Microbial metabolite butyrate modulates granzyme B in tolerogenic IL-10 producing Th1 cells to regulate intestinal inflammation.

CD4+ T cells play a critical role in regulating autoimmune diseases, and intestinal microbial metabolites control various immune responses. Granzyme B (GzmB)-producing CD4+ T cells have been recently reported to participate in the pathogenesis of autoimmune diseases. Here, we found that GzmbB-deficient CD4+ T cells induced more severe colitis in Rag1-/- mice than wild-type (WT) CD4+ T cells. Germ-free (GF) mice exhibited a lower expression of GzmB in intestinal CD4+ T cells compared to specific pathogen-free (SPF) mice. Intestinal microbial metabolite butyrate increased GzmB expression in CD4+ T cells, especially in IL-10-producing Th1 cells, through HDAC inhibition and GPR43, but not GPR41 and GPR109a. Butyrate-treated GzmB-deficient CD4+ T cells demonstrated more severe colitis compared to butyrate-treated WT CD4+ T cells in the T cell transfer model. Butyrate altered intestinal microbiota composition, but altered microbiota did not mediate butyrate induction of intestinal CD4+ T cell expression of GzmB in mice. Blimp1 was involved in the butyrate induction of GzmB in IL-10-producing Th1 cells. Glucose metabolism, including glycolysis and pyruvate oxidation, mediated butyrate induction of GzmB in Th1 cells. In addition, we found that IKZF3 and NR2F6 regulated GzmB expression induced by butyrate. Together, our studies underscored the critical role of GzmB in mediating gut bacterial metabolite butyrate regulation of T cell tolerance at the mucosal surface.

Regional trends in diagnosis of advanced lung cancer in Michigan over 33 years.

Background: Lung cancer is the most common cancer killer worldwide. Nearly 80 percent of lung cancers are diagnosed at advanced stages. Lack of access to medical care and undwerutilized lung cancer screening are key reasons for advanced diagnoses. We sought to understand the regional differences in presentation of lung cancer across Michigan. Utilizing a comprehensive cancer registry over 33 years, our goal was to examine associations between sociodemographic patient factors and diagnoses at advanced stages. Methods: The Michigan Cancer Registry was queried from 1985 to 2018 to include all new diagnoses of non-small cell lung cancer (NSCLC) using International Classification of Diseases for Oncology (ICD-O) version 3 codes. NSCLC was categorized as early, regional and distant disease. Advanced disease was considered to be any disease that was regional or distant. NSCLC rates were calculated and mapped at the zip code level using the 2010 population as the denominator and spatial empirical Bayes methodology. Regional hospital service areas were constructed using travel time to treatment from the patient's zip code centroid. Logistic regression models were estimated to investigate the significance of rural vs. urban and travel time on level of disease at presentation. Kaplan-Meier and multivariate survival analysis was performed to evaluate the association between distance from the nearest medical center and length of survival controlling for known risk factors for lung cancer. Results: From 1985 to 2018, there were 141,977 patients in Michigan diagnosed with NSCLC. In 1985, men were 2.2 times more likely than women to be diagnosed but by 2018 women and men developed disease at equal rates. Mean age was 67.8 years. Among all patients with known stage of disease, 72.5% of patients were diagnosed with advanced disease. Regional and distant NSCLC rates were both higher in the northern parts of the state. Longer drive times in rural regions also significantly increased the likelihood of advanced NSCLC diagnoses, in particular regional lung cancer. Patients with longer drive times also experienced overall worse survival after controlling for other factors. Conclusions: Regional disparities exist in Michigan for diagnoses of NSCLC at advanced stages. Factors such as lack of screening in urban regions and distances to treating institutions in rural areas likely contribute to the increased likelihood of advanced NSCLC. Future interventions should target the specific needs of residents to detect disease at earlier stages and improve overall outcomes.

Interkingdom interactions shape the fungal microbiome of mosquitoes.

