Impaired IFNγ-signaling and mycobacterial clearance in IFNγR1-deficient human iPSC-derived macrophages

Mendelian susceptibility to mycobacterial disease (MSMD) is caused by inborn errors of interferon gamma (IFNγ) immunity and is characterized by severe infections by weakly virulent mycobacteria. Although IFNγ is the macrophage-activating factor, macrophages from these patients have never been studied. We demonstrate the generation of heterozygous and compound heterozygous (iMSMD-cohet) induced pluripotent stem cells (iPSCs) from a single chimeric patient, who suffered from complete autosomal recessive IFNγR1 deficiency and received bone-marrow transplantation. Loss of IFNγR1 expression had no influence on the macrophage differentiation potential of patient-specific iPSCs. In contrast, lack of IFNγR1 in iMSMD-cohet macrophages abolished IFNγ-dependent phosphorylation of STAT1 and induction of IFNγ-downstream targets such as IRF-1, SOCS-3, and IDO. As a consequence, iMSMD-cohet macrophages show impaired upregulation of HLA-DR and reduced intracellular killing of Bacillus Calmette-Guérin. We provide a disease-modeling platform that might be suited to investigate novel treatment options for MSMD and to gain insights into IFNγ signaling in macrophages

Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria

Inborn errors of human IFN-γ-dependent macrophagic immunity underlie mycobacterial diseases, whereas inborn errors of IFN-α/β-dependent intrinsic immunity underlie viral diseases. Both types of IFNs induce the transcription factor IRF1. We describe unrelated children with inherited complete IRF1 deficiency and early-onset, multiple, life-threatening diseases caused by weakly virulent mycobacteria and related intramacrophagic pathogens. These children have no history of severe viral disease, despite exposure to many viruses, including SARS-CoV-2, which is life-threatening in individuals with impaired IFN-α/β immunity. In leukocytes or fibroblasts stimulated in vitro, IRF1-dependent responses to IFN-γ are, both quantitatively and qualitatively, much stronger than those to IFN-α/β. Moreover, IRF1-deficient mononuclear phagocytes do not control mycobacteria and related pathogens normally when stimulated with IFN-γ. By contrast, IFN-α/β-dependent intrinsic immunity to nine viruses, including SARS-CoV-2, is almost normal in IRF1-deficient fibroblasts. Human IRF1 is essential for IFN-γ-dependent macrophagic immunity to mycobacteria, but largely redundant for IFN-α/β-dependent antiviral immunity

An immune cell spray (ICS) formulation allows for the delivery of functional monocyte/macrophages

Abstract Macrophages are key cells of the innate immune system and act as tissue resident macrophages (TRMs) in the homeostasis of various tissues. Given their unique functions and therapeutic use as well as the feasibility to derive macrophages in vitro from hematopoietic stem cell (HSC) sources, we propose an “easy-to-use” immune cell spray (ICS) formulation to effectively deliver HSC-derived macrophages. To achieve this aim, we used classical pump spray devices to spray either the human myeloid cell line U937 or primary murine HSC-derived macrophages. For both cell types used, one puff could deliver cells with maintained morphology and functionality. Of note, cells tolerated the spraying process very well with a recovery of more than 90%. In addition, we used osmotic preconditioning to reduce the overall cell size of macrophages. While a 800 mosm hyperosmolar sucrose solution was able to reduce the cell size by 27%, we identified 600 mosm to be effective to reduce the cell size by 15% while maintaining macrophage morphology and functionality. Using an isolated perfused rat lung preparation, the combinatorial use of the ICS with preconditioned and genetically labeled U937 cells allowed the intra-pulmonary delivery of cells, thus paving the way for a new cell delivery platform

Patient iPSC-Derived Macrophages to Study Inborn Errors of the IFN-γ Responsive Pathway.

