Migration-induced cell shattering due to DOCK8 deficiency causes a type 2-biased helper T cell response.

Mutations that impact immune cell migration and result in immune deficiency illustrate the importance of cell movement in host defense. In humans, loss-of-function mutations in DOCK8, a guanine exchange factor involved in hematopoietic cell migration, lead to immunodeficiency and, paradoxically, allergic disease. Here, we demonstrate that, like humans, Dock8-/- mice have a profound type 2 CD4+ helper T (TH2) cell bias upon pulmonary infection with Cryptococcus neoformans and other non-TH2 stimuli. We found that recruited Dock8-/-CX3CR1+ mononuclear phagocytes are exquisitely sensitive to migration-induced cell shattering, releasing interleukin (IL)-1ß that drives granulocyte-macrophage colony-stimulating factor (GM-CSF) production by CD4+ T cells. Blocking IL-1ß, GM-CSF or caspase activation eliminated the type-2 skew in mice lacking Dock8. Notably, treatment of infected wild-type mice with apoptotic cells significantly increased GM-CSF production and TH2 cell differentiation. This reveals an important role for cell death in driving type 2 signals during infection, which may have implications for understanding the etiology of type 2 CD4+ T cell responses in allergic disease.

Regulation of age-associated B cells by IRF5 in systemic autoimmunity.

Age-associated B cells (ABCs) are a subset of B cells dependent on the transcription factor T-bet that accumulate prematurely in autoimmune settings. The pathways that regulate ABCs in autoimmunity are largely unknown. SWAP-70 and DEF6 (also known as IBP or SLAT) are the only two members of the SWEF family, a unique family of Rho GTPase-regulatory proteins that control both cytoskeletal dynamics and the activity of the transcription factor IRF4. Notably, DEF6 is a newly identified human risk variant for systemic lupus erythematosus. Here we found that the lupus syndrome that developed in SWEF-deficient mice was accompanied by the accumulation of ABCs that produced autoantibodies after stimulation. ABCs from SWEF-deficient mice exhibited a distinctive transcriptome and a unique chromatin landscape characterized by enrichment for motifs bound by transcription factors of the IRF and AP-1 families and the transcription factor T-bet. Enhanced ABC formation in SWEF-deficient mice was controlled by the cytokine IL-21 and IRF5, whose variants are strongly associated with lupus. The lack of SWEF proteins led to dysregulated activity of IRF5 in response to stimulation with IL-21. These studies thus elucidate a previously unknown signaling pathway that controls ABCs in autoimmunity.

Systemic Inflammation and Normocytic Anemia in DOCK11 Deficiency.

BACKGROUND: Increasing evidence links genetic defects affecting actin-regulatory proteins to diseases with severe autoimmunity and autoinflammation, yet the underlying molecular mechanisms are poorly understood. Dedicator of cytokinesis 11 (DOCK11) activates the small Rho guanosine triphosphatase (GTPase) cell division cycle 42 (CDC42), a central regulator of actin cytoskeleton dynamics. The role of DOCK11 in human immune-cell function and disease remains unknown. METHODS: We conducted genetic, immunologic, and molecular assays in four patients from four unrelated families who presented with infections, early-onset severe immune dysregulation, normocytic anemia of variable severity associated with anisopoikilocytosis, and developmental delay. Functional assays were performed in patient-derived cells, as well as in mouse and zebrafish models. RESULTS: We identified rare, X-linked germline mutations in DOCK11 in the patients, leading to a loss of protein expression in two patients and impaired CDC42 activation in all four patients. Patient-derived T cells did not form filopodia and showed abnormal migration. In addition, the patient-derived T cells, as well as the T cells from Dock11-knockout mice, showed overt activation and production of proinflammatory cytokines that were associated with an increased degree of nuclear translocation of nuclear factor of activated T cell 1 (NFATc1). Anemia and aberrant erythrocyte morphologic features were recapitulated in a newly generated dock11-knockout zebrafish model, and anemia was amenable to rescue on ectopic expression of constitutively active CDC42. CONCLUSIONS: Germline hemizygous loss-of-function mutations affecting the actin regulator DOCK11 were shown to cause a previously unknown inborn error of hematopoiesis and immunity characterized by severe immune dysregulation and systemic inflammation, recurrent infections, and anemia. (Funded by the European Research Council and others.).

