DOCK2-deficiency causes defects in anti-viral T cell responses and impaired control of herpes simplex virus infection.

The expanding number of rare immunodeficiency syndromes offers an opportunity to understand key genes that support immune defence against infectious diseases. However, analysis of these in patients is complicated by their treatments and co-morbid infections requiring the use of mouse models for detailed investigations. Here we develop a mouse model of DOCK2 immunodeficiency and demonstrate that these mice have delayed clearance of herpes simplex virus type 1 (HSV-1) infections. We also uncovered a critical, cell intrinsic role of DOCK2 in the priming of anti-viral CD8+ T cells and in particular their initial expansion, despite apparently normal early activation of these cells. When this defect was overcome by priming in vitro, DOCK2-deficient CD8+ T cells were surprisingly protective against HSV-1-disease, albeit not as effectively as wild type cells. These results shed light on a cellular deficiency that is likely to impact anti-viral immunity in DOCK2-deficient patients.

DOCK2 regulates MRGPRX2/B2-mediated mast cell degranulation and drug-induced anaphylaxis.

BACKGROUND: Drug-induced anaphylaxis is triggered by the direct stimulation of mast cells (MCs) via Mas-related G protein-coupled receptor X2 (MRGPRX2; mouse ortholog MRGPRB2). However, the precise mechanism that links MRGPRX2/B2 to MC degranulation is poorly understood. Dedicator of cytokinesis 2 (DOCK2) is a Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 regulates migration and activation of leukocytes, its role in MCs remains unknown. OBJECTIVE: We aimed to elucidate whether-and if so, how-DOCK2 is involved in MRGPRX2/B2-mediated MC degranulation and anaphylaxis. METHODS: Induction of drug-induced systemic and cutaneous anaphylaxis was compared between wild-type and DOCK2-deficient mice. In addition, genetic or pharmacologic inactivation of DOCK2 in human and murine MCs was used to reveal its role in MRGPRX2/B2-mediated signal transduction and degranulation. RESULTS: Induction of MC degranulation and anaphylaxis by compound 48/80 and ciprofloxacin was severely attenuated in the absence of DOCK2. Although calcium influx and phosphorylation of several signaling molecules were unaffected, MRGPRB2-mediated Rac activation and phosphorylation of p21-activated kinase 1 (PAK1) were impaired in DOCK2-deficient MCs. Similar results were obtained when mice or MCs were treated with small-molecule inhibitors that bind to the catalytic domain of DOCK2 and inhibit Rac activation. CONCLUSION: DOCK2 regulates MRGPRX2/B2-mediated MC degranulation through Rac activation and PAK1 phosphorylation, thereby indicating that the DOCK2-Rac-PAK1 axis could be a target for preventing drug-induced anaphylaxis.

DOCK2 Promotes Asthma Development by Eliciting Airway Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) contributes to airway remodeling, a predominant feature of asthma. DOCK2 (dedicator of cytokinesis 2) is an innate immune signaling molecule involved in vascular remodeling. However, it is unknown if DOCK2 plays a role in airway remodeling during asthma development. In this study, we found that DOCK2 is highly induced in both normal human bronchial epithelial cells treated with house dust mite (HDM) extract and human asthmatic airway epithelium. DOCK2 is also upregulated by TGF-ß1 (transforming growth factor ß1) during EMT of human bronchial epithelial cells. Importantly, knockdown of DOCK2 inhibits, and overexpression of DOCK2 promotes, TGF-ß1-induced EMT. Consistently, DOCK2 deficiency suppresses the EMT of airway epithelium, attenuates the subepithelial fibrosis, and improves pulmonary function in HDM-induced asthmatic lungs. These data suggest that DOCK2 plays an important role in EMT and asthma development. Mechanistically, DOCK2 interacts with transcription factor FoxM1 (forkhead box M1), which enhances FoxM1 binding to mesenchymal marker gene promoters and further promotes mesenchymal marker gene transcription and expression, leading to EMT. Taken together, our study identifies DOCK2 as a novel regulator for airway EMT in an HDM-induced asthma model, thus providing a potential therapeutic target for treatment of asthma.

