Mesangial cells are key contributors to the fibrotic damage seen in the lupus nephritis glomerulus

As studies have previously demonstrated that MCs are able to secrete pro-inflammatory cytokines in response to injurious stimuli, this was assessed in an in vitro, cytokine-based LN model. Multiplex analysis was used to determine levels of IL-6, IL-8, IL-10, and M-CSF.

Relatively high levels of IL-6 were secreted by untreated MCs (5180 pg/mL [4570-5602]), this was significantly increased in response to treatment with IL-1β (6807 pg/mL [6349-6725]; p = 0.03) and the combined treatment (6545 pg/mL [6381-6678]; p = 0.02) (Fig. 1a). IL-8 was expressed at high levels under basal conditions (2247 pg/mL [2012-2643]) and this was increased further by treatment with the combination of cytokines (3168 pg/mL [3140-3215]; p = 0.03) (Fig. 1b). In contrast, IL-10 was expressed at low levels in the absence of treatment (5.237 pg/mL [5.237–6.18]), but this was significantly increased in response to IFN-α (17.08 pg/mL [16.78–18.05]; p = 0.02), IFN-γ (17.81 pg/mL [17.31–18.5]; p = 0.001) and the combined cytokine treatment (19.44 pg/mL [19.08–20]; p < 0.0001) (Fig. 1c). Baseline expression of M-CSF was below the level of detection for the assay (and were thus set at (minimum detection level/√2): 363.45 pg/mL); however, in response to IL-1β treatment (4905 pg/mL [2739–10,545]; p = 0.02] and the combination of cytokines treatment (5781 pg/mL [4218-11,778]; p = 0.004) this significantly increased (Fig. 1d).

An attempt was made to recapitulate this effect in a more physiological model of LN using sera from patients with active disease (renal BILAG A/B), inactive disease (renal BILAG D/E) and age- and sex-matched HCs.

IL-6 was expressed at relatively high levels in untreated MCs (2396 pg/mL [2156-2475]) and this was significantly reduced following treatment with active LN sera (2249 pg/mL [2214-2308]; p = 0.03) (Fig. 2a). IL-8, however, was expressed at baseline but not affected by treatment (Fig. 2b). IL-10 is expressed by untreated MCs (62.83 pg/mL [33.61–95.29]) and following all treatments (except 1 active disease patient sera) this was reduced to below the level of detection for the assay (Fig. 2c). M-CSF was expressed by unstimulated MCs (528.1 pg/mL [381.6–829]) and this was significantly increased in response to treatment with sera from active disease patients (1090 pg/mL [835.2–1616]; p = 0.04) (Fig. 2d).

In order to exclude the sera as the source of the cytokines the levels of each cytokine were assessed in RPMI + 10% sera (to mimic that which was used to treat the cells); IL-6 and IL-8 levels in RPMI + 10% sera were below the level of detection for all groups (data not shown). IL-10 was expressed in patient sera, in all groups this was reduced to below the level of significance following treatment of MCs (Fig. 2e). M-CSF on the other hand was relatively lowly expressed in active disease patient sera (121.7 pg/mL [92.49–192.3]) and this was significantly increased in MC conditioned media following treatment (1090 pg/mL [835.2–1616]; p < 0.0001). Further M-CSF was relatively lowly expressed in healthy control (HC) sera (320.7 pg/mL [114.3–376.6]) and this was significantly increased in conditioned media following 24 h treatment (608.4 pg/mL [356.7–1467]; P = 0.016) (Fig. 2f).

