Oxalate disrupts monocyte and macrophage cellular function via Interleukin-10 and mitochondrial reactive oxygen species (ROS) signaling

Abstract

Oxalate is a small compound found in certain plant-derived foods and is a major component of calcium oxalate (CaOx) kidney stones. Individuals that consume oxalate enriched meals have an increased risk of forming urinary crystals, which are precursors to CaOx kidney stones. We previously reported that a single dietary oxalate load induces nanocrystalluria and reduces monocyte cellular bioenergetics in healthy adults. The purpose of this study was to extend these investigations to identify specific oxalate-mediated mechanisms in monocytes and macrophages. We performed RNA-Sequencing analysis on monocytes isolated from healthy subjects exposed to a high oxalate (8 mmol) dietary load. RNA-sequencing revealed 1,198 genes were altered and Ingenuity Pathway Analysis demonstrated modifications in several pathways including Interleukin-10 (IL-10) anti-inflammatory cytokine signaling, mitochondrial metabolism and function, oxalic acid downstream signaling, and autophagy. Based on these findings, we hypothesized that oxalate induces mitochondrial and lysosomal dysfunction in monocytes and macrophages via IL-10 and reactive oxygen species (ROS) signaling which can be reversed with exogenous IL-10 or Mitoquinone (MitoQ; a mitochondrial targeted antioxidant). We exposed monocytes and macrophages to oxalate in an in-vitro setting which caused oxidative stress, a decline in IL-10 cytokine levels, mitochondrial and lysosomal dysfunction, and impaired autophagy in both cell types. Administration of exogenous IL-10 and MitoQ attenuated these responses. These findings suggest that oxalate impairs metabolism and immune response via IL-10 signaling and mitochondrial ROS generation in both monocytes and macrophages which can be potentially limited or reversed. Future studies will examine the benefits of these therapies on CaOx crystal formation and growth in vivo.

Keywords: Dietary oxalate; IL-10; Lysosome; Macrophages; Metabolism; Mitochondria; Monocytes; Transcriptomics.

Fig. 1

A single dietary oxalate load alters monocyte transcriptomics in healthy subjects. (A) Venn diagram of differentially expressed genes in monocytes from healthy subjects before and after a dietary oxalate load. (B) Activation z-scores of the top canonical pathways identified using Ingenuity Pathway Analysis (IPA). (C) Gene ontology (Biological Process) enrichment analysis of the differentially expressed genes using ClusterProfiler. Enriched (D) KEGG and (E) Hallmark pathways in upregulated and downregulated genes using ClusterProfiler. Data are from n = 3 healthy subjects.

Fig. 2

Heat maps of significant pathways modified in monocytes in response to a single dietary oxalate load. Heat maps of genes involved in (A) IL-10 signaling, (B) mitochondrial metabolism and function, (C) oxalic acid downstream involving immune and inflammatory response, and (D) autophagy in monocytes from healthy subjects. Darker green shades represent high gene expression and lighter green shades represent low gene expression. Data are from paired healthy subjects (n = 3; pre-oxalate vs. post-oxalate).

Fig. 3

The effect of exogenous IL-10 and MitoQ treatment on IL-10 protein expression and mitochondrial and lysosomal markers in oxalate-treated monocytes. THP-1 monocytes were treated with oxalate (50 μM) with or without IL-10 (40 μg/mL) or MitoQ (200 nM) for 24 h. (A) Protein expression was determined for Interleukin-10 (IL-10) and TOM20, a mitochondrial protein, using western blotting. (B–C) Western blots were analyzed using ImageJ software for semi-quantitative measurement of the proteins. (D) Mitochondrial membrane potential was measured using the fluorescent dye, TMRE. (E) Mitochondrial ROS was determined using MitoSox Red. (F) LysoTracker Red was used to evaluate lysosomal activity. (G) Protein expression was determined for Ras-related protein Rab-7a (Rab7-membrane trafficking and phagosome maturation), Lysosomal-associated membrane protein 1 (LAMP1- lysosome marker), and Light Chain 3 B (LC3B – autophagy marker) using western blotting. (H–J) Western blots were analyzed using ImageJ software for semi-quantitative measurement of the proteins. Data are represented as mean ± SEM, n = 3–5. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Fig. 4

