Differences between female and male immunity may contribute to variations in response to infections and predisposition to autoimmunity. We previously reported that neutrophils from reproductive-age males are more immature and less activated than their female counterparts. To further characterize the mechanisms that drive differential neutrophil phenotypes, we performed RNA sequencing on circulating neutrophils from healthy adult females and males. Female neutrophils displayed significant up-regulation of type I IFN (IFN)-stimulated genes (ISGs). Single-cell RNA-sequencing analysis indicated that these differences are neutrophil specific, driven by a distinct neutrophil subset and related to maturation status. Neutrophil hyperresponsiveness to type I IFNs promoted enhanced responses to Toll-like receptor agonists. Neutrophils from young adult males had significantly increased mitochondrial metabolism compared to those from females and this was modulated by estradiol. Assessment of ISGs and neutrophil maturation genes in Klinefelter syndrome (47, XXY) males and in prepubescent children supported that differences in neutrophil phenotype between adult male and female neutrophils are hormonally driven and not explained by X chromosome gene dosage. Our results indicate that there are distinct sex differences in neutrophil biology related to responses to type I IFNs, immunometabolism, and maturation status that may have prominent functional and pathogenic implications.
ObjectivesRecent investigations in humans and mouse models with lupus have revealed evidence of mitochondrial dysfunction and production of mitochondrial reactive oxygen species (mROS) in T cells and neutrophils. This can provoke numerous cellular changes including oxidation of nucleic acids, proteins, lipids and even induction of cell death. We have previously observed that in T cells from patients with lupus, the increased mROS is capable of provoking oligomerisation of mitochondrial antiviral stimulator (MAVS) and production of type I interferon (IFN-I). mROS in SLE neutrophils also promotes the formation of neutrophil extracellular traps (NETs), which are increased in lupus and implicated in renal damage. As a result, in addition to traditional immunosuppression, more comprehensive treatments for lupus may also include non-immune therapy, such as antioxidants.MethodsLupus-prone MRL-lpr mice were treated from weaning for 11 weeks with the mitochondria-targeted antioxidant, MitoQ (200 µM) in drinking water. Mice were then assessed for ROS production in neutrophils, NET formation, MAVS oligomerisation, serum IFN-I, autoantibody production and renal function.ResultsMitoQ-treated mice manifested reduced neutrophil ROS and NET formation, decreased MAVS oligomerisation and serum IFN-I, and reduced immune complex formation in kidneys, despite no change in serum autoantibody .ConclusionsThese findings reveal the potential utility of targeting mROS in addition to traditional immunosuppressive therapy for lupus.
Objective A role for mitochondrial dysfunction has been proposed in the immune dysregulation and organ damage characteristic of systemic lupus erythematosus (SLE). Idebenone is a coenzyme Q10 synthetic quinone analog and an antioxidant that has been used in humans to treat diverse diseases in which mitochondrial function is impaired. This study was undertaken to assess whether idebenone ameliorates lupus in murine models. Methods Idebenone was administered orally to MRL/lpr mice at 2 different doses (1 gm/kg or 1.5 gm/kg idebenone‐containing diet) for 8 weeks. At peak disease activity, clinical, immunologic, and metabolic parameters were analyzed and compared to those in untreated mice (n = 10 per treatment group). Results were confirmed in the lupus‐prone NZM2328 mouse model. Results In MRL/lpr mice, idebenone‐treated mice showed a significant reduction in mortality incidence (P < 0.01 versus untreated mice), and the treatment attenuated several disease features, including glomerular inflammation and fibrosis (each P < 0.05 versus untreated mice), and improved renal function in association with decreased renal expression of interleukin‐17A (IL‐17A) and mature IL‐18. Levels of splenic proinflammatory cytokines and inflammasome‐related genes were significantly decreased (at least P < 0.05 and some with higher significance) in mice treated with idebenone, while no obvious drug toxicity was observed. Idebenone inhibited neutrophil extracellular trap formation in neutrophils from lupus‐prone mice (P < 0.05) and human patients with SLE. Idebenone also improved mitochondrial metabolism (30% increase in basal respiration and ATP production), reduced the extent of heart lipid peroxidation (by one‐half that of untreated mice), and significantly improved endothelium‐dependent vasorelaxation (P < 0.001). NZM2328 mice exposed to idebenone also displayed improvements in renal and systemic inflammation, reducing the kidney pathology score (P < 0.05), IgG/C3 deposition (P < 0.05), and the gene expression of interferon, proinflammatory, and inflammasome‐related genes (at least P < 0.05 and some with higher significance). Conclusion Idebenone ameliorates murine lupus disease activity and the severity of organ damage, supporting the hypothesis that agents that modulate mitochondrial biologic processes may have a therapeutic role in human SLE.
