Inducible generalized activation of hSTING-N154S expression in mice leads to lethal hypercytokinemia: a model for "cytokine storm".

Excessive levels of circulating proinflammatory mediators, known as "hypercytokinemia," that are generated by overwhelming immune system activation can lead to death due to critical organ failure and thrombotic events. Hypercytokinemia has been frequently associated with a variety of infectious and autoimmune diseases, with severe acute respiratory syndrome coronavirus 2 infection currently being the commonest cause, of what has been termed the cytokine storm. Among its various functions within the host, STING (stimulator of interferon genes) is critical in the defense against certain viruses and other pathogens. STING activation, particularly within cells of the innate immune system, triggers potent type I interferon and proinflammatory cytokine production. We thus hypothesized that generalized expression of a constitutively active STING mutant in mice would lead to hypercytokinemia. To test this, a Cre-loxP-based system was used to cause the inducible expression of a constitutively active hSTING mutant (hSTING-N154S) in any tissue or cell type. Herein, we employed a tamoxifen-inducible ubiquitin C-CreERT2 transgenic to obtain generalized expression of the hSTING-N154S protein, thereby triggering the production of IFN-ß and multiple proinflammatory cytokines. This required euthanizing the mice within 3 to 4 d after tamoxifen administration. This preclinical model will allow for the rapid identification of compounds aimed at either preventing or ameliorating the lethal effects of hypercytokinemia.

PNKP is required for maintaining the integrity of progenitor cell populations in adult mice.

DNA repair proteins are critical to the maintenance of genomic integrity. Specific types of genotoxic factors, including reactive oxygen species generated during normal cellular metabolism or as a result of exposure to exogenous oxidative agents, frequently leads to "ragged" single-strand DNA breaks. The latter exhibits abnormal free DNA ends containing either a 5'-hydroxyl or 3'-phosphate requiring correction by the dual function enzyme, polynucleotide kinase phosphatase (PNKP), before DNA polymerase and ligation reactions can occur to seal the break. Pnkp gene deletion during early murine development leads to lethality; in contrast, the role of PNKP in adult mice is unknown. To investigate the latter, we used an inducible conditional mutagenesis approach to cause global disruption of the Pnkp gene in adult mice. This resulted in a premature aging-like phenotype, characterized by impaired growth of hair follicles, seminiferous tubules, and neural progenitor cell populations. These results point to an important role for PNKP in maintaining the normal growth and survival of these murine progenitor populations.

Expression of a constitutively active human STING mutant in hematopoietic cells produces an Ifnar1-dependent vasculopathy in mice.

STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disorder characterized by blood vessel occlusions, acral necrosis, myositis, rashes, and pulmonary inflammation that are the result of activating mutations in the STimulator of Interferon Genes (STING). We generated a transgenic line that recapitulates many of the phenotypic aspects of SAVI by targeting the expression of the human STING-N154S-mutant protein to the murine hematopoietic compartment. hSTING-N154S mice demonstrated failure to gain weight, lymphopenia, progressive paw swelling accompanied by inflammatory infiltrates, severe myositis, and ear and tail necrosis. However, no significant lung inflammation was observed. X-ray microscopy imaging revealed vasculopathy characterized by arteriole occlusions and venous thromboses. Type I interferons and proinflammatory mediators were elevated in hSTING-N154S sera. Importantly, the phenotype was prevented in hSTING-N154S mice lacking the type I interferon receptor gene (Ifnar1). This model, based on a mutant human STING protein, may shed light on the pathophysiological mechanisms operative in SAVI.

Neuronal expression of the intermediate conductance calcium-activated potassium channel KCa3.1 in the mammalian central nervous system.

The expression pattern and functional roles for calcium-activated potassium channels of the KCa2.x family and KCa1.1 have been extensively examined in central neurons. Recent work indicates that intermediate conductance calcium-activated potassium channels (KCa3.1) are also expressed in central neurons of the cerebellum and spinal cord. The current study used immunocytochemistry and GFP linked to KCNN4 promoter activity in a transgenic mouse to determine the expression pattern of KCa3.1 channels in rat or mouse neocortex, hippocampus, thalamus, and cerebellum. KCa3.1 immunolabel and GFP expression were closely matched and detected in both excitatory and inhibitory cells of all regions examined. KCa3.1 immunolabel was localized primarily to the somatic region of excitatory cells in cortical structures but at the soma and over longer segments of dendrites of cells in deep cerebellar nuclei. More extensive labeling was apparent for inhibitory cells at the somatic and dendritic level with no detectable label associated with axon tracts or regions of intense synaptic innervation. The data indicate that KCa3.1 channels are expressed in the CNS with a differential pattern of distribution between cells, suggesting important functional roles for these calcium-activated potassium channels in regulating the excitability of central neurons.