Dedicator of Cytokinesis 2 (DOCK2) Deficiency Attenuates Lung Injury Associated with Chronic High-Fat and High-Fructose Diet-Induced Obesity.

Obesity is a major risk factor for lung disease development. However, little is known about the impact of chronic high-fat and high-fructose (HFHF) diet-induced obesity on lung inflammation and subsequent pulmonary fibrosis. Herein we hypothesized that dedicator of cytokinesis 2 (DOCK2) promotes a proinflammatory phenotype of lung fibroblasts (LFs) to elicit lung injury and fibrosis in chronic HFHF diet-induced obesity. An HFHF diet for 20 weeks induced lung inflammation and profibrotic changes in wild-type C57BL/6 mice. CD68 and monocyte chemoattractant protein-1 (MCP-1) expression were notably increased in the lungs of wild-type mice fed an HFHF diet. An HFHF diet further increased lung DOCK2 expression that co-localized with fibroblast-specific protein 1, suggesting a role of DOCK2 in regulating proinflammatory phenotype of LFs. Importantly, DOCK2 knockout protected mice from lung inflammation and fibrosis induced by a HFHF diet. In primary human LFs, tumor necrosis factor-α (TNF-α) and IL-1ß induced DOCK2 expression concurrent with MCP-1, IL-6, and matrix metallopeptidase 2. DOCK2 knockdown suppressed TNF-α-induced expression of these molecules and activation of phosphatidylinositol 3-kinase/AKT and NF-κB signaling pathways, suggesting a mechanism of DOCK2-mediated proinflammatory and profibrotic changes in human LFs. Taken together, these findings reveal a previously unrecognized role of DOCK2 in regulating proinflammatory phenotype of LFs, potentiation of lung inflammation, and pulmonary fibrosis in chronic HFHF diet-caused obesity.

Intracellular receptor EPAC regulates von Willebrand factor secretion from endothelial cells in a PI3K-/eNOS-dependent manner during inflammation.

Coagulopathy is associated with both inflammation and infection, including infections with novel severe acute respiratory syndrome coronavirus-2, the causative agent Coagulopathy is associated with both inflammation and infection, including infection with novel severe acute respiratory syndrome coronavirus-2, the causative agent of COVID-19. Clot formation is promoted via cAMP-mediated secretion of von Willebrand factor (vWF), which fine-tunes the process of hemostasis. The exchange protein directly activated by cAMP (EPAC) is a ubiquitously expressed intracellular cAMP receptor that plays a regulatory role in suppressing inflammation. To assess whether EPAC could regulate vWF release during inflammation, we utilized our EPAC1-null mouse model and revealed increased secretion of vWF in endotoxemic mice in the absence of the EPAC1 gene. Pharmacological inhibition of EPAC1 in vitro mimicked the EPAC1-/- phenotype. In addition, EPAC1 regulated tumor necrosis factor-α-triggered vWF secretion from human umbilical vein endothelial cells in a manner dependent upon inflammatory effector molecules PI3K and endothelial nitric oxide synthase. Furthermore, EPAC1 activation reduced inflammation-triggered vWF release, both in vivo and in vitro. Our data delineate a novel regulatory role for EPAC1 in vWF secretion and shed light on the potential development of new strategies to control thrombosis during inflammation.

Two isoforms of the RAC-specific guanine nucleotide exchange factor TIAM2 act oppositely on transmission ratio distortion by the mouse t-haplotype.