Superoxide dismutases (SODs) are metalloenzymes that catalyse the dismutation of the superoxide radical anion into O(2) and H(2)O(2) in a two-step reaction. The crystal structure of the iron superoxide dismutase from the cold-adapted and fish-pathogenic bacterium Aliivibrio salmonicida (asFeSOD) has been determined and refined to 1.7 A resolution. The protein has been characterized and compared with the closely related homologous iron superoxide dismutase from the mesophilic Escherichia coli (ecFeSOD) in an attempt to rationalize its environmental adaptation. ecFeSOD shares 75% identity with asFeSOD. Compared with the mesophilic FeSOD, the psychrophilic FeSOD has distinct temperature differences in residual activity and thermostability that do not seem to be related to structural differences such as intramolecular or intermolecular ion bonds, hydrogen bonds or cavity sizes. However, an increased net negative charge on the surface of asFeSOD may explain its lower thermostability compared with ecFeSOD. Activity measurements and differential scanning calorimetry measurements revealed that the psychrophilic asFeSOD had a thermostability that was significantly higher than the optimal growth temperature of the host organism.
Immune aggregates organized as tertiary lymphoid structures (TLS) are observed within the kidneys of patients with systemic lupus erythematosus and lupus nephritis (LN). Renal TLS was characterized in lupus-prone New Zealand black  New Zealand white F1 mice analyzing cell composition and vessel formation. RNA sequencing was performed on transcriptomes isolated from lymph nodes, macrodissected TLS from kidneys, and total kidneys of mice at different disease stages by using a personal genome machine and RNA sequencing. Formation of TLS was found in antiedouble-stranded DNA antibodyepositive mice, and the structures were organized as interconnected large networks with distinct T/B cell zones with adjacent dendritic cells, macrophages, plasma cells, high endothelial venules, supporting follicular dendritic cells network, and functional germinal centers. Comparison of gene profiles of whole kidney, renal TLS, and lymph nodes revealed a similar gene signature of TLS and lymph nodes. The up-regulated genes within the kidneys of lupus-prone mice during LN development reflected TLS formation, whereas the down-regulated genes were involved in metabolic processes of the kidney cells. A comparison with human LN gene expression revealed similar up-regulated genes as observed during the development of murine LN and TLS. In conclusion, kidney TLS have a similar cell composition, structure, and gene signature as lymph nodes and therefore may function as a kidney-specific type of lymph node.
The genome of the psychrophilic fish-pathogen Aliivibrio salmonicida encodes a putative ATPdependent DNA ligase in addition to a housekeeping NAD-dependent enzyme. In order to study the structure and activity of the ATP dependent ligase in vitro we have undertaken its recombinant production and purification from an E. coli based expression system. Expression and purification of this protein presented two significant challenges. First, the gene product was moderately toxic to E. coli cells, second it was necessary to remove the large amounts of E. coli DNA present in bacterial lysates without contamination of the protein preparation by nucleases which might interfere with future assaying. The toxicity problem was overcome by fusion of the putative ligase to large solubility tags such as maltose-binding protein (MBP) or Glutathione-stransferase (GST), and DNA was removed by treatment with a nuclease which could be inhibited by reducing agents.As the A. salmonicida ATP-dependent DNA ligase gene encodes a predicted leader peptide, both the full-length and mature forms of the protein were produced. Both possessed ATP-dependent DNA ligase activity, but the truncated form was significantly more active. Here we detail the first reported production, purification and preliminary characterization of active Aliivibrio salmonicida ATPdependent DNA ligase. Highlights A putative periplasmic DNA ligase from Aliivibrio salmonicida was recombinantly produced in E. coli Both the full-length and N-terminally truncated variants were expressed and purified Toxicity of gene products to the host cells was overcame by fusion to large solubility tags The truncated protein is more active in DNA ligation than the full-length version
Objective Gasdermin D (GSDMD) is the key executioner of an inflammatory cell death mechanism known as pyroptosis. Recent reports have also implicated GSDMD in other mechanisms of cell death, including apoptosis, necroptosis, and NETosis. Given the role of dysregulated cell death in autoimmune syndromes such as systemic lupus erythematosus (SLE), this study was undertaken in a murine lupus model to investigate whether GSDMD plays a pathogenic role in systemic autoimmunity by promoting inflammatory cell death, leading to increased generation of nuclear autoantigens and autoantibodies. Methods An imiquimod‐induced model of SLE was tested in GSDMD−/− mice (n = 30), with wild‐type (WT) mice as controls (n = 34), on a C57BL/6 background. At the time of euthanasia, the mice were examined for serum autoantibodies, immune complex deposition, organ inflammation, immune dysregulation, and type I interferon responses. A model of pristane‐induced lung injury in GSDMD−/− mice (n = 7), with WT mice as controls (n = 10), was used to confirm the pulmonary phenotype. Regulation of various mechanisms of cell death by GSDMD was investigated in the mice. Results Unexpectedly, GSDMD−/− mice developed enhanced mortality, more severe renal and pulmonary inflammation, and exacerbated autoantibody production in response to imiquimod. Pulmonary involvement was also more severe in the absence of GSDMD in mice with pristane‐induced lung injury. Compared to WT mice, lack of GSDMD was associated with increased levels of circulating nuclear autoantigens (P < 0.01), anti–double‐stranded DNA autoantibodies (P < 0.01), tissue immune complex deposition (P < 0.05), expansion of myeloid cell subsets (P < 0.05), and enhanced B cell activation and plasma cell differentiation (P = 0.001). Moreover, in the absence of GSDMD, enhanced autoantigen generation was associated with increased local induction of cell death in vivo. Conclusion GSDMD negatively regulates autoantigen generation and immune dysregulation in response to tissue injury and may play previously unappreciated protective roles in systemic autoimmunity.
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