The nuclear receptor Hr46/Hr3 is required in the blood brain barrier of mature males for courtship.

The blood brain barrier (BBB) forms a stringent barrier that protects the brain from components in the circulation that could interfere with neuronal function. At the same time, the BBB enables selective transport of critical nutrients and other chemicals to the brain. Beyond these functions, another recently recognized function is even less characterized, specifically the role of the BBB in modulating behavior by affecting neuronal function in a sex-dependent manner. Notably, signaling in the adult Drosophila BBB is required for normal male courtship behavior. Courtship regulation also relies on male-specific molecules in the BBB. Our previous studies have demonstrated that adult feminization of these cells in males significantly lowered courtship. Here, we conducted microarray analysis of BBB cells isolated from males and females. Findings revealed that these cells contain male- and female-enriched transcripts, respectively. Among these transcripts, nuclear receptor Hr46/Hr3 was identified as a male-enriched BBB transcript. Hr46/Hr3 is best known for its essential roles in the ecdysone response during development and metamorphosis. In this study, we demonstrate that Hr46/Hr3 is specifically required in the BBB cells for courtship behavior in mature males. The protein is localized in the nuclei of sub-perineurial glial cells (SPG), indicating that it might act as a transcriptional regulator. These data provide a catalogue of sexually dimorphic BBB transcripts and demonstrate a physiological adult role for the nuclear receptor Hr46/Hr3 in the regulation of male courtship, a novel function that is independent of its developmental role.

Genetic variation in P-element dysgenic sterility is associated with double-strand break repair and alternative splicing of TE transcripts.

The germline mobilization of transposable elements (TEs) by small RNA mediated silencing pathways is conserved across eukaryotes and critical for ensuring the integrity of gamete genomes. However, genomes are recurrently invaded by novel TEs through horizontal transfer. These invading TEs are not targeted by host small RNAs, and their unregulated activity can cause DNA damage in germline cells and ultimately lead to sterility. Here we use hybrid dysgenesis-a sterility syndrome of Drosophila caused by transposition of invading P-element DNA transposons-to uncover host genetic variants that modulate dysgenic sterility. Using a panel of highly recombinant inbred lines of Drosophila melanogaster, we identified two linked quantitative trait loci (QTL) that determine the severity of dysgenic sterility in young and old females, respectively. We show that ovaries of fertile genotypes exhibit increased expression of splicing factors that suppress the production of transposase encoding transcripts, which likely reduces the transposition rate and associated DNA damage. We also show that fertile alleles are associated with decreased sensitivity to double-stranded breaks and enhanced DNA repair, explaining their ability to withstand high germline transposition rates. Together, our work reveals a diversity of mechanisms whereby host genotype modulates the cost of an invading TE, and points to genetic variants that were likely beneficial during the P-element invasion.

Lung-resident CD69+ST2+ TH2 cells mediate long-term type 2 memory to inhaled antigen in mice.

BACKGROUND: Chronic airway diseases such as asthma are characterized by persistent type 2 immunity in the airways. We know little about the mechanisms that explain why type 2 inflammation continues in these diseases. OBJECTIVE: We used mouse models to investigate the mechanisms involved in long-lasting immune memory. METHODS: Naive mice were exposed intranasally to ovalbumin (OVA) antigen with Alternaria extract as an adjuvant. Type 2 memory was analyzed by parabiosis model, flow cytometry with in vivo antibody labeling, and intranasal OVA recall challenge. Gene-deficient mice were used to analyze the mechanisms. RESULTS: In the parabiosis model, mice previously exposed intranasally to OVA with Alternaria showed more robust antigen-specific immune responses and airway inflammation than mice with circulating OVA-specific T cells. After a single airway exposure to OVA with Alternaria, CD69+ST2+ TH2-type T cells, which highly express type 2 cytokine messenger RNA and lack CD62L expression, appeared in lung tissue within 5 days and persisted for at least 84 days. When exposed again to OVA in vivo, these cells produced type 2 cytokines quickly without involving circulating T cells. Development of tissue-resident CD69+ST2+ TH2 cells and long-term memory to an inhaled antigen were abrogated in mice deficient in ST2 or IL-33, but not TSLP receptor. CONCLUSION: CD69+ST2+ TH2 memory cells develop quickly in lung tissue after initial allergen exposure and persist for a prolonged period. The ST2/IL-33 pathway may play a role in the development of immune memory in lung to certain allergens.

Blocking the inhibitory receptor programmed cell death 1 prevents allergic immune response and anaphylaxis in mice.

