P2Y2 purinergic receptor gene deletion protects mice from bacterial endotoxin and sepsis-associated liver injury and mortality.

The liver plays a significant role in regulating a wide range of metabolic, homeostatic, and host-defense functions. However, the impact of liver injury on the host's ability to control bacteremia and morbidity in sepsis is not well understood. Leukocyte recruitment and activation lead to cytokine and chemokine release, which, in turn, trigger hepatocellular injury and elevate nucleotide levels in the extracellular milieu. P2Y2 purinergic receptors, G protein-coupled and activated by extracellular ATP/UTP, are expressed at the cell surface of hepatocytes and nonparenchymal cells. We sought to determine whether P2Y2 purinergic receptor function is necessary for the maladaptive host response to bacterial infection and endotoxin-mediated inflammatory liver injury and mortality in mice. We report that P2Y2 purinergic receptor knockout mice (P2Y2-/-) had attenuated inflammation and liver injury, with improved survival in response to LPS/galactosamine (LPS/GalN; inflammatory liver injury) and cecal ligation and puncture (CLP; polymicrobial sepsis). P2Y2-/- livers had attenuated c-Jun NH2-terminal kinase activation, matrix metallopeptidase-9 expression, and hepatocyte apoptosis in response to LPS/GalN and attenuated inducible nitric oxide synthase and nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 protein expression in response to CLP. Implicating liver injury in the disruption of amino acid homeostasis, CLP led to lower serum arginine and higher bacterial load and morbidity in the WT mice, whereas serum arginine levels were comparable to sham-operated controls in P2Y2-/- mice, which had attenuated bacteremia and improved survival. Collectively, our studies highlight the pathophysiological relevance of P2Y2 purinergic receptor function in inflammatory liver injury and dysregulation of systemic amino acid homeostasis with implications for sepsis-associated immune dysfunction and morbidity in mice.NEW & NOTEWORTHY Our studies provide experimental evidence for P2Y2 purinergic receptor-mediated potentiation of inflammatory liver injury, morbidity, and mortality, in two well-established animal models of inflammatory liver injury. Our findings highlight the potential to target P2Y2 purinergic signaling to attenuate the induction of "cytokine storm" and prevent its deleterious consequences on liver function, systemic amino acid homeostasis, host response to bacterial infection, and sepsis-associated morbidity and mortality.

Identification of a Halotolerant Mutant via In Vitro Mutagenesis in the Cyanobacterium Fremyella diplosiphon.

Energy metabolism and photosynthetic pigment accumulation are affected by salt stress in cyanobacteria leading to cessation of growth. In this study, the effect of salinity on the freshwater cyanobacterium, Fremyella diplosiphon, was investigated and mutagenesis-based efforts were undertaken to enhance salt tolerance. Salinity at a concentration of 10 g/L sodium chloride (NaCl) inhibited growth of wild type F. diplosiphon under white, red, and green light. Efforts to enhance halotolerance resulted in a mutant that could survive in 20 g/L NaCl for 15 generations with no significant reduction in phycobiliproteins (phycocyanin, phycoerythrin, and allophycocyanin) or chlorophyll a. Gene expression measured by quantitative reverse transcription-polymerase chain reaction revealed a three-fold increase in tripartite ATP-independent periplasmic transporters (TRAP) solute receptor transcript in the mutant compared to wild type. Our discovery of a TRAP transporter system in F. diplosiphon and its possible role in salinity response enables growth in brackish waters, which enhances its potential for biotechnological applications.

Magnetically Bioprinted Human Myometrial 3D Cell Rings as A Model for Uterine Contractility.

Deregulation in uterine contractility can cause common pathological disorders of the female reproductive system, including preterm labor, infertility, inappropriate implantation, and irregular menstrual cycle. A better understanding of human myometrium contractility is essential to designing and testing interventions for these important clinical problems. Robust studies on the physiology of human uterine contractions require in vitro models, utilizing a human source. Importantly, uterine contractility is a three-dimensionally (3D)-coordinated phenomenon and should be studied in a 3D environment. Here, we propose and assess for the first time a 3D in vitro model for the evaluation of human uterine contractility. Magnetic 3D bioprinting is applied to pattern human myometrium cells into rings, which are then monitored for contractility over time and as a function of various clinically relevant agents. Commercially available and patient-derived myometrium cells were magnetically bioprinted into rings in 384-well formats for throughput uterine contractility analysis. The bioprinted uterine rings from various cell origins and patients show different patterns of contractility and respond differently to clinically relevant uterine contractility inhibitors, indomethacin and nifedipine. We believe that the novel system will serve as a useful tool to evaluate the physiology of human parturition while enabling high-throughput testing of multiple agents and conditions.

