Increased Neutrophil H2O2 Production and Enhanced Pulmonary Clearance of Klebsiella pneumoniae in G6PD A- Mice.

The X-linked A- variant (rs1050828, Val68Met) in G6PDX accounts for glucose-6-phosphate (G6PD) deficiency in approximately 11% of African American males. This common, hypomorphic variant may impact pulmonary host defense and phagocyte function during pneumonia by altering levels of reactive oxygen species produced by host leukocytes. We used CRISPR-Cas9 technology to generate novel mouse strain with "humanized" G6PD A- variant containing non-synonymous Val68Met single nucleotide polymorphism. Male hemizygous or littermate wild-type (WT) controls were inoculated intratracheally with K. pneumoniae (KP2 serotype, ATCC 43816 strain,103 CFU inoculum). We examined leukocyte recruitment, organ bacterial burden, bone marrow neutrophil and macrophage (BMDM) phagocytic capacity, and hydrogen peroxide (H2O2) production. Unexpectedly, G6PD-deficient mice showed decreased lung bacterial burden (p=0.05) compared to controls 24-h post-infection. Extrapulmonary dissemination and bacteremia were significantly reduced in G6PD-deficient mice 48-h post-infection. Bronchoalveolar lavage fluid (BALF) IL-10 levels were elevated in G6PD-deficient mice (p=0.03) compared to controls at 24-h but were lower at 48-h (p=0.03). G6PD A- BMDMs show mildly decreased in vitro phagocytosis of pHrodo-labeled KP2 (p=0.03). Baseline, but not stimulated, H2O2 production by G6PD A- neutrophils was greater compared to WT neutrophils. G6PD A- variant demonstrate higher basal neutrophil H2O2 production and are protected against acute Klebsiella intrapulmonary infection.

Functional effects of an African glucose-6-phosphate dehydrogenase (G6PD) polymorphism (Val68Met) on red blood cell hemolytic propensity and post-transfusion recovery.

BACKGROUND: Donor genetic variation is associated with red blood cell (RBC) storage integrity and post-transfusion recovery. Our previous large-scale genome-wide association study demonstrated that the African G6PD deficient A- variant (rs1050828, Val68Met) is associated with higher oxidative hemolysis after cold storage. Despite a high prevalence of X-linked G6PD mutation in African American population (>10%), blood donors are not routinely screened for G6PD status and its importance in transfusion medicine is relatively understudied. STUDY DESIGN AND METHODS: To further evaluate the functional effects of the G6PD A- mutation, we created a novel mouse model carrying this genetic variant using CRISPR-Cas9. We hypothesize that this humanized G6PD A- variant is associated with reduced G6PD activity with a consequent effect on RBC hemolytic propensity and post-transfusion recovery. RESULTS: G6PD A- RBCs had reduced G6PD protein with ~5% residual enzymatic activity. Significantly increased in vitro hemolysis induced by oxidative stressors was observed in fresh and stored G6PD A- RBCs, along with a lower GSH:GSSG ratio. However, no differences were observed in storage hemolysis, osmotic fragility, mechanical fragility, reticulocytes, and post-transfusion recovery. Interestingly, a 14% reduction of 24-h survival following irradiation was observed in G6PD A- RBCs compared to WT RBCs. Metabolomic assessment of stored G6PD A- RBCs revealed an impaired pentose phosphate pathway (PPP) with increased glycolytic flux, decreasing cellular antioxidant capacity. DISCUSSION: This novel mouse model of the common G6PD A- variant has impaired antioxidant capacity like humans and low G6PD activity may reduce survival of transfused RBCs when irradiation is performed.

Brief Report: Hydroxychloroquine does not induce hemolytic anemia or organ damage in a "humanized" G6PD A- mouse model.