BACKGROUND: Food allergy and acute anaphylaxis can be life-threatening. While T follicular helper (Tfh) cells play a pivotal role in the allergic immune responses, the immunologic mechanisms that regulate the production of antibodies (Abs) that mediate anaphylaxis are not fully understood. OBJECTIVE: The aim of this study was to investigate the role of the inhibitory receptor programmed cell death protein 1 (PD-1), which is highly expressed on Tfh cells, in allergic immune responses using an animal model of peanut allergy and anaphylaxis. METHODS: Naive wild-type mice were exposed to peanut flour intranasally and then challenged with peanut extract to induce systemic anaphylaxis. The roles of PD-1 were examined by blocking Abs and using gene-deficient animals. A hapten model and passive cutaneous anaphylaxis were used to characterize allergen-specific Abs. RESULTS: Treatment with anti-PD-1 enhanced development of Tfh cells and germinal center B cells in mice exposed to peanut flour. Nonetheless, anti-PD-1 or its ligand fully protected mice from developing anaphylaxis. Anti-PD-1 treatment or genetic deficiency of PD-1 in CD4+ T cells inhibited production of peanut-specific IgE and increased the levels of IgG. The passive cutaneous anaphylaxis showed that peanut-specific Abs generated in anti-PD-1-treated animals prevented, rather than provoked, anaphylaxis when transferred to naive animals. Anti-PD-1 promoted production of Abs with low affinity for an antigen in the hapten model. CONCLUSION: Blockade of the pathway between PD-1 and its ligand is protective against allergic immune responses. The direct interaction between Tfh cells and B cells may play a pivotal role in controlling Ab quality and clinical manifestation of allergic diseases.

TLR3-driven IFN-ß antagonizes STAT5-activating cytokines and suppresses innate type 2 response in the lung.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are involved in type 2 immune responses in mucosal organs and are associated with various allergic diseases in humans. Studies are needed to understand the molecules and pathways that control ILC2s. OBJECTIVE: The aims of this study were to develop a mouse model that limits the innate type 2 immune response in the lung and to investigate the immunologic mechanisms involved in regulation of lung ILC2s. METHODS: Naive BALB/c mice were administered various Toll-like receptor agonists and exposed intranasally to the fungal allergen Alternaria alternata. The mechanisms were investigated using gene knockout mice as well as cultures of lung cells and isolated lung ILC2s. RESULTS: Polyinosinic-polycytidylic acid, or poly (I:C), effectively inhibited innate type 2 response to A alternata. Poly (I:C) promoted production of IFNα, -ß, and -γ, and its inhibitory effects were dependent on the IFN-α/ß receptor pathway. IFN-ß was 100 times more potent than IFN-α at inhibiting type 2 cytokine production by lung ILC2s. Signal transducer and activator of transcription 5 (STAT5)-activating cytokines, including IL-2, IL-7, and thymic stromal lymphopoietin, but not IL-33, promoted survival and proliferation of lung ILC2s in vitro, while IFN-ß blocked these effects. Expression of the transcription factor GATA3, which is critical for differentiation and maintenance of ILC2s, was inhibited by IFN-ß. CONCLUSIONS: IFN-ß blocks the effects of STAT5-activating cytokines on lung ILC2s and inhibits their survival and effector functions. Administration of IFN-ß may provide a new strategy to treat diseases involving ILC2s.

A mouse model of the LEAP study reveals a role for CTLA-4 in preventing peanut allergy induced by environmental peanut exposure.

BACKGROUND: A human study, Learning Early About Peanut Allergy (LEAP), showed that early introduction of peanut products decreases the prevalence of peanut allergy among children. However, the immunologic mechanisms mediating the protective effects of consuming peanut products are not well understood. OBJECTIVE: The objective was to develop a mouse model that simulates the LEAP study and investigate the underlying mechanisms for the study observations. METHODS: Adult naive BALB/c mice were fed a commercial peanut butter product (Skippy) or buffer control and concomitantly exposed to peanut flour through the airway or skin to mimic environmental exposure. The animals were analyzed for anaphylactic reaction and by molecular and immunologic approaches. RESULTS: After exposure to peanut flour through the airway or skin, naive mice developed peanut allergy, as demonstrated by acute and systemic anaphylaxis in response to challenge with peanut extract. Ingestion of Skippy, however, nearly abolished the increase in peanut-specific IgE and IgG and protected animals from developing anaphylaxis. Skippy-fed mice showed reduced numbers of T follicular helper (Tfh) cells and germinal center B cells in their draining lymph nodes, and single-cell RNA sequencing revealed a CD4+ T-cell population expressing cytotoxic T lymphocyte-associated protein 4 (CTLA-4) in these animals. Critically, blocking CTLA-4 with antibody increased levels of peanut-specific antibodies and reversed the protective effects of Skippy. CONCLUSION: Ingestion of a peanut product protects mice from peanut allergy induced by environmental exposure to peanuts, and the CTLA-4 pathway, which regulates Tfh cell responses, likely plays a pivotal role in this protection.