Transmission ratio distortion (TRD) by the mouse t-haplotype, a variant region on chromosome 17, is a well-studied model of non-Mendelian inheritance. It is characterized by the high transmission ratio (up to 99%) of the t-haplotype from t/+ males to their offspring. TRD is achieved by the exquisite ability of the responder (Tcr) to trigger non-Mendelian inheritance of homologous chromosomes. Several distorters (Tcd1-Tcd4), which act cumulatively, together promote the high transmission ratio of Tcr and the t-haplotype. Molecularly, TRD is brought about by deregulation of Rho signaling pathways via the distorter products, which impair sperm motility, and the t-sperm specific rescue of sperm motility by the responder. The t-sperm thus can reach the egg cells faster than +-sperm and fertilize them. Previously we have shown that the responder function is accomplished by a dominant negative form of sperm motility kinase (SMOKTCR), while the distorter functions are accomplished by the Rho G protein regulators TAGAP, FGD2 and NME3 proposed to function in two oppositely acting pathways. Here we identify the RAC1-specific guanine nucleotide exchange factor TIAM2 as modifier of t-haplotype TRD. Tiam2 is expressed in two isoforms, the full-length (Tiam2l) and a short transcript (Tiam2s). Tiam2s expression from the t-allele is strongly increased compared to the wild-type allele. By transgenic approaches we show that Tiam2s enhances t-haplotype transmission, while Tiam2l has the opposite effect. Our data show that a single modifier locus can encode different gene products exerting opposite effects on a trait. They also suggest that the expression ratio of the isoforms determines if the outcome is an enhancing or a suppressive effect on the trait.

Lineage-Specific Chimerism and Outcome After Hematopoietic Stem Cell Transplantation for DOCK8 Deficiency.

Bi-allelic variants in the dedicator of cytokinesis 8 (DOCK8) gene cause a combined immunodeficiency, characterized by recurrent sinopulmonary and skin infections, food allergies, eczema, eosinophilia, and elevated IgE. Long-term outcome is poor given susceptibility to infections, malignancy, and vascular complications. Allogeneic hematopoietic stem cell transplantation is currently the only curative treatment option and has shown promising outcome. The impact of mixed chimerism on long-term outcome is unclear. We reasoned that reversal of disease phenotype would depend on cell lineage-specific chimerism. DOCK8 variants were confirmed by Sanger and/or exome sequencing and immunoblot and/or intracellular flow cytometry. Donor chimerism was analyzed by XY-fluorescence in situ hybridization or quantitative short tandem repeat PCR. Outcome was assessed by laboratory tests, lymphocyte subsets, intracellular DOCK8 protein flow cytometry, T-cell proliferation analysis, and multiparameter immunoblot allergy screening. We report on nine patients, four of whom with mixed chimerism, with a median follow-up of 78 months after transplantation. Overall, we report successful transplantation with improvement of susceptibility to infections and allergies, and resolution of eczema in all patients. Immunological outcome in patients with mixed chimerism suggests a selective advantage for wild-type donor T-cells but lower donor B-cell chimerism possibly results in a tendency to hypogammaglobulinemia. No increased infectious and allergic complications were associated with mixed chimerism. Aware of the relatively small cohort size, we could not demonstrate a consistent detrimental effect of mixed chimerism on clinical outcomes. We nevertheless advocate aiming for complete donor chimerism in treating DOCK8 deficiency, but recommend reduced toxicity conditioning.

Daratumumab for delayed RBC engraftment following major ABO mismatched haploidentical bone marrow transplantation.

BACKGROUND: Recent case reports have described the efficacy of daratumumab to treat refractory pure red cell aplasia (PRCA) following major ABO mismatched allogeneic hematopoietic stem cell transplantation (HSCT). In this report, we describe the use of daratumumab as a first-line agent for treatment of delayed red blood cell (RBC) engraftment following a major ABO mismatched pediatric HSCT and provide a review of the literature. STUDY DESIGN AND MATERIALS: We report on a 14-year-old with DOCK8 deficiency who underwent a myeloablative, haploidentical bone marrow transplant from her major ABO mismatched sister (recipient O+, donor A+) for treatment of her primary immunodeficiency. Despite achieving full donor chimerism, she had delayed RBC engraftment requiring ongoing transfusions. Due to iron deposition, symptomatic anemia, and persistence of anti-A iso-hemagglutinins despite discontinuation of immunosuppression, treatment for delayed RBC engraftment with the CD38-targeted monoclonal antibody daratumumab was selected as a less immunosuppressive agent that could more selectively target iso-hemagglutinin producing plasma cells without causing broad B-cell aplasia. RESULTS: Clinical effect with daratumumab was demonstrated by reduced iso-hemagglutinin titer, increased reticulocytosis, normalization of her hemoglobin, and transfusion independence. In the 11-month follow-up period to date, no additional transfusions or immunosuppression have been necessary, despite persistence of low-level anti-A iso-hemagglutinin. CONCLUSION: Our experience suggests that daratumumab was an effective first-line therapy for delayed RBC engraftment and that earlier consideration for daratumumab in treatment of delayed RBC engraftment may be warranted.

