A next-generation intranasal trivalent MMS vaccine induces durable and broad protection against SARS-CoV-2 variants of concern.

As SARS-CoV-2 variants of concern (VoCs) that evade immunity continue to emerge, next-generation adaptable COVID-19 vaccines which protect the respiratory tract and provide broader, more effective, and durable protection are urgently needed. Here, we have developed one such approach, a highly efficacious, intranasally delivered, trivalent measles-mumps-SARS-CoV-2 spike (S) protein (MMS) vaccine candidate that induces robust systemic and mucosal immunity with broad protection. This vaccine candidate is based on three components of the MMR vaccine, a measles virus Edmonston and the two mumps virus strains [Jeryl Lynn 1 (JL1) and JL2] that are known to provide safe, effective, and long-lasting protective immunity. The six proline-stabilized prefusion S protein (preS-6P) genes for ancestral SARS-CoV-2 WA1 and two important SARS-CoV-2 VoCs (Delta and Omicron BA.1) were each inserted into one of these three viruses which were then combined into a trivalent "MMS" candidate vaccine. Intranasal immunization of MMS in IFNAR1-/- mice induced a strong SARS-CoV-2-specific serum IgG response, cross-variant neutralizing antibodies, mucosal IgA, and systemic and tissue-resident T cells. Immunization of golden Syrian hamsters with MMS vaccine induced similarly high levels of antibodies that efficiently neutralized SARS-CoV-2 VoCs and provided broad and complete protection against challenge with any of these VoCs. This MMS vaccine is an efficacious, broadly protective next-generation COVID-19 vaccine candidate, which is readily adaptable to new variants, built on a platform with a 50-y safety record that also protects against measles and mumps.

A highly efficacious live attenuated mumps virus-based SARS-CoV-2 vaccine candidate expressing a six-proline stabilized prefusion spike.

With the rapid increase in SARS-CoV-2 cases in children, a safe and effective vaccine for this population is urgently needed. The MMR (measles/mumps/rubella) vaccine has been one of the safest and most effective human vaccines used in infants and children since the 1960s. Here, we developed live attenuated recombinant mumps virus (rMuV)-based SARS-CoV-2 vaccine candidates using the MuV Jeryl Lynn (JL2) vaccine strain backbone. The soluble prefusion SARS-CoV-2 spike protein (preS) gene, stablized by two prolines (preS-2P) or six prolines (preS-6P), was inserted into the MuV genome at the P-M or F-SH gene junctions in the MuV genome. preS-6P was more efficiently expressed than preS-2P, and preS-6P expression from the P-M gene junction was more efficient than from the F-SH gene junction. In mice, the rMuV-preS-6P vaccine was more immunogenic than the rMuV-preS-2P vaccine, eliciting stronger neutralizing antibodies and mucosal immunity. Sera raised in response to the rMuV-preS-6P vaccine neutralized SARS-CoV-2 variants of concern, including the Delta variant equivalently. Intranasal and/or subcutaneous immunization of IFNAR1-/- mice and golden Syrian hamsters with the rMuV-preS-6P vaccine induced high levels of neutralizing antibodies, mucosal immunoglobulin A antibody, and T cell immune responses, and were completely protected from challenge by both SARS-CoV-2 USA-WA1/2020 and Delta variants. Therefore, rMuV-preS-6P is a highly promising COVID-19 vaccine candidate, warranting further development as a tetravalent MMR vaccine, which may include protection against SARS-CoV-2.