P2Y2 purinergic receptor activation is essential for efficient hepatocyte proliferation in response to partial hepatectomy.

Extracellular nucleotides via activation of P2 purinergic receptors influence hepatocyte proliferation and liver regeneration in response to 70% partial hepatectomy (PH). Adult hepatocytes express multiple P2Y (G protein-coupled) and P2X (ligand-gated ion channels) purinergic receptor subtypes. However, the identity of key receptor subtype(s) important for efficient hepatocyte proliferation in regenerating livers remains unknown. To evaluate the impact of P2Y2 purinergic receptor-mediated signaling on hepatocyte proliferation in regenerating livers, wild-type (WT) and P2Y2 purinergic receptor knockout (P2Y2-/-) mice were subjected to 70% PH. Liver tissues were analyzed for activation of early events critical for hepatocyte priming and subsequent cell cycle progression. Our findings suggest that early activation of p42/44 ERK MAPK (5 min), early growth response-1 (Egr-1) and activator protein-1 (AP-1) DNA-binding activity (30 min), and subsequent hepatocyte proliferation (24-72 h) in response to 70% PH were impaired in P2Y2-/- mice. Interestingly, early induction of cytokines (TNF-α, IL-6) and cytokine-mediated signaling (NF-κB, STAT-3) were intact in P2Y2-/- remnant livers, uncovering the importance of cytokine-independent and nucleotide-dependent early priming events critical for subsequent hepatocyte proliferation in regenerating livers. Hepatocytes isolated from the WT and P2Y2-/- mice were treated with ATP or ATPγS for 5-120 min and 12-24 h. Extracellular ATP alone, via activation of P2Y2 purinergic receptors, was sufficient to induce ERK phosphorylation, Egr-1 protein expression, and key cyclins and cell cycle progression of hepatocytes in vitro. Collectively, these findings highlight the functional significance of P2Y2 purinergic receptor activation for efficient hepatocyte priming and proliferation in response to PH.

Mesenchymal Stem Cells Suppress Inflammatory Cytokines in Lipopolysaccharide Exposed Preterm and Term Human Pregnant Myometrial Cells.

Objective The objective of this study was to determine the cytokine response in human pregnant preterm and term myometrial cells exposed to lipopolysaccharide (LPS) and cocultured with mesenchymal stem cells (MSCs). Study Design Myometrium was obtained at cesarean delivery in term and preterm patients. Human myometrial cells were exposed to 5 µg/mL LPS for 4 hours followed by 1 µg/mL LPS for 24 hours and were cocultured with MSCs for 24 hours. Culture supernatants were collected at 24 hours and expression of cytokines, including interleukin-1ß (IL-1ß), IL-6, IL-8, tumor necrosis factor-α (TNF-α), transforming growth factor-ß (TGF-ß), and IL-10, was quantified by enzyme-linked immunosorbent assay. Results There was significantly increased expression of the proinflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α in preterm myometrial cells treated with LPS compared with untreated preterm myometrial cells. Coculture with MSCs significantly suppressed the proinflammatory cytokine levels in LPS-treated preterm versus treated term myometrial cells. Moreover, MSC cocultured preterm myometrial cells expressed increased levels of the anti-inflammatory cytokines TGF-ß and IL-10 compared with treated term myometrial cells. Conclusion MSCs ameliorate LPS-mediated inflammation in preterm human myometrial cells compared with term myometrial cells. Immunomodulatory effects of MSCs mediated through anti-inflammatory cytokine regulation suggest a potential cell-based therapy for preterm birth.

Neuronal exosome proteins: novel biomarkers for predicting neonatal response to therapeutic hypothermia.

