Systems genetics uncover new loci containing functional gene candidates in Mycobacterium tuberculosis-infected Diversity Outbred mice.

Mycobacterium tuberculosis infects two billion people across the globe, and results in 8-9 million new tuberculosis (TB) cases and 1-1.5 million deaths each year. Most patients have no known genetic basis that predisposes them to disease. Here, we investigate the complex genetic basis of pulmonary TB by modelling human genetic diversity with the Diversity Outbred mouse population. When infected with M. tuberculosis, one-third develop early onset, rapidly progressive, necrotizing granulomas and succumb within 60 days. The remaining develop non-necrotizing granulomas and survive longer than 60 days. Genetic mapping using immune and inflammatory mediators; and clinical, microbiological, and granuloma correlates of disease identified five new loci on mouse chromosomes 1, 2, 4, 16; and three known loci on chromosomes 3 and 17. Further, multiple positively correlated traits shared loci on chromosomes 1, 16, and 17 and had similar patterns of allele effects, suggesting these loci contain critical genetic regulators of inflammatory responses to M. tuberculosis. To narrow the list of candidate genes, we used a machine learning strategy that integrated gene expression signatures from lungs of M. tuberculosis-infected Diversity Outbred mice with gene interaction networks to generate scores representing functional relationships. The scores were used to rank candidates for each mapped trait, resulting in 11 candidate genes: Ncf2, Fam20b, S100a8, S100a9, Itgb5, Fstl1, Zbtb20, Ddr1, Ier3, Vegfa, and Zfp318. Although all candidates have roles in infection, inflammation, cell migration, extracellular matrix remodeling, or intracellular signaling, and all contain single nucleotide polymorphisms (SNPs), SNPs in only four genes (S100a8, Itgb5, Fstl1, Zfp318) are predicted to have deleterious effects on protein functions. We performed methodological and candidate validations to (i) assess biological relevance of predicted allele effects by showing that Diversity Outbred mice carrying PWK/PhJ alleles at the H-2 locus on chromosome 17 QTL have shorter survival; (ii) confirm accuracy of predicted allele effects by quantifying S100A8 protein in inbred founder strains; and (iii) infection of C57BL/6 mice deficient for the S100a8 gene. Overall, this body of work demonstrates that systems genetics using Diversity Outbred mice can identify new (and known) QTLs and functionally relevant gene candidates that may be major regulators of complex host-pathogens interactions contributing to granuloma necrosis and acute inflammation in pulmonary TB.

Propranolol Reduces p-tau Accumulation and Improves Behavior Outcomes in a Polytrauma Murine Model.

INTRODUCTION: Traumatic brain injury (TBI) can lead to neurocognitive decline, in part due to phosphorylated tau (p-tau). Whether p-tau accumulation worsens in the setting of polytrauma remains unknown. Propranolol has shown clinical benefit in head injuries; however, the underlying mechanism is also unknown. We hypothesize that hemorrhagic shock would worsen p-tau accumulation but that propranolol would improve functional outcomes on behavioral studies. METHODS: A murine polytrauma model was developed to examine the accumulation of p-tau and whether it can be mitigated by early administration of propranolol. TBI was induced using a weight-drop model and hemorrhagic shock was achieved via controlled hemorrhage for 1 h. Mice were given intraperitoneal propranolol 4 mg/kg or saline control. The animals underwent behavioral testing at 30 d postinjury and were sacrificed for cerebral histological analysis. These studies were completed in male and female mice. RESULTS: TBI alone led to increased p-tau generation compared to sham on both immunohistochemistry and immunofluorescence (P < 0.05). The addition of hemorrhage led to greater accumulation of p-tau in the hippocampus (P < 0.007). In male mice, p-tau accumulation decreased with propranolol administration for both polytrauma and TBI alone (P < 0.0001). Male mice treated with propranolol also outperformed saline-control mice on the hippocampal-dependent behavioral assessment (P = 0.0013). These results were not replicated in female mice; the addition of hemorrhage did not increase p-tau accumulation and propranolol did not demonstrate a therapeutic effect. CONCLUSIONS: Polytrauma including TBI generates high levels of hippocampal p-tau, but propranolol may help prevent this accumulation to improve both neuropathological and functional outcomes in males.

Postinjury treatments to make early tactical aeromedical evacuation practical for the brain after TBI.

