Moderate Increase of Indoxyl Sulfate Promotes Monocyte Transition into Profibrotic Macrophages

Abstract

Objective: The uremic toxin Indoxyl-3-sulphate (IS), a ligand of Aryl hydrocarbon Receptor (AhR), raises in blood during early renal dysfunction as a consequence of tubular damage, which may be present even when eGFR is normal or only moderately reduced, and promotes cardiovascular damage and monocyte-macrophage activation. We previously found that patients with abdominal aortic aneurysms (AAAs) have higher CD14+CD16+ monocyte frequency and prevalence of moderate chronic kidney disease (CKD) than age-matched control subjects. Here we aimed to evaluate the IS levels in plasma from AAA patients and to investigate in vitro the effects of IS concentrations corresponding to mild-to-moderate CKD on monocyte polarization and macrophage differentiation.

Methods: Free IS plasma levels, monocyte subsets and laboratory parameters were evaluated on blood from AAA patients and eGFR-matched controls. THP-1 monocytes, treated with IS 1, 10, 20 μM were evaluated for CD163 expression, AhR signaling and then induced to differentiate into macrophages by PMA. Their phenotype was evaluated both at the stage of semi-differentiated and fully differentiated macrophages. AAA and control sera were similarly used to treat THP-1 monocytes and the resulting macrophage phenotype was analyzed.

Results: IS plasma concentration correlated positively with CD14+CD16+ monocytes and was increased in AAA patients. In THP-1 cells, IS promoted CD163 expression and transition to macrophages with hallmarks of classical (IL-6, CCL2, COX2) and alternative phenotype (IL-10, PPARγ, TGF-β, TIMP-1), via AhR/Nrf2 activation. Analogously, AAA sera induced differentiation of macrophages with enhanced IL-6, MCP1, TGF-β, PPARγ and TIMP-1 expression.

Conclusion: IS skews monocyte differentiation toward low-inflammatory, profibrotic macrophages and may contribute to sustain chronic inflammation and maladaptive vascular remodeling.

Fig 1. Free IS levels, CD14⁺CD16⁺ monocytes and eGFR in study populations.

(A) IS serum levels in AAA patients versus age- and e-GFR-matched control subjects, **p<0.01; (B) scattergram and regression line representing the relationship of CD14⁺CD16⁺monocyte percentages with IS serum levels (p = 0,014; r = 0,34); (C) and (D): scattergram and regression line representing the relationship between IS concentrations and eGFR in control subjects (empty circle, p = 0,02; r = -0,55) and AAA patients (grey triangle, p = 0,2; r = -0,17) respectively.

Fig 2. Effects of IS on THP-1 monocytes.

Effect of 72 h treatment with 1, 10, 20 μM IS ±10 μM CH-223191 pretreatment (dashed bars) on (A) CD163 expression; § p< 0,05; **p< 0,01 vs. CTR; ***p<0,001 vs. CTR; (B) Proliferation rate expressed as % of cells in first (grey bars) and second (white bars) generation as evaluated by flow cytometry using the CFSE-DA probe; *p<0.05 vs. IS1; **p<0.01 vs. CTR; #p<0.01 vs. the corresponding IS alone concentrations; (C) Chemotaxis during 3 hours of incubation in a Boyden chamber after a 48 h IS treatment; CCL2 (10 ng/ml) and PBS were used as chemoattractant and negative control respectively. Values are expressed by means of cell number/field on a polycarbonate polyvinylpyrrolidone-free filter (5-nm pore size). *p<0.05; § p<0.05; #p<0.05 vs 20μM IS. (D) CCR2 membrane expression in cells after 72 h IS treatment evaluated by flow cytometry. (E) CCR2 and (F) CCL2 gene expression in cells after 24 h IS treatment as revealed by RT-PCR; **p< 0,01; ***p<0,001 vs. CTR. Results are plotted as mRNA fold induction (mean ± SEM) versus control cells.(G) Immunofluorescence localization of AhR (green) and AhRR (blue) after a 72 hour IS treatment; nuclei are counterstained with Propidium Iodide (red). (H) Protein expression of CYPOR (cytochrome p450 reductase); * p <0.05; ** p <0.01 vs. CTR.

Fig 3. Gene expression in IS-treated M/2M.

RT-PCR for (A) AhR, (B) AhRR, (C) Nrf2, (D) HO1, (E) IL-6 and (F) CCL2. Results were plotted as mRNA fold induction in IS-treated versus Control cells; * p <0.05; ** p <0.01; ***p<0,001 vs. control cells.

Fig 4. Protein expression in IS treated MdM.

Western Blot analysis of total cell lysates for (A) AhR, (B) AhRR, (C) CYPOR, (D) Nrf2, (E) HO1,(F) PPARγ, (G) TGF-β, (H) TIMP-1, (I) COX2; values are normalized to GAPDH expression. (J) Gelatinolic activity of MMP-9. (K) IL-10 and (L) CCL2 content in IS-treated MdM conditioned medium. * p <0.05; ** p <0.01; *** p <0.001 vs. CTR.

Fig 5. Gene and protein expression in M/2M and MdM treated with sera from AAA patients and control subjects.

RT-PCR for (A) IL-6, and (B) CCL2 in M/2M; results were plotted as mRNA fold induction in cell treated with AAA sera vs. cells treated with Normal sera. Western Blot analysis of total cell lysates for (C) PPARγ, (D) TGF-β, (E) TIMP-1 and (F) CYPOR in MdM; values are normalized to GAPDH expression. * p <0.05; ** p <0.01 vs. CTR.

Fig 6. Proposed scheme of IS effects on monocyte differentiation.

Mild IS concentrations (1, 10, 20 μM) potentiate the detoxifying and anti-oxidant pathways of AhR and Nrf2 in monocytes (THP-1 cells), resulting in CD163 and HO1 overexpression and monocyte activation. Macrophages derived from IS-treated monocytes (IS-treated MdM) retain an upregulation of the AhR activity and features of a profibrotic, low inflammatory phenotype.