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.

Highlights

• •A dietary oxalate load alters transcriptomics in circulating monocytes from healthy subjects.
• •Oxalate alters Interleukin-10 (IL-10) and mitochondrial reactive oxygen species (ROS) levels in monocytes and macrophages.
• •Exogenous Interleukin-10 (IL-10) and Mitoquinone (MitoQ) treatment reverses oxalate-mediated outcomes in vitro.
• •Consuming meals rich in oxalate could disrupt monocyte and macrophage metabolism and immune response.

1. Introduction

Kidney stone (KS) disease is a painful condition that affects approximately ten percent of the United States population [1]. It is becoming more prevalent in children and adults, imparts a significant economic burden, and promotes a reduction in quality of life [[1], [2], [3], [4]]. KS are also associated with a number of systemic pathologies such as renal and cardiovascular disease [5]. It has been established that these conditions are heavily influenced by increased reactive oxygen species (ROS) levels and that antioxidant therapies may be useful to treat them [[6], [7], [8]]. The mechanisms leading to KS formation and its recurrence continues to remain to be undefined. Thus, understanding additional processes contributing to KS formation could lead to novel approaches for its prevention.

The majority of KS are comprised of CaOx crystals, which form when urine becomes supersaturated with calcium and oxalate [9]. Oxalate is an organic dicarboxylic acid that is produced endogenously or found in specific fruits, vegetables, nuts, or grains [10]. It is absorbed in the intestine and excreted in urine in either soluble or crystalline forms [11]. Consumption of oxalate-rich meals promotes CaOx crystal formation, which is the major component of CaOx KS [12]. The presence of CaOx crystals within the kidney has been suggested to cause oxidative stress and inflammation. In-vitro experiments have demonstrated that renal tubular cells exposed to oxalate release cytokines which are essential for inducing inflammation and immune cell recruitment [[13], [14], [15]]. In addition, inflammatory markers and macrophages have been observed in renal biopsies from patients with CaOx KS or crystal deposits [16,17]. This infers that perturbing the immune system may contribute to CaOx KS formation [18,19].

The innate immune system is essential for development, homeostasis, and the repair of tissues. Monocytes and macrophages are involved in these processes. Monocytes are derived from bone marrow and respond immediately to immune stimuli within the circulation. When needed, monocytes can enter tissue and differentiate into macrophages which exert either pro- or anti-inflammatory responses. Additionally, resident macrophages that remain in tissue following development can also respond to local stimuli [20]. Macrophages are known to rely on metabolism and ROS signaling for proper function [21]. However, if ROS production is excessive, it can lead to oxidative stress and inflammation promoting cellular dysfunction. Interleukin-10 (IL-10) is an anti-inflammatory cytokine produced by activated immune cells [22] which mitigates this inflammatory response and oxidative stress [21,23]. Macrophages, which are known to express IL-10 receptors, are primary IL-10 targets [24]. Furthermore, IL-10 is essential for inhibiting excessive ROS generation in macrophages [25].

In the context of KS formation, macrophages are thought to be critical for engulfing and removing crystals from the kidney as they have been shown to accumulate around crystal deposits in the kidney [26]. If crystals are not efficiently removed from the kidney this may induce inflammation, monocyte recruitment, oxidative stress, tissue injury, and promote KS formation [12]. In addition, monocytes and macrophages could be exposed to elevated levels of oxalate. Earlier in-vitro studies from our laboratory established that oxalate induces oxidative stress and reduces metabolism in monocytes [27]. In addition, we have demonstrated that a single moderate dietary oxalate load equivalent to consumption of a spinach salad induces nanocrystalluria and alters metabolism to varying degrees in circulating monocytes isolated from healthy subjects [27,28]. We have also shown that oxalate exposure impairs cellular bioenergetics, redox homeostasis, and immune response in macrophages [29]. Others have reported that CaOx crystals induce the differentiation of monocytes into M1 pro-inflammatory macrophages [30]. M2 macrophages which have anti-inflammatory properties are known to phagocytize CaOx crystals, which ultimately may play a role in preventing KS formation [17,31]. Thus, learning more about the balance of macrophage response to oxalate and CaOx crystals may provide useful insights into the mechanisms of CaOx KS formation.

The objective of this study was to identify specific molecular mechanisms contributing to oxalate-induced changes in monocytes and macrophages. RNA-Sequencing analysis was performed on monocytes isolated from healthy subjects exposed to a dietary oxalate load to identify specific pathways and molecules affected by oxalate. Based on the pathway analysis, several cellular pathways including IL-10 signaling, mitochondrial metabolism and function, oxalic acid downstream signaling, and autophagy were identified and further analyzed and validated in vitro. We also tested whether exogenous IL-10 or Mitoquinone (MitoQ), a mitochondrial targeted antioxidant that directly detoxifies mitochondrial superoxide, could attenuate these responses. MitoQ is commercially available and has been shown to reduce inflammation and ROS levels in macrophages [32]. It has also been tested in phase II clinical trials for Hepatitis C induced liver disease and Parkinson's disease [33,34]. To our knowledge, this is the first study demonstrating the role of IL-10 and mitochondrial ROS signaling in macrophages following oxalate exposure. This new knowledge could aid in the design and testing of novel therapeutics that could specifically enhance immune and redox signaling in macrophages to lead to the prevention of CaOx KS formation.

2. Methods

2.1. Study approval and cohort

This study was approved by the University of Alabama at Birmingham Institutional Review board and adhered to the Declaration of Helsinki. It is registered on clinicaltrials.gov (NCT03877276). All participants signed a written informed consent form prior to starting the study. Participants were 30.7 ± 2.2 years old with a BMI of 23.9 ± 3.0 kg/m² (n = 3). Sixty-seven percent of the cohort were women. All participants had no personal or family history of CaOx KS disease.