SARS-CoV-2 prefusion spike protein stabilized by six rather than two prolines is more potent for inducing antibodies that neutralize viral variants of concern.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the main target for neutralizing antibodies (NAbs). The S protein trimer is anchored in the virion membrane in its prefusion (preS) but metastable form. The preS protein has been stabilized by introducing two or six proline substitutions, to generate stabilized, soluble 2P or HexaPro (6P) preS proteins. Currently, it is not known which form is the most immunogenic. Here, we generated recombinant vesicular stomatitis virus (rVSV) expressing preS-2P, preS-HexaPro, and native full-length S, and compared their immunogenicity in mice and hamsters. The rVSV-preS-HexaPro produced and secreted significantly more preS protein compared to rVSV-preS-2P. Importantly, rVSV-preS-HexaPro triggered significantly more preS-specific serum IgG antibody than rVSV-preS-2P in both mice and hamsters. Antibodies induced by preS-HexaPro neutralized the B.1.1.7, B.1.351, P.1, B.1.427, and B.1.617.2 variants approximately two to four times better than those induced by preS-2P. Furthermore, preS-HexaPro induced a more robust Th1-biased cellular immune response than preS-2P. A single dose (104 pfu) immunization with rVSV-preS-HexaPro and rVSV-preS-2P provided complete protection against challenge with mouse-adapted SARS-CoV-2 and B.1.617.2 variant, whereas rVSV-S only conferred partial protection. When the immunization dose was lowered to 103 pfu, rVSV-preS-HexaPro induced two- to sixfold higher antibody responses than rVSV-preS-2P in hamsters. In addition, rVSV-preS-HexaPro conferred 70% protection against lung infection whereas only 30% protection was observed in the rVSV-preS-2P. Collectively, our data demonstrate that both preS-2P and preS-HexaPro are highly efficacious but preS-HexaPro is more immunogenic and protective, highlighting the advantages of using preS-HexaPro in the next generation of SARS-CoV-2 vaccines.

Caspase-4/11 exacerbates disease severity in SARS-CoV-2 infection by promoting inflammation and immunothrombosis.

Severe acute respiratory syndrome coronavirus 2 (SARS­CoV-2) is a worldwide health concern, and new treatment strategies are needed. Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive. Here, we show that human caspase-4 (CASP4) and its mouse homolog, caspase-11 (CASP11), are up-regulated in SARS­CoV-2 infections and that CASP4 expression correlates with severity of SARS­CoV-2 infection in humans. SARS­CoV-2­infected Casp11−/− mice were protected from severe weight loss and lung pathology, including blood vessel damage, compared to wild-type (WT) mice and mice lacking the caspase downstream effector gasdermin-D (Gsdmd−/−). Notably, viral titers were similar regardless of CASP11 knockout. Global transcriptomics of SARS­CoV-2­infected WT, Casp11−/−, and Gsdmd−/− lungs identified restrained expression of inflammatory molecules and altered neutrophil gene signatures in Casp11−/− mice. We confirmed that protein levels of inflammatory mediators interleukin (IL)-1ß, IL-6, and CXCL1, as well as neutrophil functions, were reduced in Casp11−/− lungs. Additionally, Casp11−/− lungs accumulated less von Willebrand factor, a marker for endothelial damage, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity. Overall, our results demonstrate that CASP4/11 promotes detrimental SARS­CoV-2­induced inflammation and coagulopathy, largely independently of GSDMD, identifying CASP4/11 as a promising drug target for treatment and prevention of severe COVID-19.

Inhibition of elastase enhances the adjuvanticity of alum and promotes anti-SARS-CoV-2 systemic and mucosal immunity.

Alum, used as an adjuvant in injected vaccines, promotes T helper 2 (Th2) and serum antibody (Ab) responses. However, it fails to induce secretory immunoglobulin (Ig) A (SIgA) in mucosal tissues and is poor in inducing Th1 and cell-mediated immunity. Alum stimulates interleukin 1 (IL-1) and the recruitment of myeloid cells, including neutrophils. We investigated whether neutrophil elastase regulates the adjuvanticity of alum, and whether a strategy targeting neutrophil elastase could improve responses to injected vaccines. Mice coadministered a pharmacological inhibitor of elastase, or lacking elastase, developed high-affinity serum IgG and IgA antibodies after immunization with alum-adsorbed protein vaccines, including the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). These mice also developed broader antigen-specific CD4+ T cell responses, including high Th1 and T follicular helper (Tfh) responses. Interestingly, in the absence of elastase activity, mucosal SIgA responses were induced after systemic immunization with alum as adjuvant. Importantly, lack or suppression of elastase activity enhanced the magnitude of anti-SARS-CoV-2 spike subunit 1 (S1) antibodies, and these antibodies reacted with the same epitopes of spike 1 protein as sera from COVID-19 patients. Therefore, suppression of neutrophil elastase could represent an attractive strategy for improving the efficacy of alum-based injected vaccines for the induction of broad immunity, including mucosal immunity.

