Specific Inhibition of Orai1-mediated Calcium Signalling Resolves Inflammation and Clears Bacteria in an Acute Respiratory Distress Syndrome Model.

Rationale: Acute respiratory distress syndrome (ARDS) has an unacceptably high mortality rate (35%) and is without effective therapy. Orai1 is a Ca2+ channel involved in store-operated Ca2+ entry (SOCE), a process that exquisitely regulates inflammation. Orai1 is considered a druggable target, but no Orai1-specific inhibitors exist to date. Objectives: To evaluate whether ELD607, a first-in-class Orai1 antagonist, can treat ARDS caused by bacterial pneumonia in preclinical models. Methods: ELD607 pharmacology was evaluated in HEK293T cells and freshly isolated immune cells from patients with ARDS. A murine acute lung injury model caused by bacterial pneumonia was then used: mice were infected with Pseudomonas aeruginosa, Staphylococcus aureus, methicillin-resistant S. aureus, or multidrug-resistant P. aeruginosa and then treated with ELD607 intranasally. Measurements and Main Results: ELD607 specifically inhibited SOCE in HEK293T cells with a half-maximal inhibitory concentration of 9 nM. ELD607 was stable in ARDS airway secretions and inhibited SOCE in ARDS immune cells. In vivo, inhaled ELD607 significantly reduced neutrophilia and improved survival. Surprisingly, Orai1 inhibition by ELD607 caused a significant reduction in lung bacteria, including methicillin-resistant S. aureus. ELD607 worked as an immunomodulator that reduced cytokine levels, reduced neutrophilia, and promoted macrophage-mediated resolution of inflammation and clearance of bacteria. Indeed, when alveolar macrophages were depleted with inhaled clodronate, ELD607 was no longer able to resolve inflammation or clear bacteria. Conclusions: These data indicate that specific Orai1 inhibition by ELD607 may be a novel approach to reduce multiorgan inflammation and treat antibiotic-resistant bacteria.

Antiviral immunity via RIG-I-mediated recognition of RNA bearing 5'-diphosphates.

Mammalian cells possess mechanisms to detect and defend themselves from invading viruses. In the cytosol, the RIG-I-like receptors (RLRs), RIG-I (retinoic acid-inducible gene I; encoded by DDX58) and MDA5 (melanoma differentiation-associated gene 5; encoded by IFIH1) sense atypical RNAs associated with virus infection. Detection triggers a signalling cascade via the adaptor MAVS that culminates in the production of type I interferons (IFN-α and ß; hereafter IFN), which are key antiviral cytokines. RIG-I and MDA5 are activated by distinct viral RNA structures and much evidence indicates that RIG-I responds to RNAs bearing a triphosphate (ppp) moiety in conjunction with a blunt-ended, base-paired region at the 5'-end (reviewed in refs 1, 2, 3). Here we show that RIG-I also mediates antiviral responses to RNAs bearing 5'-diphosphates (5'pp). Genomes from mammalian reoviruses with 5'pp termini, 5'pp-RNA isolated from yeast L-A virus, and base-paired 5'pp-RNAs made by in vitro transcription or chemical synthesis, all bind to RIG-I and serve as RIG-I agonists. Furthermore, a RIG-I-dependent response to 5'pp-RNA is essential for controlling reovirus infection in cultured cells and in mice. Thus, the minimal determinant for RIG-I recognition is a base-paired RNA with 5'pp. Such RNAs are found in some viruses but not in uninfected cells, indicating that recognition of 5'pp-RNA, like that of 5'ppp-RNA, acts as a powerful means of self/non-self discrimination by the innate immune system.

Age-dependent susceptibility to reovirus encephalitis in mice is influenced by maturation of the type-I interferon response.

BackgroundInfants and young children are particularly susceptible to viral encephalitis; however, the mechanisms are unknown. We determined the age-dependent contribution of innate and adaptive immune functions to reovirus-induced encephalitis in mice.MethodsNewborn wild-type mice, 2-20 days of age, were inoculated with reovirus or diluent and monitored for mortality, weight gain, and viral load. Four- and fifteen-day-old IFNAR-/- and RAG2-/- mice were inoculated with reovirus and similarly monitored.ResultsWeight gain was impaired in mice inoculated with reovirus at 8 days of age or less. Clinical signs of encephalitis were detected in mice inoculated at 10 days of age or less. Mortality decreased when mice were inoculated after 6 days of age. Survival was ≤15% in wild type (WT), RAG2-/-, and IFNAR-/- mice inoculated at 4 days of age. All WT mice, 92% of RAG2-/- mice, and only 48% of IFNAR-/- mice survived following inoculation at 15 days of age.ConclusionsSusceptibility of mice to reovirus-induced disease decreases between 6 and 8 days of age. Enhanced reovirus virulence in IFNAR-/- mice relative to WT and RAG2-/- mice inoculated at 15 days of age suggests that maturation of the type-I interferon response contributes to age-related mortality following reovirus infection.