2.2. Controlled diets and clinical procedures

All diets were prepared by the UAB Center for Clinical and Translational Science (CCTS) Bionutrition Core as previously described [28]. The diets contained 50 mg of oxalate and 1000 mg of calcium per day [35]. Participants consumed the diet for 3 days and fasted overnight before returning to the CCTS Clinical Research Unit (CRU) to have their blood drawn via venipuncture. Next, they consumed a low oxalate breakfast and a dietary oxalate load (blended liquid smoothie containing spinach, avocado, banana, and orange juice; 8 mmol of oxalate). Following this, participants fasted from food for 5 h after which another venous blood sample was obtained. These were immediately processed as subsequently described.

2.2.1. Circulating monocyte isolation, RNA preparation, and next-generation sequencing

Circulating monocytes were isolated using a series of centrifugations and positive CD14⁺ antibody selection as previously described [36]. RNA was isolated from monocytes using a Maxwell simply RNA cells kit (Promega, Madison, WI). RNA quality was determined by analyzing the RNA integrity number (RIN) using a Bioanalyzer 2100 instrument (Agilent, Santa Clara, CA). RNA samples with RIN value > 7 were selected to be used for RNA-Sequencing. The NEBNext Ultra II Directional RNA library prep kit was used according to the manufacturer's instructions (New England Biolabs, Ipswich MA). RNA with a RIN value > 7 was used for library preparation and RNA-Seq analysis using the Illumina NextSeq 500 platform per the manufacturer's protocol was done at the UAB Genomic Core Facility. RNA-sequencing data was validated using quantitative real-time PCR (qRT-PCR).

2.3. Bioinformatics analysis

For data processing and bioinformatics, samples were aligned to the human GRCh38 (hg38) p7 Release 25 reference genome [37] from Gencode using STAR version 2.6.1b (parameters used: -outReadsUnmapped Fastx; --outSAMtype BAMSortedByCoordinate; --outSAMattributes All) and the overall alignments to the human genome were satisfactory. Transcript abundances were estimated using HTSeq count version 0.11.0 (parameters used: r pos; -t exon; -i gene_id; -a 10; -s no; -f bam) and the differential expression and regulation between the post-oxalate group vs the pre-oxalate group were performed using DESeq2 version 1.20.0 (using default parameters). Genes with an absolute fold change ≥2.0 and a q-value <0.05 were considered significantly different in expression [38].

2.4. Pathway analyses

To generate cellular networks and processes of the RNA-Sequencing Data, differentially expressed genes containing gene identifiers and corresponding expression values were imported into Ingenuity Pathway Analysis (IPA) software. All identifiers were mapped to their corresponding object in Ingenuity's Knowledge Base. A fold change in gene expression was set to a cutoff of ± 2 and p-value <0.05 to identify differentially regulated Network Eligible molecules. These molecules were then uploaded to the Ingenuity's Knowledge Base to identify significant biological processes, biological functions and/or diseases using Gene Ontology and Functional Analysis [39]. A right-tailed Fisher's exact test was used to calculate a p-value to confirm the data set. In addition, mapping of significant differentially expressed proteins and pathway analysis were performed using Kyoto encyclopedia of genes and genomes (KEGG) and Hallmark pathway analyses using ClusterProfiler (p < 0.05 and q < 0.1). The compareCluster() function was used to perform enrichment analysis.

2.5. Quantitative RT-PCR analysis

cDNA was synthesized from RNA using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). PowerUP SYBR Green Master Mix (ABI, Waltham, MA) was used to perform real-time PCR reactions. Human primers were designed using the Primer 3 software (Primer3 input, version 0.4.0) and synthesized from Eurofins MWG Operon (Ebersberg, Germany). All of the primers used for this study are shown in Supplementary Table 1. The housekeeping gene was GAPDH and the ΔΔCt method was used to determine relative mRNA levels [40,41]. All reactions were performed in duplicates.

2.6. Cell culture models

THP-1 monocytes (a human monocytic cell line) were cultured in RPMI 1640 medium (cat # A1049101, ThermoFisher Scientific, Waltham, MA) supplemented with 10% FBS (cat# A5256801, ThermoFisher Scientific) and 20 μM β-mercaptoethanol (cat# M7522, Sigma Aldrich, St. Louis, MO) at 37 °C in a CO₂ incubator before treatment and during experiments. Monocytes were treated with oxalate (50 μM) with or without IL-10 (40 μg/mL) or MitoQ (200 nM) for 24 h. For additional experiments with macrophages, THP-1 monocytes were treated with NaOx (50 μM) for 24 h and then differentiated into macrophages using 200 nM Phorbol 12-myristate 13-acetate (PMA, cat #P8139) for 48 h [29]. The macrophages were allowed to rest for 24 h in fresh RPMI complete media without PMA before being treated with CaOx crystals (50 μM) for 48 h with or without IL-10 (40 ng/mL) and MitoQ (100 nM).

2.7. Western blotting

Following treatment, cells were lysed with 1X RIPA buffer (cat# 89900, ThermoFisher Scientific, Waltham, MA) on ice for 15 min. All lysates were centrifuged at 13000 rpm for 15 min at 4 °C and the supernatant was collected to determine the protein concentrations using a Pierce Coomassie plus assay kit (cat# PI2326, ThermoFisher Scientific, Waltham, MA). Cell lysates (30 μg) were mixed with Laemmli buffer and heated at 95 °C for 10 min before loading samples in 4–15% Tris glycine gel (cat# 4561085, Bio-Rad, Hercules, CA). The gels were run at 100 V for 1–1.5 h before transferring the protein onto PVDF membranes (cat# 10026934, Bio-Rad, Hercules, CA) using a Bio-Rad semi-dry blotting apparatus. The membrane was blocked using 5% BSA in 1X-PBST for 1 h at room temperature (RT) and subsequently incubated with primary antibodies overnight at 4 °C. The following antibodies were used: IL-10 (cat# sc8438; 1:500, Santa Cruz Biotechnology Inc., Dallas, TX), VDAC-1 (cat# PAI-954A; 1:500, ThermoFisher Scientific, Waltham, MA), TOM20 (cat# sc17764; 1:500, Santa Cruz Biotechnology Inc., Dallas, TX), Histone H3 (cat# 9715S; 1:2000, Cell Signaling Technology Inv., Danvers, MA), Rab7 (cat# 9367S; 1:500, Cell Signaling Technology Inv., Danvers, MA), LAMP1 (cat# sc20011; 1:500, Santa Cruz Biotechnology Inc., Dallas, TX), LC3B (cat# 12741S; 1:500, Cell Signaling Technology Inv., Danvers, MA), β-actin (cat# ab8227; 1:2000, Abcam, Cambridge, UK), iNOS (cat# PA1-036; 1:500, ThermoFisher Scientific, Waltham, MA), and Arg1 (cat# PA5-29645; 1:500, ThermoFisher Scientific, Waltham, MA). The following day, all blots were washed with 1X PBST and incubated with a Horseradish Peroxidase Antibody (HRP) conjugated secondary antibody (goat anti-mouse, cat# 31430; 1:5000; goat anti-rabbit, cat#31460; 1:5000; ThermoFisher Scientific, Waltham, MA) for 2 h at RT. The membranes were washed with 1X PBST and developed using clarity western ECL substrate (cat# 1705061, Bio-Rad, Hercules, CA) on ImageQuant LAS 4000 biomolecular imager chemiluminescent analyzer (GE Healthcare, Chicago, IL). The blots were analyzed using ImageJ software (National Institute of Health, Bethesda, MD).