nterferon γ (IFN-γ) was shown to be a macrophage activating factor already in 1984. Consistently, inborn errors of IFN-γ immunity underlie Mendelian Susceptibility to Mycobacterial Disease (MSMD). MSMD is characterized by genetic predisposition to disease caused by weakly virulent mycobacterial species. Paradoxically, macrophages from patients with MSMD were little tested. Here, we report a disease modeling platform for studying IFN-γ related pathologies using macrophages derived from patient specific induced pluripotent stem cells (iPSCs). We used iPSCs from patients with autosomal recessive complete- and partial IFN-γR2 deficiency, partial IFN-γR1 deficiency and complete STAT1 deficiency. Macrophages from all patient iPSCs showed normal morphology and IFN-γ-independent functionality like phagocytic uptake of bioparticles and internalization of cytokines. For the IFN-γ-dependent functionalities, we observed that the deficiencies played out at various stages of the IFN-γ pathway, with the complete IFN-γR2 and complete STAT1 deficient cells showing the most severe phenotypes, in terms of upregulation of surface markers and induction of downstream targets. Although iPSC-derived macrophages with partial IFN-γR1 and IFN-γR2 deficiency still showed residual induction of downstream targets, they did not reduce the mycobacterial growth when challenged with Bacillus Calmette-Guérin. Taken together, we report a disease modeling platform to study the role of macrophages in patients with inborn errors of IFN-γ immunity

Human Lentiviral Gene Therapy Restores the Cellular Phenotype of Autosomal Recessive Complete IFN-γR1 Deficiency.

Autosomal recessive (AR) complete interferon-γ receptor 1 (IFN-γR1) deficiency, also known as one genetic etiology of Mendelian susceptibility to mycobacterial disease (MSMD), is a life-threatening congenital disease leading to premature death. Affected patients present a pathognomonic predisposition to recurrent and severe infections with environmental mycobacteria or the Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine. Current therapeutic options are limited to antibiotic treatment and hematopoietic stem cell transplantation, however with poor outcome. Given the clinical success of gene therapy, we introduce the first lentiviral-based gene therapy approach to restore expression and function of the human IFN-γR-downstream signaling cascade. In our study, we developed lentiviral vectors constitutively expressing the human IFN-γR1 and demonstrate stable transgene expression without interference with cell viability and proliferation in transduced human hematopoietic cells. Using an IFN-γR1-deficient HeLa cell model, we show stable receptor reconstitution and restored IFN-γR1 signaling without adverse effect on cell functionality. Transduction of both SV40-immortalized and primary fibroblasts derived from IFN-γR1-deficient MSMD patients was able to recover IFN-γR1 expression and restore type II IFN signaling upon stimulation with IFN-γ. In summary, we highlight lentiviral vectors to correct the IFN-γ mediated immunity and present the first gene therapy approach for patients suffering from AR complete IFN-γR1 deficiency

Patient iPSC-Derived Macrophages to Study Inborn Errors of the IFN-γ Responsive Pathway

Interferon γ (IFN-γ) was shown to be a macrophage activating factor already in 1984. Consistently, inborn errors of IFN-γ immunity underlie Mendelian Susceptibility to Mycobacterial Disease (MSMD). MSMD is characterized by genetic predisposition to disease caused by weakly virulent mycobacterial species. Paradoxically, macrophages from patients with MSMD were little tested. Here, we report a disease modeling platform for studying IFN-γ related pathologies using macrophages derived from patient specific induced pluripotent stem cells (iPSCs). We used iPSCs from patients with autosomal recessive complete- and partial IFN-γR2 deficiency, partial IFN-γR1 deficiency and complete STAT1 deficiency. Macrophages from all patient iPSCs showed normal morphology and IFN-γ-independent functionality like phagocytic uptake of bioparticles and internalization of cytokines. For the IFN-γ-dependent functionalities, we observed that the deficiencies played out at various stages of the IFN-γ pathway, with the complete IFN-γR2 and complete STAT1 deficient cells showing the most severe phenotypes, in terms of upregulation of surface markers and induction of downstream targets. Although iPSC-derived macrophages with partial IFN-γR1 and IFN-γR2 deficiency still showed residual induction of downstream targets, they did not reduce the mycobacterial growth when challenged with Bacillus Calmette–Guérin. Taken together, we report a disease modeling platform to study the role of macrophages in patients with inborn errors of IFN-γ immunity

Impaired respiratory burst contributes to infections in PKC-deficient patients

Patients with autosomal recessive protein kinase C delta (PKC delta) deficiency suffer from childhood-onset autoimmunity, including systemic lupus erythematosus. They also suffer from recurrent infections that overlap with those seen in patients with chronic granulomatous disease (CGD), a disease caused by defects of the phagocyte NADPH oxidase and a lack of reactive oxygen species (ROS) production. We studied an international cohort of 17 PKC delta-deficient patients and found that their EBV-B cells and monocyte-derived phagocytes produced only small amounts of ROS and did not phosphorylate p40(phox) normally after PMA or opsonized Staphylococcus aureus stimulation. Moreover, the patients' circulating phagocytes displayed abnormally low levels of ROS production and markedly reduced neutrophil extracellular trap formation, altogether suggesting a role for PKC delta in activation of the NADPH oxidase complex. Our findings thus show that patients with PKC delta deficiency have impaired NADPH oxidase activity in various myeloid subsets, which may contribute to their CGD-like infectious phenotype

