Identification of miRNAs that target Fcγ receptor-mediated phagocytosis during macrophage activation syndrome

Abstract

Macrophage activation syndrome (MAS) is a life-threatening complication of systemic juvenile arthritis, accompanied by cytokine storm and hemophagocytosis. In addition, COVID-19-related hyperinflammation shares clinical features of MAS. Mechanisms that activate macrophages in MAS remain unclear. Here, we identify the role of miRNA in increased phagocytosis and interleukin-12 (IL-12) production by macrophages in a murine model of MAS. MAS significantly increased F4/80+ macrophages and phagocytosis in the mouse liver. Gene expression profile revealed the induction of Fcγ receptor-mediated phagocytosis (FGRP) and IL-12 production in the liver. Phagocytosis pathways such as High-affinity IgE receptor is known as Fc epsilon RI -signaling and pattern recognition receptors involved in the recognition of bacteria and viruses and phagosome formation were also significantly upregulated. In MAS, miR-136-5p and miR-501-3p targeted and caused increased expression of Fcgr3, Fcgr4, and Fcgr1 genes in FGRP pathway and consequent increase in phagocytosis by macrophages, whereas miR-129-1-3p and miR-150-3p targeted and induced Il-12. Transcriptome analysis of patients with MAS revealed the upregulation of FGRP and FCGR gene expression. A target analysis of gene expression data from a patient with MAS discovered that miR-136-5p targets FCGR2A and FCGR3A/3B, the human orthologs of mouse Fcgr3 and Fcgr4, and miR-501-3p targets FCGR1A, the human ortholog of mouse Fcgr1. Together, we demonstrate the novel role of miRNAs during MAS pathogenesis, thereby suggesting miRNA mimic-based therapy to control the hyperactivation of macrophages in patients with MAS as well as use overexpression of FCGR genes as a marker for MAS classification.

Keywords: Fcγ receptors; MAS; MiRNA inhibitors; MiRNA mimics; cytokine; miRNA therapeutics; phagocytosis.

Figure 1

Mice with MAS display peripheral pancytopenia, cytokine storm and hepatosplenomegaly. Mice were given CpG as described in Materials and methods to induce MAS, and, on day 10, the mice were euthanized for the analysis as follows. (A) Complete blood count analyses of control and CpG treated mice (n = 5). (B) Measurement of inflammatory cytokines using ELISA assays of blood serum collected from control and mice with MAS (n = 3). (C) Measuring ferritin in the serum using ELISA (n = 8). (D) Measurement of liver weight (n = 4). (E) Measurement of spleen weight (n = 4). Vertical bars represent mean ± SEM. MAS and control mice were compared for statistical significance using Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.0001.

Figure 2

An increase in macrophage and monocyte populations is observed in the liver MNCs of mice with MAS. Liver MNCs were isolated from control (n = 4) and MAS mice (n = 4) and subjected to flow cytometry analysis. (A) Flow plots showing staining of liver MNCs with respective antibodies. (B) Percentage of F4/80+ cells representing macrophages, LY6C+ cells indicating monocytes, and CD11C+ cells representing DCs/monocytes. (C) Total number of respective cell type. (D) Flow plots and percentage of CD11C and CD11B population. (E) Control (brown) and MAS (blue) mice t-SNE plots generated for the markers CD11B, F4/80, LY6C, LY6G, and CD11C. Vertical bars represent mean ± SEM. MAS and control mice were compared for statistical significance using Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.0001.

Figure 3

Gene and miRNA expression profiling of liver MNCs following miRNA and RNA sequencing. (A) Gene expression profile of top upregulated and downregulated genes in control and mice with MAS. Heat map showing expression pattern of genes involved in (B) Fcγ receptor–mediated phagocytosis, (C) Fcϵ RI signaling, (D) role of pattern recognition receptors in recognition of bacteria and viruses, and (E) Il-12 production by macrophages and monocytes. (F) Volcano plot of significantly altered miRNAs in MAS-bearing mice. Target predictions of miRNAs regulating (G) Fcγ receptor–mediated phagocytosis and (H) Il-12 production by macrophages.