2.8. Mitochondrial and lysosomal fluorescence assays

Monocytes were treated with sodium oxalate (50 μM) for 48 h before being plated into 6-well plates or differentiated into macrophages in 96-well black plates (30,000 cells/well) using PMA (200 nM) for 48 h. Monocytes and macrophages were treated with calcium oxalate (50 μM) with or without IL-10 (40 ng/mL) and MitoQ (100 nM) for 48 h. After treatment, the cells were washed twice in staining buffer (RPMI + 5% BSA) before being stained with the following fluorescent dyes from ThermoFisher Scientific (Waltham, MA): TMRE, a marker of mitochondrial membrane potential (1 μM, 30 min, cat# T669), MitoSOX Red, a mitochondrial superoxide indicator (2.5 μM, 15 min, cat# M36005), and LysoTracker Red DnD-99, a marker of lysosomes (1 μM, 30 min, cat# L7528). Following staining, cells were washed twice with RPMI staining buffer. The fluorescence was immediately measured using a synergy HT plate reader (BioTek, Winooski, VT).

2.9. Cytokine assays

ELISA MAX assays from BioLegend were used to measure Interleukin-6 (IL-6) pro-inflammatory (cat# 430501) and IL-10 anti-inflammatory cytokine (cat# 430601) levels according to the manufacturer's instructions. In brief, Nunc Maxisorp 96-well plates (cat#44-2404-21, ThermoFisher Scientific, Waltham, MA) were coated with IL-6 and IL-10 capture antibodies overnight at 4 °C. Plates were washed with wash buffer (1X PBST (0.05%) and subsequently blocked with assay diluent (1% BSA in PBS) prior to adding 100 μl of standards and samples (4X dilution) as well as a negative and positive control. The plate was placed on a shaker for 2 h at RT (500 RPM) before being washed 3 times with wash buffer. Next, detection antibodies were added to the plate before being placed on a shaker for 1 h (500 RPM at RT). The plate was washed again with wash buffer before being incubated with Avidin-HRP for 30 min. Subsequently, the plate was washed once more before being exposed to TMB substrate solution for 30 min in the dark. The stop solution was added and absorbance was read at 450 nm and 570 nm to calculate cytokine concentrations (Absorbance 570 nm-450nm) based on a standard curve.

2.10. Cellular bioenergetics analyses

THP-1 monocytes were plated in 96-well Seahorse plates at 70,000 cells/well. The following day, cells were treated with NaOx (50 μM) for 48 h. Cells were subsequently differentiated into macrophages using PMA (200 nM) for 48 h. Next, cells were treated with CaOx crystals (50 μM) with or without IL-10 (40 ng/mL) or MitoQ (100 nM) for 48 h. Following treatment, cellular bioenergetics was measured in macrophages using the Seahorse XFe96 analyzer (Agilent Technologies, Santa Clara, CA). The mitochondrial stress test was performed by sequentially injecting oligomycin (0.5 μg/mL), FCCP (0.6 μM), and Antimycin A (10 μM) into the cellular media as previously described to measure oxygen consumption rates (OCR) [29,42]. In additional experiments, a Real-Time ATP Rate assay was performed to determine the amount of ATP produced from mitochondrial respiration and glycolysis. Macrophages were exposed to oligomycin (1.5 μM) followed by rotenone/antimycin A (0.5 μM). After all assays were completed, samples were lysed with RIPA buffer (ThermoFisher, Waltham, MA) before determining cellular protein concentrations using a Bradford assay. All data were normalized to the protein content in each well. Reagents used for the mitochondrial stress tests were from Sigma-Aldrich (St. Louis, MO) and the reagents used for the ATP Rate assay were from Agilent (Santa Clara, CA).

2.11. Immunocytochemistry

Immunocytochemistry was used to assess protein expression in macrophages. THP1 monocytes were differentiated into macrophages on glass coverslips using PMA (200 nM) for 48 h and treated with oxalate (50 μM) with or without IL-10 (40 ng/mL) or MitoQ (200 nM) for 48 h. Cells were fixed using ice-cold 100% methanol for 15 min at 4 °C before rinsing and adding blocking buffer (1X PBS/3% BSA/0.2% Triton™ X-100 buffer) for 1 h at RT. The blocking buffer was removed from the samples prior to adding the primary antibodies in the dilution buffer (1X PBS/1% BSA/0.2% Triton™ X-100 buffer) overnight at 4 °C. The primary antibodies included: TOMM20 (cat# sc17764; 1:100, Santa Cruz Biotechnology Inc., Dallas, TX), LAMP1 (cat# sc20011; 1:100, Santa Cruz Biotechnology Inc., Dallas, TX), RAB7 (cat# 9367S; 1:100, Cell Signaling Technology Inv., Danvers, MA), and LC3B (cat# 12741S; 1:100, Cell Signaling Technology Inv., Danvers, MA). The following day, cells were rinsed with 1X PBS (3 times for 5 min) before being incubated with fluorochrome-conjugated secondary antibodies for 2 h at RT in the dark. Cells were washed with 1X PBS and counterstained with DAPI for 15 min. The samples were washed and mounted onto a glass slide using ProLong Gold Anti-fade Mountant (cat# P36930, ThermoFisher Scientific, Waltham, MA) and sealed. All slides were stored overnight at RT for curing and then imaged using a confocal microscope (A1R Confocal Microscope, Nikon, Melville, NY) at the UAB High Resolution Imaging Facility. Images were analyzed for fluorescence intensity using ImageJ software (National Institute of Health, Bethesda, MD).

