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Background: Heart failure with preserved ejection fraction (HFpEF) is a significant public health concern with limited treatment options. Dysregulated nitric oxide-mediated signaling has been implicated in HFpEF pathophysiology, however, little is known about the role of endogenous hydrogen sulfide (H2S)., Objectives: This study evaluated H2S bioavailability in patients and two animal models of cardiometabolic HFpEF and assessed the impact of H2S on HFpEF severity through alterations in endogenous H2S production and pharmacological supplementation., Methods: HFpEF patients and two rodent models of HFpEF ("two-hit" L-NAME + HFD mouse and ZSF1 obese rat) were evaluated for H2S bioavailability. Two cohorts of two-hit mice were investigated for changes in HFpEF pathophysiology: (1) endothelial cell cystathionine-γ-lyase (EC-CSE) knockout; (2) H2S donor, JK-1, supplementation., Results: H2S levels were significantly reduced (i.e., 81%) in human HFpEF patients and in both preclinical HFpEF models. This depletion was associated with reduced CSE expression and activity, and increased SQR expression. Genetic knockout of H2S -generating enzyme, CSE, worsened HFpEF characteristics, including elevated E/e' ratio and LVEDP, impaired aortic vasorelaxation and increased mortality. Pharmacologic H2S supplementation restored H2S bioavailability, improved diastolic function and attenuated cardiac fibrosis corroborating an improved HFpEF phenotype., Conclusions: H2S deficiency is evident in HFpEF patients and conserved across multiple HFpEF models. Increasing H2S bioavailability improved cardiovascular function, while knockout of endogenous H2S production exacerbated HFpEF pathology and mortality. These results suggest H2S dysregulation contributes to HFpEF and increasing H2S bioavailability may represent a novel therapeutic strategy for HFpEF., Highlights: H2S deficiency is evident in both human HFpEF patients and two clinically relevant models.Reduced H2S production by CSE and increased metabolism by SQR impair H2S bioavailability in HFpEF.Pharmacological H2S supplementation improves diastolic function and reduces cardiac fibrosis in HFpEF models.Targeting H2S dysregulation presents a novel therapeutic strategy for managing HFpEF.

Background: The renal sympathetic nervous system modulates systemic blood pressure, cardiac performance, and renal function. Pathological increases in renal sympathetic nerve activity contribute to the pathogenesis of heart failure with preserved ejection fraction (HFpEF). We investigated the effects of renal sympathetic denervation performed at early or late stages of HFpEF progression., Methods and Results: Male ZSF1 obese rats were subjected to radiofrequency renal denervation (RF-RDN) or sham procedure at either 8 weeks or 20 weeks of age and assessed for cardiovascular function, exercise capacity, and cardiorenal fibrosis. Renal norepinephrine and renal nerve tyrosine hydroxylase staining were performed to quantify denervation following RF-RDN. In addition, renal injury, oxidative stress, inflammation, and profibrotic biomarkers were evaluated to determine pathways associated with RDN. RF-RDN significantly reduced renal norepinephrine and tyrosine hydroxylase content in both study cohorts. RF-RDN therapy performed at 8 weeks of age attenuated cardiac dysfunction, reduced cardiorenal fibrosis, and improved endothelial-dependent vascular reactivity. These improvements were associated with reductions in renal injury markers, expression of renal NLR family pyrin domain containing 3/interleukin 1β, and expression of profibrotic mediators. RF-RDN failed to exert beneficial effects when administered in the 20-week-old HFpEF cohort., Conclusions: Our data demonstrate that early RF-RDN therapy protects against HFpEF disease progression in part due to the attenuation of renal fibrosis and inflammation. In contrast, the renoprotective and left ventricular functional improvements were lost when RF-RDN was performed in later HFpEF progression. These results suggest that RDN may be a viable treatment option for HFpEF during the early stages of this systemic inflammatory disease.

Background: On long-duration spaceflight, most astronauts experience persistent immune dysregulation and the reactivation of latent herpesviruses, including varicella zoster virus (VZV). To understand the clinical risk of these perturbations to astronauts, we paralleled the immunology and virology work-up of astronauts to otherwise healthy terrestrial persons with acute herpes zoster., Methods: Blood samples from 42 zoster patients - confirmed positive by PCR for VZV DNA in saliva (range from 100 to >285 million copies/mL) were analyzed for peripheral leukocyte distribution, T cell function, and plasma cytokine profiles via multi-parametric flow cytometry and multiplex bead-based immune-array assays. Patient findings were compared to normal value ranges specific for each assay that were defined in-house previously from healthy adult test subjects., Results: Compared to the healthy adult ranges, the zoster patients possess (1) a higher proportion of constitutively activated T-cells, (2) a T-cell population skewed towards a more experienced maturation state, (3) depressed general T-cell function, and (4) a higher concentration of 20 of 22 measured plasma cytokines., Discussion: The pattern of immune dysregulation in zoster patients is similar to that of astronauts during spaceflight who shed VZV DNA in their saliva. Because future deep space exploration missions will be of an unprecedented duration, prolonged immune depression and chronic viral reactivation threaten to manifest overt disease in exploration class astronauts., Competing Interests: Declaration of Competing Interest This work is completely original and has not been published elsewhere. Every author has reviewed the manuscript and has contributed to the work in a substantive and intellectual manner. Study procedures were approved by NASA Johnson Space Center's Institutional Review Board. No animals were used in the study, and all human subjects provided written informed consent. All funding information for the study has been disclosed in the “Acknowledgments” section, and there are no financial or other relationships regarding this article that may be perceived as leading to a conflict of interest., (Copyright © 2019 The Committee on Space Research (COSPAR). All rights reserved.)