MCs are heavily involved in maintenance of the ECM and in response to damage secrete proteins and enzymes that restructure the matrix. The expression of genes involved in remodelling the ECM were assessed in response to 24 h cytokine treatments. MCs expressed low levels of COL1A1 mRNA at baseline (0.708 [0.262–1.96]) and this was significantly increased in response to treatment with IFN-γ (5.089 [0.169–7.484]; p = 0.03) and the combination of cytokines (4.951 [4.299–6.628]; p = 0.03) (Fig. 3a). Relatively low levels of mRNA for COL1A2 were expressed by untreated MCs (0.0002 [0.0001–0.0003]), this was significantly increased in response to IFN-α (0.0006 [0.0003–0.001]; p = 0.03), IFN-γ (0.0006 [0.0003–0.001]; p = 0.04) and the combination of cytokines (0.006 [0.0002–0.001]; p = 0.03) (Fig. 3b). COL4A1 mRNA was expressed at low levels in control MCs (1.428 [0.945–2.335]), this was significantly increased by treatment with IL-1β (4.021 [2.375–7.703]; p = 0.05), TNF-α (4.195 [3.144–6.859]; p = 0.03), IFN-γ (6.331 [2.398–9.013]; p = 0.03) and the combination of cytokines (5.453 [3.908–8.688]; p = 0.02) (Fig. 3d). MCs expressed low levels of LAMB1 mRNA under baseline conditions (0.002 [0.001–0.008]), this was significantly increased in response to treatment with IL-1β (0.019 [0.013–0.028]; p = 0.05) and the combination of cytokines (0.025 [0.022–0.029]; p = 0.002) (Fig. 3e). MCs expressed mRNA for COL3A1 and LAMB2 however these were not affected by treatment (Fig. 3c and e).

In order to obtain a clear representation of the remodelling of the ECM it is important to assess the expression of ECM protein genes, MMPs and TIMPs. MCs have been shown to express MMP2 and MMP9, as well as TIMP1 [20, 21] and thus these were assessed following cytokine stimulation. Relatively low levels of MMP9 mRNA were expressed by untreated MCs (0.0001 [0.00006–0.0003]), this was significantly increased in response to treatment with IL-1β (0.0016 [0.0015–0.0019]; p = 0.01), TNF-α (0.0015 [0.0014–0.0019]; p = 0.02) and the combination of cytokines (0.0016 [0.0013–0.0019]; p = 0.03) (Fig. 4a). TIMP1 was expressed at relatively high levels in control MCs (0.564 [0.526–0.595]), this was significantly decreased in response to IFN-γ (0.178 [0.116–0.215]; p = 0.01) and the combination of cytokines (0.139 [0.106–0.172]; p = 0.01) (Fig. 4c). MMP2 mRNA was also expressed by MCs but was not significantly affected by any of the treatments (Fig. 4a).

This was recapitulated in the more physiological model of treating with RPMI (+ 10% patient sera). MCs expressed mRNA for COL1A1 and COL3A1 under normal conditions and these were not significantly modulated following treatment with 10% LN patient sera (Fig. 5a and c). Prior to treatment COL1A2 mRNA was expressed at relatively low levels (0.00065 [0.00022–0.0024]), this was significantly increased in response to treatment with active sera (0.0012 [0.0003–0.003]; p = 0.03) while inactive and HC sera had no effect (Fig. 5b). COL4A1 mRNA was expressed by untreated MCs (0.933 [0.181–2.307]), a trend was seen towards an increase with active sera (1.947 [1.397–4.028]; p = 0.07) however this did not reach significance. No other treatments induced a change in COL4A1 mRNA (Fig. 5d). MCs express mRNA for LAMB1 and LAMB2 however these were not affected by any of the sera treatments (Figs. 5e-f).

Levels of mRNA for remodelling enzymes were also assessed. MMP2 and TIMP1 mRNA were expressed by untreated MCs but levels were not affected by any of the sera treatments (Fig. 6a and c). MMP9 mRNA was expressed by MCs under normal conditions (0.000078 [0.000011–0.00022]) and this was significantly increased by treatment with sera from active LN patients (0.00045 [0.00026–0.00071]; p = 0.011), no other treatments induced any changes in MMP9 mRNA (Fig. 6b).