Multiple Immune Defects in Two Patients with Novel DOCK2 Mutations Result in Recurrent Multiple Infection Including Live Attenuated Virus Vaccine.

The dedicator of cytokinesis 2(DOCK2) protein, an atypical guanine nucleotide exchange factor (GEFs), is a member of the DOCKA protein subfamily. DOCK2 protein deficiency is characterized by early-onset lymphopenia, recurrent infections, and lymphocyte dysfunction, which was classified as combined immune deficiency with neutrophil abnormalities as well. The only cure is hematopoietic stem cell transplantation. Here, we report two patients harboring four novel DOCK2 mutations associated with recurrent infections including live attenuated vaccine-related infections. The patient's condition was partially alleviated by symptomatic treatment or intravenous immunoglobulin. We also confirmed defects in thymic T cell output and T cell proliferation, as well as aberrant skewing of T/B cell subset TCR-Vß repertoires. In addition, we noted neutrophil defects, the weakening of actin polymerization, and BCR internalization under TCR/BCR activation. Finally, we found that the DOCK2 protein affected antibody affinity although with normal total serum immunoglobulin. The results reported herein expand the clinical phenotype, the pathogenic DOCK2 mutation database, and the immune characteristics of DOCK2-deficient patients.

Cancer-derived cholesterol sulfate is a key mediator to prevent tumor infiltration by effector T cells.

Effective tumor immunotherapy requires physical contact of T cells with cancer cells. However, tumors often constitute a specialized microenvironment that excludes T cells from the vicinity of cancer cells, and its underlying mechanisms are still poorly understood. DOCK2 is a Rac activator critical for migration and activation of lymphocytes. We herein show that cancer-derived cholesterol sulfate (CS), a lipid product of the sulfotransferase SULT2B1b, acts as a DOCK2 inhibitor and prevents tumor infiltration by effector T cells. Using clinical samples, we found that CS was abundantly produced in certain types of human cancers such as colon cancers. Functionally, CS-producing cancer cells exhibited resistance to cancer-specific T-cell transfer and immune checkpoint blockade. Although SULT2B1b is known to sulfate oxysterols and inactivate their tumor-promoting activity, the expression levels of cholesterol hydroxylases, which mediate oxysterol production, are low in SULT2B1b-expressing cancers. Therefore, SULT2B1b inhibition could be a therapeutic strategy to disrupt tumor immune evasion in oxysterol-non-producing cancers. Thus, our findings define a previously unknown mechanism for tumor immune evasion and provide a novel insight into the development of effective immunotherapies.

DOCK2 Promotes Pleural Fibrosis by Modulating Mesothelial to Mesenchymal Transition.

Mesothelial to mesenchymal transition (MesoMT) is one of the crucial mechanisms underlying pleural fibrosis, which results in restrictive lung disease. DOCK2 (dedicator of cytokinesis 2) plays important roles in immune functions; however, its role in pleural fibrosis, particularly MesoMT, remains unknown. We found that amounts of DOCK2 and the MesoMT marker α-SMA (α-smooth muscle actin) were significantly elevated and colocalized in the thickened pleura of patients with nonspecific pleuritis, suggesting the involvement of DOCK2 in the pathogenesis of MesoMT and pleural fibrosis. Likewise, data from three different pleural fibrosis models (TGF-ß [transforming growth factor-ß], carbon black/bleomycin, and streptococcal empyema) consistently demonstrated DOCK2 upregulation and its colocalization with α-SMA in the pleura. In addition, induced DOCK2 colocalized with the mesothelial marker calretinin, implicating DOCK2 in the regulation of MesoMT. Our in vivo data also showed that DOCK2-knockout mice were protected from Streptococcus pneumoniae-induced pleural fibrosis, impaired lung compliance, and collagen deposition. To determine the involvement of DOCK2 in MesoMT, we treated primary human pleural mesothelial cells with the potent MesoMT inducer TGF-ß. TGF-ß significantly induced DOCK2 expression in a time-dependent manner, together with α-SMA, collagen 1, and fibronectin. Furthermore, DOCK2 knockdown significantly attenuated TGF-ß-induced α-SMA, collagen 1, and fibronectin expression, suggesting the importance of DOCK2 in TGF-ß-induced MesoMT. DOCK2 knockdown also inhibited TGF-ß-induced Snail upregulation, which may account for its role in regulating MesoMT. Taken together, the current study provides evidence that DOCK2 contributes to the pathogenesis of pleural fibrosis by mediating MesoMT and deposition of neomatrix and may represent a novel target for its prevention or treatment.