Epac1 (Exchange Protein Directly Activated by cAMP 1) Upregulates LOX-1 (Oxidized Low-Density Lipoprotein Receptor 1) to Promote Foam Cell Formation and Atherosclerosis Development.

OBJECTIVE: The cAMP second messenger system, a major stress-response pathway, plays essential roles in normal cardiovascular functions and in pathogenesis of heart diseases. Here, we test the hypothesis that the Epac1 (exchange protein directly activated by cAMP 1) acts as a major downstream effector of cAMP signaling to promote atherogenesis and represents a novel therapeutic target. Approach and Results: To ascertain Epac1's function in atherosclerosis development, a triple knockout mouse model (LTe) was generated by crossing Epac1-/- mice with atherosclerosis-prone LDb mice lacking both Ldlr and Apobec1. Deletion of Epac1 led to a significant reduction of atherosclerotic lesion formation as measured by postmortem staining, accompanied by attenuated macrophage/foam cell infiltrations within atherosclerotic plaques as determined by immunofluorescence staining in LTe animals compared with LDb littermates. Primary bone marrow-derived macrophages were isolated from Epac1-null and wild-type mice to investigate the role of Epac1 in lipid uptake and foam cell formation. ox-LDLs (oxidized low-density lipoproteins) stimulation of bone marrow-derived macrophages led to elevated intracellular cAMP and Epac1 levels, whereas an Epac-specific agonist, increased lipid accumulation in wild-type, but not Epac1-null, bone marrow-derived macrophages. Mechanistically, Epac1 acts through PKC (protein kinase C) to upregulate LOX-1 (ox-LDL receptor 1), a major scavenger receptor for ox-LDL uptake, exerting a feedforward mechanism with ox-LDL to increase lipid uptake and propel foam cell formation and atherogenesis. CONCLUSIONS: Our study demonstrates a fundamental role of cAMP/Epac1 signaling in vascular remodeling by promoting ox-LDL uptake and foam cell formation during atherosclerosis lesion development. Therefore, Epac1 represents a promising, unexplored therapeutic target for atherosclerosis.

Rho-GEF trio regulates osteoclast differentiation and function by Rac1/Cdc42.

Many bone diseases result from abnormal bone resorption by osteoclasts (OCs). Studying OC related regulatory genes is necessary for the development of new therapeutic strategies. Rho GTPases have been proven to regulate OC differentiation and function and only mature OCs can carry out bone resorption. Here we demonstrate that Rac1 and Cdc42 exchange factor Triple functional domain (Trio) is critical for bone resorption caused by OCs. In this study, we created LysM-Cre;Triofl/fl conditional knockout mice in which Trio was conditionally ablated in monocytes. LysM-Cre;Triofl/fl mice showed increased bone mass due to impaired bone resorption caused by OCs. Furthermore, our in vitro analysis indicated that Trio conditional deficiency significantly suppressed OC differentiation and function. At the molecular level, Trio deficiency significantly inhibited the expression of genes critical for osteoclastogenesis and OC function. Mechanistically, our researches suggested that perturbed Rac1/Cdc42-PAK1-ERK/p38 signaling could be used to explain the lower ability of bone resorption in CKO mice. Taken together, this study indicates that Trio is a regulator of OCs. Studying the role of Trio in OCs provides a potential new insight for the treatment of OC related bone diseases.

CalDAG-GEFI Deficiency in a Family with Symptomatic Heterozygous and Homozygous Carriers of a Likely Pathogenic Variant in RASGRP2.