A safe and highly efficacious measles virus-based vaccine expressing SARS-CoV-2 stabilized prefusion spike.

The current pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights an urgent need to develop a safe, efficacious, and durable vaccine. Using a measles virus (rMeV) vaccine strain as the backbone, we developed a series of recombinant attenuated vaccine candidates expressing various forms of the SARS-CoV-2 spike (S) protein and its receptor binding domain (RBD) and evaluated their efficacy in cotton rat, IFNAR-/-mice, IFNAR-/--hCD46 mice, and golden Syrian hamsters. We found that rMeV expressing stabilized prefusion S protein (rMeV-preS) was more potent in inducing SARS-CoV-2-specific neutralizing antibodies than rMeV expressing full-length S protein (rMeV-S), while the rMeVs expressing different lengths of RBD (rMeV-RBD) were the least potent. Animals immunized with rMeV-preS produced higher levels of neutralizing antibody than found in convalescent sera from COVID-19 patients and a strong Th1-biased T cell response. The rMeV-preS also provided complete protection of hamsters from challenge with SARS-CoV-2, preventing replication in lungs and nasal turbinates, body weight loss, cytokine storm, and lung pathology. These data demonstrate that rMeV-preS is a safe and highly efficacious vaccine candidate, supporting its further development as a SARS-CoV-2 vaccine.

Targeting the EGFR-ERK axis using the compatible solute ectoine to stabilize CFTR mutant F508del.

Mutations in the CFTR gene lead to cystic fibrosis, a genetic disease associated with chronic infection and inflammation and ultimately respiratory failure. The most common CF-causing mutation is F508del and CFTR modulators (correctors and potentiators) are being developed to rescue its trafficking and activity defects. However, there are currently no modulators that stabilize the rescued membrane F508del-CFTR which is endocytosed and quickly degraded resulting in a shorter half-life than wild-type (WT). We previously reported that the extracellular signal-regulated kinase (ERK) MAPK pathway is involved in CFTR degradation upon cigarette smoke exposure. Interestingly, we found that ERK phosphorylation was increased in CF human bronchial epithelial (HBE) cells (CF-HBE41o- and primary CF-HBE) compared to non-CF controls, and this was likely due to signaling by the epidermal growth factor receptor (EGFR). EGFR can be activated by several ligands, and we provide evidence that amphiregulin (AREG) is important for activating this signaling axis in CF. The natural osmolyte ectoine stabilizes membrane macromolecules. We show that ectoine decreases ERK phosphorylation, increases the half-life of rescued CFTR, and increases CFTR-mediated chloride transport in combination with the CFTR corrector VX-661. Additionally, ectoine reduces production of AREG and interleukin-8 by CF primary bronchial epithelial cells. In conclusion, EGFR-ERK signaling negatively regulates CFTR and is hyperactive in CF, and targeting this axis with ectoine may prove beneficial for CF patients.

A Methyltransferase-Defective Vesicular Stomatitis Virus-Based SARS-CoV-2 Vaccine Candidate Provides Complete Protection against SARS-CoV-2 Infection in Hamsters.