One of the most common mediators of ECM remodelling is transforming growth factor (TGF)-β1 therefore the expression of latent TGF-β1 following cytokine stimulation was assessed in our model. As we were keen to investigate the role of TGF-β1 in inducing the changes seen at 24 h the levels were assessed at 4 h and 24 h to look at temporal modulation. At 4 h post-cytokine stimulation latent TGF-β1 was expressed at relatively high levels (1201 pg/mL [1129-1257], this was significantly increased following treatment with IL-1β (2028 pg/mL [1984-2051]; p = 0.02), IFN-γ (2,076 pg/mL [1963-2096]; p = 0.02) and the combination of cytokines (2589 pg/mL [2152-2721]; p = 0.0001) (Fig. 7a). At 24 h however, latent TGF-β1 was expressed by untreated MCs (873.5 pg/mL [443.4–1130], but this was decreased following treatment with IFN-α (340 pg/mL [280.4–679.8]; p = 0.05), IFN-γ (400.7 pg/mL [285.2–438]; p = 0.02) and the combination of cytokines (391.3 pg/mL [151.5–561.7]; p = 0.03) (Fig. 7b).

As MCs treated with patient sera appeared to have a milder phenotype than those treated with cytokines the levels of latent TGF-β1 were assessed only at 24 h. When MCs were treated with patient sera for 24 h there was a significant increase in the production of latent TGF-β1 by MCs treated with active sera (1897 pg/mL [1515-2010]; p = 0.014) compared to untreated MCs (941.1 pg/mL [449.9–1150]) while no other treatments had a significant effect (Fig. 7c). The expression of latent TGF-β1 in RPMI (+ 10% patient sera) was also assessed and it was determined that although no significant difference in the expression was seen between the groups this was similar to that seen in conditioned media following MC stimulation (Fig. 7d).

TGF-β1 receptor activity blockade was induced by pre-treatment with SB-431542 and following this MCs were treated with the combination of cytokines treatment (IL-1β, TNF-α, IFN-α and IFN-γ) for 24 h as previously and the expression of mRNA for ECM genes was assessed. As demonstrated previously the combination of cytokines induced increased expression of mRNA for COL1A1, COL1A2, COL4A1, LAMB1, MMP9 and TIMP1. MCs express mRNA for COL1A1 at baseline (0.0009 [0.0002–0.005]) and this is significantly increased following stimulation with the combination of cytokines (0.01 [0.005–0.012]; p = 0.02). Pre-treatment with SB-431542 was able to reduce this close to baseline levels (0.002 [0.0005–0.005]) (Fig. 8a). COL1A2 mRNA is also expressed by MCs at baseline (0.1213 [0.0168–0.2257]) and again this is significantly increased in response to the combination of cytokines (0.6297 [0.4857–0.7428]; p = 0.02), again this was reduced to similar to baseline levels by pre-treatment with SB-431542 (0.0858 [0.0387–0.1071]) (Fig. 8b). Untreated MCs express mRNA for COL4A1 (0.7654 [0.7259–1.255]) and this is significantly increased in response to the combination of cytokines (2.501 [1.016–4.212]; p = 0.009), this was unchanged compared to control following pre-treatment with SB-431542 (0.9711 [0.8184–1.515]) (Fig. 8c). LAMB1 mRNA is expressed by untreated MCs (0.004 [0.0003–0.018]) and trended towards an increase following treatment with the combination of cytokines (0.1551 [0.098–0.243]; p = 0.08), levels returned to baseline following pre-treatment with SB-431542 (0.005 [0.0015–0.0076]) (Fig. 8d). The expression of mRNA for MMP9 was also assessed – this was at relatively low levels at baseline (0.005 [0.0001–0.018]) and was significantly increased following stimulation with the combination of cytokines (0.024 [0.014–0.0369]; p = 0.047) and again, this was reduced following pre-treatment with SB-431542 (0.001 [0.0002–0.0165]) (Fig. 8e). Finally, the expression of mRNA for TIMP1 was assessed – this was expressed by untreated MCs (20.84 [11.3–23.93]). Although it appears that a decrease in expression can be seen following treatment with the combination of cytokines this did not reach significance (9.754 [3.178–14.41]; p = 0.6). Following pre-treatment with SB-431542 the levels appeared closer to that seen in the untreated cells (20.47 [16.54–36.94]) (Fig. 8f).

