Transcriptional and Linkage Analyses Identify Loci that Mediate the Differential Macrophage Response to Inflammatory Stimuli and Infection

Macrophages display flexible activation states that range between pro-inflammatory (classical activation) and anti-inflammatory (alternative activation). These macrophage polarization states contribute to a variety of organismal phenotypes such as tissue remodeling and susceptibility to infectious and inflammatory diseases. Several macrophage- or immune-related genes have been shown to modulate infectious and inflammatory disease pathogenesis. However, the potential role that differences in macrophage activation phenotypes play in modulating differences in susceptibility to infectious and inflammatory disease is just emerging. We integrated transcriptional profiling and linkage analyses to determine the genetic basis for the differential murine macrophage response to inflammatory stimuli and to infection with the obligate intracellular parasite Toxoplasma gondii. We show that specific transcriptional programs, defined by distinct genomic loci, modulate macrophage activation phenotypes. In addition, we show that the difference between AJ and C57BL/6J macrophages in controlling Toxoplasma growth after stimulation with interferon gamma and tumor necrosis factor alpha mapped to chromosome 3, proximal to the Guanylate binding protein (Gbp) locus that is known to modulate the murine macrophage response to Toxoplasma. Using an shRNA-knockdown strategy, we show that the transcript levels of an RNA helicase, Ddx1, regulates strain differences in the amount of nitric oxide produced by macrophage after stimulation with interferon gamma and tumor necrosis factor. Our results provide a template for discovering candidate genes that modulate macrophage-mediated complex traits

Transcriptional and Linkage Analyses Identify Loci that Mediate the Differential Macrophage Response to Inflammatory Stimuli and Infection.

Macrophages display flexible activation states that range between pro-inflammatory (classical activation) and anti-inflammatory (alternative activation). These macrophage polarization states contribute to a variety of organismal phenotypes such as tissue remodeling and susceptibility to infectious and inflammatory diseases. Several macrophage- or immune-related genes have been shown to modulate infectious and inflammatory disease pathogenesis. However, the potential role that differences in macrophage activation phenotypes play in modulating differences in susceptibility to infectious and inflammatory disease is just emerging. We integrated transcriptional profiling and linkage analyses to determine the genetic basis for the differential murine macrophage response to inflammatory stimuli and to infection with the obligate intracellular parasite Toxoplasma gondii. We show that specific transcriptional programs, defined by distinct genomic loci, modulate macrophage activation phenotypes. In addition, we show that the difference between AJ and C57BL/6J macrophages in controlling Toxoplasma growth after stimulation with interferon gamma and tumor necrosis factor alpha mapped to chromosome 3, proximal to the Guanylate binding protein (Gbp) locus that is known to modulate the murine macrophage response to Toxoplasma. Using an shRNA-knockdown strategy, we show that the transcript levels of an RNA helicase, Ddx1, regulates strain differences in the amount of nitric oxide produced by macrophage after stimulation with interferon gamma and tumor necrosis factor. Our results provide a template for discovering candidate genes that modulate macrophage-mediated complex traits

Autoantibodies against podocytic UCHL1 are associated with idiopathic nephrotic syndrome relapses and induce proteinuria in mice

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shRNA- mediated <i>Ddx1</i> knockdown in C57BL/B6J immortalized macrophages relieves NO inhibition.

<p>Fold knockdown of <i>Ddx1</i> and fold change in NO levels are relative to the <i>Ddx</i>1 expression and the amount of NO in cells transduced with control shRNA (<i>LacZ</i>), respectively. Knocking down <i>E2f6</i>, the other candidate gene at this locus, did not affect the amount of NO produced. Shown are values of NO (μM) fold change obtained from two independent experiments using three different shRNA constructs. The knockdown level is indicated by the black triangles. Fold knockdown was calculated using the 2^deltadelta<i>Ct</i> method. The shRNA transductions and NO measurements were done in 3 independent replicates.</p

The transcriptional response in BMDM is regulated by stimulation-specific <i>trans</i>-loci.