BACKGROUND: The mosquito microbiome is an important modulator of vector competence and vectoral capacity. Unlike the extensively studied bacterial microbiome, fungal communities in the mosquito microbiome (the mycobiome) remain largely unexplored. To work towards getting an improved understanding of the fungi associated with mosquitoes, we sequenced the mycobiome of three field-collected and laboratory-reared mosquito species (Aedes albopictus, Aedes aegypti, and Culex quinquefasciatus). RESULTS: Our analysis showed both environment and host species were contributing to the diversity of the fungal microbiome of mosquitoes. When comparing species, Ae. albopictus possessed a higher number of diverse fungal taxa than Cx. quinquefasciatus, while strikingly less than 1% of reads from Ae. aegypti samples were fungal. Fungal reads from Ae. aegypti were < 1% even after inhibiting host amplification using a PNA blocker, indicating that this species lacked a significant fungal microbiome that was amplified using this sequencing approach. Using a mono-association mosquito infection model, we confirmed that mosquito-derived fungal isolates colonize Aedes mosquitoes and support growth and development at comparable rates to their bacterial counterparts. Strikingly, native bacterial taxa isolated from mosquitoes impeded the colonization of symbiotic fungi in Ae. aegypti suggesting interkingdom interactions shape fungal microbiome communities. CONCLUSION: Collectively, this study adds to our understanding of the fungal microbiome of different mosquito species, that these fungal microbes support growth and development, and highlights that microbial interactions underpin fungal colonization of these medically relevent species.

Establishment and comparison of human term placenta-derived trophoblast cells†.

Research on the biology of fetal-maternal barriers has been limited by access to physiologically relevant cells, including trophoblast cells. In this study, we describe the development of a human term placenta-derived cytotrophoblast immortalized cell line (hPTCCTB) derived from the basal plate. Human-term placenta-derived cytotrophoblast immortalized cell line cells are comparable to their primary cells of origin in terms of morphology, marker expression, and functional responses. We demonstrate that these can transform into syncytiotrophoblast and extravillous trophoblasts. We also compared the hPTCCTB cells to immortalized chorionic trophoblasts (hFM-CTC), trophoblasts of the chorionic plate, and BeWo cells, choriocarcinoma cell lines of conventional use. Human-term placenta-derived cytotrophoblast immortalized cell line and hFM-CTCs displayed more similarity to each other than to BeWos, but these differ in syncytialization ability. Overall, this study (1) demonstrates that the immortalized hPTCCTB generated are cells of higher physiological relevance and (2) provides a look into the distinction between the spatially distinct placental and fetal barrier trophoblasts cells, hPTCCTB and hFM-CTC, respectively.

Exclusive Enteral Nutrition Beneficially Modulates Gut Microbiome in a Preclinical Model of Crohn's-like Colitis.

Exclusive enteral nutrition (EEN) is an established dietary treatment for Crohn's disease (CD) by alleviating inflammation and inducing remission. However, the mechanisms of action of EEN are incompletely understood. As CD is associated with gut microbiome dysbiosis, we investigated the effect of EEN on the microbiome in a rat model of CD-like colitis. The rat model of CD-like colitis was established by an intracolonic instillation of TNBS at 65 mg/kg in 250 µL of 40% ethanol. Sham control rats were instilled with saline. Rats were fed ad libitum with either regular pellet food or EEN treatment with a clear liquid diet (Ensure). Rats were euthanized at 7 days. Fecal pellets were collected from the distal colon for 16S rRNA sequencing analysis of gut microbiota. In addition, colon tissues were taken for histological and molecular analyses in all the groups of rats. EEN administration to TNBS-induced CD rats significantly improved the body weight change, inflammation scores, and disease activity index. The mRNA expression of IL-17A and interferon-γ was significantly increased in the colonic tissue in TNBS rats when fed with regular food. However, EEN treatment significantly attenuated the increase in IL-17A and interferon-γ in TNBS rats. Our 16S rRNA sequencing analysis found that gut microbiota diversity and compositions were significantly altered in TNBS rats, compared to controls. However, EEN treatment improved alpha diversity and increased certain beneficial bacteria such as Lactobacillus and Dubosiella and decreased bacteria such as Bacteroides and Enterorhabdus in CD-like rats, compared to CD-like rats with the regular pellet diet. In conclusion, EEN treatment increases the diversity of gut microbiota and the composition of certain beneficial bacteria. These effects may contribute to the reduced inflammation by EEN in the rat model of CD-like colitis.