Mast cell regranulation requires a metabolic switch involving mTORC1 and a glucose-6-phosphate transporter.

Mast cells (MCs) are granulated cells implicated in inflammatory disorders because of their capacity to degranulate, releasing prestored proinflammatory mediators. As MCs have the unique capacity to reform granules following degranulation in vitro, their potential to regranulate in vivo is linked to their pathogenesis. It is not known what factors regulate regranulation, let alone if regranulation occurs in vivo. We report that mice can undergo multiple bouts of MC regranulation following successive anaphylactic reactions. mTORC1, a nutrient sensor that activates protein and lipid synthesis, is necessary for regranulation. mTORC1 activity is regulated by a glucose-6-phosphate transporter, Slc37a2, which increases intracellular glucose-6-phosphate and ATP during regranulation, two upstream signals of mTOR. Additionally, Slc37a2 concentrates extracellular metabolites within endosomes, which are trafficked into nascent granules. Thus, the metabolic switch associated with MC regranulation is mediated by the interactions of a cellular metabolic sensor and a transporter of extracellular metabolites into MC granules.

Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease.

Viral infections have been proposed to elicit pathological processes leading to the initiation of T helper 1 (TH1) immunity against dietary gluten and celiac disease (CeD). To test this hypothesis and gain insights into mechanisms underlying virus-induced loss of tolerance to dietary antigens, we developed a viral infection model that makes use of two reovirus strains that infect the intestine but differ in their immunopathological outcomes. Reovirus is an avirulent pathogen that elicits protective immunity, but we discovered that it can nonetheless disrupt intestinal immune homeostasis at inductive and effector sites of oral tolerance by suppressing peripheral regulatory T cell (pTreg) conversion and promoting TH1 immunity to dietary antigen. Initiation of TH1 immunity to dietary antigen was dependent on interferon regulatory factor 1 and dissociated from suppression of pTreg conversion, which was mediated by type-1 interferon. Last, our study in humans supports a role for infection with reovirus, a seemingly innocuous virus, in triggering the development of CeD.

Engineering Recombinant Reoviruses To Display gp41 Membrane-Proximal External-Region Epitopes from HIV-1.

The gp41 membrane-proximal external region (MPER) is a target for broadly neutralizing antibody responses against human immunodeficiency virus type 1 (HIV-1). However, replication-defective virus vaccines currently under evaluation in clinical trials do not efficiently elicit MPER-specific antibodies. Structural modeling suggests that the MPER forms an α-helical coiled coil that is required for function and immunogenicity. To maintain the native MPER conformation, we used reverse genetics to engineer replication-competent reovirus vectors that displayed MPER sequences in the α-helical coiled-coil tail domain of viral attachment protein σ1. Sequences in reovirus strain type 1 Lang (T1L) σ1 were exchanged with sequences encoding HIV-1 strain Ba-L MPER epitope 2F5 or the entire MPER. Individual 2F5 or MPER substitutions were introduced at virion-proximal or virion-distal sites in the σ1 tail. Recombinant reoviruses containing heterologous HIV-1 sequences were viable and produced progeny yields comparable to those with wild-type virus. HIV-1 sequences were retained following 10 serial passages in cell culture, indicating that the substitutions were genetically stable. Recombinant viruses engineered to display the 2F5 epitope or full-length MPER in σ1 were recognized by purified 2F5 antibody. Inoculation of mice with 2F5-containing vectors or rabbits with 2F5- or MPER-containing vectors elicited anti-reovirus antibodies, but HIV-1-specific antibodies were not detected. Together, these findings indicate that heterologous sequences that form α-helices can functionally replace native sequences in the α-helical tail domain of reovirus attachment protein σ1. However, although these vectors retain native antigenicity, they were not immunogenic, illustrating the difficulty of experimentally inducing immune responses to this essential region of HIV-1. IMPORTANCE Vaccines to protect against HIV-1, the causative agent of AIDS, are not approved for use. Antibodies that neutralize genetically diverse strains of HIV-1 bind to discrete regions of the envelope glycoproteins, including the gp41 MPER. We engineered recombinant reoviruses that displayed MPER epitopes in attachment protein σ1 (REO-MPER vectors). The REO-MPER vectors replicated with wild-type efficiency, were genetically stable, and retained native antigenicity. However, we did not detect HIV-1-specific immune responses following inoculation of the REO-MPER vectors into small animals. This work provides proof of principle for engineering reovirus to express antigenic epitopes and illustrates the difficulty in eliciting MPER-specific immune responses.

