Neutrophil extracellular traps (NETs) are implicated in autoimmunity but how they are generated and their roles in sterile inflammation remain unclear. Ribonucleoprotein immune complexes, inducers of NETosis, require mitochondrial ROS for maximal NET stimulation. During this process, mitochondria become hypopolarized and translocate to the cell surface. Extracellular release of oxidized mitochondrial DNA is proinflammatory in vitro and, when injected into mice, stimulates type-I interferon (IFN) signaling through a pathway dependent on the DNA sensor, STING. Mitochondrial ROS is also necessary for spontaneous NETosis of low-density granulocytes from individuals with systemic lupus erythematosus (SLE). This was also observed in individuals with chronic granulomatous disease (CGD), which lack NADPH-oxidase activity, but still develop autoimmunity and type I-IFN signatures. Mitochondrial ROS inhibition in vivo reduces disease severity and type-I IFN responses in a mouse model of lupus. These findings highlight a role for mitochondria in the generation not only of NETs but also of pro-inflammatory oxidized mitochondrial DNA in autoimmune diseases.

Background-Failure to generate phagocyte-derived superoxide and related reactive oxygen intermediates (ROIs) is the major defect in chronic granulomatous disease, causing recurrent infections and granulomatous complications. Chronic granulomatous disease is caused by missense, nonsense, frameshift, splice, or deletion mutations in the genes for p22 phox , p40 phox , p47 phox , p67 phox (autosomal chronic granulomatous disease), or gp91 phox (X-linked chronic granulomatous disease), which result in variable production of neutrophil-derived ROIs. We hypothesized that residual ROI production might be linked to survival in patients with chronic granulomatous disease.

Fungal infections remain a major determinant of survival in CGD. X-linked patients generally had more severe disease, and this was generally in those with lower residual superoxide production. Survival in CGD has increased over the years, but infections are still major causes of morbidity and mortality.

X-linked severe combined immunodeficiency (SCID-X1) is a profound deficiency of T, B, and natural killer (NK) cell immunity caused by mutations in IL2RG encoding the common chain (γc) of several interleukin receptors. Gamma-retroviral (γRV) gene therapy of SCID-X1 infants without conditioning restores T cell immunity without B or NK cell correction, but similar treatment fails in older SCID-X1 children. We used a lentiviral gene therapy approach to treat five SCID-X1 patients with persistent immune dysfunction despite haploidentical hematopoietic stem cell (HSC) transplant in infancy. Follow-up data from two older patients demonstrate that lentiviral vector γc transduced autologous HSC gene therapy after nonmyeloablative busulfan conditioning achieves selective expansion of gene-marked T, NK, and B cells, which is associated with sustained restoration of humoral responses to immunization and clinical improvement at 2 to 3 years after treatment. Similar gene marking levels have been achieved in three younger patients, albeit with only 6 to 9 months of follow-up. Lentiviral gene therapy with reduced-intensity conditioning appears safe and can restore humoral immune function to posthaploidentical transplant older patients with SCID-X1.

BACKGROUND Allogeneic hematopoietic stem-cell transplantation for X-linked severe combined immunodeficiency (SCID-X1) often fails to reconstitute immunity associated with T cells, B cells, and natural killer (NK) cells when matched sibling donors are unavailable unless high-dose chemotherapy is given. In previous studies, autologous gene therapy with γ-retroviral vectors failed to reconstitute B-cell and NK-cell immunity and was complicated by vector-related leukemia. METHODS We performed a dual-center, phase 1–2 safety and efficacy study of a lentiviral vector to transfer IL2RG complementary DNA to bone marrow stem cells after low-exposure, targeted busulfan conditioning in eight infants with newly diagnosed SCID-X1. RESULTS Eight infants with SCID-X1 were followed for a median of 16.4 months. Bone marrow harvest, busulfan conditioning, and cell infusion had no unexpected side effects. In seven infants, the numbers of CD3+, CD4+, and naive CD4+ T cells and NK cells normalized by 3 to 4 months after infusion and were accompanied by vector marking in T cells, B cells, NK cells, myeloid cells, and bone marrow progenitors. The eighth infant had an insufficient T-cell count initially, but T cells developed in this infant after a boost of gene-corrected cells without busulfan conditioning. Previous infections cleared in all infants, and all continued to grow normally. IgM levels normalized in seven of the eight infants, of whom four discontinued intravenous immune globulin supplementation; three of these four in-fants had a response to vaccines. Vector insertion-site analysis was performed in seven infants and showed polyclonal patterns without clonal dominance in all seven. CONCLUSIONS Lentiviral vector gene therapy combined with low-exposure, targeted busulfan conditioning in infants with newly diagnosed SCID-X1 had low-grade acute toxic effects and resulted in multilineage engraftment of transduced cells, reconstitution of functional T cells and B cells, and normalization of NK-cell counts during a median follow-up of 16 months. (Funded by the American Lebanese Syrian Associated Charities and others; LVXSCID-ND ClinicalTrials.gov number, .)