A-MYB and BRDT-dependent RNA Polymerase II pause release orchestrates transcriptional regulation in mammalian meiosis.

During meiotic prophase I, spermatocytes must balance transcriptional activation with homologous recombination and chromosome synapsis, biological processes requiring extensive changes to chromatin state. We explored the interplay between chromatin accessibility and transcription through prophase I of mammalian meiosis by measuring genome-wide patterns of chromatin accessibility, nascent transcription, and processed mRNA. We find that Pol II is loaded on chromatin and maintained in a paused state early during prophase I. In later stages, paused Pol II is released in a coordinated transcriptional burst mediated by the transcription factors A-MYB and BRDT, resulting in ~3-fold increase in transcription. Transcriptional activity is temporally and spatially segregated from key steps of meiotic recombination: double strand breaks show evidence of chromatin accessibility earlier during prophase I and at distinct loci from those undergoing transcriptional activation, despite shared chromatin marks. Our findings reveal mechanisms underlying chromatin specialization in either transcription or recombination in meiotic cells.

Histone deacetylase 3 represses cholesterol efflux during CD4+ T-cell activation.

After antigenic activation, quiescent naive CD4+ T cells alter their metabolism to proliferate. This metabolic shift increases production of nucleotides, amino acids, fatty acids, and sterols. Here, we show that histone deacetylase 3 (HDAC3) is critical for activation of murine peripheral CD4+ T cells. HDAC3-deficient CD4+ T cells failed to proliferate and blast after in vitro TCR/CD28 stimulation. Upon T-cell activation, genes involved in cholesterol biosynthesis are upregulated while genes that promote cholesterol efflux are repressed. HDAC3-deficient CD4+ T cells had reduced levels of cellular cholesterol both before and after activation. HDAC3-deficient cells upregulate cholesterol synthesis appropriately after activation, but fail to repress cholesterol efflux; notably, they overexpress cholesterol efflux transporters ABCA1 and ABCG1. Repression of these genes is the primary function for HDAC3 in peripheral CD4+ T cells, as addition of exogenous cholesterol restored proliferative capacity. Collectively, these findings demonstrate HDAC3 is essential during CD4+ T-cell activation to repress cholesterol efflux.

Uninvited guests: how transposable elements take advantage of Drosophila germline stem cells, and how stem cells fight back.

Transposable elements (TEs) are mobile genetic parasites that spread through host genomes by replicating in germline cells. New TE copies that arise in the genomes of germline stem cells (GSCs) are of particular value, because they are potentially transmitted to multiple offspring through the plethora of gametes arising from the same progenitor GSC. However, the fidelity of GSC genomes is also of utmost importance to the host in ensuring the production of abundant and fit offspring. Here we review tactics that TEs employ to replicate in Drosophila female GSCs, as well as mechanisms those cells use to defend against TEs. We also discuss the relationship between transposition and GSC loss, which is arbitrated through reduced signaling for self renewal, increased signaling for differentiation, and DNA damage response pathways.

Detection of Brucella suis, Campylobacter jejuni, and Escherichia coli Strains in Feral Pig (Sus scrofa) Communities of Georgia.

Feral pigs (Sus scrofa) are an environmentally destructive invasive species that act as a reservoir for zoonotic pathogens. The aim of this study was to determine the presence of Brucella suis, Campylobacter jejuni, and of Escherichia coli in feces of feral pigs from Georgia. Fecal samples were collected from 87 feral pigs from forested and agricultural regions of Georgia. DNA was extracted from the fecal samples and quantitative PCR (qPCR) was used to screen for each of the four pathogens. The qPCR assays indicated that B. suis and eaeA-containing strains of E. coli was present in about 22% and 28% of the samples, respectively. C. jejuni was undetected in any of the feral pig fecal samples. The incidence of B. suis was higher in the pigs from forested region, whereas E. coli strains possessing eaeA gene incidence was higher in the pigs from agricultural regions. In Georgia, feral pigs harbor infectious agents and are a growing threat to the transmission of pathogens to native wildlife, humans, and food crops.