2.12. Phagocytosis assay

The impact of oxalate on macrophage phagocytic activity was assessed using a phagocytosis assay. THP1 monocytes were differentiated into macrophages on glass coverslips and treated with oxalate alone or in combination with IL-10 or MitoQ as described above. Escherichia coli (E. coli) was grown overnight in Lysogeny broth (LB broth) to a concentration at optical density 0.7–0.8 before being incubated with BacLight green bacterial stain (ThermoFisher Scientific, cat#B3500, 0.1 μM) for 15 min at RT. Subsequently, the bacteria were washed with 1X PBS and fixed with 4% formaldehyde solution for 15 min at RT before being washed and resuspended in 1X PBS. Following treatment, macrophages were exposed to stained and fixed E.coli (1:100 MOI) for 30 min, washed with 1X PBS, and fixed using ice-cold 100% ethanol for 15 min at 4 °C. Next, the cells were rinsed and blocked for 1 h at RT with blocking buffer (1XBSA + 3% BSA + 0.2% Triton X-100) and treated with LAMP1 antibody (1:100) overnight at 4 °C. The following day cells were treated with a secondary antibody and mounted on a glass slide as mentioned above. All samples were analyzed using a confocal microscope (Nikon A1R confocal) after curing. Confocal images were analyzed for fluorescence intensity using advanced NIS elements. Experiments were repeated 4 times for each treatment in triplicate and 10 images were analyzed for each replicate.

2.13. Statistical analyses

GraphPad Prism (version 9.2.0; La Jolla, CA) was used to perform statistical analyses and to generate graphs of the data. Descriptive statistics including means and standard errors (SEM) were calculated using n = 3 or more determinations. Comparisons of different groups were performed using a one-way ANOVA. All statistical tests were two-sided and a p value less than 0.05 was considered statistically significant.

3. Results

3.1. Dietary oxalate alters human monocyte transcriptomics

A total of 1,197 genes were determined to be differentially expressed in monocytes following the oxalate load (false discovery rate, FDR ≤0.01, Fig. 1A). Of these genes, 418 were upregulated and 779 were downregulated. Ingenuity pathway analysis identified five top canonical pathways altered in monocytes following the dietary oxalate load. These pathways included: 1) Role of pattern recognition receptor in recognition of bacteria and viruses; 2) Circadian rhythm signaling; 3) Th2 pathway; 4) IL-10 signaling; and 5) Neuro-inflammation signaling pathway (Table 1). The activation z-scores of these pathways are shown in Fig. 1B. Activation z-scores >2, were considered significantly activated (orange color); whereas those < −2, were considered significantly inhibited (blue color). We next performed Gene Ontology analyses and determined that several genes were involved in a number of biological processes (Fig. 1C), which included DNA replication, regulation of autophagy, and macroautophagy. We also analyzed signaling pathways impacted in these cells using Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses (Fig. 1D) and the Hallmark Pathway analyses (Fig. 1E). A number of the pathways were involved in immune related pathways including: cytokine receptor interaction and chemokine signaling pathways (Fig. 1D) as well as Interferon pathways and Tumor Necrosis Factor-alpha (TNF-α) signaling via nuclear factor (NF)-kappa B (Fig. 1E).

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.

Table 1.

Top canonical pathways identified in monocytes from healthy subjects following a dietary oxalate load using Ingenuity Pathway Analysis.

Top Canonical Pathways
	P Value	Overlap
Role of pattern recognition receptor in recognition of bacteria and viruses	3.06E-04	8.0% (11/137)
Circadian Rhythm Signaling	9.65E-04	14.7% (5/34)
Th2 Pathway	2.34E-03	6.7% (10/150)
IL-10 Signaling	4.82E-03	8.7% (6/69)
Neuroinflammation Signaling Pathway	5.35E-03	4.8% (15/311)

To gain additional insight about the cause and effects of these differentially expressed genes, further ingenuity pathway analysis revealed the top regulator effect networks (Table 2). The top networks were mainly involved in the clearance of virus, immune response and activation of leukocytes, and cellular infiltration by leukocytes. In addition, the dietary oxalate load modified top diseases and biofunctions in monocytes as shown in Table 3. Of note, 118 genes were related to the inflammatory response. Genes involved in important molecular and cellular functions were also identified including cell-to-cell signaling and interaction (n = 106), cellular growth and proliferation (n = 105), cellular development (n = 86), cell death and survival (n = 54), and protein synthesis (n = 50) (Table 3).

Table 2.

Top regulator effect networks identified in monocytes from healthy subjects following a dietary oxalate load using Ingenuity Pathway Analysis.

Top Regulator Effect Networks
Regulators	Disease & Function	Consistency Score
CREB1, EPAS1, HGF, HIF1A, PTX3, RETNLB	Clearance of virus, Immune response of leukocyte	16.678
EPAS1, HDL, HIF1A, PTX3	Immune response of leukocyte	16.086
CD44, EPAS1, HIF1A, PTX3, RETNLB	Clearance of virus, Quality of bone cells	16.008
BMP6, CREBBP, CTNNB1, DICER1	Activation of leukocytes	15.513
EPAS1, HIF1A, RETNLB	Cellular infiltration by leukocytes	10.958

Table 3.

Top diseases and biofunctions identified to be modified in monocytes from healthy subjects following a dietary oxalate load using Ingenuity Pathway Analysis.

