Immunodeficiency and severe susceptibility to bacterial infection associated with a loss-of-function homozygous mutation of MKL1.

Megakaryoblastic leukemia 1 (MKL1), also known as MAL or myocardin-related transcription factor A (MRTF-A), is a coactivator of serum response factor, which regulates transcription of actin and actin cytoskeleton-related genes. MKL1 is known to be important for megakaryocyte differentiation and function in mice, but its role in immune cells is unexplored. Here we report a patient with a homozygous nonsense mutation in the MKL1 gene resulting in immunodeficiency characterized predominantly by susceptibility to severe bacterial infection. We show that loss of MKL1 protein expression causes a dramatic loss of filamentous actin (F-actin) content in lymphoid and myeloid lineage immune cells and widespread cytoskeletal dysfunction. MKL1-deficient neutrophils displayed reduced phagocytosis and almost complete abrogation of migration in vitro. Similarly, primary dendritic cells were unable to spread normally or to form podosomes. Silencing of MKL1 in myeloid cell lines revealed that F-actin assembly was abrogated through reduction of globular actin (G-actin) levels and disturbed expression of multiple actin-regulating genes. Impaired migration of these cells was associated with failure of uropod retraction likely due to altered contractility and adhesion, evidenced by reduced expression of the myosin light chain 9 (MYL9) component of myosin II complex and overexpression of CD11b integrin. Together, our results show that MKL1 is a nonredundant regulator of cytoskeleton-associated functions in immune cells and fibroblasts and that its depletion underlies a novel human primary immunodeficiency.

p63RhoGEF regulates auto- and paracrine signaling in cardiac fibroblasts.

Cardiac remodeling, a hallmark of heart disease, is associated with intense auto- and paracrine signaling leading to cardiac fibrosis. We hypothesized that the specific mediator of Gq/11-dependent RhoA activation p63RhoGEF, which is expressed in cardiac fibroblasts, plays a role in the underlying processes. We could show that p63RhoGEF is up-regulated in mouse hearts subjected to transverse aortic constriction (TAC). In an engineered heart muscle model (EHM), p63RhoGEF expression in cardiac fibroblasts increased resting and twitch tensions, and the dominant negative p63ΔN decreased both. In an engineered connective tissue model (ECT), p63RhoGEF increased tissue stiffness and its knockdown as well as p63ΔN reduced stiffness. In 2D cultures of neonatal rat cardiac fibroblasts, p63RhoGEF regulated the angiotensin II (Ang II)-dependent RhoA activation, the activation of the serum response factor, and the expression and secretion of the connective tissue growth factor (CTGF). All these processes were inhibited by the knockdown of p63RhoGEF or by p63ΔN likely based on their negative influence on the actin cytoskeleton. Moreover, we show that p63RhoGEF also regulates CTGF in engineered tissues and correlates with it in the TAC model. Finally, confocal studies revealed a closely related localization of p63RhoGEF and CTGF in the trans-Golgi network.

Inhibition of calpain-1 stabilizes TCF11/Nrf1 but does not affect its activation in response to proteasome inhibition.

Protein degradation is essential to compensate for the damaging effects of proteotoxic stress. To ensure protein and redox homeostasis in response to proteasome inhibition, the cleavage and nuclear translocation of the endoplasmic reticulum (ER)-bound transcription factor TCF11/Nrf1 (NFE2L1) is crucial for the activation of rescue factors including the synthesis of new proteasomal subunits. Even though TCF11/Nrf1 is an essential transcription factor, the exact mechanisms by which it is activated and stabilized are not fully understood. It was previously shown that the calcium-dependent protease calpain-1 interacts with TCF11/Nrf1 and the TCF11/Nrf1 cleavage site is a potential calpain target. Here, we tested the hypothesis that calpain-1 or -2 cleave TCF11/Nrf1. However, we did not find a role for calpain-1 or -2 in the activation of TCF11/Nrf1 after proteasome inhibition neither by using chemical inhibitors nor siRNA-mediated knockdown or overexpression of calpain subunits. Instead, we found that TCF11/Nrf1 is digested by calpain-1 in vitro and that calpain-1 inhibition slows down the degradation of membrane-bound TCF11/Nrf1 by the proteasome in cultured cells. Thus, we provide evidence that calpain-1 is involved in the degradation of TCF11/Nrf1. Furthermore, we confirmed DDI2 as an essential factor for TCF11/Nrf1 activation and demonstrate an undefined role of DDI2 and calpain-1 in TCF11/Nrf1 stability.