OBJECTIVE: Central nervous system (CNS) derived exosomes can be purified from peripheral blood and have been used widely in adult neurological disease. Application to neonatal neurological disease deserves investigation in the setting of hypoxic-ischaemic encephalopathy (HIE). DESIGN: Observational cohort. SETTING: Level III neonatal intensive care unit. PARTICIPANTS: Term/near-term neonates undergoing therapeutic hypothermia (TH) for HIE. INTERVENTIONS: Blood samples were collected at 0-6, 12, 24, 48 and 96 hours of life. MAIN OUTCOMES AND MEASURES: CNS exosomes were purified from serum using previously described methods. Biomarker protein levels were quantified using standard ELISA methods and normalised to exosome marker CD-81. The slope of change for biomarker levels was calculated for each time interval. Our primary outcome was MRI basal ganglia/watershed score of ≥3. RESULTS: 26 subjects were included (umbilical artery pH range 6.6-7.29; 35% seizures). An increasing MRI injury score was significantly associated with decreasing levels of synaptopodin between 0-6 and 12 hours (p=0.03) and increasing levels of lipocalin-2 (NGAL) between 12 and 48 hours (p<0.0001). Neuronal pentraxin was not significant. The negative predictive values for increasing synaptopodin and decreasing NGAL was 70.0% and 90.9%, respectively. CONCLUSIONS AND RELEVANCE: Our results indicate that CNS exosome cargo has the potential to act as biomarkers of the severity of brain injury and response to TH as well as quantify pharmacological response to neuroactive therapeutic/adjuvant agents. Rigorous prospective trials are critical to evaluate potential clinical use of exosome biomarkers.

Human M1 macrophages express unique innate immune response genes after mycobacterial infection to defend against tuberculosis.

Mycobacterium tuberculosis (Mtb) is responsible for approximately 1.5 million deaths each year. Though 10% of patients develop tuberculosis (TB) after infection, 90% of these infections are latent. Further, mice are nearly uniformly susceptible to Mtb but their M1-polarized macrophages (M1-MΦs) can inhibit Mtb in vitro, suggesting that M1-MΦs may be able to regulate anti-TB immunity. We sought to determine whether human MΦ heterogeneity contributes to TB immunity. Here we show that IFN-γ-programmed M1-MΦs degrade Mtb through increased expression of innate immunity regulatory genes (Inregs). In contrast, IL-4-programmed M2-polarized MΦs (M2-MΦs) are permissive for Mtb proliferation and exhibit reduced Inregs expression. M1-MΦs and M2-MΦs express pro- and anti-inflammatory cytokine-chemokines, respectively, and M1-MΦs show nitric oxide and autophagy-dependent degradation of Mtb, leading to increased antigen presentation to T cells through an ATG-RAB7-cathepsin pathway. Despite Mtb infection, M1-MΦs show increased histone acetylation at the ATG5 promoter and pro-autophagy phenotypes, while increased histone deacetylases lead to decreased autophagy in M2-MΦs. Finally, Mtb-infected neonatal macaques express human Inregs in their lymph nodes and macrophages, suggesting that M1 and M2 phenotypes can mediate immunity to TB in both humans and macaques. We conclude that human MФ subsets show unique patterns of gene expression that enable differential control of TB after infection. These genes could serve as targets for diagnosis and immunotherapy of TB.

Mesenchymal Stem Cells Attenuate Lipopolysaccharide-Induced Inflammatory Response in Human Uterine Smooth Muscle Cells.

Objective The aim of this study was to determine if mesenchymal stem cells (MSCs) would suppress the inflammatory response in human uterine cells in an in vitro lipopolysaccharide (LPS)-based preterm birth (PTB) model. Study Design Cocultures of human uterine smooth muscle cells (HUtSMCs) and MSCs were exposed to 5 µg/mL LPS for 4 hours and further challenged with 1 µg/mL LPS for a subsequent 24 hours. Key elements of the parturition cascade regulated by toll-like receptors (TLRs) through activation of mitogen-activated protein kinases (MAPKs) were quantified in culture supernatant as biomarkers of MSC modulation. Results Coculture with MSCs significantly attenuated TLR-4, p-JNK, and p- extracellular signal-regulated kinase 1/2 (ERK1/2) protein levels compared with HUtSMCs monoculture ( p = 0.05). In addition, coculture was associated with significant inhibition of proinflammatory cytokines interleukin (IL)-6 and IL-8 ( p = 0.0001) and increased production of anti-inflammatory cytokines IL-10 and transforming growth factor (TGF)-ß1 ( p = 0.0001). Conclusion MSCs appear to play a role in significantly attenuating LPS-mediated inflammation via alteration of down-stream MAPKs. MSCs may represent a novel, cell-based therapy in women with increased risk of inflammatory-mediated preterm birth.