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to dramatic economic and health burdens. Although the worldwide SARS-CoV-2 vaccination campaign has begun, exploration of other vaccine candidates is needed due to uncertainties with the current approved vaccines, such as durability of protection, cross-protection against variant strains, and costs of long-term production and storage. In this study, we developed a methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidate. We generated mtdVSVs expressing SARS-CoV-2 full-length spike (S) protein, S1, or its receptor-binding domain (RBD). All of these recombinant viruses grew to high titers in mammalian cells despite high attenuation in cell culture. The SARS-CoV-2 S protein and its truncations were highly expressed by the mtdVSV vector. These mtdVSV-based vaccine candidates were completely attenuated in both immunocompetent and immunocompromised mice. Among these constructs, mtdVSV-S induced high levels of SARS-CoV-2-specific neutralizing antibodies (NAbs) and Th1-biased T-cell immune responses in mice. In Syrian golden hamsters, the serum levels of SARS-CoV-2-specific NAbs triggered by mtdVSV-S were higher than the levels of NAbs in convalescent plasma from recovered COVID-19 patients. In addition, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 replication in lung and nasal turbinate tissues, cytokine storm, and lung pathology. Collectively, our data demonstrate that mtdVSV expressing SARS-CoV-2 S protein is a safe and highly efficacious vaccine candidate against SARS-CoV-2 infection. IMPORTANCE Viral mRNA cap methyltransferase (MTase) is essential for mRNA stability, protein translation, and innate immune evasion. Thus, viral mRNA cap MTase activity is an excellent target for development of live attenuated or live vectored vaccine candidates. Here, we developed a panel of MTase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidates expressing full-length S, S1, or several versions of the RBD. These mtdVSV-based vaccine candidates grew to high titers in cell culture and were completely attenuated in both immunocompetent and immunocompromised mice. Among these vaccine candidates, mtdVSV-S induces high levels of SARS-CoV-2-specific neutralizing antibodies (Nabs) and Th1-biased immune responses in mice. Syrian golden hamsters immunized with mtdVSV-S triggered SARS-CoV-2-specific NAbs at higher levels than those in convalescent plasma from recovered COVID-19 patients. Furthermore, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 challenge. Thus, mtdVSV is a safe and highly effective vector to deliver SARS-CoV-2 vaccine.

In Vivo Tumorigenesis, Osteolytic Sarcomas, and Tumorigenic Cell Lines from Transgenic Mice Expressing the Human T-Lymphotropic Virus Type 1 (HTLV-1) Tax Viral Oncogene.

Human T-lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukemia, a disease commonly associated with hypercalcemia and osteolysis. There is no effective treatment for HTLV-1, and the osteolytic mechanisms are not fully understood. Mice expressing the HTLV-1 oncogene Tax, driven by the human granzyme B promoter (Tax+), develop osteolytic tumors. To investigate the progression of the bone-invasive malignancies, wild-type, Tax+, and Tax+/interferon-γ-/- mice were assessed using necropsy, histologic examination, IHC analysis, flow cytometry, and advanced imaging. Tax+ and Tax+/interferon-γ-/- malignancies of the ear, tail, and foot comprised poorly differentiated, round to spindle-shaped cells with prominent neutrophilic infiltrates. Tail tumors originated from muscle, nerve, and/or tendon sheaths, with frequent invasion into adjacent bone. F4/80+ and anti-mouse CD11b (Mac-1)+ histiocytic cells predominated within the tumors. Three Tax+/interferon-γ-/- cell lines were generated for in vivo allografts, in vitro gene expression and bone resorption assays. Two cell lines were of monocyte/macrophage origin, and tumors formed in vivo in all three. Differences in Pthrp, Il6, Il1a, Il1b, and Csf3 expression in vitro were correlated with differences in in vivo plasma calcium levels, tumor growth, metastasis, and neutrophilic inflammation. Tax+ mouse tumors were classified as bone-invasive histiocytic sarcomas. The cell lines are ideal for further examination of the role of HTLV-1 Tax in osteolytic tumor formation and the development of hypercalcemia and tumor-associated inflammation.

