Loss of DAP12 and FcRγ drives exaggerated IL-12 production and CD8(+) T cell response by CCR2(+) Mo-DCs

Dap12 and FcRγ, the two transmembrane ITAM-containing signaling adaptors expressed in dendritic cells (DC), are implicated in the regulation of DC function. Several activating and adhesion receptors including integrins require these chains for their function in triggering downstream signaling and effector pathways, however the exact role(s) for Dap12 and FcRγ remains elusive as their loss can lead to both attenuating and enhancing effects. Here, we report that mice congenitally lacking both Dap12 and FcRγ chains (DF) show a massively enhanced effector CD8(+) T cell response to protein antigen immunization or West Nile Virus (WNV) infection. Thus, immunization of DF mice with MHCI-restricted OVA peptide leads to accumulation of IL-12-producing monocyte-derived dendritic cells (Mo-DC) in draining lymph nodes, followed by vastly enhanced generation of antigen-specific IFNγ-producing CD8(+) T cells. Moreover, DF mice show increased viral clearance in the WNV infection model. Depletion of CCR2+ monocytes/macrophages in vivo by administration anti-CCR2 antibodies or clodronate liposomes completely prevents the exaggerated CD8+ T cell response in DF mice. Mechanistically, we show that the loss of Dap12 and FcRγ-mediated signals in Mo-DC leads to a disruption of GM-CSF receptor-induced STAT5 activation resulting in upregulation of expression of IRF8, a transcription factor. Consequently, Dap12- and FcRγ-deficiency exacerbates GM-CSF-driven monocyte differentiation and production of inflammatory Mo-DC. Our data suggest a novel cross-talk between DC-ITAM and GM-CSF signaling pathways, which controls Mo-DC differentiation, IL-12 production, and CD8(+) T cell responses

Genome-wide association meta-analyses and fine-mapping elucidate pathways influencing albuminuria

Abstract: Increased levels of the urinary albumin-to-creatinine ratio (UACR) are associated with higher risk of kidney disease progression and cardiovascular events, but underlying mechanisms are incompletely understood. Here, we conduct trans-ethnic (n = 564,257) and European-ancestry specific meta-analyses of genome-wide association studies of UACR, including ancestry- and diabetes-specific analyses, and identify 68 UACR-associated loci. Genetic correlation analyses and risk score associations in an independent electronic medical records database (n = 192,868) reveal connections with proteinuria, hyperlipidemia, gout, and hypertension. Fine-mapping and trans-Omics analyses with gene expression in 47 tissues and plasma protein levels implicate genes potentially operating through differential expression in kidney (including TGFB1, MUC1, PRKCI, and OAF), and allow coupling of UACR associations to altered plasma OAF concentrations. Knockdown of OAF and PRKCI orthologs in Drosophila nephrocytes reduces albumin endocytosis. Silencing fly PRKCI further impairs slit diaphragm formation. These results generate a priority list of genes and pathways for translational research to reduce albuminuria

Genome-wide association meta-analyses and fine-mapping elucidate pathways influencing albuminuria

Increased levels of the urinary albumin-to-creatinine ratio (UACR) are associated with higher risk of kidney disease progression and cardiovascular events, but underlying mechanisms are incompletely understood. Here, we conduct trans-ethnic (n = 564,257) and European-ancestry specific meta-analyses of genome-wide association studies of UACR, including ancestry- and diabetes-specific analyses, and identify 68 UACR-associated loci. Genetic correlation analyses and risk score associations in an independent electronic medical records database (n = 192,868) reveal connections with proteinuria, hyperlipidemia, gout, and hypertension. Fine-mapping and trans-Omics analyses with gene expression in 47 tissues and plasma protein levels implicate genes potentially operating through differential expression in kidney (including TGFB1, MUC1, PRKCI, and OAF), and allow coupling of UACR associations to altered plasma OAF concentrations. Knockdown of OAF and PRKCI orthologs in Drosophila nephrocytes reduces albumin endocytosis. Silencing fly PRKCI further impairs slit diaphragm formation. These results generate a priority list of genes and pathways for translational research to reduce albuminuria

Genome-wide association meta-analyses and fine-mapping elucidate pathways influencing albuminuria

Publisher Copyright: © 2019, The Author(s).Increased levels of the urinary albumin-to-creatinine ratio (UACR) are associated with higher risk of kidney disease progression and cardiovascular events, but underlying mechanisms are incompletely understood. Here, we conduct trans-ethnic (n = 564,257) and European-ancestry specific meta-analyses of genome-wide association studies of UACR, including ancestry- and diabetes-specific analyses, and identify 68 UACR-associated loci. Genetic correlation analyses and risk score associations in an independent electronic medical records database (n = 192,868) reveal connections with proteinuria, hyperlipidemia, gout, and hypertension. Fine-mapping and trans-Omics analyses with gene expression in 47 tissues and plasma protein levels implicate genes potentially operating through differential expression in kidney (including TGFB1, MUC1, PRKCI, and OAF), and allow coupling of UACR associations to altered plasma OAF concentrations. Knockdown of OAF and PRKCI orthologs in Drosophila nephrocytes reduces albumin endocytosis. Silencing fly PRKCI further impairs slit diaphragm formation. These results generate a priority list of genes and pathways for translational research to reduce albuminuria.Peer reviewe

Dendritic cells utilize the evolutionarily conserved WASH and retromer complexes to promote MHCII recycling and helper T cell priming.