Human mesenchymal stem cell based intracellular dormancy model of Mycobacterium tuberculosis.

Understanding the biology of the tuberculosis pathogen during dormant asymptomatic infection, called latent tuberculosis is crucial to decipher a resilient therapeutic strategy for the disease. Recent discoveries exhibiting presence of pathogen's DNA and bacilli in mesenchymal stem cells (MSCs) of human and mouse despite completion of antitubercular therapy, indicates that these specific cells could be one of the niches for dormant Mycobacterium tuberculosis in humans. To determine if in vitro infection of human MSCs could recapitulate the in vivo characteristics of dormant M. tuberculosis, we examined survival, phenotype, and drug susceptibility of the pathogen in MSCs. When a very low multiplicity of infection (1:1) was used, M. tuberculosis could survive in human bone marrow derived MSCs for more than 22 days without any growth. At this low level of infection, the pathogen did not cause any noticeable host cell death. During the later phase of infection, MSC-residing M. tuberculosis exhibited increased expression of HspX (a 16-kDa alpha-crystallin homolog) with a concurrent increase in tolerance to the frontline antitubercular drugs Rifampin and isoniazid. These results present a human MSC-based intracelllular model of M. tuberculosis infection to dissect the mechanisms through which the pathogen acquires and maintains dormancy in the host.

Overexpression of hlyB and mdh genes confers halotolerance in Fremyella diplosiphon, a freshwater cyanobacterium.

Fremyella diplosiphon is a freshwater cyanobacterium that has great potential as a biofuel agent due to its ability to grow in low light intensity and acclimation to different wavelengths. To enhance its halotolerance for growth in 35gL-1 sodium chloride (NaCl), plasmids harboring hemolysin B (hlyB) and malate dehydrogenase (mdh) genes were transformed into wild type F. diplosiphon (WT-Fd33). Electroporation-mediated overexpression of the genes resulted in two transformants, HSF33-1 and HSF33-2, with 9- and 20-fold increases in hlyB and mdh transcript levels. In addition, up-regulation of proteins at the expected size ranges of 50-60kDa for HlyB and 40-50kDa for MDH was observed. Two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry revealed a protein spot corresponding to HlyB in HSF33-1 with a significant MOWSE score of 164 and 3% sequence coverage, and a spot corresponding to MDH in HSF33-2 gave a significant MOWSE score of 124 with 10% sequence coverage. Physiological evaluation in BG11/HEPES medium and seawater adjusted to 35gL-1 NaCl confirmed that the transformants could thrive in high salinity with no loss of photosynthetic pigments. Results of the study indicate that overexpression of hlyB and mdh genes confer halotolerance in F. diplosiphon, thus maximizing its potential as a large-scale biofuel agent.

Phenotypic and functional analysis of immune CD8+ T cell responses induced by a single injection of a HIV DNA vaccine in mice.

HIV DNA vaccines are potent inducers of cell-mediated immune (CMI) response in mice but elicit poor HIV-specific IFN-gamma-producing T cells in monkeys and humans. In this study, we performed kinetic analyses on splenocytes of BALB/c mice that were immunized by a single injection with a unique DNA vaccine. Using IFN-gamma-ELISPOT and multiparametric FACS analysis, we characterized the induced CMI response. We found that the response was detectable for at least 63 wk. ELISPOT detection of IFN-gamma-producing T cells showed a profile with two waves separated by a long period of minimal response. Multiparametric FACS analysis showed two populations of CD3(+)CD8(+) T cells that were specific for all HIV Ags. These cells had similar robust proliferation abilities and contained granzyme B. However, only a few produced IFN-gamma. Both IFN-gamma-producing and non-IFN-gamma-producing HIV-specific CD8(+) T cells were detected in the early stage (week (W)1 and W2 postimmunization (PI)), in the prolonged intermediate period of minimal response (W4-W26 PI), and in the final late phase of increased response (W30-W63 PI). Our longitudinal characterization showed that both subsets of cells underwent expansion, contraction, and memory generation/maintenance phases throughout the lifespan of the animal. Altogether, these findings bring insight to the heterogeneity of the immune T cell response induced by a single immunization with this DNA and strengthen the concept that used of the IFN-gamma-ELISPOT assay alone may be insufficient to detect critical T cell responses to candidate HIV vaccines.