Alternative splicing of interleukin-33 and type 2 inflammation in asthma.

Type 2 inflammation occurs in a large subgroup of asthmatics, and novel cytokine-directed therapies are being developed to treat this population. In mouse models, interleukin-33 (IL-33) activates lung resident innate lymphoid type 2 cells (ILC2s) to initiate airway type 2 inflammation. In human asthma, which is chronic and difficult to model, the role of IL-33 and the target cells responsible for persistent type 2 inflammation remain undefined. Full-length IL-33 is a nuclear protein and may function as an "alarmin" during cell death, a process that is uncommon in chronic stable asthma. We demonstrate a previously unidentified mechanism of IL-33 activity that involves alternative transcript splicing, which may operate in stable asthma. In human airway epithelial cells, alternative splicing of the IL-33 transcript is consistently present, and the deletion of exons 3 and 4 (Δ exon 3,4) confers cytoplasmic localization and facilitates extracellular secretion, while retaining signaling capacity. In nonexacerbating asthmatics, the expression of Δ exon 3,4 is strongly associated with airway type 2 inflammation, whereas full-length IL-33 is not. To further define the extracellular role of IL-33 in stable asthma, we sought to determine the cellular targets of its activity. Comprehensive flow cytometry and RNA sequencing of sputum cells suggest basophils and mast cells, not ILC2s, are the cellular sources of type 2 cytokines in chronic asthma. We conclude that IL-33 isoforms activate basophils and mast cells to drive type 2 inflammation in chronic stable asthma, and novel IL-33 inhibitors will need to block all biologically active isoforms.

Polerovirus N-terminal readthrough domain structures reveal molecular strategies for mitigating virus transmission by aphids.

Poleroviruses, enamoviruses, and luteoviruses are icosahedral, positive sense RNA viruses that cause economically important diseases in food and fiber crops. They are transmitted by phloem-feeding aphids in a circulative manner that involves the movement across and within insect tissues. The N-terminal portion of the viral readthrough domain (NRTD) has been implicated as a key determinant of aphid transmission in each of these genera. Here, we report crystal structures of the NRTDs from the poleroviruses turnip yellow virus (TuYV) and potato leafroll virus (PLRV) at 1.53-Å and 2.22-Å resolution, respectively. These adopt a two-domain arrangement with a unique interdigitated topology and form highly conserved dimers that are stabilized by a C-terminal peptide that is critical for proper folding. We demonstrate that the PLRV NRTD can act as an inhibitor of virus transmission and identify NRTD mutant variants that are lethal to aphids. Sequence conservation argues that enamovirus and luteovirus NRTDs will follow the same structural blueprint, which affords a biological approach to block the spread of these agricultural pathogens in a generalizable manner.

Larval Diet Abundance Influences Size and Composition of the Midgut Microbiota of Aedes aegypti Mosquitoes.

The midgut microbiota of the yellow fever mosquito Aedes aegypti impacts pathogen susceptibility and transmission by this important vector species. However, factors influencing the composition and size of the microbiome in mosquitoes are poorly understood. We investigated the impact of larval diet abundance during development on the composition and size of the larval and adult microbiota by rearing Aedes aegypti under four larval food regimens, ranging from nutrient deprivation to nutrient excess. We assessed the persistent impacts of larval diet availability on the microbiota of the larval breeding water, larval mosquitoes, and adult mosquitoes under sugar and blood fed conditions using qPCR and high-throughput 16S amplicon sequencing to determine bacterial load and microbiota composition. Bacterial loads in breeding water increased with increasing larval diet. Larvae reared with the lowest diet abundance had significantly fewer bacteria than larvae from two higher diet treatments, but not from the highest diet abundance. Adults from the lowest diet abundance treatment had significantly fewer bacteria in their midguts compared to all higher diet abundance treatments. Larval diet amount also had a significant impact on microbiota composition, primarily within larval breeding water and larvae. Increasing diet correlated with increased relative levels of Enterobacteriaceae and Flavobacteriaceae and decreased relative levels of Sphingomonadaceae. Multiple individual OTUs were significantly impacted by diet including one mapping to the genus Cedecea, which increased with higher diet amounts. This was consistent across all sample types, including sugar fed and blood fed adults. Taken together, these data suggest that availability of diet during development can cause lasting shifts in the size and composition of the microbiota in the disease vector Aedes aegypti.

