The virulence antigen (V-antigen, LcrV) of Yersinia pestis, the causative agent of bubonic plague, is an established protective antigen known to regulate, target, and mediate type III translocation of cytotoxic yersiniae outer proteins termed Yops; LcrV also prompts TLR2-dependent upregulation of anti-inflammatory IL-10. In this study, we determined the parameters of specific interaction of LcrV with TLR2 expressed on human transfected HEK293 cells (TLR2+/CD14-), VTEC2.HS cells (TLR2+/CD14-), primary monocytes (TLR2+/CD14+), and THP-1 cells (TLR2+/CD14+). The IRRL314-317 motif of the extracellular domain of human and mouse TLR2 accounted for high-affinity binding of LcrV. The CD14 co-receptor did not influence this interaction. LcrV did not bind to human U937 (TLR2-/CD14-) and alveolar macrophages (TLR2-/CD14+) in the absence of receptor-bound human IFN-γ or a synthetic C-terminal fragment (hIFN-γ132-143). The latter, but not mouse IFN-γ (or synthetic control peptides), shared a GRRA138-141 site necessary for high-affinity specific binding. LcrV of Y. pestis shares the N-terminal LEEL32-35 binding site of Yersinia enterocolitica and also has an exposed internal DEEI203-206 binding site. Comparison of binding constants and consideration of steric restrictions indicate that binding is not cooperative and only the internal site binds LcrV to target cells. Both the LEEL32-35 and DEEI203-206 binding sites are removed by five amino acids from DKN residues associated with biological activity of bound LcrV. LcrV of Y. pestis promoted both TLR2/CD14-dependent and TLR2/CD14-independent amplification of IL-10 and concomitant downregulation of TNF-α in human target cells. The ability of LcrV to utilize human IFN-γ (a major inflammatory effector of innate immunity) to minimize inflammation is insidious and may account in part for the severe symptoms of plague in man.
Research Containing: Mice
Coactivator-Dependent Oscillation of Chromatin Accessibility Dictates Circadian Gene Amplitude via REV-ERB Loading
A central mechanism for controlling circadian gene amplitude remains elusive. We present evidence for a "facilitated repression (FR)" model that functions as an amplitude rheostat for circadian gene oscillation. We demonstrate that ROR and/or BMAL1 promote global chromatin decondensation during the activation phase of the circadian cycle to actively facilitate REV-ERB loading for repression of circadian gene expression. Mechanistically, we found that SRC-2 dictates global circadian chromatin remodeling through spatial and temporal recruitment of PBAF members of the SWI/SNF complex to facilitate loading of REV-ERB in the hepatic genome. Mathematical modeling highlights how the FR model sustains proper circadian rhythm despite fluctuations of REV-ERB levels. Our study not only reveals a mechanism for active communication between the positive and negative limbs of the circadian transcriptional loop but also establishes the concept that clock transcription factor binding dynamics is perhaps a central tenet for fine-tuning circadian rhythm.
Attachment of LcrV from Yersinia pestis at dual binding sites to human TLR-2 and human IFN-gamma receptor.