Autophagy Determines Distinct Cell Fates in Human Amnion and Chorion Cells.

Human fetal membranes (amniochorion) that line the intrauterine cavity consist of two distinct cell layers; single-layer amnion epithelial cells (AEC) and multilayer chorion trophoblast cells (CTC). These layers are connected through a collagen-rich extracellular matrix. Cellular remodeling helps support membrane growth and integrity during gestation and helps to maintain pregnancy. Preterm prelabor rupture of the human amniochorionic (fetal) membrane (pPROM) is antecedent to 40% of all spontaneous preterm birth. Oxidative stress (OS) induced activation of the p38 MAPK due to various maternal risk exposures and the amniochorion cells' senescence are reported pathological features of pPROM. Our transcriptomics analysis implicated dysregulated autophagy and epithelial-mesenchymal transition (EMT) in fetal membranes from pPROM. The molecular interplay between OS-induced p38 MAPK activation, autophagy, and EMT was investigated in AECs and CTCs to better understand the involvement of autophagy and EMT. We report the differential impact of OS on the autophagic machinery in AECs and CTCs, resulting in distinct cell fates. In AECs, OS-induced p38 MAPK activation causes autophagosome accumulation and reduced autophagic flux mediated by decreased ULK1 activity and kinase activity, leading to senescence. In CTCs, induction of autophagy has a limited effect; however, inhibition of autophagy led to SQSTM1-mediated EMT of trophoblast cells. Autophagy, EMT, and senescence were associated with proinflammatory changes. Thus, AECs and CTCs respond differently to OS via differential autophagy response, partly mediated via p38 MAPK. Besides senescence, OS-induced autophagy dysregulation in amniochorion cells may play a mechanistic role in pPROM pathophysiology.

The influence of the larval microbiome on susceptibility to Zika virus is mosquito genotype-dependent.

The microbiome of the mosquito Aedes aegypti is largely determined by the environment and influences mosquito susceptibility for arthropod-borne viruses (arboviruses). Larval interactions with different bacteria can have carry-over effects on adult Ae. aegypti replication of arboviruses, but little is known about the role that mosquito host genetics play in determining how larval-bacterial interactions shape Ae aegypti susceptibility to arboviruses. To address this question, we isolated single bacterial isolates and complex microbiomes from Ae. aegypti larvae from various field sites in Senegal. Either single bacterial isolates or complex microbiomes were added to two different genetic backgrounds of Ae. aegypti in a gnotobiotic larval system. Using 16S amplicon sequencing we showed that the bacterial community structure differs between the two genotypes of Ae. aegypti when given identical microbiomes, and the abundance of single bacterial taxa differed between Ae. aegypti genotypes. Using single bacterial isolates or the entire preserved complex microbiome, we tested the ability of specific larval microbiomes to drive differences in infection rates for Zika virus in different genetic backgrounds of Ae. aegypti. We observed that the proportion of Zika virus-infected adults was dependent on the interaction between the larval microbiome and Ae. aegypti host genetics. By using the larval microbiome as a component of the environment, these results demonstrate that interactions between the Ae. aegypti genotype and its environment can influence Zika virus infection. As Ae. aegypti expands and adapts to new environments under climate change, an understanding of how different genotypes interact with the same environment will be crucial for implementing arbovirus transmission control strategies.

Transition Mutations in the hTERT Promoter Are Unrelated to Potential i-motif Formation in the C-Rich Strand.

Increased expression of the human telomere reverse transcriptase (hTERT) in tumors promotes tumor cell survival and diminishes the survival of patients. Cytosine-to-thymine (C-to-T) transition mutations (C250T or C228T) in the hTERT promoter create binding sites for transcription factors, which enhance transcription. The G-rich strand of the hTERT promoter can form G-quadruplex structures, whereas the C-rich strand can form an i-motif in which multiple cytosine residues are protonated. We considered the possibility that i-motif formation might promote cytosine deamination to uracil and C-to-T mutations. We computationally probed the accessibility of cytosine residues in an i-motif to attack by water. We experimentally examined regions of the C-rich strand to form i-motifs using pH-dependent UV and CD spectra. We then incubated the C-rich strand with and without the G-rich complementary strand DNA under various conditions, followed by deep sequencing. Surprisingly, deamination rates did not vary substantially across the 46 cytosines examined, and the two mutation hotspots were not deamination hotspots. The appearance of mutational hotspots in tumors is more likely the result of the selection of sequences with increased promoter binding affinity and hTERT expression.

