Human ß-Defensin-3 is Associated With Platelet-Derived Extracellular Vesicles and is a Potential Contributor to Endothelial Dysfunction.

While platelets are the essential mediators of hemostasis, they are being increasingly recognized for their potential of contributing to host defenses. Here, using immunofluorescent microscopy, western blot, and ELISA, we found that human ß-defensin 3 (hBD-3), an important antimicrobial peptide produced by epithelial cells, can be detected in human platelets and megakaryocytes. Flow cytometry and immuno-electron microscopy revealed hBD-3 on the surface of thrombin activated platelets. Moreover, hBD-3 was also found in platelet derived extracellular vesicles (p-EVs), isolated from platelet poor plasma and from platelet supernatants following thrombin stimulation. Incubation of platelets with hBD-3 peptide resulted in modest platelet activation and pre-incubation of platelets with synthetic hBD-3 prior to exposure to thrombin appeared to increase hBD-3 content in platelet lysates as well as in p-EVs, suggesting that hBD-3 can be initially taken up by platelets, perhaps via their open canalicular system. Interestingly, in vitro exposure of primary human endothelial cells to either hBD-3 peptide or purified p-EVs, caused significant endothelial dysfunction as documented by diminished levels of phosphorylated endothelial nitric oxide synthase (eNOS), Krüppel like factor-2 (KLF-2), and elevated relative expression of von Willebrand Factor (vWF). Pre-incubation of platelets with hBD-3 appeared to augment endothelial dysfunction caused by p-EVs. Overall, the current study provides evidence that hBD-3 enriched EVs can be released by activated platelets and may play a role in positive feedback of platelet activation as well as in endothelial dysfunction. Theoretically, these effects could contribute to both cellular recruitment to the endothelium creating a pro-thrombotic vascular microenvironment which serve as a bridge between innate immunity and hemostasis.

CX3CL1 and IL-15 Promote CD8 T cell chemoattraction in HIV and in atherosclerosis.

Atherosclerotic cardiovascular disease (ASCVD) remains an important cause of morbidity in the general population and risk for ASCVD is increased approximately 2-fold in persons living with HIV infection (PLWH). This risk is linked to elevated CD8 T cell counts that are abundant in atherosclerotic plaques and have been implicated in disease pathogenesis yet the mechanisms driving T cell recruitment to and activation within plaques are poorly defined. Here we investigated the role of CD8 T cells in atherosclerosis in a non-human primate model of HIV infection and in the HIV-uninfected elderly; we sought to identify factors that promote the activation, function, and recruitment to endothelium of CX3CR1+ CD8 T cells. We measured elevated expression of CX3CL1 and IL-15, and increased CD8 T cell numbers in the aortas of rhesus macaques infected with SIV or SHIV, and demonstrated similar findings in atherosclerotic vessels of HIV-uninfected humans. We found that recombinant TNF enhanced the production and release of CX3CL1 and bioactive IL-15 from aortic endothelial cells, but not from aortic smooth muscle cells. IL-15 in turn promoted CX3CR1 surface expression on and TNF synthesis by CD8 T cells, and IL-15-treated CD8 T cells exhibited enhanced CX3CL1-dependent chemoattraction toward endothelial cells in vitro. Finally, we show that CD8 T cells in human atherosclerotic plaques have an activated, resident phenotype consistent with in vivo IL-15 and CX3CL1 exposure. In this report, we define a novel model of CD8 T cell involvement in atherosclerosis whereby CX3CL1 and IL-15 operate in tandem within the vascular endothelium to promote infiltration by activated CX3CR1+ memory CD8 T cells that drive further endothelial activation via TNF. We propose that these interactions are prevalent in aging and in PLWH, populations where circulating activated CX3CR1+ CD8 T cell numbers are often expanded.

Cytomegalovirus Coinfection Is Associated with Increased Vascular-Homing CD57+ CD4 T Cells in HIV Infection.

Cytotoxic CD4 T cells are linked to cardiovascular morbidities and accumulate in both HIV and CMV infections, both of which are associated with increased risk of cardiovascular disease (CVD). In this study, we identify CMV coinfection as a major driver of the cytotoxic phenotype, characterized by elevated CD57 expression and reduced CD28 expression, in circulating CD4 T cells from people living with HIV infection, and investigate potential mechanisms linking this cell population to CVD. We find that human CD57+ CD4 T cells express high levels of the costimulatory receptor CD2 and that CD2/LFA-3 costimulation results in a more robust and polyfunctional effector response to TCR signals, compared with CD28-mediated costimulation. CD57+ CD4 T cells also express the vascular endothelium-homing receptor CX3CR1 and migrate toward CX3CL1-expressing endothelial cells in vitro. IL-15 promotes the cytotoxic phenotype, elevates CX3CR1 expression, and enhances the trafficking of CD57+ CD4 T cells to endothelium and may therefore be important in linking these cells to cardiovascular complications. Finally, we demonstrate the presence of activated CD57+ CD4 T cells and expression of CX3CL1 and LFA-3 in atherosclerotic plaque tissues from HIV-uninfected donors. Our findings are consistent with a model in which cytotoxic CD4 T cells contribute to CVD in HIV/CMV coinfection and in atherosclerosis via CX3CR1-mediated trafficking and CD2/LFA-3-mediated costimulation. This study identifies several targets for therapeutic interventions and may help bridge the gap in understanding how CMV infection and immunity are linked to increased cardiovascular risk in people living with HIV infection.

Outcomes in Critically Ill Patients With Traumatic Brain Injury: Ethnicity, Documentation, and Insurance Status.

