Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them via mucociliary clearance (MCC)1,2. However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases1. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus1,3. Genetic variants are linked to diverse lung diseases4-6, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in the lungs. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally7. Apoptotic macrophages accumulated, phagocytosis was impaired, and IL-23 production was reduced inMuc5b−/− mice. By contrast, in Muc5b transgenic (Tg) mice, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum1,8. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%9-11. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.

The main regulators of leukocyte trafficking during inflammatory responses are chemokines. However, another class of recently identified chemotactic agents is extracellular cyclophilins, the proteins mostly known as receptors for the immunosuppressive drug, cyclosporine A. Cyclophilins can induce leukocyte chemotaxis in vitro and have been detected at elevated levels in inflamed tissues, suggesting that they might contribute to inflammatory responses. We recently identified CD147 as the main signaling receptor for cyclophilin A. In the current study we examined the contribution of cyclophilin-CD147 interactions to inflammatory responses in vivo using a mouse model of acute lung injury. Blocking cyclophilin-CD147 interactions by targeting CD147 (using anti-CD147 Ab) or cyclophilin (using nonimmunosuppressive cyclosporine A analog) reduced tissue neutrophilia by up to 50%, with a concurrent decrease in tissue pathology. These findings are the first to demonstrate the significant contribution of cyclophilins to inflammatory responses and provide a potentially novel approach for reducing inflammation-mediated diseases.

Primary biliary cirrhosis (PBC) is a classical autoimmune liver disease for which effective immunomodulatory therapy is lacking. Here we perform meta-analyses of discovery datasets from genome-wide association studies of European subjects (n=2,764 cases and 10,475 controls) followed by validation genotyping in an independent cohort (n=3,716 cases and 4261 controls). We discover and validate six previously unknown risk loci for PBC (Pcombined<5×10−8) and used pathway analysis to identify JAK-STAT/IL12/IL27 signaling and cytokine-cytokine pathways, for which relevant therapies exist.

f Dengue virus (DENV), a flavivirus of global importance, is transmitted to humans by mosquitoes. In this study, we developed in vitro and in vivo models of saliva-mediated enhancement of DENV infectivity. Serine protease activity in Aedes aegypti saliva augmented virus infectivity in vitro by proteolyzing extracellular matrix proteins, thereby increasing viral attachment to heparan sulfate proteoglycans and inducing cell migration. A serine protease inhibitor reduced saliva-mediated enhancement of DENV in vitro and in vivo, marked by a 100-fold reduction in DENV load in murine lymph nodes. A saliva-mediated infectivity enhancement screen of fractionated salivary gland extracts identified serine protease CLIPA3 as a putative cofactor, and short interfering RNA knockdown of CLIPA3 in mosquitoes demonstrated its role in influencing DENV infectivity. Molecules in mosquito saliva that facilitate viral infectivity in the vertebrate host provide novel targets that may aid in the prevention of disease.

Engineered alphavirus vectors expressing reporters of infection have been used for a number of years due to their relatively low costs for analysis of virus replication and the capacity to utilize imaging systems for longitudinal measurements of growth within single animals. In general, these vectors have been derived from Old World alphaviruses using a second viral subgenomic promoter to express the transgenes, placed either immediately after the nonstructural proteins or at the 3= end of the viral coding sequences. However, the relevance of these vectors to natural infections is questionable, as they have not been rigorously tested for virulence in vivo in comparison with parental viruses or for the retention of the reporter during replication. Here, we report construction of new expression vectors for two Old World arthritogenic alphaviruses (Sindbis and Chikungunya viruses) and two New World encephalitic alphaviruses (eastern and Venezuelan equine encephalitis viruses) based upon either fusion of the reporter protein in frame within nonstructural protein 3 (nsP3) or insertion of the reporter as a cleavable element between the capsid and PE2 structural proteins. We have compared these with a traditional 3= double subgenomic promoter virus expressing either a large, firefly luciferase (fLuc; 1,650 nucleotides), or small, NanoLuc (nLuc; 513 nucleotides), luminescent reporter protein. Results indicate that the nLuc is substantially more stable than fLuc during repeated rounds of infection regardless of the transgene location. However, the capsid-PE2 insertion and nsP3 fusion viruses exhibit the most authentic mimicking of parental virus infection regardless of expressed protein. IMPORTANCEAs more antiviral therapeutics and vaccines are developed, rapid and accurate in vivo modeling of their efficacy will be required. However, current alphavirus vectors expressing reporters of infection have not been extensively tested for accurate mimicking of the infection characteristics of unmodified parental viruses. Additionally, use of in vivo imaging systems detecting light emitted from luciferase reporters can significantly decrease costs associated with efficacy studies by minimizing numbers of animals. Herein we report development and testing of new expression vectors for Sindbis, Chikungunya, and eastern and Venezuelan equine encephalitis viruses and demonstrate that a small (ϳ500-nucleotide) reporter gene (NanoLuc; Promega) is very stable and causes a disease severity similar to that caused by unmodified parental viruses. In contrast, expression of larger reporters is very rapidly lost with virus replication and can be significantly attenuating. The utility of NanoLuc for in vivo imaging is also demonstrated.

A gold standard of antiviral vaccination has been the safe and effective live-attenuated 17D-based yellow fever virus (YFV) vaccines. Among more than 500 million vaccinees, only a handful of cases have been reported in which vaccinees developed a virulent wild type YFV infection. This efficacy is presumed to be the result of both neutralizing antibodies and a robust T cell response. However, the particular immune components required for protection against YFV have never been evaluated. An understanding of the immune mechanisms that underlie 17D-based vaccine efficacy is critical to the development of next-generation vaccines against flaviviruses and other pathogens. Here we have addressed this question for the first time using a murine model of disease. Similar to humans, vaccination elicited long-term protection against challenge, characterized by high neutralizing antibody titers and a robust T cell response that formed long-lived memory. Both CD4+ and CD8+ T cells were polyfunctional and cytolytic. Adoptive transfer of immune sera or CD4+ T cells provided partial protection against YFV, but complete protection was achieved by transfer of both immune sera and CD4+ T cells. Thus, robust CD4+ T cell activity may be a critical contributor to protective immunity elicited by highly effective live attenuated vaccines.

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Aedes aegypti is the primary vector of several medically relevant arboviruses including dengue virus (DENV) types 1–4. Ae. aegypti transmits DENV by inoculating virus-infected saliva into host skin during probing and feeding. Ae. aegypti saliva contains over one hundred unique proteins and these proteins have diverse functions, including facilitating blood feeding. Previously, we showed that Ae. aegypti salivary gland extracts (SGEs) enhanced dissemination of DENV to draining lymph nodes. In contrast, HPLC-fractionation revealed that some SGE components inhibited infection. Here, we show that D7 proteins are enriched in HPLC fractions that are inhibitory to DENV infection, and that recombinant D7 protein can inhibit DENV infection in vitro and in vivo. Further, binding assays indicate that D7 protein can directly interact with DENV virions and recombinant DENV envelope protein. These data reveal a novel role for D7 proteins, which inhibits arbovirus transmission to vertebrates through a direct interaction with virions.