RASGRP2 encodes the calcium and diacylglycerol (DAG)-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) identified as a Rap1-activating molecule. Pathogenic variants previously identified in RASGRP2 allowed the characterization of CalDAG-GEFI deficiency as a non-syndromic, autosomal recessive platelet function disease. We report on the clinical manifestations and laboratory features of a Portuguese family with a likely pathogenic variant in RASGRP2 (c.999G>C leading to a p.Lys333Asn change in the CDC25 catalytic domain of CalDAG-GEFI) and discuss the contribution of this variant to the disease manifestations. Based on the study of this family with one homozygous patient and five heterozygous carriers and on a critical analysis of the literature, we challenge previous knowledge that CalDAG-GEFI deficiency only manifests in homozygous patients. Our data suggest that at least for the RASGRP2 variant reported herein, there is a phenotypic expression, albeit milder, in heterozygous carriers.

Proteomic Analysis of Marinesco-Sjogren Syndrome Fibroblasts Indicates Pro-Survival Metabolic Adaptation to SIL1 Loss.

Marinesco-Sjogren syndrome (MSS) is a rare multisystem pediatric disorder, caused by loss-of-function mutations in the gene encoding the endoplasmic reticulum cochaperone SIL1. SIL1 acts as a nucleotide exchange factor for BiP, which plays a central role in secretory protein folding. SIL1 mutant cells have reduced BiP-assisted protein folding, cannot fulfil their protein needs, and experience chronic activation of the unfolded protein response (UPR). Maladaptive UPR may explain the cerebellar and skeletal muscle degeneration responsible for the ataxia and muscle weakness typical of MSS. However, the cause of other more variable, clinical manifestations, such as mild to severe mental retardation, hypogonadism, short stature, and skeletal deformities, is less clear. To gain insights into the pathogenic mechanisms and/or adaptive responses to SIL1 loss, we carried out cell biological and proteomic investigations in skin fibroblasts derived from a young patient carrying the SIL1 R111X mutation. Despite fibroblasts not being overtly affected in MSS, we found morphological and biochemical changes indicative of UPR activation and altered cell metabolism. All the cell machineries involved in RNA splicing and translation were strongly downregulated, while protein degradation via lysosome-based structures was boosted, consistent with an attempt of the cell to reduce the workload of the endoplasmic reticulum and dispose of misfolded proteins. Cell metabolism was extensively affected as we observed a reduction in lipid synthesis, an increase in beta oxidation, and an enhancement of the tricarboxylic acid cycle, with upregulation of eight of its enzymes. Finally, the catabolic pathways of various amino acids, including valine, leucine, isoleucine, tryptophan, lysine, aspartate, and phenylalanine, were enhanced, while the biosynthetic pathways of arginine, serine, glycine, and cysteine were reduced. These results indicate that, in addition to UPR activation and increased protein degradation, MSS fibroblasts have profound metabolic alterations, which may help them cope with the absence of SIL1.

Evaluation of Genotypic Antiviral Resistance Testing as an Alternative to Phenotypic Testing in a Patient with DOCK8 Deficiency and Severe HSV-1 Disease.

Antiviral resistance frequently complicates the treatment of herpes simplex virus (HSV) infections in immunocompromised patients. Here we present the case of an adolescent boy with dedicator of cytokinesis 8 (DOCK8) deficiency, who experienced recurrent infections with resistant HSV-1. We used both phenotypic and genotypic methodologies to characterize the resistance profile of HSV-1 in the patient and conclude that genotypic testing outperformed phenotypic testing. We also present the first analysis of intrahost HSV-1 evolution in an immunocompromised patient. While HSV-1 can remain static in an immunocompetent individual for decades, the virus from this patient rapidly acquired genetic changes throughout its genome. Finally, we document a likely case of transmitted resistance in HSV-1 between the patient and his brother, who also has DOCK8 deficiency. This event demonstrates that resistant HSV-1 is transmissible among immunocompromised persons.

DOCK8 is expressed in microglia, and it regulates microglial activity during neurodegeneration in murine disease models.

