Emily Morris, Rezvan Parvizi, Patricia Pioli and Michael Whitfield, Geisel School of Medicine at Dartmouth, Hanover, NH
Background/Purpose: Systemic Sclerosis (SSc) is a rare autoimmune connective tissue disease characterized by inflammation and fibrosis. Treatment with mycophenolate mofetil (MMF), an inhibitor of lymphocyte guanine nucleotide synthesis that targets the enzyme inosine monophosphate dehydrogenase (IMPDH), is associated with clinical benefit in many SSc patients. Although MMF is thought to target lymphocytes exclusively, we have previously shown that MMF also reduces dermal myeloid cell numbers and CCL2 expression in the skin & sera of MMF-responsive SSc patients. Given macrophage plasticity, we hypothesized that, in addition to restricting macrophage skin infiltration, MMF might also mediate direct effects on dermal macrophage pro-fibrotic activation.
Methods: Clinical data from SSc patient cohorts were analyzed for myeloid gene signatures to identify changes in myeloid cell populations during MMF treatment. Primary human monocytes were cultured in clinically relevant serum concentrations of the active metabolite of MMF, mycophenolic acid (MPA), and analyzed for mRNA and surface marker expression, nitric oxide and cytokine production, cell viability and caspase activity in response to MPA. To assess MMF-mediated effects on pro-fibrotic macrophage activation, 3D tissue models of healthy and SSc skin were constructed and treated with or without MPA (0.5-10 µg/mL).
Results: SSc patients on MMF showed a significant decrease in myeloid cell populations in skin biopsies. The clinical target range for serum MPA levels are 1-3µg/mL for MMF patients; the IC50 of MPA in primary human monocytes in our experiments was found to be between 0.9-2.5 µg/mL (Fig. 1A), well within the clinical range. MPA dose-dependently induced apoptosis in primary human monocytes at concentrations as low as 0.5 µg/mL (Fig. 1B). Intriguingly, a subset of monocytes not killed by MPA upregulated surface expression and mRNA levels of markers associated with alternative activation (Fig. 1C). MPA treatment of monocytes in 2D co-cultures with fibroblasts and 3D fabricated skin models showed similar results. Addition of guanosine to cultures significantly attenuated the ability of MPA to induce apoptosis in and modulate activation of monocytes (Fig. 1D).
Conclusion: We now demonstrate here that MMF inhibits primary human macrophage viability and alters macrophage function. Our work suggests that IMPDH activity is required for macrophage viability and mediates alternative activation, as treatment with guanosine rescues cell death and significantly attenuates macrophage phenotype. We thus propose that clinical benefit observed with MMF in the treatment of fibrotic diseases such as SSc may be attributable, at least in part, to effects on myeloid cells.
Figure 1: Mycophenolic acid reduces viability of primary human monocytes in vitro and upregulates mRNA expression of alternative activation markers through a guanosine dependent mechanism. (A) Primary human monocytes (n= 4 biological replicates; n = 4 technical replicates per biological replicate) were grown in the following concentrations of MPA for 7 days with or without guanosine supplementation (100µM): 0 µg/mL, 0.5 µg/mL, 1 µg/mL, 2 µg/mL, 3 µg/mL, 5 µg/mL, and 10µg/mL. A CellTiter Blue assay was performed viability of cells after 7 days in culture. Decreased absorbance indicates decreased mitochondrial function and viability; the average IC50 was 1.26 µg/mL MPA for unsupplemented monocytes. (B) Primary human monocytes were grown in 0 µg/mL, 2 µg/mL, or 4 µg/mL MPA for 4 days (n=3 biological replicates) with our without guanosine supplementation (100µM) and assessed for Annexin V (FITC) positivity via flow cytometry as an indicator of apoptosis; increasing concentrations of MPA induced higher levels of Annexin V positive monocytes only in the unsupplemented conditions (representative of 3 independent experiments). (C) and (D) Primary human monocytes were grown in 0 µg/mL, 2 µg/mL, or 4 µg/mL MPA for 4 days (n=4 biological replicates) without (C) and with (D) guanosine supplementation (100µM) for 4 days and assessed for mRNA expression of genes associated with classical and alternative activation. MPA dose-dependently upregulated expression of Arginase-1, CCL2, IL-10, IL-6, and IRF4 in unsupplemented monocytes (C). Supplementation with guanosine (D) consistently blocked this effect (significance of fold change in mRNA expression was assessed by Wilcoxon Ranked Sum test; * p < 0.05, ** p < 0.005).
E. Morris: None; R. Parvizi: None; P. Pioli: Boehringer-Ingelheim, 1, Bristol-Myers Squibb(BMS), 1, 2, 5, Celdara Medical LLC, 2, 5, Pfizer, 5; M. Whitfield: Boehringer-Ingelheim, 1, Bristol-Myers Squibb(BMS), 2, 5, Celdara Medical, 2, 5, 12, Scientific Founder.