Vesicular Stomatitis Virus and DNA Vaccines Expressing Zika Virus Nonstructural Protein 1 Induce Substantial but Not Sterilizing Protection against Zika Virus Infection.

The nonstructural protein 1 (NS1) of several flaviviruses, including West Nile, dengue, and yellow fever viruses, is capable of inducing variable degrees of protection against flavivirus infection in animal models. However, the immunogenicity of NS1 protein of Zika virus (ZIKV) is less understood. Here, we determined the efficacy of ZIKV NS1-based vaccine candidates using two delivery platforms, methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV) and a DNA vaccine. We first show that expression of ZIKV NS1 could be significantly enhanced by optimizing the signal peptide. A single dose of mtdVSV-NS1-based vaccine or two doses of DNA vaccine induced high levels of NS1-specfic antibody and T cell immune responses but provided only partial protection against ZIKV viremia in BALB/c mice. In Ifnar1-/- mice, neither NS1-based vaccine provided protection against a lethal high dose (105 PFU) ZIKV challenge, but mtdVSV-NS1-based vaccine prevented deaths from a low dose (103 PFU) challenge, though they experienced viremia and body weight loss. We conclude that ZIKV NS1 alone conferred substantial, but not complete, protection against ZIKV infection. Nevertheless, these results highlight the value of ZIKV NS1 for vaccine development.IMPORTANCE Most Zika virus (ZIKV) vaccine research has focused on the E or prM-E proteins and the induction of high levels of neutralizing antibodies. However, these ZIKV neutralizing antibodies cross-react with other flaviviruses, which may aggravate the disease via an antibody-dependent enhancement (ADE) mechanism. ZIKV NS1 protein may be an alternative antigen for vaccine development, since antibodies to NS1 do not bind to the virion, thereby eliminating the risk of ADE. Here, we show that recombinant VSV and DNA vaccines expressing NS1, alone, confer partial protection against ZIKV infection in both immunocompetent and immunodeficient mice, highlighting the value of NS1 as a potential vaccine candidate.

Caspase-11 regulates lung inflammation in response to house dust mites.

Asthma is an inflammatory lung disorder characterized by mucus hypersecretion, cellular infiltration, and bronchial hyper-responsiveness. House dust mites (HDM) are the most prevalent cause of allergic sensitization. Canonical and noncanonical inflammasomes are multiprotein complexes that assemble in response to pathogen or danger-associated molecular patterns (PAMPs or DAMPs). Murine caspase-11 engages the noncanonical inflammasome. We addressed the role of caspase-11 in mediating host responses to HDM and subsequent allergic inflammation using caspase-11-/- mice, which lack caspase-11 while express caspase-1. We found that HDM induce caspase-11 expression in vitro. The presence of IL-4 and IL-13 promote caspase-11 expression. Additionally, caspase-11-/- macrophages show reduced release of IL-6, IL-12, and KC, and express lower levels of costimulatory molecules (e.g., CD40, CD86 and MHCII) in response to HDM stimulation. Notably, HDM sensitization of caspase-11-/- mice resulted in similar levels of IgE responses and hypothermia in response to nasal HDM challenge compared to WT. However, analysis of cell numbers and cytokines in bronchiolar alveolar lavage fluid (BALF) and histopathology of representative lung segments demonstrate altered inflammatory responses and reduced neutrophilia in the airways of the caspase-11-/- mice. These findings indicate that caspase-11 regulates airway inflammation in response to HDM exposure.

Inhibitors of elastase stimulate murine B lymphocyte differentiation into IgG- and IgA-producing cells.