Detailed Analyses of Zika Virus Tropism in Culex quinquefasciatus Reveal Systemic Refractoriness.

The role of Culex quinquefasciatus in Zika virus transmission has been debated since the epidemic of Zika occurred in the Americas in 2015 to 2016. The majority of studies have found no evidence that C. quinquefasciatus or other Culex species are competent vectors of Zika virus, and the few studies that have proposed Zika vector status for C. quinquefasciatus have relied predominantly on quantitative real-time PCR (qRT-PCR) for viral detection. We assessed the infectious range of pre- and post-epidemic Zika virus isolates in order to classify mosquito samples based on titer infectiousness and demonstrated that two strains of C. quinquefasciatus, including one previously found to be competent, are highly resistant to infection with these Zika isolates compared to Aedes aegypti and are not competent for virus transmission. Further dissection of the dynamics of Zika exposure in both A. aegypti and C. quinquefasciatus revealed that while virus transmission by C. quinquefasciatus is blocked at the levels of the midgut and salivary glands, viral RNA persists in these tissues for prolonged periods post-exposure. We assessed Zika entry dynamics in both Aedes and Culex cells, and our results suggest that Zika virus infection in Culex cells may be blocked downstream of cell entry. These findings strongly suggest that C. quinquefasciatus is not a vector of Zika virus and additionally inform the use of qRT-PCR in vector competence assays as well as our understanding of barriers to arbovirus infection in non-susceptible mosquito species.IMPORTANCE Understanding which mosquito species transmit an emerging arbovirus is critical to effective vector control. During the Zika virus epidemic in 2015 to 2016, Aedes mosquitoes were confirmed as vectors. However, studies addressing the vector status of Culex quinquefasciatus mosquitoes presented conflicting evidence and remain an outstanding source of confusion in the field. Here, we established a robust cell-based assay to identify infectious titers of Zika virus and assessed the virus titers in C. quinquefasciatus by quantitative real-time PCR (qRT-PCR). We found that while low levels of virus were detected in C. quinquefasciatus, these titers did not correspond to infectious virus, and these mosquitoes did not transmit virus in the saliva. We also present evidence that the virus may enter Culex cells before infection is disrupted. Our findings are important for future studies incriminating vector species using qRT-PCR for virus detection and offer new information on how virus transmission is blocked by mosquitoes.

Hydrogen cyanide produced by the soil bacterium Chromobacterium sp. Panama contributes to mortality in Anopheles gambiae mosquito larvae.

Mosquito larvae continuously encounter microbes in their aquatic environment, which serve as food and play a critical role in successful development. In previous work, we isolated a Chromobacterium sp. (C.sp_P) with larvicidal activity from the midgut of dengue vector Aedes mosquitoes in Panama. In this study, we found a positive correlation between initial concentrations of C.sp_P and larval mortality rates, and that C.sp_P is more efficient at inducing larval mortality in a high nutrient environment. Multiple Chromobacterium species induce larval mortality with similar efficacy to C.sp_P except for C. subtsugae. We also found that a non-lethal dose of C.sp_P lengthens development time and increases mortality over multiple developmental stages, suggesting persistent effects of exposure. Additionally, we showed that larvicidal activity persists in the larval breeding water after removal of live bacteria, and that the larvicidal factor in C.sp_P-treated water is smaller than 3 kDa, heat resistant to 90 °C, and lost after vacuum centrifugation. We showed that C.sp_P produces hydrogen cyanide in culture and in larval water at concentrations sufficient to kill An. gambiae larvae, and treatment of the larval water with a cyanide antidote eliminated larvicidal activity. We conclude that a potential mechanism by which C.sp_P can induce larval mortality is via production of hydrogen cyanide.

Cadherin-26 (CDH26) regulates airway epithelial cell cytoskeletal structure and polarity.

