Genetic dissection of TLR9 reveals complex regulatory and cryptic proinflammatory roles in mouse lupus.

In lupus, Toll-like receptor 7 (TLR7) and TLR9 mediate loss of tolerance to RNA and DNA, respectively. Yet, TLR7 promotes disease, while TLR9 protects from disease, implying differences in signaling. To dissect this 'TLR paradox', we generated two TLR9 point mutants (lacking either ligand (TLR9K51E) or MyD88 (TLR9P915H) binding) in lupus-prone MRL/lpr mice. Ameliorated disease of Tlr9K51E mice compared to Tlr9-/- controls revealed a TLR9 'scaffold' protective function that is ligand and MyD88 independent. Unexpectedly, Tlr9P915H mice were more protected than both Tlr9K51E and Tlr9WT mice, suggesting that TLR9 also possesses ligand-dependent, but MyD88-independent, regulatory signaling and MyD88-mediated proinflammatory signaling. Triple-mixed bone marrow chimeras showed that TLR9-MyD88-independent regulatory roles were B cell intrinsic and restrained differentiation into pathogenic age-associated B cells and plasmablasts. These studies reveal MyD88-independent regulatory roles of TLR9, shedding light on the biology of endosomal TLRs.

BOK Is a Non-canonical BCL-2 Family Effector of Apoptosis Regulated by ER-Associated Degradation.

The mitochondrial pathway of apoptosis is initiated by mitochondrial outer membrane permeabilization (MOMP). The BCL-2 family effectors BAX and BAK are thought to be absolutely required for this process. Here, we report that BCL-2 ovarian killer (BOK) is a bona fide yet unconventional effector of MOMP that can trigger apoptosis in the absence of both BAX and BAK. However, unlike the canonical effectors, BOK appears to be constitutively active and unresponsive to antagonistic effects of the antiapoptotic BCL-2 proteins. Rather, BOK is controlled at the level of protein stability by components of the endoplasmic reticulum (ER)-associated degradation pathway. BOK is ubiquitylated by the AMFR/gp78 E3 ubiquitin ligase complex and targeted for proteasomal degradation in a VCP/p97-dependent manner, which allows survival of the cell. When proteasome function, VCP, or gp78 activity is compromised, BOK is stabilized to induce MOMP and apoptosis independently of other BCL-2 proteins.

DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome.

The cellular stress response has a vital role in regulating homeostasis by modulating cell survival and death. Stress granules are cytoplasmic compartments that enable cells to survive various stressors. Defects in the assembly and disassembly of stress granules are linked to neurodegenerative diseases, aberrant antiviral responses and cancer1-5. Inflammasomes are multi-protein heteromeric complexes that sense molecular patterns that are associated with damage or intracellular pathogens, and assemble into cytosolic compartments known as ASC specks to facilitate the activation of caspase-1. Activation of inflammasomes induces the secretion of interleukin (IL)-1ß and IL-18 and drives cell fate towards pyroptosis-a form of programmed inflammatory cell death that has major roles in health and disease6-12. Although both stress granules and inflammasomes can be triggered by the sensing of cellular stress, they drive contrasting cell-fate decisions. The crosstalk between stress granules and inflammasomes and how this informs cell fate has not been well-studied. Here we show that the induction of stress granules specifically inhibits NLRP3 inflammasome activation, ASC speck formation and pyroptosis. The stress granule protein DDX3X interacts with NLRP3 to drive inflammasome activation. Assembly of stress granules leads to the sequestration of DDX3X, and thereby the inhibition of NLRP3 inflammasome activation. Stress granules and the NLRP3 inflammasome compete for DDX3X molecules to coordinate the activation of innate responses and subsequent cell-fate decisions under stress conditions. Induction of stress granules or loss of DDX3X in the myeloid compartment leads to a decrease in the production of inflammasome-dependent cytokines in vivo. Our findings suggest that macrophages use the availability of DDX3X to interpret stress signals and choose between pro-survival stress granules and pyroptotic ASC specks. Together, our data demonstrate the role of DDX3X in driving NLRP3 inflammasome and stress granule assembly, and suggest a rheostat-like mechanistic paradigm for regulating live-or-die cell-fate decisions under stress conditions.