Dedicator of cytokinesis 8 (DOCK8) is a guanine nucleotide exchange factor whose loss of function results in immunodeficiency, but its role in the central nervous system (CNS) has been unclear. Microglia are the resident immune cells of the CNS and are implicated in the pathogenesis of various neurodegenerative diseases, including multiple sclerosis (MS) and glaucoma, which affects the visual system. However, the exact roles of microglia in these diseases remain unknown. Herein, we report that DOCK8 is expressed in microglia but not in neurons or astrocytes and that its expression is increased during neuroinflammation. To define the role of DOCK8 in microglial activity, we focused on the retina, a tissue devoid of infiltrating T cells. The retina is divided into distinct layers, and in a disease model of MS/optic neuritis, DOCK8-deficient mice exhibited a clear reduction in microglial migration through these layers. Moreover, neuroinflammation severity, indicated by clinical scores, visual function, and retinal ganglion cell (RGC) death, was reduced in the DOCK8-deficient mice. Furthermore, using a glaucoma disease model, we observed impaired microglial phagocytosis of RGCs in DOCK8-deficient mice. Our data demonstrate that DOCK8 is expressed in microglia and regulates microglial activity in disease states. These findings contribute to a better understanding of the molecular pathways involved in microglial activation and implicate a role of DOCK8 in several neurological diseases.

Oleate disrupts cAMP signaling, contributing to potent stimulation of pancreatic ß-cell autophagy.

Autophagy is critical for maintaining cellular function via clearance of excess nutrients and damaged organelles. In pancreatic ß-cells, it helps counter the endoplasmic reticulum (ER) stress that impairs insulin secretory capacity during Type 2 diabetes. Chronic exposure of ß-cells to saturated fatty acids (FAs) such as palmitate stimulates ER stress and modulates autophagy, but the effects of unsaturated FAs such as oleate, which are also elevated during obesity, are less well understood. We therefore treated MIN6 cells and mouse islets for 8-48 h with either palmitate or oleate, and then monitored autophagic flux, signaling pathways, lysosomal biology, and phospholipid profiles. Compared with palmitate, oleate more effectively stimulated both autophagic flux and clearance of autophagosomes. The flux stimulation occurred independently of ER stress, nutrient-sensing (mTOR) and signaling pathways (protein kinases A, C, and D). Instead the mechanism involved the exchange factor directly activated by cAMP 2 (EPAC2). Oleate reduced cellular cAMP, and its effects on autophagic flux were reproduced or inhibited, respectively, by Epac2 knockdown or activation. Oleate also increased lysosomal acidity and increased phospholipid saturation, consistent with improved autophagosomal fusion with lysosomes. We conclude that a potent stimulation of autophagy might help explain the known benefits of unsaturated FAs in countering the toxicity of saturated FAs in ß-cells during obesity and lipid loading.

Novel loss-of-function variants in TRIO are associated with neurodevelopmental disorder: case report.

BACKGROUND: Damaging variants in TRIO have been associated with moderate to severe neurodevelopmental disorders in humans. While recent work has delineated the positional effect of missense variation on the resulting phenotype, the clinical spectrum associated with loss-of-function variation has yet to be fully defined. CASE PRESENTATION: We report on two probands with novel loss-of-function variants in TRIO. Patient 1 presents with a severe neurodevelopmental disorder and macrocephaly. The TRIO variant is inherited from his affected mother. Patient 2 presents with moderate developmental delays, microcephaly, and cutis aplasia with a frameshift variant of unknown inheritance. CONCLUSIONS: We describe two patients with neurodevelopmental disorder, macro/microcephaly, and cutis aplasia in one patient. Both patients have loss-of-function variants, helping to further characterize how these types of variants affect the phenotypic spectrum associated with TRIO. We also present the third reported case of autosomal dominant inheritance of a damaging variant in TRIO.

Brefeldin A-sensitive ER-Golgi vesicle trafficking contributes to NLRP3-dependent caspase-1 activation.