Polarization of the airway epithelial cells (AECs) in the airway lumen is critical to the proper function of the mucociliary escalator and maintenance of lung health, but the cellular requirements for polarization of AECs are poorly understood. Using human AECs and cell lines, we demonstrate that cadherin-26 (CDH26) is abundantly expressed in differentiated AECs, localizes to the cell apices near ciliary membranes, and has functional cadherin domains with homotypic binding. We find a unique and non-redundant role for CDH26, previously uncharacterized in AECs, in regulation of cell-cell contact and cell integrity through maintaining cytoskeletal structures. Overexpression of CDH26 in cells with a fibroblastoid phenotype increases contact inhibition and promotes monolayer formation and cortical actin structures. CDH26 expression is also important for localization of planar cell polarity proteins. Knockdown of CDH26 in AECs results in loss of cortical actin and disruption of CRB3 and other proteins associated with apical polarity. Together, our findings uncover previously unrecognized functions for CDH26 in the maintenance of actin cytoskeleton and apicobasal polarity of AECs.

Amino acid metabolic signaling influences Aedes aegypti midgut microbiome variability.

The mosquito midgut microbiota has been shown to influence vector competence for multiple human pathogens. The microbiota is highly variable in the field, and the sources of this variability are not well understood, which limits our ability to understand or predict its effects on pathogen transmission. In this work, we report significant variation in female adult midgut bacterial load between strains of A. aegypti which vary in their susceptibility to dengue virus. Composition of the midgut microbiome was similar overall between the strains, with 81-92% of reads coming from the same five bacterial families, though we did detect differences in the presence of some bacterial families including Flavobacteriaceae and Entobacteriaceae. We conducted transcriptomic analysis on the two mosquito strains that showed the greatest difference in bacterial load, and found that they differ in transcript abundance of many genes implicated in amino acid metabolism, in particular the branched chain amino acid degradation pathway. We then silenced this pathway by targeting multiple genes using RNA interference, which resulted in strain-specific bacterial proliferation, thereby eliminating the difference in midgut bacterial load between the strains. This suggests that the branched chain amino acid (BCAA) degradation pathway controls midgut bacterial load, though the mechanism underlying this remains unclear. Overall, our results indicate that amino acid metabolism can act to influence the midgut microbiota. Moreover, they suggest that genetic or physiological variation in BCAA degradation pathway activity may in part explain midgut microbiota variation in the field.

Aedes aegypti Molecular Responses to Zika Virus: Modulation of Infection by the Toll and Jak/Stat Immune Pathways and Virus Host Factors.

Zika (ZIKV) and dengue virus (DENV) are transmitted to humans by Aedes mosquitoes. However, the molecular interactions between the vector and ZIKV remain largely unexplored. In this work, we further investigated the tropism of ZIKV in two different Aedes aegypti strains and show that the virus infection kinetics, tissue migration, and susceptibility to infection differ between mosquito strains. We also compare the vector transcriptome changes upon ZIKV or DENV infection demonstrating that 40% of the mosquito's midgut infection-responsive transcriptome is virus-specific at 7 days after virus ingestion. Regulated genes included key factors of the mosquito's anti-viral immunity. Comparison of the ZIKV and DENV infection-responsive transcriptome data to those available for yellow fever virus and West Nile virus identified 26 genes likely to play key roles in virus infection of Aedes mosquitoes. Through reverse genetic analyses, we show that the Toll and the Jak/Stat innate immune pathways mediate increased resistance to ZIKV infection, and the conserved DENV host factors vATPase and inosine-5'-monophosphate dehydrogenase are also utilized for ZIKV infection.

IL1RL1 asthma risk variants regulate airway type 2 inflammation.

Genome-wide association studies of asthma have identified genetic variants in the IL1RL1 gene, but the molecular mechanisms conferring risk are unknown. IL1RL1 encodes the ST2 receptor (ST2L) for IL-33 and an inhibitory decoy receptor (sST2). IL-33 promotes type 2 inflammation, which is present in some but not all asthmatics. We find that two single nucleotide polymorphisms (SNPs) in IL1RL1 - rs1420101 and rs11685480 - are strongly associated with plasma sST2 levels, though neither is an expression quantitative trait locus (eQTL) in whole blood. Rather, rs1420101 and rs11685480 mark eQTLs in airway epithelial cells and distal lung parenchyma, respectively. We find that the genetically determined plasma sST2 reservoir, derived from the lung, neutralizes IL-33 activity, and these eQTL SNPs additively increase the risk of airway type 2 inflammation among asthmatics. These risk variants define a population of asthmatics at risk of IL-33-driven type 2 inflammation.