Identification of Novel Fibrosis Modifiers by In Vivo siRNA Silencing.

Fibrotic diseases contribute to 45% of deaths in the industrialized world, and therefore a better understanding of the pathophysiological mechanisms underlying tissue fibrosis is sorely needed. We aimed to identify novel modifiers of tissue fibrosis expressed by myofibroblasts and their progenitors in their disease microenvironment through RNA silencing in vivo. We leveraged novel biology, targeting genes upregulated during liver and kidney fibrosis in this cell lineage, and employed small interfering RNA (siRNA)-formulated lipid nanoparticles technology to silence these genes in carbon-tetrachloride-induced liver fibrosis in mice. We identified five genes, Egr2, Atp1a2, Fkbp10, Fstl1, and Has2, which modified fibrogenesis based on their silencing, resulting in reduced Col1a1 mRNA levels and collagen accumulation in the liver. These genes fell into different groups based on the effects of their silencing on a transcriptional mini-array and histological outcomes. Silencing of Egr2 had the broadest effects in vivo and also reduced fibrogenic gene expression in a human fibroblast cell line. Prior to our study, Egr2, Atp1a2, and Fkbp10 had not been functionally validated in fibrosis in vivo. Thus, our results provide a major advance over the existing knowledge of fibrogenic pathways. Our study is the first example of a targeted siRNA assay to identify novel fibrosis modifiers in vivo

Leucine-rich repeat containing 8A (LRRC8A) is essential for T lymphocyte development and function

Lrrc8a is a ubiquitously expressed gene that encodes a leucine-rich repeat (LRR)–containing protein detected at higher levels on the surface of thymocytes than on other immune cells. We generated Lrrc8a−/− mice to investigate the role of LRRC8A in lymphocyte development and function. Lrrc8a−/− mice had increased prenatal and postnatal mortality, growth retardation, and multiple tissue abnormalities. Lrrc8a−/− mice displayed a modest block in B cell development but intact intrinsic B cell function. In contrast, both Lrrc8a−/− mice and Lrrc8a−/−→Rag2−/− bone marrow chimeras exhibited a severe cell-intrinsic block in early thymic development, with decreased proliferation and increased apoptosis of thymocytes, and impaired peripheral T cell function. Thymic epithelial cells expressed an LRRC8A ligand that was critical for double-negative to double-positive thymocyte differentiation and survival in vitro. LRRC8A constitutively associated with the GRB2–GAB2 complex and lymphocyte-specific protein tyrosine kinase (LCK) in thymocytes. LRRC8A ligation activated AKT via the LCK–ZAP–70–GAB2–PI3K pathway, and AKT phosphorylation was markedly reduced in the thymus of Lrrc8a−/− mice. These findings reveal an essential role for LRRC8A in T cell development, survival, and function

RGS4 inhibits angiotensin II signaling and macrophage localization during renal reperfusion injury independent of vasospasm

Vascular inflammation is a major contributor to the severity of acute kidney injury. In the context of vasospasm-independent reperfusion injury we studied the potential anti-inflammatory role of the Gα-related RGS protein, RGS4. Transgenic RGS4 mice were resistant to 25 minute injury, although post-ischemic renal arteriolar diameter was equal to the wild type early after injury. A 10 minute unilateral injury was performed to study reperfusion without vasospasm. Eighteen hours after injury blood flow was decreased in the inner cortex of wild type mice with preservation of tubular architecture. Angiotensin II levels in the kidneys of wild type and transgenic mice were elevated in a sub-vasoconstrictive range 12 and 18 hours after injury. Angiotensin II stimulated pre-glomerular vascular smooth muscle cells (VSMC) to secrete the macrophage chemoattractant, RANTES; a process decreased by angiotensin II R2 (AT2) inhibition. However, RANTES increased when RGS4 expression was suppressed implicating Gα protein activation in an AT2-RGS4-dependent pathway. RGS4 function, specific to VSMC, was tested in a conditional VSMC-specific RGS4 knockout showing high macrophage density by T2 MRI compared to transgenic and non-transgenic mice after the 10 minute injury. Arteriolar diameter of this knockout was unchanged at successive time points after injury. Thus, RGS4 expression, specific to renal VSMC, inhibits angiotensin II-mediated cytokine signaling and macrophage recruitment during reperfusion, distinct from vasomotor regulation