Endoplasmic reticulum (ER)-Golgi vesicle trafficking plays a pivotal role in the conventional secretory pathway of many cytokines; however, the precise release mechanism of a major inflammasome mediator, IL-1ß, is not thought to follow the conventional ER-Golgi route and remains elusive. Here, we found that perturbation of ER-Golgi trafficking by brefeldin A (BFA) treatment attenuated nucleotide-binding oligomerization domain-like receptor family, pyrin-domain-containing 3 (NLRP3) inflammasome activation in mouse bone marrow-derived macrophages (BMDMs). BFA treatment inhibited NLRP3-mediated inflammasome assembly and caspase-1 activation but did not block IL-1ß secretion from BMDMs following BFA administration after NLRP3 inflammasome activation. Consistently, short-hairpin RNA-dependent knockdown of BFA-inhibited guanine nucleotide-exchange protein 1 (BIG1), a molecular target of BFA and an initiator of Golgi-specific vesicle trafficking, abolished NLRP3-dependent apoptosis-associated speck-like protein containing a caspase-recruitment domain oligomerization and caspase-1 activation in BMDMs. Similarly, knockdown of Golgi-specific BFA-resistance guanine nucleotide exchange factor 1, another target of BFA, clearly attenuated NLRP3-mediated caspase-1 activation in BMDMs. Mechanistically, inhibition of BIG1-mediated vesicle trafficking did not impair NLRP3-activating signal 2-promoted events, such as potassium efflux and mitochondrial rearrangement, but caused significant impairment of signal 1-triggered priming steps, including NF-κB-mediated pathways. These data suggest that BFA-targeted vesicle trafficking at the Golgi contributes to activation of the NLRP3 inflammasome signaling.-Hong, S., Hwang, I., Gim, E., Yang, J., Park, S., Yoon, S.-H., Lee, W.-W., Yu, J.-W. Brefeldin A-sensitive ER-Golgi vesicle trafficking contributes to NLRP3-dependent caspase-1 activation.

Clinical, immunological features and follow up of 20 patients with dedicator of cytokinesis 8 (DOCK8) deficiency.

Biallelic mutations in the dedicator of cytokinesis 8 gene (DOCK8) cause a progressive combined immunodeficiency (CID) characterized by susceptibility to severe viral skin infections, atopic diseases, recurrent respiratory infections, and malignancy. Hematopoietic stem cell transplantation (HSCT) is only curative treatment for the disease. However, there is limited information about long-term outcome of HSCT and its effect to protect against cancer development in DOCK8-deficient patients. In this study, we retrospectively evaluated clinical and immunologic characteristics of 20 DOCK8-deficient patients and outcome of 11 patients who underwent HSCT. We aimed to report the experience of our center and the result of the largest transplantation series of DOCK8 deficiency in our country. Median follow-up time is 71 months (min-max: 16-172) in all patients and 48 months (min-max: 5-84) in transplanted patients. Atopic dermatitis (18/20), recurrent respiratory tract infections (17/20), and food allergy (14/20) were the most frequent clinical manifestations. Failure to thrive (13/20), liver problems (12/20), bronchiectasis (11/20), chronic diarrhea (10/21), and autism spectrum disorders (3/20) were remarkable findings in our series. Elevated IgE level (20/20) and eosinophilia (17/20), low IgM level (15/20), and decreased CD3+ T (10/20) and CD4+ T (11/20) cell count were prominent laboratory findings. HSCT was performed in 11 patients. All patients achieved adequate engraftment and showed improvement in their clinical and immunologic findings. Atopic dermatitis and food allergies improved in all patients, and their dietary restriction was stopped except one patient who was transplanted recently. The frequency of infections was decreased. The overall survival is 91% in HSCT-received patients and 80% in all. HSCT at the earliest possible period with most suitable donor- and patient-specific appropriate conditioning regimen and GvHD prophylaxis is lifesaving for DOCK8 deficiency cases.

Small GTP-Binding Protein GDP Dissociation Stimulator Prevents Thoracic Aortic Aneurysm Formation and Rupture by Phenotypic Preservation of Aortic Smooth Muscle Cells.