MDM2 Regulation of HIF Signaling Causes Microvascular Dysfunction in Hypertrophic Cardiomyopathy.

BACKGROUND: Microvasculature dysfunction is a common finding in pathologic remodeling of the heart and is thought to play an important role in the pathogenesis of hypertrophic cardiomyopathy (HCM), a disease caused by sarcomere gene mutations. We hypothesized that microvascular dysfunction in HCM was secondary to abnormal microvascular growth and could occur independent of ventricular hypertrophy. METHODS: We used multimodality imaging methods to track the temporality of microvascular dysfunction in HCM mouse models harboring mutations in the sarcomere genes Mybpc3 (cardiac myosin binding protein C3) or Myh6 (myosin heavy chain 6). We performed complementary molecular methods to assess protein quantity, interactions, and post-translational modifications to identify mechanisms regulating this response. We manipulated select molecular pathways in vivo using both genetic and pharmacological methods to validate these mechanisms. RESULTS: We found that microvascular dysfunction in our HCM models occurred secondary to reduced myocardial capillary growth during the early postnatal time period and could occur before the onset of myocardial hypertrophy. We discovered that the E3 ubiquitin protein ligase MDM2 (murine double minute 2) dynamically regulates the protein stability of both HIF1α (hypoxia-inducible factor 1 alpha) and HIF2α (hypoxia-inducible factor 2 alpha)/EPAS1 (endothelial PAS domain protein 1) through canonical and noncanonical mechanisms. The resulting HIF imbalance leads to reduced proangiogenic gene expression during a key period of myocardial capillary growth. Reducing MDM2 protein levels by genetic or pharmacological methods normalized HIF protein levels and prevented the development of microvascular dysfunction in both HCM models. CONCLUSIONS: Our results show that sarcomere mutations induce cardiomyocyte MDM2 signaling during the earliest stages of disease, and this leads to long-term changes in the myocardial microenvironment.

Influence of proteolytic cleavage of ENaC's γ subunit upon Na+ and K+ handling.

The epithelial Na+ channel (ENaC) γ subunit is essential for homeostasis of Na+, K+, and body fluid. Dual γ subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (PO), in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence (143RKRR146, in the mouse γ subunit). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143RKRR146 mutation to 143QQQQ146 (γQ4) in 129/Sv mice would reduce ENaC PO, impair flow-stimulated flux of Na+ (JNa) and K+ (JK) in perfused collecting ducts, reduce colonic amiloride-sensitive short-circuit current (ISC), and impair Na+, K+, and body fluid homeostasis. Immunoblot of γQ4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, γQ4/Q4 male mice on a low Na+ diet did not exhibit altered ENaC PO or flow-induced JNa, though flow-induced JK modestly decreased. Colonic amiloride-sensitive ISC in γQ4/Q4 mice was not altered. γQ4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na+ diet. Blood Na+ and K+ were unchanged on a regular, low Na+, or high K+ diet. These findings suggest that biochemical evidence of γ subunit cleavage should not be used in isolation to evaluate ENaC activity. Furthermore, factors independent of γ subunit cleavage modulate channel PO and the influence of ENaC on Na+, K+, and fluid volume homeostasis in 129/Sv mice, in vivo.NEW & NOTEWORTHY The epithelial Na+ channel (ENaC) is activated in vitro by post-translational proteolysis. In vivo, low Na+ or high K+ diets enhance ENaC proteolysis, and proteolysis is hypothesized to contribute to channel activation in these settings. Using a mouse expressing ENaC with disruption of a key proteolytic cleavage site, this study demonstrates that impaired proteolytic activation of ENaC's γ subunit has little impact upon channel open probability or the ability of mice to adapt to low Na+ or high K+ diets.

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.

Mouse genome engineering via CRISPR-Cas9 for study of immune function.

Clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas9) technology has proven a formidable addition to our armory of approaches for genomic editing. Derived from pathways in archaea and bacteria that mediate the resistance to exogenous genomic material, the CRISPR-Cas9 system utilizes a short single guide RNA (sgRNA) to direct the endonuclease Cas9 to virtually anywhere in the genome. Upon targeting, Cas9 generates DNA double-strand breaks (DSBs) and facilitates the repair or insertion of mutations, insertion of recombinase recognition sites, or large DNA elements. Here, we discuss the practical advantages of the CRISPR-Cas9 system over conventional and other nuclease-based targeting technologies and provide suggestions for the use of this technology to address immunological questions.

Arid5a Mediates an IL-17-Dependent Pathway That Drives Autoimmunity but Not Antifungal Host Defense.

IL-17 contributes to the pathogenesis of certain autoimmune diseases, but conversely is essential for host defense against fungi. Ab-based biologic drugs that neutralize IL-17 are effective in autoimmunity but can be accompanied by adverse side effects. Candida albicans is a commensal fungus that is the primary causative agent of oropharyngeal and disseminated candidiasis. Defects in IL-17 signaling cause susceptibility to candidiasis in mice and humans. A key facet of IL-17 receptor signaling involves RNA-binding proteins, which orchestrate the fate of target mRNA transcripts. In tissue culture models we showed that the RNA-binding protein AT-rich interaction domain 5A (Arid5a) promotes the stability and/or translation of multiple IL-17-dependent mRNAs. Moreover, during oropharyngeal candidiasis, Arid5a is elevated within the oral mucosa in an IL-17-dependent manner. However, the contribution of Arid5a to IL-17-driven events in vivo is poorly defined. In this study, we used CRISPR-Cas9 to generate mice lacking Arid5a. Arid5a -/- mice were fully resistant to experimental autoimmune encephalomyelitis, an autoimmune setting in which IL-17 signaling drives pathology. Surprisingly, Arid5a -/- mice were resistant to oropharyngeal candidiasis and systemic candidiasis, similar to immunocompetent wild-type mice and contrasting with mice defective in IL-17 signaling. Therefore, Arid5a-dependent signals mediate pathology in autoimmunity and yet are not required for immunity to candidiasis, indicating that selective targeting of IL-17 signaling pathway components may be a viable strategy for development of therapeutics that spare IL-17-driven host defense.

Dysregulation of Lipid and Glucose Homeostasis in Hepatocyte-Specific SLC25A34 Knockout Mice.

Nonalcoholic fatty liver disease (NAFLD) is an epidemic affecting 30% of the US population. It is characterized by insulin resistance, and by defective lipid metabolism and mitochondrial dysfunction in the liver. SLC25A34 is a major repressive target of miR-122, a miR that has a central role in NAFLD and liver cancer. However, little is known about the function of SLC25A34. To investigate SLC25A34 in vitro, mitochondrial respiration and bioenergetics were examined using hepatocytes depleted of Slc25a34 or overexpressing Slc25a34. To test the function of SLC25A34 in vivo, a hepatocyte-specific knockout mouse was generated, and loss of SLC25A34 was assessed in mice maintained on a chow diet and a fast-food diet (FFD), a model for NAFLD. Hepatocytes depleted of Slc25a34 displayed increased mitochondrial biogenesis, lipid synthesis, and ADP/ATP ratio; Slc25a34 overexpression had the opposite effect. In the knockout model on chow diet, SLC25A34 loss modestly affected liver function (altered glucose metabolism was the most pronounced defect). RNA-sequencing revealed changes in metabolic processes, especially fatty acid metabolism. After 2 months on FFD, knockouts had a more severe phenotype, with increased lipid content and impaired glucose tolerance, which was attenuated after longer FFD feeding (6 months). This work thus presents a novel model for studying SLC25A34 in vivo in which SLC25A34 plays a role in mitochondrial respiration and bioenergetics during NAFLD.