Top Diseases and Biofunctions
	P Value	# Molecules
Top Disease and disorders
Organismal injury and Abnormalities	2.83E-03 - 7.02E-09	459
Cancer	2.82E-03 - 7.02E-09	454
Inflammatory Response	2.83E-03 - 3.85E-07	118
Neurological Disease	1.69E-03 - 6.81E-08	64
Hematological Diseases	2.83E-03 - 6.99E-07	19
Physiological system Development and Function
Organismal Survival	2.70E-03 - 2.33E-07	133
Lymphoid tissue structure & development	2.83E-03 - 8.49E-08	106
Tissue morphology	2.82E-03 - 8.49E-08	91
Hematological system development & function	2.83E-03 - 6.08E-09	76
Humoral immune response	1.14E-03 - 1.73E-10	40
Molecular and Cellular Functions
Cell-to-cell signaling and interaction	2.83E-03 - 3.25E-08	106
Cellular growth & Proliferation	2.80E-03 - 3.25E-08	105
Cellular development	2.66E-03 - 3.25E-08	86
Cell death and survival	1.00E-07 - 1.00E-07	54
Protein synthesis	1.53E-03 - 1.73E-10	50

Additional analyses showed that the dietary oxalate load regulated a number of genes as well as their mediators in monocytes. Fig. 2 shows the heat-map distribution of differentially regulated genes in post-oxalate samples compared to pre-oxalate samples from three individuals. These genes were categorized into four key pathways including IL-10 signaling (Fig. 2A), mitochondrial metabolism and function (Fig. 2B), oxalic acid downstream signaling (immune and inflammatory response) (Fig. 2C), and autophagy (Fig. 2D). Many genes involved in IL-10 upstream signaling (e.g. ARG1, CCL2, CCR2, TLR4, PDCD1, TLR7, CCR5) were modified in monocytes (Supplementary Fig. 1 and Supplementary Table 2). Specifically, oxalate upregulated the expression of several important surface receptors (i.e. TLR4, CCR2, CCR5) (Fig. 2A); whereas, the expression of a few vital transcription regulators were downregulated (i.e. HIF1α, ID3, REL) (Fig. 2B). Some genes related to mitochondrial pathways were also downregulated in monocytes following the oxalate load (Fig. 2B and Supplementary Fig. 2). In addition, oxalic acid signaling was determined to impact several genes associated with redox homeostasis (i.e. ROS and glutathione metabolism) (Fig. 2C; Supplementary Fig. 3), which could affect the immune and inflammatory response in monocytes. Genes related to autophagy, a process essential for the degradation and removal of proteins or substances from the cell, were also downregulated following the oxalate load (i.e. TOM55, GABARAPL1, UBE2V1) (Fig. 2D). Lastly, several key genes (i.e. NDFUA7, NDFUS7, CCL2, CDKN1A, CXCR4, HIF1a, NLRC4, and NLRP6) were selected from the RNA-Seq data and validated using qRT-PCR (Supplementary Fig. 4). The RNA expression levels from both qRT-PCR and RNA-sequencing analyses were similar.

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).

Oxalate-mediated mitochondrial and lysosomal dysfunction in THP1 monocytes is prevented with exogenous IL-10 and MitoQ treatment.

To confirm the impact of oxalate on IL-10 in monocytes, we assessed IL-10 protein expression using THP1 cells, a human monocytic cell line. As shown in Fig. 3A and B, oxalate caused a decreased IL-10 protein expression in monocytes compared to control cells. In addition, we tested whether treating cells with exogenous IL-10 or MitoQ would elicit any protective effects. Both IL-10 and MitoQ treatment significantly increased IL-10 protein expression in monocytes compared to cells treated with oxalate alone (Fig. 3A and B). The optimal concentrations for both IL-10 and MitoQ were determined using dose-response curves (data not shown).

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.

We further studied the effect of oxalate on THP1 monocyte mitochondrial markers to validate whether the changes observed in human monocytes were oxalate-specific. We first examined the expression of the mitochondrial protein, translocase of the outer mitochondrial membrane 20 (TOM20). TOM20 plays a critical role in importing nuclear-encoded mitochondrial proteins [43] and is considered an indicator of mitochondrial abundance. Oxalate was determined to significantly reduce TOM20 protein expression in monocytes and this was prevented with exogenous IL-10 and MitoQ treatment (Fig. 3A and C). Additionally, we examined mitochondrial membrane potential in monocytes using the fluorescent dye, TMRE. As shown in Fig. 3D, oxalate significantly reduced mitochondrial membrane potential compared to control cells and this was prevented with IL-10 or MitoQ. Alterations in mitochondrial membrane potential and mitochondrial protein expression suggested that ROS could be elevated. Thus, mitochondrial ROS levels were evaluated using the fluorescent dye, MitoSOX Red, a mitochondrial ROS indicator. Oxalate caused monocytes to have a significant increase in mitochondrial ROS generation (Fig. 3E). Importantly, both IL-10 and MitoQ significantly reduced mitochondrial ROS levels in monocytes exposed to oxalate (Fig. 3E).

Increased ROS generation and reduced mitochondrial function in cells can impair the structure and function of lysosomes [44]. To explore this phenomenon further and to support our RNA-sequencing autophagy findings, we assessed the impact of oxalate on several lysosomal markers. As shown in Fig. 3F, LysoTracker Red, an acidotropic fluorescent dye, was used to detect lysosomes. Oxalate significantly reduced LysoTracker Red staining in monocytes and this was prevented with exogenous IL-10 and MitoQ treatment (Fig. 3F). We also evaluated Rab7, a key protein important for regulating phagosome maturation in cells. Oxalate alone and oxalate plus MitoQ treatment did not alter Rab7 protein expression in monocytes (Fig. 3G and H). However, exogenous IL-10 did induce Rab7 protein expression in monocytes exposed to oxalate (Fig. 3G and H). Lastly, oxalate significantly decreased LAMP1 (lysosomal marker) (Fig. 3G and I) and LC3B (autophagy marker) (Fig. 3G and J) protein expression in monocytes. Only exogenous IL-10 was able to preserve both LAMP1 and LC3B protein levels (Fig. 3G, I, and 3J).