DOCK2 contributes to pulmonary fibrosis by promoting lung fibroblast to myofibroblast transition.

Idiopathic pulmonary fibrosis (IPF) is the most common chronic interstitial lung disease and is characterized by progressive scarring of the lung. Transforming growth factor-ß (TGF-ß) signaling plays an essential role in IPF and drives fibroblast to myofibroblast transition (FMT). Dedicator of cytokinesis 2 (DOCK2) is known to regulate diverse immune functions by activating Rac and has been recently implicated in pleural fibrosis. We now report a novel role of DOCK2 in pulmonary fibrosis development by mediating FMT. In primary normal and IPF human lung fibroblasts (HLFs), TGF-ß induced DOCK2 expression concurrent with FMT markers, smooth muscle α-actin (α-SMA), collagen-1, and fibronectin. Knockdown of DOCK2 significantly attenuated TGF-ß-induced expression of these FMT markers. In addition, we found that the upregulation of DOCK2 by TGF-ß is dependent on both Smad3 and ERK pathways as their respective inhibitors blocked TGF-ß-mediated induction. TGF-ß also stabilized DOCK2 protein, which contributes to increased DOCK2 expression. In addition, DOCK2 was also dramatically induced in the lungs of patients with IPF and in bleomycin, and TGF-ß induced pulmonary fibrosis in C57BL/6 mice. Furthermore, increased lung DOCK2 expression colocalized with the FMT marker α-SMA in the bleomycin-induced pulmonary fibrosis model, implicating DOCK2 in the regulation of lung fibroblast phenotypic changes. Importantly, DOCK2 deficiency also attenuated bleomycin-induced pulmonary fibrosis and α-SMA expression. Taken together, our study demonstrates a novel role of DOCK2 in pulmonary fibrosis by modulating FMT and suggests that targeting DOCK2 may present a potential therapeutic strategy for the prevention or treatment of IPF.

Pharmacological intervention of cholesterol sulfate-mediated T cell exclusion promotes antitumor immunity.

Effective cancer immunotherapy requires physical contact of T cells with cancer cells. However, tumors often constitute special microenvironments that exclude T cells and resist immunotherapy. Cholesterol sulfate (CS) is a product of sulfotransferase SULT2B1b and acts as an endogenous inhibitor of DOCK2, a Rac activator essential for migration and activation of lymphocytes. We have recently shown that cancer-derived CS prevents tumor infiltration by effector T cells. Therefore, SULT2B1b may be a therapeutic target to dampen CS-mediated immune evasion. Here, we identified 3ß-hydroxy-5-cholenoic acid (3ß-OH-5-Chln) as a cell-active inhibitor of SULT2B1b. 3ß-OH-5-Chln inhibited the cholesterol sulfotransferase activity of SULT2B1b in vitro and suppressed CS production from cancer cells expressing SULT2B1b. In vivo administration of 3ß-OH-5-Chln locally reduced CS level in murine CS-producing tumors and increased infiltration of CD8+ T cells. When combined with immune checkpoint blockade or antigen-specific T cell transfer, 3ß-OH-5-Chln suppressed the growth of CS-producing tumors. These results demonstrate that pharmacological inhibition of SULT2B1b can promote antitumor immunity through suppressing CS-mediated T cell exclusion.

Comprehensive characterization of ubiquitinome of human colorectal cancer and identification of potential survival-related ubiquitination.