Variable microbiomes between mosquito lines are maintained across different environments.

The composition of the microbiome is shaped by both environment and host in most organisms, but in the mosquito Aedes aegypti the role of the host in shaping the microbiome is poorly understood. Previously, we had shown that four lines of Ae. aegypti harbored different microbiomes when reared in the same insectary under identical conditions. To determine whether these lines differed from each other across time and in different environments, we characterized the microbiome of the same four lines of Ae. aegypti reared in the original insectary and at another institution. While it was clear that the environment influenced the microbiomes of these lines, we did still observe distinct differences in the microbiome between lines within each insectary. Clear differences were observed in alpha diversity, beta diversity, and abundance of specific bacterial taxa. To determine if the line specific differences in the microbiome were maintained across environments, pair-wise differential abundances of taxa was compared between insectaries. Lines were most similar to other lines from the same insectary than to the same line reared in a different insectary. Additionally, relatively few differentially abundant taxa identified between pairs of lines were shared across insectaries, indicating that line specific properties of the microbiome are not conserved across environments, or that there were distinct microbiota within each insectary. Overall, these results demonstrate that mosquito lines under the same environmental conditions have different microbiomes across microbially- diverse environments and host by microbe interactions affecting microbiome composition and abundance is dependent on environmentally available bacteria.

Identification of multivariable Boolean patterns in microbiome and microbial gene composition data.

Virtually every biological system is governed by complex relations among its components. Identifying such relations requires a rigorous or heuristics-based search for patterns among variables/features of a system. Various algorithms have been developed to identify two-dimensional (involving two variables) patterns employing correlation, covariation, mutual information, etc. It seems obvious, however, that comprehensive descriptions of complex biological systems need also to include more complicated multivariable relations, which can only be described using patterns that simultaneously embrace 3, 4, and more variables. The goal of this manuscript is to (a) introduce a novel type of associations (multivariable Boolean patterns) that can be manifested between features of complex systems but cannot be identified (described) by traditional pair-vise metrics; (b) propose patterns classification method, and (c) provide a novel definition of the pattern's strength (pattern's score) able to accommodate heterogeneous multi-omics data. To demonstrate the presence of such patterns, we performed a search for all possible 2-, 3-, and 4-dimensional patterns in historical data from the Human Microbiome Project (15 body sites) and collection of H. pylori genomes associated with gastric ulcers, gastritis, and duodenal ulcers. In all datasets under consideration, we were able to identify hundreds of statistically significant multivariable patterns. These results suggest that such patterns can be common in microbial genomics/microbiomics systems.

Amplification of microbial DNA from bacterial extracellular vesicles from human placenta.

Introduction: The placenta is essential for fetal growth and survival and maintaining a successful pregnancy. The sterility of the placenta has been challenged recently; however, the presence of a placental microbiome has been controversial. We tested the hypothesis that the bacterial extracellular vesicles (BEVs) from Gram-negative bacteria as an alternate source of microbial DNA, regardless of the existence of a microbial community in the placenta. Methods: Placentae from the term, not in labor Cesareans deliveries, were used for this study, and placental specimens were sampled randomly from the fetal side. We developed a protocol for the isolation of BEVs from human tissues and this is the first study to isolate the BEVs from human tissue and characterize them. Results: The median size of BEVs was 130-140 nm, and the mean concentration was 1.8-5.5 × 1010 BEVs/g of the wet placenta. BEVs are spherical and contain LPS and ompA. Western blots further confirmed ompA but not human EVs markers ALIX confirming the purity of preparations. Taxonomic abundance profiles showed BEV sequence reads above the levels of the negative controls (all reagent controls). In contrast, the sequence reads in the same placenta were substantially low, indicating nothing beyond contamination (low biomass). Alpha-diversity showed the number of detected genera was significantly higher in the BEVs than placenta, suggesting BEVs as a likely source of microbial DNA. Beta-diversity further showed significant overlap in the microbiome between BEV and the placenta, confirming that BEVs in the placenta are likely a source of microbial DNA in the placenta. Uptake studies localized BEVs in maternal (decidual) and placental cells (cytotrophoblast), confirming their ability to enter these cells. Lastly, BEVs significantly increased inflammatory cytokine production in THP-1 macrophages in a high-dose group but not in the placental or decidual cells. Conclusion: We conclude that the BEVs are normal constituents during pregnancy and likely reach the placenta through hematogenous spread from maternal body sites that harbor microbiome. Their presence may result in a low-grade localized inflammation to prime an antigen response in the placenta; however, insufficient to cause a fetal inflammatory response and adverse pregnancy events. This study suggests that BEVs can confound placental microbiome studies, but their low biomass in the placenta is unlikely to have any immunologic impact.