3.2. Oxalate suppresses IL-10 signaling and induces inflammation and M1 polarization in THP1 macrophages

Next, we extended our studies to determine whether oxalate could alter the pathways described above in macrophages. Consistent with the monocyte data, we determined that oxalate reduces the expression and secretion of IL-10 in macrophages (Fig. 4A, B & 4C). Both exogenous IL-10 and MitoQ treatment increased IL-10 protein expression and secretion compared to cells treated with oxalate alone (Fig. 4A, B & 4C). In contrast, oxalate significantly upregulated the secretion of the pro-inflammatory cytokine, IL-6, in macrophages (Fig. 4D). Only MitoQ was able to significantly reduce IL-6 secretion (Fig. 4D). We evaluated the effect of oxalate on macrophage polarization since it has been reported that M2 macrophages play an important role in preventing crystal formation [18]. We found an increase in iNOS (M1 marker) expression in oxalate exposed macrophages, which was reversed by exogenous IL-10 treatment (Fig. 4A & E). In contrast, Arginase-1 (Arg1, M2 marker) was significantly reduced by oxalate (Fig. 4A & F). Both IL-10 and MitoQ enhanced Arg1 protein expression. Collectively, these results suggest that oxalate alters IL-10 signaling and macrophage polarization.

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.

Exogenous IL-10 and MitoQ treatment preserves mitochondrial function and ATP levels in oxalate-treated THP1 macrophages.

It is well established that increased inflammation can promote oxidative stress altering mitochondrial quality and function in cells. We previously demonstrated that oxalate reduces redox homeostasis, metabolism, and ATP levels in macrophages [29]. To determine if we could prevent these outcomes, we investigated whether exogenous IL-10 and MitoQ could elicit protective benefits. As expected, oxalate caused increased mitochondrial ROS generation in macrophages and this was inhibited by both exogenous IL-10 and MitoQ treatment (Fig. 5A). In addition, oxalate reduced the protein expression of 2 key mitochondrial proteins, voltage-dependent anion channel (VDAC1) and TOM20 (Fig. 5B, C, & 5D). VDAC1 is located on the outer mitochondrial membrane and is essential for transporting metabolites and mitochondrial-mediated cell death [45]. Exogenous IL-10 preserved both VDAC1 and TOM20 protein expression levels in macrophages during oxalate exposure; whereas, MitoQ only significantly increased VDAC1 protein levels (Fig. 5B, C, & 5D). To further characterize the effect of oxalate on metabolism and ATP, we used the mitochondrial stress test, ATP rate kit, and the Seahorse Analyzer. We determined that oxalate impaired oxygen consumption rate (OCR) in macrophages and this was reversed with both IL-10 and MitoQ treatment (Fig. 5E). We also demonstrated that these two compounds significantly improved OCR under stressed conditions (Fig. 5F). Lastly, we used the ATP rate kit to evaluate whether ATP was generated from either glycolytic or mitochondrial sources. As shown in Fig. 5G, cells exposed to oxalate had significantly reduced ATP levels from both mitochondrial and glycolytic sources compared to control cells. In contrast, cells treated with either exogenous IL-10 or MitoQ had a significant increase in ATP generated from the mitochondria compared to cells treated with oxalate alone (Fig. 5G). These findings suggest both IL-10 and MitoQ exert protective benefits against oxalate-induced mitochondrial dysfunction in macrophages.

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.

Exogenous IL-10 and MitoQ treatment improves lysosomal integrity, phagocytosis, and autophagy in oxalate-treated THP1 macrophages.

Impaired mitochondrial function can disrupt lysosomal integrity and phagocytosis in macrophages. Thus, we investigated the effect of oxalate on lysosomal markers in THP1 macrophages as these organelles are central to phagocytosis. As shown in Fig. 6A, Rab7 was reduced in oxalate-treated macrophages. However, cells treated with oxalate plus exogenous IL-10 had a significant increase in Rab7 protein expression (Fig. 6A and B) whereas, MitoQ did not. In addition, the protein expression of LAMP1 was significantly reduced in macrophages exposed to oxalate (Fig. 6A and C). Both exogenous IL-10 and MitoQ restored LAMP1 protein to levels observed in the control cells (Fig. 6A and C). These findings were further confirmed by staining cells with LAMP1 antibody using immunocytochemistry (Fig. 6D and F). In additional experiments, macrophages were co-stained with LysoTracker Red (red staining) and Rab7 (green staining) to examine lysosome and endolysosomal co-localization, respectively (Fig. 6E). Exogenous IL-10 treatment induced both LysoTracker Red and Rab7 staining compared to macrophages treated with oxalate alone (Fig. 6E, G and 6H). However, MitoQ only increased LysoTracker Red staining compared to cells treated with oxalate alone (Fig. 6E, G and 6H).

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.

We also investigated whether microtubule-associated protein 1A/1B-light chain 3 (LC3B), an autophagy marker, would be impacted by oxalate. As shown in Fig. 7A and B, oxalate significantly reduced LC3B protein expression in macrophages. Interestingly, LC3B protein expression was significantly increased by exogenous IL-10 but reduced in macrophages treated with oxalate and MitoQ (Fig. 7A and B). We also co-stained cells with TOM20and LC3B antibodies to determine whether oxalate affects mitophagy in macrophages. Oxalate caused a significant decrease in TOM20 and LC3B expression (Fig. 7C and D). Both IL-10 and MitoQ increased TOM20 and LC3B protein levels in macrophages. These findings led us to hypothesize that oxalate impairs phagocytosis in macrophages. Thus, we opted to perform a phagocytosis assay using inactive E. coli. The ability of oxalate-treated macrophages to phagocytose inactive E. coli (green staining) cells was significantly reduced compared to control cells (Fig. 7E and F). In contrast, macrophages treated with oxalate and exogenous IL-10 or MitoQ had enhanced phagocytosis as evidenced by an accumulation of E. coli (green staining) in the cells (Fig. 7E and F).

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.