<p>Expression quantitative trait loci (eQTL) in the RI mice were mapped in A) non-stimulated, B) IFNG+TNF-stimulated, C) <i>Toxoplasma</i>-infected, and D) CpG-Stimulated BMDM. Each dot represents a single eQTL (transcript). Significant eQTL located ≤ 10 Mb from the start of the physical location of the corresponding gene were designated as <i>cis</i> mapping (diagonal lines). All other eQTL were designated as <i>trans</i>-mapping (vertical lines). eQTL significance was calculated after 1000 permutations and reported at genome-wide thresholds corresponding to FDR ≤ 10%. Red spots identify genes mapping to a <i>trans</i>-eQTL hotpot (<i>trans</i>-band).</p

BMDM from AJ and B6 mice differ in their response to various stimuli.

<p>A) Stimulation of macrophages for ~ 18 hrs with IFNG+TNF resulted in high production of NO by AJ macrophages (black bars), while B) stimulation with LPS led to higher production of IL-12 in C57BL/6J (B6) macrophages (white bars). C) Stimulation of murine macrophages with LPS or CPG resulted in significantly higher production of IL-10 in B6 macrophages compared to the AJ macrophages. D) B6 macrophages stimulated with IL-4 produced significantly more urea, compared to AJ macrophages, while LPS stimulation induced low amounts of urea in both AJ and B6 BMDM. E) AJ macrophages stimulated with CPG or LPS produced significantly higher amounts of the chemokine CCL22 compared to B6 macrophages. F) The IFNG+TNF stimulated macrophages have increased toxoplasmacidal activity, which is slightly reduced in the presence of aminoguanidine (AG), an inducible nitric oxide synthase inhibitor. Where there was no detectable amount of cytokine/chemokine measured, including in all control (non-stimulated) BMDM, we do not include cytokine/chemokine data in the figures. Three independent replicates; Mean (SD) * <i>p < 0</i>.<i>05</i> (Student’s t-test).</p

Shifting FcγRIIA-ITAM from activation to inhibitory configuration ameliorates arthritis.

International audienceRheumatoid arthritis-associated (RA-associated) inflammation is mediated through the interaction between RA IgG immune complexes and IgG Fc receptors on immune cells. Polymorphisms within the gene encoding the human IgG Fc receptor IIA (hFcγRIIA) are associated with an increased risk of developing RA. Within the hFcγRIIA intracytoplasmic domain, there are 2 conserved tyrosine residues arranged in a noncanonical immunoreceptor tyrosine-based activation motif (ITAM). Here, we reveal that inhibitory engagement of the hFcγRIIA ITAM either with anti-hFcγRII F(ab')2 fragments or intravenous hIgG (IVIg) ameliorates RA-associated inflammation, and this effect was characteristic of previously described inhibitory ITAM (ITAMi) signaling for hFcαRI and hFcγRIIIA, but only involves a single tyrosine. In hFcγRIIA-expressing mice, arthritis induction was inhibited following hFcγRIIA engagement. Moreover, hFcγRIIA ITAMi-signaling reduced ROS and inflammatory cytokine production through inhibition of guanine nucleotide exchange factor VAV-1 and IL-1 receptor-associated kinase 1 (IRAK-1), respectively. ITAMi signaling was mediated by tyrosine 304 (Y304) within the hFcγRIIA ITAM, which was required for recruitment of tyrosine kinase SYK and tyrosine phosphatase SHP-1. Anti-hFcγRII F(ab')2 treatment of inflammatory synovial cells from RA patients inhibited ROS production through induction of ITAMi signaling. These data suggest that shifting constitutive hFcγRIIA-mediated activation to ITAMi signaling could ameliorate RA-associated inflammation

Modulation of the microbiota by oral antibiotics treats immunoglobulin A nephropathy in humanized mice

Immunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis worldwide. IgA is mainly produced by the gut-associated lymphoid tissue (GALT). Both experimental and clinical data suggest a role of the gut microbiota in this disease. We aimed to determine if an intervention targeting the gut microbiota could impact the development of disease in a humanized mouse model of IgAN, the α1KI-CD89Tg mice