Identification of common sequence motifs shared exclusively among selectively packed exosomal pathogenic microRNAs during rickettsial infections.

We previously reported that microRNA (miR)23a and miR30b are selectively sorted into exosomes derived from rickettsia-infected endothelial cells (R-ECExos). Yet, the mechanism remains unknown. Cases of spotted fever rickettsioses have been increasing, and infections with these bacteria cause life-threatening diseases by targeting brain and lung tissues. Therefore, the goal of the present study is to further dissect the molecular mechanism underlying R-ECExos-induced barrier dysfunction of normal recipient microvascular endothelial cells (MECs), depending on their exosomal RNA cargos. Infected ticks transmit the rickettsiae to human hosts following a bite and injections of the bacteria into the skin. In the present study, we demonstrate that treatment with R-ECExos, which were derived from spotted fever group R parkeri infected human dermal MECs, induced disruptions of the paracellular adherens junctional protein VE-cadherin, and breached the paracellular barrier function in recipient pulmonary MECs (PMECs) in an exosomal RNA-dependent manner. We did not detect different levels of miRs in parent dermal MECs following rickettsial infections. However, we demonstrated that the microvasculopathy-relevant miR23a-27a-24 cluster and miR30b are selectively enriched in R-ECExos. Bioinformatic analysis revealed that common sequence motifs are shared exclusively among the exosomal, selectively-enriched miR23a cluster and miR30b at different levels. Taken together, these data warrant further functional identification and characterization of a monopartition, bipartition, or tripartition among ACA, UCA, and CAG motifs that guide recognition of microvasculopathy-relevant miR23a-27a-24 and miR30b, and subsequently results in their selective enrichments in R-ECExos.

The influence of the larval microbiome on susceptibility to Zika virus is mosquito genotype dependent.

The microbiome of the mosquito Aedes aegypti is largely determined by the environment and influences mosquito susceptibility for arthropod-borne viruses (arboviruses). Larval interactions with different bacteria can influence adult Ae. aegypti replication of arboviruses, but little is known about the role that mosquito host genetics play in determining how larval-bacterial interactions shape Ae aegypti susceptibility to arboviruses. To address this question, we isolated single bacterial isolates and complex microbiomes from Ae. aegypti larvae from various field sites in Senegal. Either single bacterial isolates or complex microbiomes were added to two different genetic backgrounds of Ae. aegypti in a gnotobiotic larval system. Using 16S amplicon sequencing we show that similarities in bacterial community structures when given identical microbiomes between different genetic backgrounds of Ae. aegypti was dependent on the source microbiome, and the abundance of single bacterial taxa differed between Ae. aegypti genotypes. Using single bacterial isolates or the entire preserved complex microbiome, we tested the ability of specific microbiomes to drive differences in infection rates for Zika virus in different genetic backgrounds of Ae. aegypti . We observed that the proportion of Zika virus-infected adults was dependent on the interaction between the larval microbiome and Ae. aegypti host genetics. By using the larval microbiome as a component of the environment, these results demonstrate that interactions between the Ae. aegypti genotype and its environment can influence Zika virus infection. As Ae. aegypti expands and adapts to new environments under climate change, an understanding of how different genotypes interact with the same environment will be crucial for implementing arbovirus transmission control strategies.

The relationship of social determinants and distress in newly diagnosed cancer patients.