BACKGROUND: Traumatic brain injury (TBI) is common in civilians and military personnel. No potential therapeutics have been evaluated to prevent secondary injury induced by the hypobaric hypoxia (HH) environment integral to postinjury aeromedical evacuation (AE). We examined the role of allopurinol, propranolol, adenosine/lidocaine/magnesium (ALM), or amitriptyline administration prior to simulated flight following murine TBI. METHODS: Mice underwent TBI and were given allopurinol, propranolol, amitriptyline, or ALM prior to simulated AE or normobaric normoxia (NN) control. Heart rate (HR), respiratory rate, and oxygen saturation (Spo2) were recorded throughout simulated AE. Mice were sacrificed at 24 hours, 7 days, or 30 days. Serum and cerebral cytokines were assessed by enzyme-linked immunosorbent assay. Motor function testing was performed with Rotarod ambulation. Immunohistochemistry was conducted to examine phosphorylated tau (p-tau) accumulation in the hippocampus at 30 days. RESULTS: While all treatments improved oxygen saturation, propranolol, amitriptyline, and allopurinol improved AE-induced tachycardia. At 24 hours, both propranolol and amitriptyline reduced tumor necrosis factor alpha levels while allopurinol and ALM reduced tumor necrosis factor alpha levels only in NN mice. Propranolol, amitriptyline, and ALM demonstrated lower serum monocyte chemoattractant protein-1 7 days after AE. Both amitriptyline and allopurinol improved Rotarod times for AE mice while only allopurinol improved Rotarod times for NN mice. Propranolol was able to reduce p-tau accumulation under both HH and NN conditions while ALM only reduced p-tau in hypobaric hypoxic conditions. CONCLUSION: Propranolol lowered post-TBI HR with reduced proinflammatory effects, including p-tau reduction. Amitriptyline-induced lower post-TBI HR and improved functional outcomes without affecting inflammatory response. Allopurinol did not affect vital signs but improved late post-TBI systemic inflammation and functional outcomes. Adenosine/lidocaine/magnesium provided no short-term improvements but reduced p-tau accumulation at 30 days in the HH cohort. Allopurinol may be the best of the four treatments to help prevent short-term functional deficits while propranolol may address long-term effects. LEVEL OF EVIDENCE: Basic science article.

Post-TBI splenectomy may exacerbate coagulopathy and platelet activation in a murine model.

INTRODUCTION: Traumatic brain injury (TBI) induces acute hypocoagulability, subacute hypercoagulability, and persistently elevated risk for thromboembolic events. Splenectomy is associated with increased mortality in patients with moderate or severe TBI. We hypothesized that the adverse effects of splenectomy in TBI patients may be secondary to the exacerbation of pathologic coagulation and platelet activation changes. METHODS: An established murine weight-drop TBI model was utilized and a splenectomy was performed immediately following TBI. Sham as well as TBI and splenectomy alone mice were used as injury controls. Mice were sacrificed for blood draws at 1, 6, and 24 h, as well as 7 days post-TBI. Viscoelastic coagulation parameters were assessed by rotational thromboelastometry (ROTEM) and platelet activation was measured by expression of P-selectin via flow cytometry analysis of platelet rich plasma samples. RESULTS: At 6 h following injury, TBI/splenectomy demonstrated hypocoagulability with prolonged clot formation time (CFT) compared to TBI alone. By 24 h following injury, TBI/splenectomy and splenectomy mice were hypercoagulable with a shorter CFT, a higher alpha angle, and increased MCF, despite a lower percentage of platelet contribution to clot compared to TBI alone. However, only the TBI/splenectomy continued to display this hypercoagulable state at 7 days. While TBI/splenectomy had greater P-selectin expression at 1-h post-injury, TBI alone had significantly greater P-selectin expression at 24 h post-injury compared to TBI/splenectomy. Interestingly, P-selectin expression remained elevated only in TBI/splenectomy at 7 days post-injury. CONCLUSION: Splenectomy following TBI exacerbates changes in the post-injury coagulation state. The combination of TBI and splenectomy induces an acute hypocoagulable state that could contribute to an increase in intracranial bleeding. Subacutely, the addition of splenectomy to TBI exacerbates post-injury hypercoagulability and induces persistent platelet activation. These polytrauma effects on coagulation may contribute to the increased mortality observed in patients with combined brain and splenic injuries.

Oxygenation extremes after traumatic brain injury transiently affect coagulation.

INTRODUCTION: Trauma patients may become hypoxic or iatrogenically hyperoxic in the early post-injury period. While both extremes of oxygenation may be harmful following injury, the mechanism has yet to be elucidated. We hypothesized that hypoxia or hyperoxia would induce changes in coagulation, creating a secondary insult exacerbating the primary injury. MATERIALS AND METHODS: Mice underwent traumatic brain injury (TBI) or sham and were subsequently exposed to room air or brief hypoxia (15% FiO2) then sacrificed at intervals. Another cohort of uninjured mice underwent more prolonged hypoxia (10% FiO2) or hyperoxia (60% FiO2). Platelet function was evaluated by ADP- or ASPi-induced impedance aggregometry and viscoelastic coagulation parameters were assessed with rotational thromboelastometry. RESULTS: In uninjured mice, ADP-induced platelet aggregation was acutely increased after prolonged hypoxia, but this difference did not persist at 6 h. Hypoxic and hyperoxic TBI mice had increased ADP induced platelet aggregation at 1 h compared to the normoxia group. TBI mice demonstrated an early increased platelet aggregation after hypoxia or hyperoxia. Viscoelastic coagulation parameters were similar between hypoxic, hyperoxic and room air groups at 1 h and 6 h. However, ROTEM clotting time and clot formation time were prolonged and maximal clot firmness were lower in sham TBI/hypoxia mice at 24 h. CONCLUSION: Post-TBI hypoxia and hyperoxia resulted in transiently increased platelet aggregation in response to ADP, without lasting effecting on platelet function or coagulation. Hypoxia also induced delayed hypocoagulability after platelet aggregation normalized. Brief changes in oxygenation may temporally affect coagulation and platelet aggregation, which could contribute to physiologic secondary injury after trauma.