BACKGROUND: According to the Global Cancer Statistics in 2020, the incidence and mortality of colorectal cancer (CRC) rank third and second among all tumors. The disturbance of ubiquitination plays an important role in the initiation and development of CRC, but the ubiquitinome of CRC cells and the survival-relevant ubiquitination are poorly understood. METHODS: The ubiquitinome of CRC patients (n = 6) was characterized using our own data sets of proteomic and ubiquitin-proteomic examinations. Then, the probable survival-relevant ubiquitination was searched based on the analyses of data sets from public databases. RESULTS: For the ubiquitinomic examination, we identified 1690 quantifiable sites and 870 quantifiable proteins. We found that the highly-ubiquitinated proteins (n ≥ 10) were specifically involved in the biological processes such as G-protein coupling, glycoprotein coupling, and antigen presentation. Also, we depicted five motif sequences frequently recognized by ubiquitin. Subsequently, we revealed that the ubiquitination content of 1172 proteins were up-regulated and 1700 proteins were down-regulated in CRC cells versus normal adjacent cells. We demonstrated that the differentially ubiquitinated proteins were relevant to the pathways including metabolism, immune regulation, and telomere maintenance. Then, integrated with the proteomic datasets from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) (n = 98), we revealed that the increased ubiquitination of FOCAD at Lys583 and Lys587 was potentially associated with patient survival. Finally, we depicted the mutation map of FOCAD and elucidated its potential functions on RNA localization and translation in CRC. CONCLUSIONS: The findings of this study described the ubiquitinome of CRC cells and identified abnormal ubiquitination(s) potentially affecting the patient survival, thereby offering new probable opportunities for clinical treatment.

The role of Dock2 on macrophage migration and functions during Citrobacter rodentium infection.

Dedicator of cytokinesis 2 (Dock2), an atypical guanine exchange factor, is specifically expressed on immune cells and mediates cell adhesion and migration by activating Rac and regulates actin cytoskeleton remodeling. It plays a crucial role in the migration, formation of immune synapses, cell proliferation, activation of T and B lymphocytes and chemotaxis of pDCs and neutrophils. However, in-vivo physiological functions of Dock2 have been relatively seldom studied. Our previous studies showed that Dock2-/- mice were highly susceptible to colitis induced by Citrobacter rodentium infection, and in early infection, Dock2-/- mice had defects in macrophage migration. However, the specific roles of Dock2 in the migration and functions of macrophages are not clear. In this study, we found that the expression of chemokines such as chemokine (C-C motif) ligand (CCL)4 and CCL5 and chemokine receptors such as chemokine (C-C motif) receptor (CCR)4 and CCR5 in bone marrow-derived macrophages (BMDM) of Dock2-/- mice decreased after infection, which were supported by the in-vivo infection experimental results; the Transwell experiment results showed that Dock2-/- BMDM had a defect in chemotaxis. The bacterial phagocytic and bactericidal experiment results also showed that Dock2-/- BMDM had the defects of bacterial phagocytosis and killing. Furthermore, the adoptive transfer of wild-type BMDM alleviated the susceptibility of Dock2-/- mice to C. rodentium infection. Our results show that Dock2 affects migration and phagocytic and bactericidal ability of macrophages by regulating the expression of chemokines, chemokine receptors and their responses to chemokine stimulation, thus playing an essential role in the host defense against enteric bacterial infection.

DOCK2 couples with LEF-1 to regulate B cell metabolism and memory response.

Dedicator of cytokinesis 2 (DOCK2) is essential for the B cell differentiation, BCR signaling and humoral immune response. However, the role of DOCK2 in the memory response of B cell is unknown. By using two DOCK2 deficient patients, we found that the memory B cells were decreased and the early activation of DOCK2 deficient memory B cells was abolished to the degree of naïve B cells due to the decreased expression of CD19 and CD21 mechanistically. Interestingly the expression of LEF-1, a negative regulator of CD21, was increased in DOCK2 deficient B cells. This was linked to the increased expression of HIF-1α and cell metabolism, which in turn affected the ER structure. Finally, the reduction of memory B cells in DOCK2 patients was due to the increased apoptosis, which might be related with the increased metabolism.

miR-16 exhibits protective function in LPS-treated cardiomyocytes by targeting DOCK2 to repress cell apoptosis and exert anti-inflammatory effect.