Patients with a new cancer diagnosis can experience distress when diagnosed. There are disparities in treatment of cancer patients based on social determinants, but minimal research exists on the relationship of those social determinants and distress after a new cancer diagnosis. Our goals were to determine the social determinants associated with distress after a new cancer diagnosis and determine the relationship of distress with outcome. Patients with a new cancer diagnosis at one institution from January 2019 to December 2020 were analyzed. Patients were given the National Comprehensive Cancer Network (NCCN) distress thermometer during their first visit. Demographics, tumor characteristics, clinical variables and survival were recorded. Patients were also asked to share specific factors that led to distress, including: (1) financial, (2) transportation, (3) childcare and (4) religious. A total of 916 patients returned distress thermometers. Mean age was 59.1 years. Females comprised 71.3 (653/916) percent of the cohort. On Dunn's multiple comparison, the following factors were associated with increased distress level: female (p < 0.01), ages 27 to 45 (p < 0.01), uninsured (p < 0.01) and unemployed (p < 0.01). Patients with higher distress scores also experienced worse overall survival (p < 0.05). Females, young patients, uninsured patients and unemployed patients experience more distress after a new cancer diagnosis. Increased distress is independently associated with worse overall survival. Social determinants can be used to predict which patients may require focused interventions to reduce distress after a new cancer diagnosis.

Identification of common sequence motifs shared exclusively among selectively packed exosomal pathogenic microRNAs during rickettsial infections.

We previously reported that microRNA (miR)23a and miR30b are selectively sorted into rickettsia-infected, endothelial cell-derived exosomes ( R -ECExos). Yet, the mechanism remains unknown. The number of cases of spotted fever rickettsioses has been increasing in recent years, and infections with these bacteria cause life-threatening diseases by targeting brain and lung tissues. Therefore, the aim of the present study is to continue to dissect the molecular mechanism underlying R -ECExos-induced barrier dysfunction of normal recipient microvascular endothelial cells (MECs), depending on their exosomal RNA cargos. Rickettsiae are transmitted to human hosts by the bite of an infected tick into the skin. In the present study we demonstrate that treatment with R -ECExos, which were derived from spotted fever group R parkeri infected human dermal MECs, induced disruptions of the paracellular adherens junctional protein VE-cadherin and breached the paracellular barrier function in recipient pulmonary MECs (PMECs) in an exosomal RNA-dependent manner. Similarly, we did not detect different levels of miRs in parent dermal MECs following rickettsial infections. However, we demonstrated that the microvasculopathy-relevant miR23a-27a-24 cluster and miR30b are selectively enriched in R -ECExos. Bioinformatic analysis revealed that common sequence motifs are shared exclusively among the exosomal, selectively-enriched miR23a cluster and miR30b at different levels. Taken together, these data warrant further functional identification and characterization of a single, bipartition, or tripartition among ACA, UCA, and CAG motifs that guide recognition of microvasculopathy-relevant miR23a-27a-24 and miR30b, and subsequently results in their selective enrichments in R -ECExos.

Expression of CYP450 enzymes in human fetal membranes and its implications in xenobiotic metabolism during pregnancy.

BACKGROUND: Environmental exposure to toxicants is a major risk factor for spontaneous preterm birth (PTB, <37 weeks). Toxicants and drugs administered to patients are metabolized primarily by the cytochrome P450 (CYP450) system. Along with the adult and fetal liver, the placenta, a critical feto-maternal interface organ, expresses CYP450 enzymes that metabolize these xenobiotics. However, the contribution of the fetal membranes, another tissue of the feto-maternal interface, to the expression of CYP450 enzymes and the detoxification of xenobiotics remains unknown. AIMS: This study characterized CYP450 expression and determined the functional activity of CYP450 enzymes in fetal membranes. MAIN METHODS: RNA sequencing (RNA-Seq) of placental and fetal membrane tissues and cells was done. Differential expressions of CYP450 genes were compared and validated via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) between the two tissues. The functional activity of major CYP450 enzymes was determined using a fluorophore-based enzymatic assay in the presence and absence of their corresponding inhibitors. KEY FINDINGS: With the exception of genes that regulate cholesterol metabolism, the expression profile of CYP450 genes was similar between placental and fetal membranes tissues/cells. RT-qPCR analysis confirmed these findings with significant levels of mRNA for major CYP450 genes being detectable in amnion epithelial cells (AECs) and chorion trophoblasts cells (CTCs). Biochemical analyses revealed significant CYP450 enzymatic activities that were sensitive to specific inhibitors for both AECs and CTCs, suggesting that the genes were expressed as functional enzymes. SIGNIFICANCE: This is the first study to determine global expression of CYP450 enzymes in fetal membranes which may play a role in xenobiotic metabolism during pregnancy. Given that many women are exposed to environmental toxins or require medications during pregnancy, a better understanding of their role in metabolism is required to develop safer therapeutics and prevent adverse outcomes.