The virulence antigen (V-antigen, LcrV) of Yersinia pestis, the causative agent of bubonic plague, is an established protective antigen known to regulate, target, and mediate type III translocation of cytotoxic yersiniae outer proteins termed Yops; LcrV also prompts TLR2-dependent upregulation of anti-inflammatory IL-10. In this study, we determined the parameters of specific interaction of LcrV with TLR2 expressed on human transfected HEK293 cells (TLR2+/CD14-), VTEC2.HS cells (TLR2+/CD14-), primary monocytes (TLR2+/CD14+), and THP-1 cells (TLR2+/CD14+). The IRRL314-317 motif of the extracellular domain of human and mouse TLR2 accounted for high-affinity binding of LcrV. The CD14 co-receptor did not influence this interaction. LcrV did not bind to human U937 (TLR2-/CD14-) and alveolar macrophages (TLR2-/CD14+) in the absence of receptor-bound human IFN-gamma or a synthetic C-terminal fragment (hIFN-gamma132-143). The latter, but not mouse IFN-gamma (or synthetic control peptides), shared a GRRA138-141 site necessary for high-affinity specific binding. LcrV of Y. pestis shares the N-terminal LEEL32-35 binding site of Yersinia enterocolitica and also has an exposed internal DEEI203-206 binding site. Comparison of binding constants and consideration of steric restrictions indicate that binding is not cooperative and only the internal site binds LcrV to target cells. Both the LEEL32-35 and DEEI203-206 binding sites are removed by five amino acids from DKN residues associated with biological activity of bound LcrV. LcrV of Y. pestis promoted both TLR2/CD14-dependent and TLR2/CD14-independent amplification of IL-10 and concomitant downregulation of TNF-alpha in human target cells. The ability of LcrV to utilize human IFN-gamma (a major inflammatory effector of innate immunity) to minimize inflammation is insidious and may account in part for the severe symptoms of plague in man.
Guidelines for Dual Energy X-Ray Absorptiometry Analysis of Trabecular Bone-Rich Regions in Mice: Improved Precision, Accuracy, and Sensitivity for Assessing Longitudinal Bone Changes
Trabecular bone is frequently studied in osteoporosis research because changes in trabecular bone are the most common cause of osteoporotic fractures. Dual energy X-ray absorptiometry (DXA) analysis specific to trabecular bone-rich regions is crucial to longitudinal osteoporosis research. The purpose of this study is to define a novel method for accurately analyzing trabecular bone-rich regions in mice via DXA. This method will be utilized to analyze scans obtained from the International Space Station in an upcoming study of microgravity-induced bone loss. Thirty 12-week-old BALB/c mice were studied. The novel method was developed by preanalyzing trabecular bone-rich sites in the distal femur, proximal tibia, and lumbar vertebrae via high-resolution X-ray imaging followed by DXA and micro-computed tomography (micro-CT) analyses. The key DXA steps described by the novel method were (1) proper mouse positioning, (2) region of interest (ROI) sizing, and (3) ROI positioning. The precision of the new method was assessed by reliability tests and a 14-week longitudinal study. The bone mineral content (BMC) data from DXA was then compared to the BMC data from micro-CT to assess accuracy. Bone mineral density (BMD) intra-class correlation coefficients of the new method ranging from 0.743 to 0.945 and Levene’s test showing that there was significantly lower variances of data generated by new method both verified its consistency. By new method, a Bland-Altman plot displayed good agreement between DXA BMC and micro-CT BMC for all sites and they were strongly correlated at the distal femur and proximal tibia (r=0.846, p<0.01; r=0.879, p<0.01, respectively). The results suggest that the novel method for site-specific analysis of trabecular bone-rich regions in mice via DXA yields more precise, accurate, and repeatable BMD measurements than the conventional method.
Synchrony of the mammalian circadian clock is achieved by complex transcriptional and translational feedback loops centered on the BMAL1:CLOCK heterodimer. Modulation of circadian feedback loops is essential for maintaining rhythmicity, yet the role of transcriptional coactivators in driving BMAL1:CLOCK transcriptional networks is largely unexplored. Here, we show diurnal hepatic steroid receptor coactivator 2 (SRC-2) recruitment to the genome that extensively overlaps with the BMAL1 cistrome during the light phase, targeting genes that enrich for circadian and metabolic processes. Notably, SRC-2 ablation impairs wheel-running behavior, alters circadian gene expression in several peripheral tissues, alters the rhythmicity of the hepatic metabolome, and deregulates the synchronization of cell-autonomous metabolites. We identify SRC-2 as a potent coregulator of BMAL1:CLOCK and find that SRC-2 targets itself with BMAL1:CLOCK in a feedforward loop. Collectively, our data suggest that SRC-2 is a transcriptional coactivator of the BMAL1:CLOCK oscillators and establish SRC-2 as a critical positive regulator of the mammalian circadian clock.