Male Microchimerism at High Levels in Peripheral Blood Mononuclear Cells from Women with End Stage Renal Disease before Kidney Transplantation

Patients with end stage renal diseases (ESRD) are generally tested for donor chimerism after kidney transplantation for tolerance mechanism purposes. But, to our knowledge, no data are available on natural and/or iatrogenic microchimerism (Mc), deriving from pregnancy and/or blood transfusion, acquired prior to transplantation. In this context, we tested the prevalence of male Mc using a real time PCR assay for DYS14, a Y-chromosome specific sequence, in peripheral blood mononuclear cells (PBMC) from 55 women with ESRD, prior to their first kidney transplantation, and compared them with results from 82 healthy women. Male Mc was also quantified in 5 native kidney biopsies obtained two to four years prior to blood testing and in PBMC from 8 women collected after female kidney transplantation, several years after the initial blood testing. Women with ESRD showed statistically higher frequencies (62%) and quantities (98 genome equivalent cells per million of host cells, gEq/M) of male Mc in their PBMC than healthy women (16% and 0.3 gEq/M, p<0.00001 and p = 0.0005 respectively). Male Mc was increased in women with ESRD whether they had or not a history of male pregnancy and/or of blood transfusion. Three out of five renal biopsies obtained a few years prior to the blood test also contained Mc, but no correlation could be established between earlier Mc in a kidney and later presence in PBMC. Finally, several years after female kidney transplantation, male Mc was totally cleared from PBMC in all women tested but one. This intriguing and striking initial result of natural and iatrogenic male Mc persistence in peripheral blood from women with ESRD raises several hypotheses for the possible role of these cells in renal diseases. Further studies are needed to elucidate mechanisms of recruitment and persistence of Mc in women with ESRD

Mechanisms and Consequences of Dendritic Cell Migration

Dendritic cells (DCs) are critical for adaptive immunity and tolerance. Most DCs are strategically positioned as immune sentinels poised to respond to invading pathogens in tissues throughout the body. Differentiated DCs and their precursors also circulate in blood and can get rapidly recruited to sites of challenge. Within peripheral tissues, DCs collect antigenic material and then traffic to secondary lymphoid organs, where they communicate with lymphocytes to orchestrate adaptive immune responses. Hence, the migration and accurate positioning of DCs is indispensable for immune surveillance. Here, we review the molecular traffic signals that govern the migration of DCs throughout their life cycle

Identification of Novel Fibrosis Modifiers by In Vivo siRNA Silencing.

Fibrotic diseases contribute to 45% of deaths in the industrialized world, and therefore a better understanding of the pathophysiological mechanisms underlying tissue fibrosis is sorely needed. We aimed to identify novel modifiers of tissue fibrosis expressed by myofibroblasts and their progenitors in their disease microenvironment through RNA silencing in vivo. We leveraged novel biology, targeting genes upregulated during liver and kidney fibrosis in this cell lineage, and employed small interfering RNA (siRNA)-formulated lipid nanoparticles technology to silence these genes in carbon-tetrachloride-induced liver fibrosis in mice. We identified five genes, Egr2, Atp1a2, Fkbp10, Fstl1, and Has2, which modified fibrogenesis based on their silencing, resulting in reduced Col1a1 mRNA levels and collagen accumulation in the liver. These genes fell into different groups based on the effects of their silencing on a transcriptional mini-array and histological outcomes. Silencing of Egr2 had the broadest effects in vivo and also reduced fibrogenic gene expression in a human fibroblast cell line. Prior to our study, Egr2, Atp1a2, and Fkbp10 had not been functionally validated in fibrosis in vivo. Thus, our results provide a major advance over the existing knowledge of fibrogenic pathways. Our study is the first example of a targeted siRNA assay to identify novel fibrosis modifiers in vivo

Supplementary Table 1 from Single-Cell Analysis Identifies NOTCH3-Mediated Interactions between Stromal Cells That Promote Microenvironment Remodeling and Invasion in Lung Adenocarcinoma

The summary of clinical features of the cohort A patient samples for scRNA-seq study and cohort B patient samples for IMC</p