Exosomally Targeting microRNA23a Ameliorates Microvascular Endothelial Barrier Dysfunction Following Rickettsial Infection.

Spotted fever group rickettsioses caused by Rickettsia (R) are devastating human infections, which mainly target microvascular endothelial cells (ECs) and can induce lethal EC barrier dysfunction in the brain and lungs. Our previous evidence reveals that exosomes (Exos) derived from rickettsial-infected ECs, namely R-ECExos, can induce disruption of the tight junctional (TJ) protein ZO-1 and barrier dysfunction of human normal recipient brain microvascular endothelial cells (BMECs). However, the underlying mechanism remains elusive. Given that we have observed that microRNA23a (miR23a), a negative regulator of endothelial ZO-1 mRNA, is selectively sorted into R-ECExos, the aim of the present study was to characterize the potential functional role of exosomal miR23a delivered by R-ECExos in normal recipient BMECs. We demonstrated that EC-derived Exos (ECExos) have the capacity to deliver oligonucleotide RNAs to normal recipient BMECs in an RNase-abundant environment. miR23a in ECExos impairs normal recipient BMEC barrier function, directly targeting TJ protein ZO-1 mRNAs. In separate studies using a traditional in vitro model and a novel single living-cell biomechanical assay, our group demonstrated that miR23a anti-sense oligonucleotide-enriched ECExos ameliorate R-ECExo-provoked recipient BMEC dysfunction in association with stabilization of ZO-1 in a dose-dependent manner. These results suggest that Exo-based therapy could potentially prove to be a promising strategy to improve vascular barrier function during bacterial infection and concomitant inflammation.

AhR promotes phosphorylation of ARNT isoform 1 in human T cell malignancies as a switch for optimal AhR activity.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor present in immune cells as a long and short isoform, referred to as isoforms 1 and 3, respectively. However, investigation into potential ARNT isoform­specific immune functions is lacking despite the well-established heterodimerization requirement of ARNT with, and for the activity of, the aryl hydrocarbon receptor (AhR), a critical mediator of immune homeostasis. Here, using global and targeted transcriptomics analyses, we show that the relative ARNT isoform 1:3 ratio in human T cell lymphoma cells dictates the amplitude and direction of AhR target gene regulation. Specifically, shifting the ARNT isoform 1:3 ratio lower by suppressing isoform 1 enhances, or higher by suppressing isoform 3 abrogates, AhR responsiveness to ligand activation through preprograming a cellular genetic background that directs explicit gene expression patterns. Moreover, the fluctuations in gene expression patterns that accompany a decrease or increase in the ARNT isoform 1:3 ratio are associated with inflammation or immunosuppression, respectively. Molecular studies identified the unique casein kinase 2 (CK2) phosphorylation site within isoform 1 as an essential parameter to the mechanism of ARNT isoform­specific regulation of AhR signaling. Notably, CK2-mediated phosphorylation of ARNT isoform 1 is dependent on ligand-induced AhR nuclear translocation and is required for optimal AhR target gene regulation. These observations reveal ARNT as a central modulator of AhR activity predicated on the status of the ARNT isoform ratio and suggest that ARNT-based therapies are a viable option for tuning the immune system to target immune disorders.

Heightened cocaine-seeking in male rats associates with a distinct transcriptomic profile in the medial prefrontal cortex.