It is well established that dendritic cells and macrophages play a role in antigen presentation to B and T cells and in shaping B and T cell responses via cytokines they produce. We have previously reported that depletion of neutrophils improves the production of mucosal IgA after sublingual immunization with Bacillus anthracis edema toxin as adjuvant. These past studies also demonstrated that an inverse correlation exists between the number of neutrophils and production of IgA by B cells. Using specific inhibitors of elastase, we addressed whether the elastase activity of neutrophil could be the factor that interferes with production of IgA and possibly other immunoglobulin isotypes. We found that murine splenocytes and mesenteric lymph node cells cultured for 5 days in the presence of neutrophil elastase inhibitors secreted higher levels of IgG and IgA than cells cultured in the absence of inhibitors. The effect of the inhibitors was dose-dependent and was consistent with increased frequency of CD138+ cells expressing IgG or IgA. Finally, neutrophil elastase inhibitors increased transcription of mRNA for AID, IL-10, BAFF and APRIL, factors involved in B cell differentiation. These findings identify inhibitors of elastase as potential adjuvants for increasing production of antibodies.

Reduced expression of the Ion channel CFTR contributes to airspace enlargement as a consequence of aging and in response to cigarette smoke in mice.

Chronic Obstructive Pulmonary Disease (COPD) is a complex disease resulting in respiratory failure and represents the third leading cause of global death. The two classical phenotypes of COPD are chronic bronchitis and emphysema. Owing to similarities between chronic bronchitis and the autosomal-recessive disease Cystic Fibrosis (CF), a significant body of research addresses the hypothesis that dysfunctional CF Transmembrane Conductance Regulator (CFTR) is implicated in the pathogenesis of COPD. Much less attention has been given to emphysema in this context, despite similarities between the two diseases. These include early-onset cellular senescence, similar comorbidities, and the finding that CF patients develop emphysema as they age. To determine a potential role for CFTR dysfunction in the development of emphysema, Cftr+/+ (Wild-type; WT), Cftr+/- (heterozygous), and Cftr-/- (knock-out; KO) mice were aged or exposed to cigarette smoke and analyzed for airspace enlargement. Aged knockout mice demonstrated increased alveolar size compared to age-matched wild-type and heterozygous mice. Furthermore, both heterozygous and knockout mice developed enlarged alveoli compared to their wild-type counterparts following chronic smoke exposure. Taken into consideration with previous findings that cigarette smoke leads to reduced CFTR function, our findings suggest that decreased CFTR expression sensitizes the lung to the effects of cigarette smoke. These findings may caution normally asymptomatic CF carriers against exposure to cigarette smoke; as well as highlight emphysema as a future challenge for CF patients as they continue to live longer. More broadly, our data, along with clinical findings, may implicate CFTR dysfunction in a pathology resembling accelerated aging.

Inducing Mucosal IgA: A Challenge for Vaccine Adjuvants and Delivery Systems.

Mucosal IgA or secretory IgA (SIgA) are structurally equipped to resist chemical degradation in the harsh environment of mucosal surfaces and enzymes of host or microbial origin. Production of SIgA is finely regulated, and distinct T-independent and T-dependent mechanisms orchestrate Ig α class switching and SIgA responses against commensal and pathogenic microbes. Most infectious pathogens enter the host via mucosal surfaces. To provide a first line of protection at these entry ports, vaccines are being developed to induce pathogen-specific SIgA in addition to systemic immunity achieved by injected vaccines. Mucosal or epicutaneous delivery of vaccines helps target the inductive sites for SIgA responses. The efficacy of such vaccines relies on the identification and/or engineering of vaccine adjuvants capable of supporting the development of SIgA alongside systemic immunity and delivery systems that improve vaccine delivery to the targeted anatomic sites and immune cells.

Intranasal delivery of influenza antigen by nanoparticles, but not NKT-cell adjuvant differentially induces the expression of B-cell activation factors in mice and swine.