Absence of γ-Chain in Keratinocytes Alters Chemokine Secretion, Resulting in Reduced Immune Cell Recruitment.

Loss-of-function mutations in the common gamma (γc) chain cytokine receptor subunit give rise to severe combined immunodeficiency characterized by lack of T and natural killer cells and infant death from infection. Hematopoietic stem cell transplantation or gene therapy offer a cure, but despite successful replacement of lymphoid immune lineages, a long-term risk of severe cutaneous human papilloma virus infections persists, possibly related to persistent γc-deficiency in other cell types. Here we show that keratinocytes, the only cell type directly infected by human papilloma virus, express functional γc and its co-receptors. After stimulation with the γc-ligand IL-15, γc-deficient keratinocytes show significantly impaired secretion of specific chemokines including CXCL1, CXCL8, and CCL20, resulting in reduced chemotaxis of dendritic cells and CD4+ T cells. Furthermore, γc-deficient keratinocytes also exhibit defective induction of T-cell chemotaxis in a model of stable human papilloma virus-18 infection. These findings suggest that persistent γc-deficiency in keratinocytes alters immune cell recruitment to the skin, which may contribute to the development and persistence of warts in this condition and would require different treatment approaches.

Autoinflammatory periodic fever, immunodeficiency, and thrombocytopenia (PFIT) caused by mutation in actin-regulatory gene WDR1.

The importance of actin dynamics in the activation of the inflammasome is becoming increasingly apparent. IL-1ß, which is activated by the inflammasome, is known to be central to the pathogenesis of many monogenic autoinflammatory diseases. However, evidence from an autoinflammatory murine model indicates that IL-18, the other cytokine triggered by inflammasome activity, is important in its own right. In this model, autoinflammation was caused by mutation in the actin regulatory gene WDR1 We report a homozygous missense mutation in WDR1 in two siblings causing periodic fevers with immunodeficiency and thrombocytopenia. We found impaired actin dynamics in patient immune cells. Patients had high serum levels of IL-18, without a corresponding increase in IL-18-binding protein or IL-1ß, and their cells also secreted more IL-18 but not IL-1ß in culture. We found increased caspase-1 cleavage within patient monocytes indicative of increased inflammasome activity. We transfected HEK293T cells with pyrin and wild-type and mutated WDR1 Mutant protein formed aggregates that appeared to accumulate pyrin; this could potentially precipitate inflammasome assembly. We have extended the findings from the mouse model to highlight the importance of WDR1 and actin regulation in the activation of the inflammasome, and in human autoinflammation.

Biallelic JAK1 mutations in immunodeficient patient with mycobacterial infection.

Mutations in genes encoding components of the immune system cause primary immunodeficiencies. Here, we study a patient with recurrent atypical mycobacterial infection and early-onset metastatic bladder carcinoma. Exome sequencing identified two homozygous missense germline mutations, P733L and P832S, in the JAK1 protein that mediates signalling from multiple cytokine receptors. Cells from this patient exhibit reduced JAK1 and STAT phosphorylation following cytokine stimulations, reduced induction of expression of interferon-regulated genes and dysregulated cytokine production; which are indicative of signalling defects in multiple immune response pathways including Interferon-γ production. Reconstitution experiments in the JAK1-deficient cells demonstrate that the impaired JAK1 function is mainly attributable to the effect of the P733L mutation. Further analyses of the mutant protein reveal a phosphorylation-independent role of JAK1 in signal transduction. These findings clarify JAK1 signalling mechanisms and demonstrate a critical function of JAK1 in protection against mycobacterial infection and possibly the immunological surveillance of cancer.

Nesprin-1 and nesprin-2 regulate endothelial cell shape and migration.

Nesprins are large multi-domain proteins that link the nuclear envelope to the cytoskeleton and nucleoskeleton. Here we show that nesprin-1 and nesprin-2 play important roles in regulating cell shape and migration in endothelial cells. Nesprin-1 or nesprin-2 depletion by RNAi increased endothelial cell spread area and the length of cellular protrusions, as well as stimulating stress fibre assembly which correlated with an increase in F-actin levels. Nuclear area was also increased by nesprin depletion, and localization of the inner nuclear membrane protein emerin to the nuclear envelope was reduced. Depletion of nesprin-1 or nesprin-2 reduced migration of endothelial cells into a cell-free area, and decreased loop formation in an in vitro angiogenesis assay. Taken together, our results indicate that nesprin-1 and nesprin-2 both regulate nuclear and cytoplasmic architecture, which we propose leads to their effects on endothelial cell migration and angiogenic loop formation.