Inherited deficiency of stress granule ZNFX1 in patients with monocytosis and mycobacterial disease

Human inborn errors of IFN-γ underlie mycobacterial disease, due to insufficient IFN-γ production by lymphoid cells, impaired myeloid cell responses to this cytokine, or both. We report four patients from two unrelated kindreds with intermittent monocytosis and mycobacterial disease, including bacillus Calmette-Guérin-osis and disseminated tuberculosis, and without any known inborn error of IFN-γ. The patients are homozygous for ZNFX1 variants (p.S959* and p.E1606Rfs*10) predicted to be loss of function (pLOF). There are no subjects homozygous for pLOF variants in public databases. ZNFX1 is a conserved and broadly expressed helicase, but its biology remains largely unknown. It is thought to act as a viral double-stranded RNA sensor in mice, but these patients do not suffer from severe viral illnesses. We analyze its subcellular localization upon overexpression in A549 and HeLa cell lines and upon stimulation of THP1 and fibroblastic cell lines. We find that this cytoplasmic protein can be recruited to or even induce stress granules. The endogenous ZNFX1 protein in cell lines of the patient homozygous for the p.E1606Rfs*10 variant is truncated, whereas ZNFX1 expression is abolished in cell lines from the patients with the p.S959* variant. Lymphocyte subsets are present at normal frequencies in these patients and produce IFN-γ normally. The hematopoietic and nonhematopoietic cells of the patients tested respond normally to IFN-γ. Our results indicate that human ZNFX1 is associated with stress granules and essential for both monocyte homeostasis and protective immunity to mycobacteria

Preexisting autoantibodies to type I IFNs underlie critical COVID-19 pneumonia in patients with APS-1

Patients with biallelic loss-of-function variants of AIRE suffer from autoimmune polyendocrine syndrome type-1 (APS-1) and produce a broad range of autoantibodies (auto-Abs), including circulating auto-Abs neutralizing most type I interferons (IFNs). These auto-Abs were recently reported to account for at least 10% of cases of life-threatening COVID-19 pneumonia in the general population. We report 22 APS-1 patients from 21 kindreds in seven countries, aged between 8 and 48 yr and infected with SARS-CoV-2 since February 2020. The 21 patients tested had auto-Abs neutralizing IFN-α subtypes and/or IFN-ω; one had anti–IFN-β and another anti–IFN-ε, but none had anti–IFN-κ. Strikingly, 19 patients (86%) were hospitalized for COVID-19 pneumonia, including 15 (68%) admitted to an intensive care unit, 11 (50%) who required mechanical ventilation, and four (18%) who died. Ambulatory disease in three patients (14%) was possibly accounted for by prior or early specific interventions. Preexisting auto-Abs neutralizing type I IFNs in APS-1 patients confer a very high risk of life-threatening COVID-19 pneumonia at any age

Preexisting autoantibodies to type I IFNs underlie critical COVID-19 pneumonia in patients with APS-1.

Patients with biallelic loss-of-function variants of AIRE suffer from autoimmune polyendocrine syndrome type-1 (APS-1) and produce a broad range of autoantibodies (auto-Abs), including circulating auto-Abs neutralizing most type I interferons (IFNs). These auto-Abs were recently reported to account for at least 10% of cases of life-threatening COVID-19 pneumonia in the general population. We report 22 APS-1 patients from 21 kindreds in seven countries, aged between 8 and 48 yr and infected with SARS-CoV-2 since February 2020. The 21 patients tested had auto-Abs neutralizing IFN-α subtypes and/or IFN-ω; one had anti-IFN-β and another anti-IFN-ε, but none had anti-IFN-κ. Strikingly, 19 patients (86%) were hospitalized for COVID-19 pneumonia, including 15 (68%) admitted to an intensive care unit, 11 (50%) who required mechanical ventilation, and four (18%) who died. Ambulatory disease in three patients (14%) was possibly accounted for by prior or early specific interventions. Preexisting auto-Abs neutralizing type I IFNs in APS-1 patients confer a very high risk of life-threatening COVID-19 pneumonia at any age