Intranasal vaccination of pigs with poly lactic-co-glycolic acid and polyanhydride nanoparticles delivered inactivated influenza virus provides cross-reactive T-cell response, but not antibody response, resulting in incomplete protection and no reduction in nasal virus shedding. Expression of BAFF and Th2 transcription factor GATA-3 were downregulated in lungs of pigs vaccinated with influenza nanovaccine, but in mice it upregulated the expression of BAFF and cytokine TGFß in cervical lymph nodes. However, the intranasal iNKT cell adjuvant, α-Galctosylceramide upregulates the expression of BAFF in pig lungs. In conclusion, expression of BAFF is differentially regulated by intranasal nanovaccine and α-Galctosylceramide in pig respiratory tract.

The psychoactive substance of cannabis Δ9-tetrahydrocannabinol (THC) negatively regulates CFTR in airway cells.

BACKGROUND: Marijuana consumption is on the rise in the US but the health benefits of cannabis smoking are controversial and the impact of cannabis components on lung homeostasis is not well-understood. Lung function requires a fine regulation of the ion channel CFTR, which is responsible for fluid homeostasis and mucocilliary clearance. The goal of this study was to assess the effect that exposure to Δ9-tetrahydrocannabinol (THC), the psychoactive substance present in marijuana, has on CFTR expression and function. METHODS: Cultures of human bronchial epithelial cell line 16HBE14o- and primary human airway epithelial cells were exposed to THC. The expression of CFTR protein was determined by immunoblotting and CFTR function was measured using Ussing chambers. We also used specific pharmacological inhibitors of EGFR and ERK to determine the role of this pathway in THC-induced regulation of CFTR. RESULTS: THC decreased CFTR protein expression in primary human bronchial epithelial cells. This decrease was associated with reduced CFTR-mediated short-circuit currents. THC also induced activation of the ERK MAPK pathway via activation of EGFR. Inhibition of EGFR or MEK/ERK prevented THC-induced down regulation of CFTR protein expression. CONCLUSIONS AND GENERAL SIGNIFICANCE: THC negatively regulates CFTR and this is mediated through the EGFR/ERK axis. This study provides the first evidence that THC present in marijuana reduces the expression and function of CFTR in airway epithelial cells.

IL-17A promotes susceptibility during experimental visceral leishmaniasis caused by Leishmania donovani.

Leishmania donovani is an intracellular parasite that infects professional phagocytes and causes visceral leishmaniasis (VL). The immune response during VL has been extensively studied in the context of T-helper (Th)1 and Th2 responses. Immunity against this parasite is dependent on IFN-γ production and subsequent macrophage activation, and the Th2 response promotes granuloma formation. The cytokine IL-17A is associated with neutrophilic inflammation. Depletion of neutrophils during experimental VL results in enhanced parasitic loads. Furthermore, although patients resistant to VL showed enhanced levels of IL-17A in circulation, little is known about the role of IL-17A during VL infection. Here, we used IL-17A-deficient mice and IL-17A reporter mice to address the role of IL-17A during VL. IL-17A(-/-) mice were highly resistant to VL infection, showing decreased parasites in the liver and spleen. This unexpected phenotype was associated with enhanced IFN-γ production by T cells and decreased accumulation of neutrophils and monocytes, resulting in reduced number of granulomas. We also found γδ T and Th17 cells as the main IL-17A(+) cells during VL infection. Our data reveal an unexpected role of IL-17A rendering susceptibility against L. donovani by regulating the IFN-γ response and promoting detrimental inflammation.

Cigarette smoke exposure reveals a novel role for the MEK/ERK1/2 MAPK pathway in regulation of CFTR.