This study aims to investigate the effects of microRNA (miR)-16/dedicator of cytokinesis 2 (DOCK2) on myocarditis. The differences in the expression of genes in acute myocarditis were filtered out across Gene Expression Omnibus (GEO) database. Myocarditis cell model was established by lipopolysaccharide (LPS) stimulation in cardiomyocytes. The association between miR-16 and DOCK2 was predicted by bioinformatics software and confirmed by dual-luciferase assay. Polymerase chain reaction and western blot analysis were employed to assess the expression levels of miR-16 and DOCK2 under different conditions. Cells viability, apoptosis, and inflammatory reaction were evaluated by Cell Counting Kit-8, flow cytometry, and enzyme-linked immunosorbent assays. miR-16, as an upstream regulator of DOCK2, exhibited lower expression in LPS-induced myocarditis model. More importantly, we revealed that a marked augmentation of miR-16 promoted the growth of LPS-stimulated cardiomyocytes, and attenuated cell apoptosis and inflammatory response. However, an increasing expression of DOCK2 inhibited the remission of LPS-induced myocardial injury caused by miR-16 mimic. Herein, our results highlighted that upregulation of miR-16 resulted in the protective effects on LPS-induced myocardial injury by reducing DOCK2 expression, affording a pair of novel target molecules for ameliorating the symptoms of myocarditis.

Human DOCK2 Deficiency: Report of a Novel Mutation and Evidence for Neutrophil Dysfunction.

DOCK2 is a guanine-nucleotide-exchange factor for Rac proteins. Activated Rac serves various cellular functions including the reorganization of the actin cytoskeleton in lymphocytes and neutrophils and production of reactive oxygen species in neutrophils. Since 2015, six unrelated patients with combined immunodeficiency and early-onset severe viral infections caused by bi-allelic loss-of-function mutations in DOCK2 have been described. Until now, the function of phagocytes, specifically neutrophils, has not been assessed in human DOCK2 deficiency. Here, we describe a new kindred with four affected siblings harboring a homozygous splice-site mutation (c.2704-2 A > C) in DOCK2. The mutation results in alternative splicing and a complete loss of DOCK2 protein expression. The patients presented with leaky severe combined immunodeficiency or Omenn syndrome. The novel mutation affects EBV-B cell migration and results in NK cell dysfunction similar to previous observations. Moreover, both cytoskeletal rearrangement and reactive oxygen species production are partially impaired in DOCK2-deficient neutrophils.

Dock2 in the development of inflammation and cancer.

An atypical guanine exchange factor, Dock2 is specifically expressed in hematopoietic cells and regulates activation and migration of immune cells through activating Ras-related C3 botulinum toxin substrate (Rac). Dock2 was shown to be critical in the development of various inflammatory diseases, including allergic diseases, HIV infection, and graft rejection in organ transplantation. DOCK2 mutation in infants was recently identified to be associated with T and B cell combined immunodeficiency. Furthermore, Dock2 is involved in host protection during enteric bacterial infection and is also associated with the proliferation of cancer cells. It was also shown that patients with digestive tract cancer had high frequency mutation of DOCK2. This review summarizes the latest research progresses on the role of Dock2 for the development of various inflammatory diseases and cancers, and discusses the potential application of Dock2 modulators for patient treatment.

Human inborn errors of the actin cytoskeleton affecting immunity: way beyond WAS and WIP.

Inherited defects in genes encoding for proteins that are involved in the assembly and dynamics of the actin skeleton have increasingly been identified in patients presenting with primary immunodeficiencies. In this review, we summarize a subset of the recently described conditions, emphasizing the clinical features as well as the immunophenotype and pathophysiology.

DOCK8 deficiency: Insights into pathophysiology, clinical features and management.

Dedicator of cytokinesis 8 (DOCK8) deficiency is a combined immunodeficiency that exemplifies the broad clinical features of primary immunodeficiencies (PIDs), extending beyond recurrent infections to include atopy, autoimmunity and cancer. It is caused by loss of function mutations in DOCK8, encoding a guanine nucleotide exchange factor highly expressed in lymphocytes that regulates the actin cytoskeleton. Additional roles of DOCK8 have also emerged, including regulating MyD88-dependent Toll-like receptor signaling and the activation of the transcription factor STAT3. DOCK8 deficiency impairs immune cell migration, function and survival, and it impacts both innate and adaptive immune responses. Clinically, DOCK8 deficiency is characterized by allergic inflammation as well as susceptibility towards infections, autoimmunity and malignancy. This review details the pathophysiology, clinical features and management of DOCK8 deficiency. It also surveys the recently discovered combined immunodeficiency due to DOCK2 deficiency, highlighting in the process the emerging spectrum of PIDs resulting from DOCK protein family abnormalities.