Viral infection. Prevention and cure of rotavirus infection via TLR5/NLRC4-mediated production of IL-22 and IL-18.

Activators of innate immunity may have the potential to combat a broad range of infectious agents. We report that treatment with bacterial flagellin prevented rotavirus (RV) infection in mice and cured chronically RV-infected mice. Protection was independent of adaptive immunity and interferon (IFN, type I and II) and required flagellin receptors Toll-like receptor 5 (TLR5) and NOD-like receptor C4 (NLRC4). Flagellin-induced activation of TLR5 on dendritic cells elicited production of the cytokine interleukin-22 (IL-22), which induced a protective gene expression program in intestinal epithelial cells. Flagellin also induced NLRC4-dependent production of IL-18 and immediate elimination of RV-infected cells. Administration of IL-22 and IL-18 to mice fully recapitulated the capacity of flagellin to prevent or eliminate RV infection and thus holds promise as a broad-spectrum antiviral agent.

Serotype-specific differences in inhibition of reovirus infectivity by human-milk glycans are determined by viral attachment protein σ1.

Human milk contains many bioactive components, including secretory IgA, oligosaccharides, and milk-associated proteins. We assessed the antiviral effects of several components of milk against mammalian reoviruses. We found that glucocerebroside (GCB) inhibited the infectivity of reovirus strain type 1 Lang (T1L), whereas gangliosides GD3 and GM3 and 3'-sialyllactose (3SL) inhibited the infectivity of reovirus strain type 3 Dearing (T3D). Agglutination of erythrocytes mediated by T1L and T3D was inhibited by GD3, GM3, and bovine lactoferrin. Additionally, α-sialic acid, 3SL, 6'-sialyllactose, sialic acid, human lactoferrin, osteopontin, and α-lactalbumin inhibited hemagglutination mediated by T3D. Using single-gene reassortant viruses, we found that serotype-specific differences segregate with the gene encoding the viral attachment protein. Furthermore, GD3, GM3, and 3SL inhibit T3D infectivity by blocking binding to host cells, whereas GCB inhibits T1L infectivity post-attachment. These results enhance an understanding of reovirus cell attachment and define a mechanism for the antimicrobial activity of human milk.

A rapid, automated approach for quantitation of rotavirus and reovirus infectivity.

Current microscopy-based approaches for immunofluorescence detection of viral infectivity are time consuming and labor intensive and can yield variable results subject to observer bias. To circumvent these problems, we developed a rapid and automated infrared immunofluorescence imager-based infectivity assay for both rotavirus and reovirus that can be used to quantify viral infectivity and infectivity inhibition. For rotavirus, monolayers of MA104 cells were infected with simian strain SA-11 or SA-11 preincubated with rotavirus-specific human IgA. For reovirus, monolayers of either HeLa S3 cells or L929 cells were infected with strains type 1 Lang (T1L), type 3 Dearing (T3D), or either virus preincubated with a serotype-specific neutralizing monoclonal antibody (mAb). Infected cells were fixed and incubated with virus-specific polyclonal antiserum, followed by an infrared fluorescence-conjugated secondary antibody. Well-to-well variation in cell number was normalized using fluorescent reagents that stain fixed cells. Virus-infected cells were detected by scanning plates using an infrared imager, and results were obtained as a percent response of fluorescence intensity relative to a virus-specific standard. An expected dose-dependent inhibition of both SA-11 infectivity with rotavirus-specific human IgA and reovirus infectivity with T1L-specific mAb 5C6 and T3D-specific mAb 9BG5 was observed, confirming the utility of this assay for quantification of viral infectivity and infectivity blockade. The imager-based viral infectivity assay fully automates data collection and provides an important advance in technology for applications such as screening for novel modulators of viral infectivity. This basic platform can be adapted for use with multiple viruses and cell types.