which were strongly reduced by IFX but not by MTX, in both untreated and treated patients. A plausible explanation for the prominent effect of MTX on PBMCs as opposed to SFMCs may be related to the MTX-induced adenosine signalling in PBMCs, as adenosine acts as a major mediator in the down-regulation of T-lymphocyte activation and proliferation (24). Importantly, the present study did not demonstrate any additive impact of the IFX + MTX combination in reduction of intermediate synovial monocytes, as used in clinical practice for RA treatment.

We also documented different effects of the drugs on SFMC gene expression. IFX alone and the combination of IFX + MTX reduced the inflammatory IL-1β and MMP-9 expression and increased the apoptotic caspase-3, with a concomitant increase in the anti-inflammatory IL-10 expression, while none of these genes was modulated by MTX alone. Since MTX and IFX concentrations used in the assays were equivalent to their therapeutic range in patients’ circulation, it can be speculated that they act similarly in vivo as well. The difference in the activity of MTX and IFX may derive from their different modes of action. For example, the mechanism of action of MTX involves: (i) inhibition of the dihydrofolate reductase enzyme and interference with DNA synthesis, leading to cell-cycle arrest and apoptosis in dividing T cells (16); and (ii) intracellular accumulation of aminoimidazolecarboxamido ribonucleotide transformylase, leading to increased adenosine transport out of the cell (25). Adenosine interacts with cells and limits the inflammatory response through the induction of immunomodulatory properties, and down-regulates T-cell activation and proliferation (26). The anti-proliferative and anti-inflammatory effects of MTX in monocytes were found to be dose dependent (27). There was a significant inhibition of proliferation and apoptosis mediated by MTX at a concentration of 75–500 µg/mL, but not at lower concentrations (5 ng/mL to 5 µg/mL), as used in rheumatic disease therapy, in human monocytic myeloid cell line (THP-1) and in synovial macrophages of RA patients (27). This suggests that MTX used at the therapeutic dosage may not possess anti-inflammatory effects on monocyte populations.

Indeed, MTX has a completely different mode of action from that of TNFis. MTX penetrates the cells (28), while a TNFi induces signals through binding to transmembrane tumour necrosis factor (tmTNF) on monocytes (29). Given that intermediate monocytes (CD14⁺CD16⁺ cells) are recruited from the blood to joints in RA (30) and that they express elevated levels of tmTNF on their surface (31), it was suggested that TNFi agents induce cell death in tmTNF-expressing cells via binding to tmTNF (32, 33). Destruction of tmTNF-expressing cells via a TNFi is mediated by antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and apoptosis via reverse signals through tmTNF (29, 33, 34). TNFi binding to tmTNF leads to inhibition of intrinsic nuclear factor-κB (NF-κB) activation and IL-1β secretion, inducing apoptosis in monocytes (35–37). IL-1β aggravates synovial inflammation, enhances cartilage breakdown, and increases the expression of MMPs, including MMP-9 (38), while IL-10 exerts a protective action via an increase in anti-inflammatory mediators in the synovium (39). Our previous study demonstrated that TNFis reduced the CD14⁺CD16⁺ cells in PsA and RA patients’ SFMCs compared to glucocorticoids, delivered directly to synovial joints to treat synovial inflammation. That study also found that TNFis reduced IL-1β in PsA patients’ SFMCs, while glucocorticoids led to increased IL-1β gene expression, indicating that TNFi agents possess unique anti-inflammatory activity compared to glucocorticoids (18).

We chose to investigate the effect of MTX and IFX separately on PBMC and SFMC populations. Two other studies have tested the activity of MTX or TNFi agents separately and in combination in vitro: (i) on a human jurkat T-cell line expressing tmTNF (40); and (ii) on synoviocytes and PBMCs in an in vitro model imitating the local synovium environment (23). In accordance with our results, the study on jurkat T cells showed that a combination of MTX and IFX was the most effective in apoptosis induction (40). This effect could explain the synergistic effect of MTX and IFX in RA therapy. However, MTX had almost no effect on pro-inflammatory cytokine production in synoviocytes, whereas IFX down-regulated these cytokines (23). Although these studies examined different cell populations, the results are in line with ours, showing an additive effect of MTX and IFX on a T-cell line (PBMC lymphocytes in our study) and a lower ability of MTX than IFX to modulate synovial cells.

Our study has several limitations. First, we analysed the intermediate monocytes (CD14⁺CD16⁺ cells) and did not look more deeply into the effect of the drugs on classical and non-classical monocytes. In addition, CD14⁺CD16⁺ cells (assumed to reflect the inflammatory synovial cell subset) and RNA expression analyses were performed on total SFMCs. It will be interesting to investigate the immunosuppressive effects of the drugs on separated synovial monocyte populations. Secondly, we analysed the effects of the drugs on changes in CD14⁺CD16⁺ cells after 7 days and gene expression after 48 h, whereas the effects across other time-points were not investigated in this study. At the clinical level, our study focused on synovial cells derived only from the knee joint. It could be hypothesized that cellular populations, activation pathways, and effects of drug treatment may be somewhat different in other joints. Furthermore, the results of the study may be skewed by the current anti-rheumatic therapy in the treated versus untreated patients. The effects observed with IFX may be drug specific and not applicable to other TNFi drugs, in view of differences in their structure, signalling, immunogenicity, and pharmacokinetics. Finally, the lack of data on duration of rheumatic disease, C-reactive protein levels, and patient-reported outcomes precludes clinical correlation of the study findings.

Conclusion

We demonstrated the distinct effect of MTX and IFX on different immune cell populations. Both MTX and the TNFi inhibited PBMC proliferation and IFN-γ secretion, indicating their ability to suppress circulating effector T cells and their cytokine production. The combination of IFX + MTX significantly increased the inhibition of PBMC proliferation and IFN-γ secretion. In SFMCs, there was a significant difference between IFX and MTX in their ability to modulate CD14⁺CD16⁺ monocytes, which were efficiently reduced by IFX but not by MTX. Remarkably, suppression of the pro-inflammatory genes IL-1β and MMP-9 was mediated by IFX but not by MTX. The opposite trend was shown for caspase-3 and IL-10, which were up-regulated by IFX and not by MTX. IFX + MTX had the most pronounced and synergistic up-regulating effect on the anti-inflammatory gene IL-10. Thus, this ex vivo study contributes towards a better understanding of the cellular targets of each anti-rheumatic drug. Further elucidation of the underlying mechanisms of various anti-rheumatic drugs on the cellular immune response in the periphery and synovial inflammatory microenvironment awaits future research.

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Ethical approval

The experiments were performed according to the protocols approved by Hospital Tel Aviv Sourasky Medical Center Ethics Committee (0182-18-TLV).

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Authors’ contributions

SG and MR were responsible for performing the experiments and drawing the figures. SG and VF wrote the manuscript. MR, AP, IL, MA, and OE edited the manuscript. All authors read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

There was no external funding for this study.