Novel DOCK2-selective inhibitory peptide that suppresses B-cell line migration.

Dedicator of cytokinesis 2 (DOCK2) is a key molecule for lymphocyte activation and migration. DOCK2 interacts with Ras-related C3 botulinus toxin substrate 1 (Rac1, GTPase) and mediates the GDP-GTP exchange reaction, indicating that inhibitors against protein-protein interaction (PPI) between DOCK2 and Rac1 would be good drug candidates for treating immune-related disorders. Here, we report DOCK2-selective PPI inhibitory peptides discovered using random peptide T7 phage display technology. These peptides inhibited DOCK2 activity at nanomolar concentrations and were delivered to intracellular compartments by combination with cell-penetrating peptide (CPP). Consequently, one peptide, R4-DCpep-2(V2W/K4R/ox)-NH2 (Ac-RRRRCWARYHGYPWCRRRR-NH2), inhibited migration in human B lymphocyte MINO cell line at IC50 = 120 nM. To our knowledge, this is the first report of a DOCK2-selective peptide inhibitor; this study will contribute to the development of novel DOCK2-targeting immunosuppressive drugs.

DOCK2 is critical for CD8(+) TCR(-) graft facilitating cells to enhance engraftment of hematopoietic stem and progenitor cells.

CD8(+) TCR(-) graft facilitating cells (FCs) enhance engraftment of hematopoietic stem cells (HSCs) in allogeneic and syngeneic recipients. The mechanisms by which FCs promote HSC engraftment and tolerance induction have not been fully elucidated. Here, we provide data to support a critical role for dedicator of cytokinesis 2 (DOCK2) in multiple aspects of FCs function. DOCK2(-/-) FCs exhibit compromised facilitative function in vivo as evidenced by the loss of engraftment-enhancing capability for c-Kit(+) Sca-1(+) lineage(-) (KSL) cells, and compromised ability to promote KSL cell homing and lodgment in hematopoietic niche. Deletion of DOCK2 abrogates the ability of FCs to induce differentiation of naïve CD4(+) CD25(-) T cells into FoxP3(+) regulatory T cells and interleukin-10-producing type 1 regulatory T cells in vitro. Moreover, DOCK2(-/-) FCs are unable to promote survival of KSL cells when cocultured with KSL cells. DOCK2(-/-) FCs also exhibit compromised migration to stroma-derived factor-1 in vitro and impaired homing to the bone marrow in vivo. In conclusion, our results demonstrate that DOCK2 is critical for FCs to maintain its immunomodulatory function and exert its trophic effects on KSL cells. These findings may have direct clinical relevance to promote HSC engraftment for treatment of autoimmunity, hemoglobinopathies, and to induce transplantation tolerance.

Silencing DOCK2 Attenuates Cardiac Fibrosis Following Myocardial Infarction in Mice Via Targeting PI3K/Akt and Wnt/ß-Catenin Pathways.

Cardiac fibrosis following myocardial infarction (MI) seriously affects the prognosis and survival rate of patients. This study aimed to determine the effect and regulation mechanism of the dedicator of cytokinesis 2 (DOCK2) during this process. Experiments were carried out in mice in vivo, and in Ang II treated cardiac fibroblasts (CFs) in vitro. DOCK2 was increased in mouse myocardial tissues after MI and Ang II-treated CFs. In MI mice, DOCK2 silencing improved cardiac function, and ameliorated cardiac fibrosis. DOCK2 knockdown suppressed the activation of CFs and decreased the expression of α-SMA, collagen I, and collagen III. Suppression of DOCK2 mitigated Ang II induced migration of CFs. DOCK2 inhibition reduced the activity of the PI3K/Akt and Wnt/ß-catenin pathways, while this change could be reversed by the pathway activators, SC79 and SKL2001. In summary, DOCK2 suppression improves cardiac dysfunction and attenuates cardiac fibrosis after MI via attenuating PI3K/Akt and Wnt/ß-catenin pathways.