An IL-17F.S65L Knock-In Mouse Reveals Similarities and Differences in IL-17F Function in Oral Candidiasis: A New Tool to Understand IL-17F.

Oropharyngeal candidiasis (OPC) is an opportunistic infection of the oral mucosa caused by the commensal fungus Candida albicans IL-17R signaling is essential to prevent OPC in mice and humans, but the individual roles of its ligands, IL-17A, IL-17F, and IL-17AF, are less clear. A homozygous IL-17F deficiency in mice does not cause OPC susceptibility, whereas mice lacking IL-17A are moderately susceptible. In humans, a rare heterozygous mutation in IL-17F (IL-17F.S65L) was identified that causes chronic mucocutaneous candidiasis, suggesting the existence of essential antifungal pathways mediated by IL-17F and/or IL-17AF. To investigate the role of IL-17F and IL-17AF in more detail, we exploited this "experiment of nature" by creating a mouse line bearing the homologous mutation in IL-17F (Ser65Leu) by CRISPR/Cas9. Unlike Il17f-/- mice that are resistant to OPC, Il17fS65L/S65L mice showed increased oral fungal burdens similar to Il17a -/- mice. In contrast to humans, however, disease was only evident in homozygous, not heterozygous, mutant mice. The mutation was linked to modestly impaired CXC chemokine expression and neutrophil recruitment to the infected tongue but not to alterations in oral antimicrobial peptide expression. These findings suggest mechanisms by which the enigmatic cytokine IL-17F contributes to host defense against fungi. Moreover, because these mice do not phenocopy Il17f-/- mice, they may provide a valuable tool to interrogate IL-17F and IL-17AF function in vivo in other settings.

Successful surgical weight loss with laparoscopic sleeve gastrectomy for morbid obesity prior to kidney transplantation.

Morbid obesity in kidney transplant (KT) candidates is associated with increased complications and graft failure. Multiple series have demonstrated rapid and significant weight loss after laparoscopic sleeve gastrectomy (LSG) in this population. Long-term and post-transplant weight evolutions are still largely unknown. A retrospective review was performed in eighty patients with end-stage kidney disease (ESKD) who underwent LSG in preparation for KT. From a median initial BMI of 43.7 kg/m2 , the median change at 1-year was -10.0 kg/m2 . Successful surgical weight loss (achieving a BMI < 35 kg/m2 or an excess body weight loss >50%) was attained in 76.3% and was associated with male gender, predialysis status, lower obesity class and lack of coronary artery disease. Thirty-one patients subsequently received a KT with a median delay of 16.7 months. Weight regain (increase in BMI of 5 kg/m2 postnadir) and recurrent obesity (weight regain + BMI > 35) remain a concern, occurring post-KT in 35.7% and 17.9%, respectively. Early LSG should be considered for morbidly obese patients with ESKD for improved weight loss outcomes. Early KT after LSG does not appear to affect short-term surgical weight loss. Candidates with a BMI of up to 45 kg/m2 can have a reasonable expectation to achieve the limit within 1 year.

Overlapping Role of SCYL1 and SCYL3 in Maintaining Motor Neuron Viability.