Gene repair of CD34 hematopoietic stem and progenitor cells (HSPCs) may avoid problems associated with gene therapy, such as vector-related mutagenesis and dysregulated transgene expression. We used CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated 9) to repair a mutation in the CYBB gene of CD34 HSPCs from patients with the immunodeficiency disorder X-linked chronic granulomatous disease (X-CGD). Sequence-confirmed repair of >20% of HSPCs from X-CGD patients restored the function of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and superoxide radical production in myeloid cells differentiated from these progenitor cells in vitro. Transplant of gene-repaired X-CGD HSPCs into NOD (nonobese diabetic) SCID (severe combined immunodeficient) γc mice resulted in efficient engraftment and production of functional mature human myeloid and lymphoid cells for up to 5 months. Whole-exome sequencing detected no indels outside of the CYBB gene after gene correction. CRISPR-mediated gene editing of HSPCs may be applicable to other CGD mutations and other monogenic disorders of the hematopoietic system.

Chronic Granulomatous Disease (CGD) still causes significant morbidity and mortality. The difficulty in considering high-risk yet curative treatments, such as allogeneic bone marrow transplantation, lies in the unpredictable courses of both CGD and bone marrow transplantation in different patients. Some CGD patients may have frequent infections and/or suffer from granulomatous or autoimmune disorders necessitating immunosuppressive therapy, but also experience long periods of relative good health. However, the risk of death is clearly higher in CGD of all types, and the complications of CGD short of death can still cause significant morbidity. Therefore, with recent developments and improvements, bone marrow transplantation, previously considered an experimental or high-risk procedure, has emerged as an important option for patients with CGD. We will discuss the complications of CGD that result in significant morbidity and mortality, in particular the most common infections and autoimmune/inflammatory complications as well as their typical management. We will then discuss the status of bone marrow transplantation.

Chronic granulomatous disease (CGD) is characterized by recurrent infections and granuloma formation. In addition, we have observed a number of diverse autoimmune conditions in our CGD population, suggesting that patients with CGD are at an elevated risk for development of autoimmune (AI) disorders. In this report, we describe antiphospholipid syndrome (aPL), recurrent pericardial effusion, juvenile idiopathic arthritis (JIA), IgA nephropathy, cutaneous lupus erythematosus, and autoimmune pulmonary disease in the setting of CGD. The presence and type of autoimmune disease has important treatment implications for patients with CGD. KeywordsChronic granulomatous disease; autoimmune; antiphospholipid syndrome; IgA nephropathy; lupus; juvenile idiopathic nephropathy Chronic granulomatous disease (CGD) is a primary immunodeficiency (PID) resulting from a defect in the multicomponent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, which is responsible for production of bactericidal reactive oxygen species (ROS) in phagocytes. As a result, CGD patients are at increased susceptibility to certain catalasepositive bacteria and fungi, and Aspergillus species 1 . The primary clinical features of CGD are recurrent infections and granuloma formation. However, reports of sarcoidosis 2 , JIA 3 , IgA nephropathy 4 , pericardial effusion 5 , and severe Crohn's-like inflammatory bowel disease 6 suggest that the breadth of altered immune regulation extends beyond recurrent infections and granulomas. We propose, in addition, that CGD patients are at significant risk for development of autoimmune disease (AI), and provide a series of case reports and a review of the literature to support that hypothesis. Specifically we report here the following AI in our CGD patients; antiphospholipid syndrome (aPL), juvenile idiopathic arthritis (JIA), IgA nephropathy; steroidresponsive recurrent pericardial effusions, cutaneous lupus erythematosus, and lastly, a case with 'geographic pulmonary lesions', a finding we have observed in four other CGD patients. A 14.5 year-old Caucasian male was diagnosed with X-linked (gp91 phox -deficient) chronic granulomatous disease (CGD) following the development of Serratia marcescens abscesses of his neck and mesentery at 3 years of age. Long-term prophylaxis consisting of trimethoprimsulfamethoxazole, itraconazole and interferon-γ (IFN-γ) was commenced. Of interest, his mother (a CGD carrier) and maternal aunt (not a carrier) were both diagnosed with discoid lupus. At 10 years, he developed cellulitis of his arm following a minor skin abrasion. Treatment with intravenous antibiotics was complicated by venous thrombosis in his affected arm, which was treated with a 3-month course of warfarin. At age 14, he described acute swelling, pain and redness of the left thigh, with no other associated symptoms, fever, or history of trauma. His laboratory tests were unremarkable, other than an ESR of 50 mm/hr (NIH range 0-25mm/hr). Doppler ultrasound revealed a left femoral deep venous thromb...