Astronaut intestinal health may be impacted by microgravity, radiation, and diet. The aim of this study was to characterize how high and low linear energy transfer (LET) radiation, microgravity, and elevated dietary iron affect colon microbiota (determined by 16S rDNA pyrosequencing) and colon function. Three independent experiments were conducted to achieve these goals: 1) fractionated low LET gamma radiation (137Cs, 3 Gy, RAD), high Fe diet (IRON) (650 mg/kg diet), and a combination of low LET gamma radiation and high Fe diet (IRON+RAD) in male Sprague-Dawley rats; 2) high LET 38Si particle exposure (0.050 Gy), 1/6 G partial weight bearing (PWB), and a combination of high LET38Si particle exposure and PWB in female BalbC/ByJ mice; and 3) 13 d spaceflight in female C57BL/6 mice. Low LET radiation, IRON and spaceflight increased Bacteroidetes and decreased Firmicutes. RAD and IRON+RAD increased Lactobacillales and lowered Clostridiales compared to the control (CON) and IRON treatments. Low LET radiation, IRON, and spaceflight did not significantly affect diversity or richness, or elevate pathogenic genera. Spaceflight increased Clostridiales and decreased Lactobacillales, and similar trends were observed in the experiment using a ground-based model of microgravity, suggesting altered gravity may affect colonic microbiota. Although we noted no differences in colon epithelial injury or inflammation, spaceflight elevated TGFbeta gene expression. Microbiota and mucosal characterization in these models is a first step in understanding the impact of the space environment on intestinal health.
Bone loss associated with microgravity exposure poses a significant barrier to long-duration spaceflight. Osteoprotegerin-Fc (OPG-Fc) is a receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitor that causes sustained inhibition of bone resorption after a single subcutaneous injection. We tested the ability of OPG-Fc to preserve bone mass during 12 days of spaceflight (SF). 64-day-old female C57BL/6J mice (n=12/group) were injected subcutaneously with OPG-Fc (20mg/kg) or an inert vehicle (VEH), 24h prior to launch. Ground control (GC) mice (VEH or OPG-Fc) were maintained under environmental conditions that mimicked those in the space shuttle middeck. Age-matched baseline (BL) controls were sacrificed at launch. GC/VEH, but not SF/VEH mice, gained tibia BMD and trabecular volume fraction (BV/TV) during the mission (P<0.05 vs. BL). SF/VEH mice had lower BV/TV vs. GC/VEH mice, while SF/OPG-Fc mice had greater BV/TV than SF/VEH or GC/VEH. SF reduced femur elastic and maximum strength in VEH mice, with OPG-Fc increasing elastic strength in SF mice. Serum TRAP5b was elevated in SF/VEH mice vs. GC/VEH mice. Conversely, SF/OPG-Fc mice had lower TRAP5b levels, suggesting that OPG-Fc preserved bone during spaceflight via inhibition of osteoclast-mediated bone resorption. Decreased bone formation also contributed to the observed osteopenia, based on the reduced femur periosteal bone formation rate and serum osteocalcin level. Overall, these observations suggest that the beneficial effects of OPG-Fc during SF are primarily due to dramatic and sustained suppression of bone resorption. In growing mice, this effect appears to compensate for the SF-related inhibition of bone formation, while preventing any SF-related increase in bone resorption. We have demonstrated that the young mouse is an appropriate new model for SF-induced osteopenia, and that a single pre-flight treatment with OPG-Fc can effectively prevent the deleterious effects of SF on mouse bone.