Members of the SCY1-like (SCYL) family of protein kinases are evolutionarily conserved and ubiquitously expressed proteins characterized by an N-terminal pseudokinase domain, centrally located Huntingtin, elongation factor 3, protein phosphatase 2A, yeast kinase TOR1 repeats, and an overall disorganized C-terminal segment. In mammals, three family members encoded by genes Scyl1, Scyl2, and Scyl3 have been described. Studies have pointed to a role for SCYL1 and SCYL2 in regulating neuronal function and viability in mice and humans, but little is known about the biological function of SCYL3. Here, we show that the biochemical and cell biological properties of SCYL3 are similar to those of SCYL1 and both proteins work in conjunction to maintain motor neuron viability. Specifically, although lack of Scyl3 in mice has no apparent effect on embryogenesis and postnatal life, it accelerates the onset of the motor neuron disorder caused by Scyl1 deficiency. Growth abnormalities, motor dysfunction, hindlimb paralysis, muscle wasting, neurogenic atrophy, motor neuron degeneration, and loss of large-caliber axons in peripheral nerves occurred at an earlier age in Scyl1/Scyl3 double-deficient mice than in Scyl1-deficient mice. Disease onset also correlated with the mislocalization of TDP-43 in spinal motor neurons, suggesting that SCYL1 and SCYL3 regulate TDP-43 proteostasis. Together, our results demonstrate an overlapping role for SCYL1 and SCYL3 in vivo and highlight the importance the SCYL family of proteins in regulating neuronal function and survival. Only male mice were used in this study.SIGNIFICANCE STATEMENT SCYL1 and SCYL2, members of the SCY1-like family of pseudokinases, have well established roles in neuronal function. Herein, we uncover the role of SCYL3 in maintaining motor neuron viability. Although targeted disruption of Scyl3 in mice had little or no effect on embryonic development and postnatal life, it accelerated disease onset associated with the loss of Scyl1, a novel motor neuron disease gene in humans. Scyl1 and Scyl3 double-deficient mice had neuronal defects characteristic of amyotrophic lateral sclerosis, including TDP-43 pathology, at an earlier age than did Scyl1-deficient mice. Thus, we show that SCYL1 and SCYL3 play overlapping roles in maintaining motor neuronal viability in vivo and confirm that SCYL family members are critical regulators of neuronal function and survival.

Keratinocytes contribute intrinsically to psoriasis upon loss of Tnip1 function.

Psoriasis is a chronic inflammatory skin disease with a clear genetic contribution, characterized by keratinocyte proliferation and immune cell infiltration. Various closely interacting cell types, including innate immune cells, T cells, and keratinocytes, are known to contribute to inflammation. Innate immune cells most likely initiate the inflammatory process by secretion of IL-23. IL-23 mediates expansion of T helper 17 (Th17) cells, whose effector functions, including IL-17A, activate keratinocytes. Keratinocyte activation in turn results in cell proliferation and chemokine expression, the latter of which fuels the inflammatory process through further immune cell recruitment. One question that remains largely unanswered is how genetic susceptibility contributes to this process and, specifically, which cell type causes disease due to psoriasis-specific genetic alterations. Here we describe a mouse model based on the human psoriasis susceptibility locus TNIP1, also referred to as ABIN1, whose gene product is a negative regulator of various inflammatory signaling pathways, including the Toll-like receptor pathway in innate immune cells. We find that Tnip1-deficient mice recapitulate major features of psoriasis on pathological, genomic, and therapeutic levels. Different genetic approaches, including tissue-specific gene deletion and the use of various inflammatory triggers, reveal that Tnip1 controls not only immune cells, but also keratinocyte biology. Loss of Tnip1 in keratinocytes leads to deregulation of IL-17-induced gene expression and exaggerated chemokine production in vitro and overt psoriasis-like inflammation in vivo. Together, the data establish Tnip1 as a critical regulator of IL-17 biology and reveal a causal role of keratinocytes in the pathogenesis of psoriasis.

SCYL2 Protects CA3 Pyramidal Neurons from Excitotoxicity during Functional Maturation of the Mouse Hippocampus.