All recombinant cytokines were purchased from Peprotech, London, UK. All primers were purchased from Eurofins Genomics, Ebersberg, Germany.

Human conditionally immortalised MCs were kindly provided by Professor Moin Saleem (Children’s Renal Unit and Academic Renal Unit, University of Bristol, Southmead Hospital, Bristol, UK). These cells were conditionally immortalised using the temperature sensitive large T antigen-SV-40 transgene as previously described [32]. These cells have been shown to differentiate fully by 7–10 days after switching from 33 °C to 37 °C. Cell passages between 15 and 30 were used in all experiments, for all experiments n = 5–6 independent experiments were used. MCs were routinely cultured in RPMI-1640 medium with L-glutamine (Lonza, Leeds, UK) supplemented with 10% foetal calf serum (ThermoScientific) and insulin transferrin selenium (Sigma-Aldrich, Dorset, UK).

After 7–10 days of differentiation conditionally immortalised MCs were treated with cytokines designed to model the inflammatory environment of the kidney in LN patients, namely: IL-1β, TNF-α, IFN-α and IFN-γ (all known to be involved in the pathogenesis of LN) at 10 ng/mL each alone and in combination (i.e. 10 ng/mL each of IL-1β, TNF-α, IFN-α and IFN-γ altogether). These were chosen as being key cytokines known to be upregulated in the sera of patients with active LN compared to inactive disease and healthy control [16‐19]. Following 24 h incubation conditioned media were collected, and RNA was extracted using Trizol (ThermoScientific).

Upon routine clinical visits, patients within the UK JSLE Cohort Study are assessed according to the British Isles Lupus Assessment Group (BILAG) 2004 index [33, 34]. Following differentiation MCs were also treated with 10% sera from patients with active LN (renal BILAG A/B), inactive LN (renal BILAG D/E) and age- and sex- matched HCs (Table 1). Following 24 h incubation conditioned media were collected, and RNA was extracted using Trizol (ThermoScientific).

Cells were pre-treated for 30 mins with SB-431542 (ALK5 receptor blocker) to inhibit TGF-β1 binding as previously described [35] before stimulation with the combined cytokine treatment. Following this conditioned media was collected and RNA was extracted using Trizol.

A Luminex magnetic bead assay was purchased from R&D Systems, Abingdon UK which was able to detect IL-6, IL-8, IL-10 and M-CSF. The assay was performed on conditioned media collected from cells treated with cytokines for 24 h according to manufacturer’s instructions to assay protein levels in conditioned media from cytokine-treated MCs. The plate was read using a Merck Millipore Luminex MAGPIX® analyser.

TGF-β1, IL-6, IL-8 and M-CSF DuoSets were purchased from R&D Systems, Abingdon, UK. The assays were performed on conditioned media from cells that had been stimulated with cytokines or patient sera for 24 h according to the manufacturer’s instructions to determine protein levels in conditioned media from treated MCs.

RNA was extracted from MCs treated with cytokines for 24 h using the RNeasy miniprep kit (Qiagen, Manchester, UK) following the manufacturer’s instructions. The RNA concentration was determined by Nanodrop and 200-500 ng RNA was transcribed into cDNA using either the AffinityScript multi-temp cDNA synthesis kit (Agilent Technologies, Cheshire, UK) following the manufacturer’s instructions for 24 h cytokine treatments or the Primerdesign all-in-one Reverse Transcription mix (Primerdesign, York, UK) following manufacturer’s instructions (for sera and TGF-β1 blocking assays). qRT-PCR was performed using the primers described in (Table 2) with the Brilliant III Ultra-fast SYBR QPCR mastermix kit (Agilent Technologies) following the manufacturer’s instructions (for 24 h cytokine treatments) or the Primerdesign PrecisionPLUS qPCR Master Mix kit (for sera and TGF-β1 blocking assays). The geometric mean of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein Zeta (YWHAZ), β-actin (ACTB) and TUBB was used as an internal reference control for normalisation and used to calculate the ΔΔCt value.