Background: Although a state of anemia is perceived to be associated with spaceflight, to date a peripheral blood hematologic assessment of red blood cell (RBC) indices has not been performed during long-duration space missions., Methods: This investigation collected whole blood samples from astronauts participating in up to 6-months orbital spaceflight, and returned those samples (ambient storage) to Earth for analysis. As samples were always collected near undock of a returning vehicle, the delay from collection to analysis never exceeded 48 h. As a subset of a larger immunologic investigation, a complete blood count was performed. A parallel stability study of the effect of a 48 h delay on these parameters assisted interpretation of the in-flight data., Results: We report that the RBC and hemoglobin were significantly elevated during flight, both parameters deemed stable through the delay of sample return. Although the stability data showed hematocrit to be mildly elevated at +48 h, there was an in-flight increase in hematocrit that was ~3-fold higher in magnitude than the anticipated increase due to the delay in processing., Conclusions: While susceptible to the possible influence of dehydration or plasma volume alterations, these results suggest astronauts do not develop persistent anemia during spaceflight.

Three-dimensional models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by two-dimensional monolayers and respond to Salmonella in key ways that reflect in vivo infections. To further enhance the physiological relevance of three-dimensional models to more closely approximate in vivo intestinal microenvironments encountered by Salmonella , we developed and validated a novel three-dimensional co-culture infection model of colonic epithelial cells and macrophages using the NASA Rotating Wall Vessel bioreactor. First, U937 cells were activated upon collagen-coated scaffolds. HT-29 epithelial cells were then added and the three-dimensional model was cultured in the bioreactor until optimal differentiation was reached, as assessed by immunohistochemical profiling and bead uptake assays. The new co-culture model exhibited in vivo-like structural and phenotypic characteristics, including three-dimensional architecture, apical-basolateral polarity, well-formed tight/adherens junctions, mucin, multiple epithelial cell types, and functional macrophages. Phagocytic activity of macrophages was confirmed by uptake of inert, bacteria-sized beads. Contribution of macrophages to infection was assessed by colonization studies of Salmonella pathovars with different host adaptations and disease phenotypes (Typhimurium ST19 strain SL1344 and ST313 strain D23580; Typhi Ty2). In addition, Salmonella were cultured aerobically or microaerobically, recapitulating environments encountered prior to and during intestinal infection, respectively. All Salmonella strains exhibited decreased colonization in co-culture (HT-29-U937) relative to epithelial (HT-29) models, indicating antimicrobial function of macrophages. Interestingly, D23580 exhibited enhanced replication/survival in both models following invasion. Pathovar-specific differences in colonization and intracellular co-localization patterns were observed. These findings emphasize the power of incorporating a series of related three-dimensional models within a study to identify microenvironmental factors important for regulating infection.

Genomic and epigenomic studies require the precise transfer of microliter volumes among different types of tubes in order to purify DNA, RNA, or protein from biological samples and subsequently perform analyses of DNA methylation, RNA expression, and chromatin modifications on a genome-wide scale. Epigenomic and transcriptional analyses of human blood cells, for example, require separation of purified cell types to avoid confounding contributions of altered cellular proportions, and long-term preservation of these cells requires their isolation and transfer into appropriate freezing media. There are currently no protocols for these cellular isolation procedures on the International Space Station (ISS). Currently human blood samples are either frozen as mixed cell populations (within the CPT collection tubes) with poor yield of viable cells required for cell-type isolations, or returned under ambient conditions, which requires timing with Soyuz missions. Here we evaluate the feasibility of translating terrestrial cell purification techniques to the ISS. Our evaluations were performed in microgravity conditions during parabolic atmospheric flight. The pipetting of open liquids in microgravity was evaluated using analog-blood fluids and several types of pipette hardware. The best-performing pipettors were used to evaluate the pipetting steps required for peripheral blood mononuclear cell (PBMC) isolation following terrestrial density-gradient centrifugation. Evaluation of actual blood products was performed for both the overlay of diluted blood, and the transfer of isolated PBMCs. We also validated magnetic purification of cells. We found that positive-displacement pipettors avoided air bubbles, and the tips allowed the strong surface tension of water, glycerol, and blood to maintain a patent meniscus and withstand robust pipetting in microgravity. These procedures will greatly increase the breadth of research that can be performed on board the ISS, and allow improvised experimentation by astronauts on extraterrestrial missions., Competing Interests: The authors declare no conflict of interest.