OBJECTIVES: Disparities in traumatic brain injury outcomes for ethnic minorities and the uninsured have previously been demonstrated; however, outcomes in undocumented immigrants have not been examined. We wanted to determine whether ethnicity, insurance, and documentation status served as risk factors for disparities in traumatic brain injury outcomes between undocumented immigrants and documented residents. DESIGN: Retrospective study. SETTING: Patients diagnosed with traumatic brain injury admitted to the surgical/trauma ICU at a level 1 trauma center serving a large immigrant population in New York City from 2009 to 2016. PATIENTS: Four-hundred seventy-one traumatic brain injury patients requiring surgical/trauma ICU admission. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Undocumented immigrants constituted 29% of the population, were younger (39 vs 57 yr old, respectively; p < 0.0001), Hispanic (83%; p < 0.0001), and uninsured (87%; p < 0.0001). Falls resulted in the majority of traumatic brain injuries in the total population, however, undocumented immigrants were almost twice as likely to be assaulted (p = 0.0032). There was no difference in presence of midline shifts, Injury Severity Score, Glasgow Coma Score, hypotension, hypoxia, and pupillary reactions between undocumented immigrants and documented residents. Undocumented immigrants presented with significantly more effaced basilar cisterns (p = 0.0008). There was no difference in hospital care between undocumented immigrants and documented residents as determined by emergency department to surgical/trauma ICU transfer times (p = 0.967). Undocumented immigrants were more likely to be discharged home (53% vs 33%, respectively; p = 0.0009) and less likely to be sent to rehabilitation (25% vs 32%, respectively; p = 0.0009). After adjusting length of stay and mortality for covariates, undocumented immigrants had shorter length of stay (p < 0.05) and there was no difference in hospital mortality between undocumented immigrants and documented residents. CONCLUSIONS: Undocumented immigrants with traumatic brain injuries were more likely to be younger, have shorter length of stay, and experience similar mortality rates to documented residents. Social economic status may play a role in events prior to hospitalization and likely does in disposition outcomes.

Trauma care in a multiethnic population: effects of being undocumented.

BACKGROUND: Epidemiologic studies have shown that undocumented immigrants (UIs) display characteristics of having a low socioeconomic status and are primarily of ethnic minorities. These social determinants of health are known to be associated with diminished health care access and poor clinical outcomes. We therefore investigated the impact of documentation status on the clinical outcomes of patients with traumatic injuries. MATERIALS AND METHODS: We conducted a retrospective review of the trauma registry at our safety net institution for all adult patients who were admitted from 2010 to 2014. UIs were identified by the absence of a valid social security number within their medical records. Multivariate regression analysis was used to determine the impact of documentation status on in-hospital mortality, length of stay (LOS), and the odds of rehab placement. RESULTS: 4924 trauma patients met the study criteria, of which 1050 (21.3%) were UIs. There was no significant difference in mortality rates between the two populations. Multivariate regression analyses revealed a longer average LOS and a decreased likelihood for placement in an in-patient rehabilitation facility following hospitalization for UIs, even after accounting for insurance, age, injury severity, and other possible confounders known to affect these outcomes. CONCLUSIONS: There was no association between in-hospital mortality and documentation status; however, UIs had a longer average LOS and were less likely to be placed into rehab following their hospitalization. A longer LOS and a decreased likelihood for rehabilitation placement suggest that disparities in trauma care exist for UIs, putting them at risk for worse clinical and functional outcomes.

Gtpbp2 is a positive regulator of Wnt signaling and maintains low levels of the Wnt negative regulator Axin.

BACKGROUND: Canonical Wnt signals, transduced by stabilized ß-catenin, play similar roles across animals in maintaining stem cell pluripotency, regulating cell differentiation, and instructing normal embryonic development. Dysregulated Wnt/ß-catenin signaling causes diseases and birth defects, and a variety of regulatory processes control this pathway to ensure its proper function and integration with other signaling systems. We previously identified GTP-binding protein 2 (Gtpbp2) as a novel regulator of BMP signaling, however further exploration revealed that Gtpbp2 can also affect Wnt signaling, which is a novel finding reported here. RESULTS: Knockdown of Gtpbp2 in Xenopus embryos causes severe axial defects and reduces expression of Spemann-Mangold organizer genes. Gtpbp2 knockdown blocks responses to ectopic Wnt8 ligand, such as organizer gene induction in ectodermal tissue explants and induction of secondary axes in whole embryos. However, organizer gene induction by ectopic Nodal2 is unaffected by Gtpbp2 knockdown. Epistasis tests, conducted by activating Wnt signal transduction at sequential points in the canonical pathway, demonstrate that Gtpbp2 is required downstream of Dishevelled and Gsk3ß but upstream of ß-catenin, which is similar to the previously reported effects of Axin1 overexpression in Xenopus embryos. Focusing on Axin in Xenopus embryos, we find that knockdown of Gtpbp2 elevates endogenous or exogenous Axin protein levels. Furthermore, Gtpbp2 fusion proteins co-localize with Dishevelled and co-immunoprecipitate with Axin and Gsk3b. CONCLUSIONS: We conclude that Gtpbp2 is required for canonical Wnt/ß-catenin signaling in Xenopus embryos. Our data suggest a model in which Gtpbp2 suppresses the accumulation of Axin protein, a rate-limiting component of the ß-catenin destruction complex, such that Axin protein levels negatively correlate with Gtpbp2 levels. This model is supported by the similarity of our Gtpbp2-Wnt epistasis results and previously reported effects of Axin overexpression, the physical interactions of Gtpbp2 with Axin, and the correlation between elevated Axin protein levels and lost Wnt responsiveness upon Gtpbp2 knockdown. A wide variety of cancer-causing Wnt pathway mutations require low Axin levels, so development of Gtpbp2 inhibitors may provide a new therapeutic strategy to elevate Axin and suppress aberrant ß-catenin signaling in cancer and other Wnt-related diseases.