BACKGROUND: Thoracic aortic aneurysm (TAA) and dissection are fatal diseases that cause aortic rupture and sudden death. The small GTP-binding protein GDP dissociation stimulator (SmgGDS) is a crucial mediator of the pleiotropic effects of statins. Previous studies revealed that reduced force generation in aortic smooth muscle cells (AoSMCs) causes TAA and thoracic aortic dissection. METHODS: To examine the role of SmgGDS in TAA formation, we used an angiotensin II (1000 ng·min-1·kg-1, 4 weeks)-induced TAA model. RESULTS: We found that 33% of Apoe-/- SmgGDS+/- mice died suddenly as a result of TAA rupture, whereas there was no TAA rupture in Apoe-/- control mice. In contrast, there was no significant difference in the ratio of abdominal aortic aneurysm rupture between the 2 genotypes. We performed ultrasound imaging every week to follow up the serial changes in aortic diameters. The diameter of the ascending aorta progressively increased in Apoe-/- SmgGDS+/- mice compared with Apoe-/- mice, whereas that of the abdominal aorta remained comparable between the 2 genotypes. Histological analysis of Apoe-/- SmgGDS+/- mice showed dissections of major thoracic aorta in the early phase of angiotensin II infusion (day 3 to 5) and more severe elastin degradation compared with Apoe-/- mice. Mechanistically, Apoe-/- SmgGDS+/- mice showed significantly higher levels of oxidative stress, matrix metalloproteinases, and inflammatory cell migration in the ascending aorta compared with Apoe-/- mice. For mechanistic analyses, we primary cultured AoSMCs from the 2 genotypes. After angiotensin II (100 nmol/L) treatment for 24 hours, Apoe-/- SmgGDS+/- AoSMCs showed significantly increased matrix metalloproteinase activity and oxidative stress levels compared with Apoe-/- AoSMCs. In addition, SmgGDS deficiency increased cytokines/chemokines and growth factors in AoSMCs. Moreover, expressions of fibrillin-1 ( FBN1), α-smooth muscle actin ( ACTA2), myosin-11 ( MYH11), MYLLK, and PRKG1, which are force generation genes, were significantly reduced in Apoe-/- SmgGDS+/- AoSMCs compared with Apoe-/- AoSMCs. A similar tendency was noted in AoSMCs from patients with TAA compared with those from control subjects. Finally, local delivery of the SmgGDS gene construct reversed the dilation of the ascending aorta in Apoe-/- SmgGDS+/- mice. CONCLUSIONS: These results suggest that SmgGDS is a novel therapeutic target for the prevention and treatment of TAA.

SIL1 deficiency causes degenerative changes of peripheral nerves and neuromuscular junctions in fish, mice and human.

BACKGROUND: Marinesco-Sjögren Syndrome (MSS) is a rare neuromuscular condition caused by recessive mutations in the SIL1 gene resulting in the absence of functional SIL1 protein, a co-chaperone for the major ER chaperone, BiP. As BiP is decisive for proper protein processing, loss of SIL1 results in the accumulation of misshaped proteins. This accumulation likely damages and destroys cells in vulnerable tissues, leading to congenital cataracts, cerebellar ataxia, vacuolar myopathy and other MSS phenotypes. Whether the peripheral nervous system (PNS) is affected in MSS has not been conclusively shown. METHODS: To study PNS vulnerability in MSS, intramuscular nerves fibres from MSS patients and from SIL1-deficient mice (woozy) as well as sciatic nerves and neuromuscular junctions (NMJ) from these mice have been investigated via transmission electron microscopic and immunofluorescence studies accompanied by transcript studies and unbiased proteomic profiling. In addition, PNS and NMJ integrity were analyzed via immunofluorescence studies in an MSS-zebrafish model which has been generated for that purpose. RESULTS: Electron microscopy revealed morphological changes indicative of impaired autophagy and mitochondrial maintenance in distal axons and in Schwann cells. Moreover, changes of the morphology of NMJs as well as of transcripts encoding proteins important for NMJ function were detected in woozy mice. These findings were in line with a grossly abnormal structure of NMJs in SIL1-deficient zebrafish embryos. Proteome profiling of sciatic nerve specimens from woozy mice revealed altered levels of proteins implicated in neuronal maintenance suggesting the activation of compensatory mechanisms. CONCLUSION: Taken together, our combined data expand the spectrum of tissues affected by SIL1-loss and suggest that impaired neuromuscular transmission might be part of MSS pathophysiology.