The effect of exogenous IL-10 and MitoQ treatment on IL-10 protein expression and secretion, and polarization in oxalate-treated macrophages. THP1 monocytes were treated with sodium oxalate (50 μM) for 48 h followed by differentiation using PMA (200 nM) for 48 h. THP1 macrophages were then treated with CaOx crystals (50 μM) with or without Interleukin-10 (IL-10; 40 μg/mL) or MitoQ (100 nM) for 48 h. (A) Protein expression was determined for IL-10 and macrophage markers, inducible nitric oxide synthase (iNOS) and arginase-1 (Arg1) using western blotting. (B, E, F) Western blots were analyzed using ImageJ software for semi-quantitative measurement of the proteins. (C, D)The secretion of anti-inflammatory IL-10 and pro-inflammatory Interleukin-6 (IL-6) cytokines were determined using ELISAs. Data are represented as mean ± SEM, n = 3–5. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Fig. 5

The effect of exogenous IL-10 and MitoQ treatment on mitochondrial ROS generation, mitochondrial markers, and ATP levels in oxalate-treated macrophages. THP1 monocytes were treated with sodium oxalate (50 μM) for 48 h followed by differentiation using PMA (200 nM) for 48 h. THP1 macrophages were then treated with CaOx crystals (50 μM) with or without Interleukin-10 (IL-10; 40 μg/mL) or MitoQ (100 nM) for 48 h. (A) Mitochondrial ROS levels were determined using a MitoSox Red fluorescence assay. (B) Protein expression was determined for mitochondrial proteins, VDAC-1 and TOM20, using western blotting. (C–D) Western blots were analyzed using ImageJ software for semi-quantitative measurement of the proteins. (E) The oxygen consumption rate and (F) baseline and stressed OCR parameters were determined using the Mitochondrial Stress Test. (G) The ATP rate assay kit was used to determine mitochondrial and glycolytic sources of ATP using the Seahorse Analyzer. Data are represented as mean ± SEM, n = 3–5. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 6

The effect of exogenous IL-10 and MitoQ treatment on lysosomal and phagolysosomal markers in oxalate-treated macrophages. (A) Protein expression was determined for lysosomal proteins, Rab7 and LAMP1, using western blotting. (B–C) Western blots were analyzed using ImageJ software for semi-quantitative measurement of the proteins. (D) LAMP1 protein expression and nuclei staining (DAPI) was further examined using immunocytochemistry. (E) LysoTracker Red staining was performed to examine co-localization with Rab7. DAPI staining was used to assess nuclei. (F–H) Quantification of the fluorescent staining was determined using ImageJ software. Data are represented as mean ± SEM, n = 3–5. *p < 0.05 and **p < 0.01.

Fig. 7

The effect of exogenous IL-10 and MitoQ treatment on autophagy and phagocytosis in oxalate-treated macrophages. (A) The expression and (B) quantification of LC3B, an autophagy marker was determined using western blotting and ImageJ software, respectively. (C) TOM20 and LC3B protein expression was further examined using immunocytochemistry to examine co-localization of mitophagy. DAPI staining was used to assess nuclei. (D) Quantification of the fluorescent staining was determined using ImageJ software. (E) Phagocytosis of fluorescently labeled Escherichia coli (E. coli) and co-localization to LAMP1 was examined using a fluorescent phagocytosis assay. DAPI staining was used to assess nuclei. (F) Quantification of the number of E. coli per cell. Data are represented as mean ± SEM, n = 3–5. *p < 0.05, **p < 0.01, and ***p < 0.001.