Immature dendritic cells (DCs) maintain a highly dynamic pool of recycling MHCII that promotes sampling of environmental antigens for presentation to T helper cells. However, the molecular basis of MHCII recycling and the cellular machinery that orchestrates MHCII trafficking are incompletely understood. Using a mouse model we show that WASH, an actin regulatory protein that facilitates retromer function, is essential for MHCII recycling and efficient priming of T helper cells. We further demonstrate that WASH deficiency results in impaired MHCII surface levels, recycling, and an accumulation of polyubiquitinated MHCII complexes, which are subsequently slated for premature lysosomal degradation. Consequently, conditional deletion of the Wash gene in DCs impairs priming of both conventional and autoimmune T helper cells in vivo and attenuates disease progression in a model of experimental autoimmune encephalitis (EAE). Thus, we identify a novel mechanism in which DCs employ the evolutionarily conserved WASH and retromer complex for MHCII recycling in order to regulate T helper cell priming

WASH is required for efficient antigen presentation and T cell priming.

<p>(A) BMDCs from <i>WASH^f/f LysM-Cre</i> mice and control <i>LysM-Cre</i> mice were cultured with OT-II T cells and ovalbumin-derived peptide antigen at the indicated doses. After three days in culture, T cell proliferation was determined by flow cytometry and cell count. (B) Alternatively, BMDCs were first pulsed with peptide antigen, then washed and cultured at the indicated cell numbers with OT-II T cells. T cell proliferation was determined by flow cytometry and cell count after three days. (C) <i>WASH^f/f CD11c-Cre</i> mice and control <i>WASH^f/f</i> mice were immunized by subcutaneous injection of ovalbumin peptide in CFA. Seven days later, draining lymph nodes were harvested and restimulated <i>in vitro</i> with ovalbumin peptide. Antigen-specific T cells producing IL-2 upon restimulation were enumerated by ELISPOT.</p

WASH prevents the localization of MHCII into lysosomes following endocytosis.

<p>BMDCs derived from (A) <i>Vav-Cre</i> and (B) <i>WASH^f/f Vav-Cre</i> were cultured with an antibody against MHCII following the endocytosis assay then fixed and labeled with antibodies against WASH and LAMP1 for microscopic analysis. (C) Images from A and B were analyzed for MHCII co-localization with LAMP1 using Pearson's co-localization coefficient in ZEN (Carl Zeiss). Zoomed images are demarcated by the white box and dashed lines in the adjacent images. For each condition, >20 individual cells were imaged. Images were collected with 100× oil objective. Scale bars, 10 µm. Bars represent mean ≥ SEM. Horizontal lines indicate statistical comparison between indicated groups, *<i>p</i>≤0.05. (D) Ubiquitinated MHCII was detected in BMDCs by immunoprecipitation of total MHCII followed by immunoblot for ubiquitin (Ub). Blots were subsequently stripped and reprobed with I-A^b antibody as a loading control.</p

VPS35 localizes with MHCII and Vps35 is required in MHCII cell surface retention.

<p>(A) BMDCs were transduced with shRNA constructs targeting VPS35 (shVPS35) or luciferase (shLuc) as a control. Cells were treated with the Golgi transport inhibitor Brefeldin-A for a 5-hour chase, and MHCII cell surface expression was determined by flow cytometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098606#s2" target="_blank">Materials and Methods</a> section for details). The percent of initial MHCII remaining on the plasma membrane after chase was calculated for three independent samples and plotted as mean ±s.d. To confirm efficient knockdown, VPS35 expression relative to β-actin was determined by quantitative PCR. (B) BMDCs were fixed and labeled with antibodies against MHCII and VPS35 for microscopic analysis. (C) Following the MHCII endocytosis assay, BMDCs were fixed and labeled with anti-VPS35. Zoomed images are demarcated by the white box and dashed lines in the adjacent image. For each condition, >20 individual cells were imaged. Images were collected with 100× oil objective. Scale bars, 10 µm and 1 µm.</p

WASH-deficiency impairs priming of autoreactive T cells and attenuates disease progression in EAE.

<p>(A) <i>WASH^f/f CD11c-Cre</i> mice and control <i>WASH^f/f</i> mice were immunized by subcutaneous injection with MOG peptide in CFA to induce experimental autoimmune encephalitis (EAE). Disease progression was monitored over time using the following scoring system: grade 1 = Tail weakness, grade 2 =  hind limb weakness sufficient to impair righting, grade 3 =  one limb plegic, grade 4 =  hind limb paralysis, grade 5 =  moribund. (B) Mice were immunized with MOG peptide as above and sacrificed at day seven. Draining lymph nodes were harvested and restimulated <i>in vitro</i> with MOG peptide to enumerate antigen-specific T cells by IL-2 ELISPOT.</p