BACKGROUND: Cystic fibrosis transmembrane conductance regulator plays a key role in maintenance of lung fluid homeostasis. Cigarette smoke decreases CFTR expression in the lung but neither the mechanisms leading to CFTR loss, nor potential ways to prevent its loss have been identified to date. METHODS: The molecular mechanisms leading to down-regulation of CFTR by cigarette smoke were determined using pharmacologic inhibitors and silencing ribonucleic acids (RNAs). RESULTS: Using human bronchial epithelial cells, here we show that cigarette smoke induces degradation of CFTR that is attenuated by lysosomal inhibitors, but not proteasome inhibitors. Cigarette smoke can activate multiple signaling pathways in airway epithelial cells, including the MEK/Erk1/2 MAPK (MEK: mitogen-activated protein kinase/ERK kinase Erk1/2: extracellular signal-regulated kinase 1/2 MAPK: Mitogen-activated protein kinase) pathway regulating cell survival. Interestingly, pharmacological inhibition of the MEK/Erk1/2 MAPK pathway prevented the loss of plasma membrane CFTR upon cigarette smoke exposure. Similarly, decreased expression of Erk1/2 using silencing RNAs prevented the suppression of CFTR protein by cigarette smoke. Conversely, specific inhibitors of the c-Jun N-terminal kinase (JNK) or p38 MAPK pathways had no effect on CFTR decrease after cigarette smoke exposure. In addition, inhibition of the MEK/Erk1/2 MAPK pathway prevented the reduction of the airway surface liquid observed upon cigarette smoke exposure of primary human airway epithelial cells. Finally, addition of the antioxidant N-acetylcysteine inhibited activation of Erk1/2 by cigarette smoke and precluded the cigarette smoke-induced decrease of CFTR. CONCLUSIONS: These results show that the MEK/Erk1/2 MAPK pathway regulates plasma membrane CFTR in human airway cells. GENERAL SIGNIFICANCE: The MEK/Erk1/2 MAPK pathway should be considered as a target for strategies to maintain/restore CFTR expression in the lung of smokers.

Use of Attenuated but Metabolically Competent Salmonella as a Probiotic To Prevent or Treat Salmonella Infection.

Salmonella enterica is among the most burdensome of foodborne disease agents. There are over 2,600 serovars that cause a range of disease manifestations ranging from enterocolitis to typhoid fever. While there are two vaccines in use in humans to protect against typhoid fever, there are none that prevent enterocolitis. If vaccines preventing enterocolitis were to be developed, they would likely protect against only one or a few serovars. In this report, we tested the hypothesis that probiotic organisms could compete for the preferred nutrient sources of Salmonella and thus prevent or treat infection. To this end, we added the fra locus, which encodes a utilization pathway for the Salmonella-specific nutrient source fructose-asparagine (F-Asn), to the probiotic bacterium Escherichia coli Nissle 1917 (Nissle) to increase its ability to compete with Salmonella in mouse models. We also tested a metabolically competent, but avirulent, Salmonella enterica serovar Typhimurium mutant for its ability to compete with wild-type Salmonella The modified Nissle strain became more virulent and less able to protect against Salmonella in some instances. On the other hand, the modified Salmonella strain was safe and effective in preventing infection with wild-type Salmonella While we tested for efficacy only against Salmonella Typhimurium, the modified Salmonella strain may be able to compete metabolically with most, if not all, Salmonella serovars, representing a novel approach to control of this pathogen.

Fructose-asparagine is a primary nutrient during growth of Salmonella in the inflamed intestine.

Salmonella enterica serovar Typhimurium (Salmonella) is one of the most significant food-borne pathogens affecting both humans and agriculture. We have determined that Salmonella encodes an uptake and utilization pathway specific for a novel nutrient, fructose-asparagine (F-Asn), which is essential for Salmonella fitness in the inflamed intestine (modeled using germ-free, streptomycin-treated, ex-germ-free with human microbiota, and IL10-/- mice). The locus encoding F-Asn utilization, fra, provides an advantage only if Salmonella can initiate inflammation and use tetrathionate as a terminal electron acceptor for anaerobic respiration (the fra phenotype is lost in Salmonella SPI1- SPI2- or ttrA mutants, respectively). The severe fitness defect of a Salmonella fra mutant suggests that F-Asn is the primary nutrient utilized by Salmonella in the inflamed intestine and that this system provides a valuable target for novel therapies.