4. Discussion

A number of experimental and clinical studies suggest oxidative stress, inflammation, and myeloid cells contribute to KS formation [14,[46], [47], [48], [49], [50], [51], [52], [53], [54], [55]]. We previously established that monocyte function is altered in a cohort of CaOx stone formers [56] and that oxalate may contribute to this process by altering redox homeostasis and cellular bioenergetic health [27,29]. We have also reported that dietary oxalate can impair monocyte metabolism, stimulate pro-inflammatory cytokine secretion, and increase urinary markers of kidney injury in healthy subjects [28]. In addition, we have determined that oxalate can negatively impact macrophages [29]. A combination of such responses could compromise the ability of macrophages to resolve inflammation and to phagocytose crystals. As a result, this could influence KS formation. Understanding the physiological impact of oxalate on immunity and ROS signaling in the context of CaOx KS disease has not been investigated extensively and is highly warranted. The purpose of this study was to extend our previous investigations to identify specific oxalate-mediated mechanisms that influence monocyte and macrophage function, and to test whether these outcomes could be attenuated with anti-inflammatory or anti-oxidant agents.

To accomplish these objectives, we evaluated the transcriptomic profile of human monocytes isolated from healthy non-stone forming adults fed controlled low oxalate diets and subsequent administration of a dietary oxalate load to elucidate pathways modified by oxalate. Several canonical pathways were dysregulated in monocytes including the role of pattern recognition receptors in recognition of bacteria and viruses and IL-10 signaling. IL-10 acts as a potent negative feedback regulator that influences the resolution of inflammation via autocrine and paracrine mechanisms [22,23]. Monocytes and macrophages express IL-10 receptors and are the primary targets of IL-10 [24,57]. Importantly, loss of IL-10 anti-inflammatory signaling has been shown to reduce macrophage function and bacterial killing [58]. In the current study, we established that genes involved in the recognition of bacteria and viruses and IL-10 signaling, were also downregulated following the oxalate load, suggesting high dietary oxalate intake compromises the immune response via IL-10 signaling.

To investigate this further, we performed a functional analysis. We determined several genes known to regulate functional processes such as clearance of viruses, the immune response of leukocytes, activation of leukocytes, and cellular infiltration were negatively impacted. Ingenuity pathway analysis predicted several biofunctions modulated by oxalate in monocytes, including organismal injury and survival, and cell signaling, death, and survival. Additional analyses of the transcriptomic data, revealed oxalate modified several networks associated with mitochondria, oxalic acid downstream signaling, oxidative stress, and autophagy. Genes related to mitochondrial respiration, mitochondrial membrane potential, and mitochondrial dysfunction were also significantly modified following the oxalate load, which may explain why we previously observed a decline in monocyte cellular bioenergetics in humans [28,56]. Other genes downstream of oxalic acid, ROS, and glutathione pathways were also associated with inflammatory and immune responses (e.g. chemokine ligand 2 (CCL2), also known as monocyte chemoattract protein-1). CCL2 is a key chemokine responsible for regulating monocyte and macrophage migration and infiltration [59,60]. It has been previously reported that renal cells exposed to CaOx crystals have increased CCL2 expression [15]. Consistent with these findings, we demonstrated that dietary oxalate loading induces CCL2 expression in human monocytes.

Based on the transcriptomic data, we postulated that oxalate dysregulates IL-10 and ROS signaling and this leads to mitochondrial and lysosomal dysfunction, and impairs autophagy in monocytes. We also decided to study macrophages in vitro since monocytes are precursors to macrophages, and macrophages are essential for crystal removal. Previous studies have shown monocytes exposed to oxalate crystals in vitro can differentiate into macrophages and that the number of infiltrating macrophages increases in rat kidneys in response to oxalate [30,61]. Therefore, it was important to investigate if the responses observed in human monocytes extended to macrophages. Lastly, we tested whether IL-10 and MitoQ could prevent these responses. We used exogenous IL-10 because it is an important immunoregulatory cytokine and has been previously reported to induce IL-10 in human primary cells [62]. We also tested MitoQ because it has been successfully tested in phase II clinical trials and reported to reduce oxidative stress in several experimental models [33,34,[63], [64], [65], [66], [67]]. MitoQ is also currently available as a dietary supplement. Further, it has been reported to reduce inflammation and ROS in LPS-stimulated macrophages [32] and to rescue lysosomal defects in fibroblasts [44].

We also demonstrated that monocytes exposed to oxalate had decreased IL-10 protein expression and secretion as well as reduced TOM20 gene expression and mitochondrial membrane potential. These data suggested that oxalate may impact mitochondrial protein transport and cell death. Importantly, mitochondrial superoxide levels were elevated in monocytes which could serve as another source for mitochondrial dysfunction [68,69]. Our results are consistent with previous findings that have demonstrated that oxalate induces ROS levels in renal tubular cells and KS animal models [70]. Increased mitochondrial ROS levels have also been shown to impact lysosomal function, which could impair autophagy, phagocytosis, energy supply, cellular function, immune response, and viability [44,71,72]. We determined that oxalate reduced several lysosomal and autophagy markers in monocytes suggesting that oxalate impairs mitophagy which could lead to the accumulation of damaged mitochondria within a cell. This could ultimately eventuate in a highly oxidative, pro-inflammatory environment and cell death.

To confirm the role of inflammation and oxidative stress in these cells, we treated monocytes with exogenous IL-10 or MitoQ. Both compounds induced IL-10 protein expression as well as restored the expression of mitochondrial proteins. As expected, both IL-10 and MitoQ restored mitochondrial membrane potential and reduced mitochondrial superoxide levels with greater evidence of this present in MitoQ treated cells. MitoQ has been previously shown to directly decrease mitochondrial oxidative damage in experimental models and clinical trials [63,73]. Both exogenous IL-10 and MitoQ also improved the acidification of lysosomes. However, only exogenous IL-10 was able to induce endolysosomal and autophagy markers in these cells suggesting that IL-10 specifically plays an important role in maintaining the endolysosomal and phagocytic system in monocytes during oxalate exposure.