Figure 4

Validation of selected miRNAs and their targets by qRT-PCR. (A) Relative expression of miRNAs modulating Fcγ receptor–mediated phagocytosis and Il-12 production by macrophages and monocytes. (B) Relative expression of targets of miR-136 (Fcgr3 and Fcgr4) and miR-501 (Fcgr1) involved in Fcγ receptor–mediated phagocytosis and targets of miR-129-1 (Il12b) and miR-150 (Il12a) involved in Il-12 production. Vertical bars represent mean ± SEM. MAS and control mice were compared for statistical significance using Student’s t-test. *p < 0.05, **p < 0.01, and ****p < 0.0001.

Figure 5

Transfection of mouse macrophages using miRNA mimics and inhibitors and its effect on target genes. Mouse macrophages isolated from MAS mice liver were transfected with miRNA mimics, inhibitors, or respective negative controls. Relative expression of target/s of each miRNA mimic or inhibitor was calculated against the respective negative control following qRT-PCR analysis. Relative expression of targets of (A) miR-136 and miR-501 involved Fcγ receptor–mediated phagocytosis and (B) miR-129-1and miR-150 involved in Il-12 production. Vertical bars represent mean ± SEM. Mimic and inhibitor were compared for statistical significance using Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.005 and ****p < 0.0001.

Figure 6

Modulation of IgG-mediated phagocytosis by miR-136 and miR-501. RAW cell line was transfected with miRNA mimics, inhibitors, or the respective controls, and phagocytosis assay was performed using FITC-conjugated IgG. Fluorescence was quantified at excitation/emission of 465/540 and represented as radiant efficiency. Live cells were imaged following the addition of IgG and staining with NucBlue. Fluorescent microscopy images of cells stained with NucBlue and FITC and merged following transfection with (A) mimics and (B) inhibitors (20×; scale bar, 100 µm). Radiant efficiency for (C) miR-136 mimic and inhibitor and (D) miR-501 mimic and inhibitor. Vertical bars represent mean ± SEM. Mimic/inhibitor control and treated as well as MAS and control mice were compared for statistical significance using Student’s t-test. *p < 0.05.

Figure 7

In vivo phagocytosis in mice with MAS and its regulation by miRNA in vitro. RAW cell line was transfected with miRNA mimics, inhibitors, or the respective controls, and phagocytosis assay was performed using pHrodo deep red E coli bioparticles. Fluorescence was quantified at 640/680 excitation/emission and represented as radiant efficiency. Live cells were imaged following the addition of bioparticles and staining with NucBlue. Fluorescent microscopy images of cells stained with NucBlue and deep red and merged following transfection with (A) mimics and (B) inhibitors (20×; scale bar, 100 µm). Radiant efficiency for (C) miR-136 mimic and inhibitor and (D) miR-501 mimic and inhibitor. Control and MAS mice were injected with pHrodo deep red E coli bioparticles intravenously, and fluorescence was imaged and quantified using IVIS Spectrum in vivo imaging system. Respective images of (E) normal liver without particles injected, liver from (F) control and (G) MAS mouse following bioparticles injection, and (H) quantification of fluorescence. Vertical bars represent mean ± SEM. Mimic/inhibitor control and treated as well as MAS and control mice were compared for statistical significance using Student’s t-test. *p < 0.05 and **p < 0.01.

Figure 8

Gene expression profiling and pathway enrichment analysis of normal control (C) and patients with MAS (M). (A) Canonical pathways following comparison of gene expression profiles and pathway enrichment analysis of normal controls and patients with MAS. (B) Heat map demonstrating fold change expression of genes in controls and patients with MAS in FGRP. (C) Sequence similarity of human and mouse miR-136-5p and miR-501-3p. (D) Selected targets of miR-136-5p and (E) miR-501-3p in patient with MAS. (F) TargetScan analysis of miR-136 and miR-501 binding to the respective 3′ UTR of target genes.

Figure 9

Bubble chart derived from the target analysis of (A) miR-136-5p and (B) miR-501-3p against a MAS patient gene expression data.