Drug overdose deaths involving cocaine have skyrocketed, an outcome attributable in part to the lack of FDA-approved medications for the treatment of cocaine use disorder (CUD), highlighting the need to identify new pharmacotherapeutic targets. Vulnerability to cocaine-associated environmental contexts and stimuli serves as a risk factor for relapse in CUD recovery, with individual differences evident in the motivational aspects of these cues. The medial prefrontal cortex (mPFC) provides top-down control of striatal circuitry to regulate the incentive-motivational properties of cocaine-associated stimuli. Clinical and preclinical studies have identified genetic variations that impact the degree of executive restraint over drug-motivated behaviors, and we designed the present study to employ next-generation sequencing to identify specific genes associated with heightened cue-evoked cocaine-seeking in the mPFC of male, outbred rats. Rats were trained to stably self-administer cocaine, and baseline cue-reinforced cocaine-seeking was established. Rats were phenotyped as either high cue (HC) or low cue (LC) responders based upon lever pressing for previously associated cocaine cues and allowed 10 days of abstinence in their home cages prior to mPFC collection for RNA-sequencing. The expression of 309 genes in the mPFC was significantly different in HC vs. LC rats. Functional gene enrichment analyses identified ten biological processes that were overrepresented in the mPFC of HC vs. LC rats. The present study identifies distinctions in mPFC mRNA transcripts that characterizes individual differences in relapse-like behavior and provides prioritized candidates for future pharmacotherapeutics aimed to help maintain abstinence in CUD. In particular the Htr2c gene, which encodes the serotonin 5-HT2C receptor (5-HT2CR), is expressed to a lower extent in HC rats, relative to LC rats. These findings build on a plethora of previous studies that also point to the 5-HT2CR as an attractive target for the treatment of CUD.

Endothelial Exosome Plays a Functional Role during Rickettsial Infection.

Spotted fever group rickettsioses (SFRs) are devastating human infections. Vascular endothelial cells (ECs) are the primary targets of rickettsial infection. Edema resulting from EC barrier dysfunction occurs in the brain and lungs in most cases of lethal SFR, but the underlying mechanisms remain unclear. The aim of the study was to explore the potential role of Rickettsia-infected, EC-derived exosomes (Exos) during infection. Using size exclusion chromatography (SEC), we purified Exos from conditioned, filtered, bacterium-free media collected from Rickettsia parkeri-infected human umbilical vein ECs (HUVECs) (R-ECExos) and plasma of Rickettsia australis- or R. parkeri-infected mice (R-plsExos). We observed that rickettsial infection increased the release of heterogeneous plsExos, but endothelial exosomal size, morphology, and production were not significantly altered following infection. Compared to normal plsExos and ECExos, both R-plsExos and R-ECExos induced dysfunction of recipient normal brain microvascular ECs (BMECs). The effect of R-plsExos on mouse recipient BMEC barrier function is dose dependent. The effect of R-ECExos on human recipient BMEC barrier function is dependent on the exosomal RNA cargo. Next-generation sequencing analysis and stem-loop quantitative reverse transcription-PCR (RT-qPCR) validation revealed that rickettsial infection triggered the selective enrichment of endothelial exosomal mir-23a and mir-30b, which potentially target the endothelial barrier. To our knowledge, this is the first report on the functional role of extracellular vesicles following infection by obligately intracellular bacteria.IMPORTANCE Spotted fever group rickettsioses are devastating human infections. Vascular endothelial cells are the primary targets of infection. Edema resulting from endothelial barrier dysfunction occurs in the brain and lungs in most cases of lethal rickettsioses, but the underlying mechanisms remain unclear. The aim of the study was to explore the potential role of Rickettsia-infected, endothelial cell-derived exosomes during infection. We observed that rickettsial infection increased the release of heterogeneous plasma Exos, but endothelial exosomal size, morphology, and production were not significantly altered following infection. Rickettsia-infected, endothelial cell-derived exosomes induced dysfunction of human recipient normal brain microvascular endothelial cells. The effect is dependent on the exosomal RNA cargo. Next-generation sequencing analysis revealed that rickettsial infection triggered the selective enrichment of endothelial exosomal mir-23a and mir-30b, which potentially target the endothelial barrier. To our knowledge, this is the first report on the functional role of extracellular vesicles following infection by obligately intracellular bacteria.