It has been proposed that macrophages could serve as potential targets to control disease by reprogramming the macrophages to shift from a M1 to M2 phenotype or vice versa [74]. Thus, we investigated whether oxalate modified immune response and polarization in macrophages. Consistent with monocytes, macrophages exposed to oxalate had a significant decline in IL-10 protein expression and secretion. In addition, IL-6 cytokine levels were significantly elevated in macrophages in response to oxalate. These data were consistent with prior findings that demonstrated IL-6 is elevated in the urine and plasma of stone formers [49,56]. In addition, macrophages exposed to oxalate displayed a more M1 pro-inflammatory phenotype as evidenced by an increase in iNOS protein expression. This data corroborates a previous report showing CaOx crystals can shift macrophages to become M1-like [30]. Macrophages that are classified as M1 phenotypes typically have heightened ROS and pro-inflammatory signaling. We propose that oxalate causes macrophages to shift to a more M1 pro-inflammatory phenotype. We further observed a decrease in M2 macrophage marker, Arginase 1, which suggests that oxalate inhibits the ability of macrophages to shift to M2 macrophage polarization via a reduction in IL-10 signaling. Importantly, it has been reported that IL-10 is essential for inducing M2 macrophage polarization [75]. Thus, these data suggest that oxalate may influence macrophages to shift from M2-like to M1-like macrophages.

A major driving factor for reprogramming macrophages is intact redox signaling and metabolic function. In the current study, macrophages exposed to oxalate had increased mitochondrial superoxide levels, impaired mitochondrial function, and reduced ATP levels, which is consistent with M1-like macrophages [76]. This is significant since mitochondria are essential for integrating metabolism and inflammatory responses in immune cells. We were able to specifically determine that mitochondrial ROS levels could be reduced and mitochondrial markers and ATP levels could be restored with IL-10 and MitoQ treatments. These findings were supported by a study that has shown that IL-10 is essential for regulating macrophage metabolism [77]. In addition, it appears MitoQ's ability to detoxify superoxide is essential to restore mitochondrial function in these cells.

Lastly, we examined the cumulative effects of these mechanisms on macrophage cellular function. IL-10 has been previously reported to be important for inducing mitophagy in macrophages to remove dysfunctional mitochondria [77]. Consistent with the monocyte data, we demonstrated that oxalate exposure reduces expression of mitophagy markers inferring that it attenuates mitophagy in macrophages. IL-10 administration attenuated this response suggesting that it enhances the removal of dysfunctional mitochondria and reduces mitochondrial superoxide generation. Thus, we postulate that IL-10 signaling may be important in regulating mitochondrial function in macrophages.

Phagocytosis is another important function of macrophages and involves the production of superoxide to resolve inflammation [78]. We evaluated the ability of macrophages to phagocytose inactivated bacteria and determined that oxalate reduced phagocytic uptake of inactivated bacteria in macrophages which was consistent with our observation of reduced lysosomal and phagosomal proteins. However, IL-10 and MitoQ treatment improved the phagocytic ability of macrophages by inducing lysosomal as well as phagosome protein expression. These data also support the association between M2 macrophage polarization and phagocytic activity of macrophages as observed in earlier studies [18]. These findings suggest that oxalate induces M1 macrophage polarization by impairing IL-10 signaling mediated mitochondrial and lysosomal dysfunction. The decrease in IL-10 secretion and IL-10 upstream signaling may be responsible for the decline in mitochondrial metabolism as well as inhibiting M2 macrophage polarization, autophagy, and phagocytosis in macrophages.

Taken together, we have demonstrated that oxalate exposure induces molecular changes in primary monocytes from humans as well as monocytes and macrophages in vitro. We determined that oxalate suppresses IL-10 expression, secretion and signaling, and induces mitochondrial superoxide generation in both monocytes and macrophages. We propose these events lead to mitochondrial and lysosomal dysfunction and impaired phagocytosis. This is important as oxalate could compromise the ability of these cells to respond properly to crystals. Importantly, we determined that exogenous IL-10 and MitoQ, could prevent oxalate-induced responses in both cell types by targeting inflammation and mitochondrial superoxide signaling. These data further confirm the critical role of IL-10 and ROS signaling in maintaining macrophage metabolism, polarization, inflammatory response, and autophagic processes. Furthermore, it highlights how oxalate induces ROS in monocytes and macrophages, which is consistent with prior studies using renal cells [70]. It is also important to mention that elevated ROS and inflammatory cytokines produced by renal cells exposed to oxalate could also contribute to the responses observed in this study in vivo. This study has identified specific oxalate-mediated cellular mechanisms that influence monocyte and macrophage immune responses, which may play a vital role in CaOx KS formation. The potential to reverse or alter such responses with agents commercially available agents such as MitoQ may open up a novel therapeutic pasture for CaOx KS prevention.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Michael P. Murphy is on the Scientific Advisory Board of MitoQ, Inc. and holds stock in the company.

All other authors declare no conflicts of interest.

ABBREVIATIONS

AXL

AXL receptor tyrosine kinase

CaOx

Calcium oxalate

CCL2

C–C Motif Chemokine Ligand 2

CDKN1A

Cyclin Dependent Kinase Inhibitor 1A

CXCR4

C-X-C Motif Chemokine Receptor 4

HBEGF

Heparin binding EGF like growth factor

HIF1-ɑ

Hypoxia-inducible factor 1-alpha

IL-1β

Interleukin-1 beta

IL-6

Interleukin-6

IL-10

Interleukin-10

KEGG

Kyoto encyclopedia of genes and genomes

LC3B

Microtubule-associated protein 1A/1B-light chain 3

NDUA7

NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 7

NDUFS7

NADH:Ubiquinone Oxidoreductase Core Subunit S7

NLRC4

NLR Family CARD Domain Containing 4

NLRP6

NLR Family Pyrin Domain Containing 6

NF-kappa B

nuclear factor kappa B

OCR

Oxygen consumption rate

ROS

Reactive oxygen species

TNF-α

Tumor Necrosis Factor-alpha

TOM20

Translocase of the outer mitochondrial membrane 20

VDAC1

Voltage-dependent anion channel

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.