Neuronal death caused by excessive excitatory signaling, excitotoxicity, plays a central role in neurodegenerative disorders. The mechanisms regulating this process, however, are still incompletely understood. Here we show that the coated vesicle-associated kinase SCYL2/CVAK104 plays a critical role for the normal functioning of the nervous system and for suppressing excitotoxicity in the developing hippocampus. Targeted disruption of Scyl2 in mice caused perinatal lethality in the vast majority of newborn mice and severe sensory-motor deficits in mice that survived to adulthood. Consistent with a neurogenic origin of these phenotypes, neuron-specific deletion of Scyl2 also caused perinatal lethality in the majority of newborn mice and severe neurological defects in adult mice. The neurological deficits in these mice were associated with the degeneration of several neuronal populations, most notably CA3 pyramidal neurons of the hippocampus, which we analyzed in more detail. The loss of CA3 neurons occurred during the functional maturation of the hippocampus and was the result of a BAX-dependent apoptotic process. Excessive excitatory signaling was present at the onset of degeneration, and inhibition of excitatory signaling prevented the degeneration of CA3 neurons. Biochemical fractionation reveals that Scyl2-deficient mice have an altered composition of excitatory receptors at synapses. Our findings demonstrate an essential role for SCYL2 in regulating neuronal function and survival and suggest a role for SCYL2 in regulating excitatory signaling in the developing brain. Significance statement: Here we examine the in vivo function of SCYL2, an evolutionarily conserved and ubiquitously expressed protein pseudokinase thought to regulate protein trafficking along the secretory pathway, and demonstrate its importance for the normal functioning of the nervous system and for suppressing excitatory signaling in the developing brain. Together with recent studies demonstrating a role of SCYL1 in preventing motor neuron degeneration, our findings clearly establish the SCY1-like family of protein pseudokinases as key regulators of neuronal function and survival.

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.

Endothelial cell expression of a STING gain-of-function mutation initiates pulmonary lymphocytic infiltration.

Patients afflicted with Stimulator of interferon gene (STING) gain-of-function mutations frequently present with debilitating interstitial lung disease (ILD) that is recapitulated in mice expressing the STINGV154M mutation (VM). Prior radiation chimera studies revealed an unexpected and critical role for non-hematopoietic cells in initiating ILD. To identify STING-expressing non-hematopoietic cell types required for the development of ILD, we use a conditional knockin (CKI) model and direct expression of the VM allele to hematopoietic cells, fibroblasts, epithelial cells, or endothelial cells. Only endothelial cell-targeted VM expression results in enhanced recruitment of immune cells to the lung associated with elevated chemokine expression and the formation of bronchus-associated lymphoid tissue, as seen in the parental VM strain. These findings reveal the importance of endothelial cells as instigators of STING-driven lung disease and suggest that therapeutic targeting of STING inhibitors to endothelial cells could potentially mitigate inflammation in the lungs of STING-associated vasculopathy with onset in infancy (SAVI) patients or patients afflicted with other ILD-related disorders.

Proteolytic Cleavage of the ENaC γ Subunit - Impact Upon Na + and K + Handling.

The ENaC gamma subunit is essential for homeostasis of Na + , K + , and body fluid. Dual subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (P O ), in vitro . Cleavage proximal to the tract occurs at a furin recognition sequence ( 143 RKRR 146 in mouse). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143 RKRR 146 mutation to 143 QQQQ 146 ( Q4 ) in 129/Sv mice would reduce ENaC P O , impair flow-stimulated flux of Na + (J Na ) and K + (J K ) in perfused collecting ducts, reduce colonic amiloride-sensitive short circuit current (I SC ), and impair Na + , K + , and body fluid homeostasis. Immunoblot of Q4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, Q4/Q4 male mice on a low Na + diet did not exhibit altered ENaC P O or flow-induced J Na , though flow-induced J K modestly decreased. Colonic amiloride-sensitive I SC in Q4/Q4 mice was not altered. Q4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na + diet. Blood Na + and K + were unchanged on a regular, low Na + , or high K + diet. These findings suggest that biochemical evidence of gamma subunit cleavage should not be used in isolation to evaluate ENaC activity. Further, factors independent of gamma subunit cleavage modulate channel P O and the influence of ENaC on Na + , K + , and fluid volume homeostasis in 129/Sv mice, in vivo .

An early onset progressive motor neuron disorder in Scyl1-deficient mice is associated with mislocalization of TDP-43.