Secretory proteins produced by salivary glands are stored in granules and released into saliva. Rodent salivary glands are a reliable experimental model because they are morphologically and functionally similar to those of humans. To determine if the effects of microgravity on secretory proteins are increased on extended flights, their expression in mouse parotid glands, morphological, immuno- cytochemical, and biochemical/molecular methods were employed. Acinar cells of STS-135 (13 day) and Bion-M1 (30 day) flight animals showed an increase of autophagy and apoptosis, while duct cells contained vacuoles with endocytosed proteins. In STS-135, decreases were seen in the regulatory subunit of type II protein kinase A (RII) by Western blotting, and demilune cell and parotid protein (DCPP) and α- amylase (p<0.01) by immunogold labeling, while proline-rich proteins (PRPs, p<0.001) and parotid secretory protein (PSP, p<0.05) were increased. These results suggest microgravity effects on secretion are function-dependent. Microarray analyses showed significant changes in the expression of a number of genes, including components of the cyclic-3',5',-adenosine monophosphate (cyclic AMP) signaling pathway. Compared to habitat ground controls, mice from both flights exhibited altered expression of cyclic AMP-specific phosphodiesterases, adenylate cyclase isoforms, and several A-kinase anchoring proteins. Bion-M1 flight mice showed increases in gene expression for lysozyme and amylase, a decrease in PRPs, and RII expression was unchanged from control values. Secretory protein expression is altered by travel in space, representing a reversible adjustment to microgravity conditions. Ultimately, the goal is to develop a test kit using saliva — an easily obtained body fluid — to assess the physiologic effects of travel in space.
Evaluation of rodent spaceflight in the NASA animal enclosure module for an extended operational period (up to 35 days)
The National Aeronautics and Space Administration Animal Enclosure Module (AEM) was developed as a self-contained rodent habitat for shuttle flight missions that provides inhabitants with living space, food, water, ventilation, and lighting, and this study reports whether, after minimal hardware modification, the AEM could support an extended term up to 35 days for Sprague-Dawley rats and C57BL/6 female mice for use on the International Space Station. Success was evaluated based on comparison of AEM housed animals to that of vivarium housed and to normal biological ranges through various measures of animal health and well-being, including animal health evaluations, animal growth and body masses, organ masses, rodent food bar consumption, water consumption, and analysis of blood contents. The results of this study confirmed that the AEMs could support 12 adult female C57BL/6 mice for up to 35 days with self-contained RFB and water, and the AEMs could also support 5 adult male Sprague-Dawley rats for 35 days with external replenishment of diet and water. This study has demonstrated the capability and flexibility of the AEM to operate for up to 35 days with minor hardware modification. Therefore, with modifications, it is possible to utilize this hardware on the International Space Station or other operational platforms to extend the space life science research use;of mice and rats.
Increased postflight carotid artery stiffness and inflight insulin resistance resulting from 6-mo spaceflight in male and female astronauts
Removal of the normal head-to-foot gravity vector and chronic weightlessness during spaceflight might induce cardiovascular and metabolic adaptations related to changes in arterial pressure and reduction in physical activity. We tested hypotheses that stiffness of arteries located above the heart would be increased postflight, and that blood biomarkers inflight would be consistent with changes in vascular function. Possible sex differences in responses were explored in four male and four female astronauts who lived on the International Space Station for 6 mo. Carotid artery distensibility coefficient (P = 0.005) and beta-stiffness index (P = 0.006) reflected 17-30% increases in arterial stiffness when measured within 38 h of return to Earth compared with preflight. Spaceflight-by-sex interaction effects were found with greater changes in beta-stiffness index in women (P = 0.017), but greater changes in pulse wave transit time in men (P = 0.006). Several blood biomarkers were changed from preflight to inflight, including an increase in an index of insulin resistance (P < 0.001) with a spaceflight-by-sex term suggesting greater change in men (P = 0.034). Spaceflight-by-sex interactions for renin (P = 0.016) and aldosterone (P = 0.010) indicated greater increases in women than men. Six-month spaceflight caused increased arterial stiffness. Altered hydrostatic arterial pressure gradients as well as changes in insulin resistance and other biomarkers might have contributed to alterations in arterial properties, including sex differences between male and female astronauts.