Background: It is currently unknown whether immune system alterations persist during long-duration spaceflight. In this study various adaptive immune parameters were assessed in astronauts at three intervals during 6-month spaceflight on board the International Space Station (ISS)., Aims: To assess phenotypic and functional immune system alterations in astronauts participating in 6-month orbital spaceflight., Methods: Blood was collected before, during, and after flight from 23 astronauts participating in 6-month ISS expeditions. In-flight samples were returned to Earth within 48 h of collection for immediate analysis. Assays included peripheral leukocyte distribution, T-cell function, virus-specific immunity, and mitogen-stimulated cytokine production profiles., Results: Redistribution of leukocyte subsets occurred during flight, including an elevated white blood cell (WBC) count and alterations in CD8 + T-cell maturation. A reduction in general T-cell function (both CD4 + and CD8 + ) persisted for the duration of the 6-month spaceflights, with differential responses between mitogens suggesting an activation threshold shift. The percentage of CD4 + T cells capable of producing IL-2 was depressed after landing. Significant reductions in mitogen-stimulated production of IFNγ, IL-10, IL-5, TNFα, and IL-6 persisted during spaceflight. Following lipopolysaccharide (LPS) stimulation, production of IL-10 was reduced, whereas IL-8 production was increased during flight., Conclusions: The data indicated that immune alterations persist during long-duration spaceflight. This phenomenon, in the absence of appropriate countermeasures, has the potential to increase specific clinical risks for crewmembers during exploration-class deep space missions., Competing Interests: DP is a NASA Virologist and CS and BC are NASA Immunologists. All remaining authors possess positions (contractor scientist) at, or are funded by, NASA. The remaining authors declare no conflict of interest.

Background: Post-flight data suggests immunity is dysregulated immediately following spaceflight, however this data may be influenced by the stress effects of high-G entry and readaptation to terrestrial gravity. It is unknown if immunity is altered during spaceflight., Methods: Blood samples were collected from 19 US Astronauts onboard the Space Shuttle ~24 h prior to landing and returned for terrestrial analysis. Assays consisted of leukocyte distribution, T cell blastogenesis and cytokine production profiles., Results: Most bulk leukocyte subsets (WBC, differential, lymphocyte subsets) were unaltered during spaceflight, but were altered following landing. CD8+ T cell subsets, including cytotoxic, central memory and senescent were altered during spaceflight. T cell early blastogenesis varied by culture mitogen. Functional responses to staphylococcal enterotoxin were reduced during and following spaceflight, whereas response to anti-CD3/28 antibodies was elevated post-flight. The level of virus specific T cells were generally unaltered, however virus specific T cell function was depressed both during and following flight. Plasma levels of IFNα, IFNγ, IL-1β, IL-4, IL-10, IL-12, and TNFα were significantly elevated in-flight, while IL-6 was significantly elevated at R + 0. Cytokine production profiles following mitogenic stimulation were significantly altered both during, and following spaceflight. Specifically, production of IFNγ, IL-17 and IL-10 were reduced, but production of TNFα and IL-8 were elevated during spaceflight., Conclusions: This study indicates that specific parameters among leukocyte distribution, T cell function and cytokine production profiles are altered during flight. These findings distinguish in-flight dysregulation from stress-related alterations observed immediately following landing.

Introduction: Immune system dysregulation has been demonstrated to occur during and immediately following spaceflight. As the initial bias and magnitude for an immune response is heavily influenced by monocyte/macrophage secreted cytokines, this study investigated monocyte phenotype and cytokine production patterns following short-duration spaceflight., Methods: Secreted cytokine profiles were examined by cytometric bead array analysis of culture supernatants following whole blood culture activation with LPS or PMA+ionomycin. Nine short-duration Space Shuttle crewmembers participated in this study., Results: Peripheral monocyte percentages were unaltered postflight. Constitutive monocyte expression of both CD62L and HLA-DR was reduced following spaceflight in a mission-specific fashion. Loss of either molecule indicates a functional disability of monocytes, either by inhibition of adhesion and tissue migration (CD62L) or by impaired antigen presentation (HLA-DR). Following LPS stimulation of monocytes, postflight expression of IL-6, TNFalpha, and IL-10 were significantly reduced (by 43%, 44%, and 41%, respectively) and expression of IL-1b was elevated (65%). IL-8 production was either elevated or reduced in a mission-specific fashion. Following PMA+ionomycin stimulation of all leukocyte populations, only expression of IL-6 was significantly reduced postflight., Discussion: These data indicate that changes in monocyte constitutive phenotype and inflammatory cytokine production occur following short-duration spaceflight, which may impact overall crewmember immunocompetence. Also, monocyte/macrophage function may be highly sensitive to mission specific parameters.