The molecular and cellular bases of motor neuron diseases (MNDs) are still poorly understood. The diseases are mostly sporadic, with ~10% of cases being familial. In most cases of familial motor neuronopathy, the disease is caused by either gain-of-adverse-effect mutations or partial loss-of-function mutations in ubiquitously expressed genes that serve essential cellular functions. Here we show that deletion of Scyl1, an evolutionarily conserved and ubiquitously expressed gene encoding the COPI-associated protein pseudokinase SCYL1, causes an early onset progressive MND with characteristic features of amyotrophic lateral sclerosis (ALS). Skeletal muscles of Scyl1(-/-) mice displayed neurogenic atrophy, fiber type switching, and disuse atrophy. Peripheral nerves showed axonal degeneration. Loss of lower motor neurons (LMNs) and large-caliber axons was conspicuous in Scyl1(-/-) animals. Signs of neuroinflammation were seen throughout the CNS, most notably in the ventral horn of the spinal cord. Neural-specific, but not skeletal muscle-specific, deletion of Scyl1 was sufficient to cause motor dysfunction, indicating that SCYL1 acts in a neural cell-autonomous manner to prevent LMN degeneration and motor functions. Remarkably, deletion of Scyl1 resulted in the mislocalization and accumulation of TDP-43 (TAR DNA-binding protein of 43 kDa) and ubiquilin 2 into cytoplasmic inclusions within LMNs, features characteristic of most familial and sporadic forms of ALS. Together, our results identify SCYL1 as a key regulator of motor neuron survival, and Scyl1(-/-) mice share pathological features with many human neurodegenerative conditions.

Stat5 tetramer formation is associated with leukemogenesis.

Activation of Stat5 is frequently found in leukemias. To study the mechanism and role of Stat5 activation, we introduced a constitutively activated Stat5a mutant, cS5F, into murine bone marrow (BM) cells. BM transplantation with cS5F-transfected cells caused development of multilineage leukemias in lethally irradiated wild-type or nonirradiated Rag2(-/-) mice. The leukemic cells showed strongly enhanced levels of cS5F tetramers but unchanged cS5F dimer levels in a DNA binding assay. Moreover, Stat5a mutants engineered to form only dimers, but not tetramers, failed to induce leukemias. In addition, Stat5 tetramers were found to accumulate in excess compared to dimers in various human leukemias. These data suggest that Stat5 tetramers are associated with leukemogenesis.

Differentiating primary from secondary lung cancer with FDG PET/CT and extra-pulmonary tumor grade.

OBJECTIVE: Assess feasibility of differentiating primary from secondary lung cancer in patients with a solid solitary malignant pulmonary lesion (SMPL) and a previously resected extrapulmonary tumor. METHODS: Patients with pathology proven primary or secondary lung cancer from a solitary pulmonary lesion and known histopathology of extrapulmonary tumor were included. Patients with a small pulmonary lesion size, multiple malignant pulmonary nodules or an active infectious/inflammatory process were excluded. Extrapulmonary tumor grade was categorized as low, intermediate and high and was matched to FDG uptake intensity of SMPL, with FDG uptake range (SMPL/Liver SUVmax) of <0.9 for low, 0.91-1.99 for intermediate and >2.0 for high extrapulmonary tumor grade. RESULTS: Of 274 patients, 62 met the study criteria. 46 are primary and 16 are secondary lung cancer. There are 19 low, 27 intermediate and 16 high grade extrapulmonary tumors. Mean SMPL SUVmax is 8.2 ± 4.5 and SMPL/liver SUVmax is 2.4 ± 1.4. There are 37 cases (60%) with mismatched results (e.g., low FDG SMPL with intermediate or high grade extrapulmonary tumor or vice versa) and 25 matched cases (40%) that are inconclusive (e.g., low FDG with low tumor grade or high FDG with high tumor grade). Of the mismatched cases, we correctly predicted 30 cases (81%) as primary lung cancers. CONCLUSION: A mismatch between the SMPL SUVmax and the extrapulmonary tumor grade could be used to differentiate a primary lung cancer from a metastasis with reasonable accuracy. Our preliminary results support the hypothesis that FDG uptake intensity of a metastatic pulmonary lesion mirrors the tumor aggressiveness of its extrapulmonary neoplasm of origin.