BACKGROUND: Hydroxychloroquine (HCQ) is widely used in the treatment of malaria, rheumatologic disease such as lupus, and most recently, COVID-19. These uses raise concerns about its safe use in the setting of glucose-6-phosphate dehydrogenase (G6PD) deficiency, especially as 11% of African American men carry the G6PD A- variant. However, limited data exist regarding the safety of HCQ in this population. STUDY DESIGN AND METHODS: Recently, we created a novel "humanized" mouse model containing the G6PD deficiency A- variant (Val68Met) using CRISPR technology. We tested the effects of high-dose HCQ administration over 5 days on hemolysis in our novel G6PD A- mice. In addition to standard hematologic parameters including plasma hemoglobin, erythrocyte methemoglobin, and reticulocytes, hepatic and renal function were assessed after HCQ. RESULTS: Residual erythrocyte G6PD activity in G6PD A- mice was ~6% compared to wild-type (WT) littermates. Importantly, we found no evidence of clinically significant intravascular hemolysis, methemoglobinemia, or organ damage in response to high-dose HCQ. CONCLUSIONS: Though the effects of high doses over prolonged periods was not assessed, this study provides early, novel safety data of the use of HCQ in the setting of G6PD deficiency secondary to G6PD A-. In addition to novel safety data for HCQ, to our knowledge, we are the first to present the creation of a "humanized" murine model of G6PD deficiency.

Apaf-1 deficiency confers resistance to ultraviolet-induced apoptosis in mouse embryonic fibroblasts by disrupting reactive oxygen species amplification production and mitochondrial pathway.

Apoptosis requires tightly regulated cell death pathways. The signaling pathways that trigger a cell to undergo apoptosis after UV radiation are cell type specific and are currently being defined. Here, we have used pharmacological and genetic tools to demonstrate the decisive part of the mitochondrial pathway in UVC-induced apoptosis in mouse embryo fibroblasts (MEFs). UVC-induced apoptosis proceeded independent of the activation of death receptor components. In contrast, soon after UV radiation, MAPK activation and generation of reactive oxygen species (ROS) increased, followed by a decline in mitochondrial membrane potential (MMP) and cytochrome c release, as well as activation of caspase-9 and -3 and the upregulation of p47-phox. Deficiency of apaf-1, a critical member of the apoptosome, dramatically abolished all the UV-induced signal deterioration and cell death. In parallel, UVC-induced apoptosis was largely attenuated by either DN-caspase-9 or Bcl-X(L) overexpression. Pretreatment of cells with N-acetylcysteine or catalase but not Tempol decreased UVC-induced MAPK activation and apoptosis. Inhibition of JNK and caspase attenuated p47-phox upregulation. Altogether, we have for the first time demonstrated the critical role of Apaf-1 in the regulation of MAPK, ROS, and MMP in UVC-radiated MEFs and propose that the amplification feedback loop among mitochondrial signal molecules culminates in the demise of the cell.

Suppression of ES cell differentiation by retinol (vitamin A) via the overexpression of Nanog.

Embryonic stem cells (ESCs) derived from the inner cell mass of blastocysts maintain their pluripotency through a complex interplay of different signaling pathways and transcription factors including Leukemia Inhibitory Factor (LIF), homeo-domain protein Nanog and POU-domain-containing transcription factor Oct3/4. LIF can maintain the self-renewal of mouse ESCs by activating the Jak/Stat3 pathway; however, it is dispensable for human ESCs. Nanog, a homeo-domain transcription factor alone is sufficient for sustaining the self-renewal of ESCs. Overexpression of Nanog by heterologous promoters can maintain self-renewal of human and mouse ESCs in the absence of LIF/Stat3 pathway. The mechanisms that control the expression of Nanog, however, remain poorly understood. In this report we demonstrate that retinol, the alcohol form of Vitamin A, can suppress the differentiation of ESCs by up-regulating the expression of Nanog. Retinol is mainly associated with differentiation through its active metabolite retinoic acid during early development of the embryo. The activation of Nanog by retinol is not mediated via retinoic acid signaling and appears to be independent of previously described LIF/Stat3, bone morphogenic proteins, Wnt/beta-catenin, and Oct3/4-Sox2 pathways. These studies therefore, reveal a previously unknown function of retinol and offer a model system to define alternate regulatory pathways that control the self-renewal of ESCs as well as to identify upstream "master" regulatory factors that are responsible for maintaining the integrity of stem cells.