Rhinovirus (RV), a ssRNA virus of the picornavirus family, is a major cause of the common cold as well as asthma and chronic obstructive pulmonary disease exacerbations. Viral dsRNA produced during replication may be recognized by the host pattern recognition receptors TLR-3, retinoic acid-inducible gene (RIG)-I, and melanoma differentiation-associated gene (MDA)-5. No study has yet identified the receptor required for sensing RV dsRNA. To examine this, BEAS-2B human bronchial epithelial cells were infected with intact RV-1B or replication-deficient UV-irradiated virus, and IFN and IFN-stimulated gene expression was determined by quantitative PCR. The separate requirements of RIG-I, MDA5, and IFN response factor (IRF)-3 were determined using their respective small interfering RNAs (siRNA). The requirement of TLR3 was determined using siRNA against the TLR3 adaptor molecule Toll/IL-1R homologous region-domain-containing adapter-inducing IFN-β (TRIF). Intact RV-1B, but not UV-irradiated RV, induced IRF3 phosphorylation and dimerization, as well as mRNA expression of IFN-β, IFN-λ1, IFN-λ2/3, IRF7, RIG-I, MDA5, 10-kDa IFN-γ-inducible protein/CXCL10, IL-8/CXCL8, and GM-CSF. siRNA against IRF3, MDA5, and TRIF, but not RIG-I, decreased RV-1B-induced expression of IFN-β, IFN-λ1, IFN-λ2/3, IRF7, RIG-I, MDA5, and inflammatory protein-10/CXCL10 but had no effect on IL-8/CXCL8 and GM-CSF. siRNAs against MDA5 and TRIF also reduced IRF3 dimerization. Finally, in primary cells, transfection with MDA5 siRNA significantly reduced IFN expression, as it did in BEAS-2B cells. These results suggest that TLR3 and MDA5, but not RIG-I, are required for maximal sensing of RV dsRNA and that TLR3 and MDA5 signal through a common downstream signaling intermediate, IRF3.

Human rhinovirus is responsible for the majority of virus-induced asthma exacerbations. To determine the immunologic mechanisms underlying rhinovirus-induced asthma exacerbations, we combined mouse models of allergic airways disease and human rhinovirus infection. We inoculated ovalbumin-sensitized and challenged BALB/c mice with rhinovirus serotype 1B, a minor group strain capable of infecting mouse cells. Compared to sham-infected, ovalbumin-treated mice, virus-infected mice showed increased lung infiltration with neutrophils, eosinophils and macrophages, airway cholinergic hyperresponsiveness, and increased lung expression of cytokines including eotaxin-1/CCL11, IL-4, IL-13 and IFN-γ. Administration of anti-eotaxin-1 attenuated rhinovirus-induced airway eosinophilia and responsiveness. Immunohistochemistry showed eotaxin-1 in the lung macrophages of virus-infected, ovalbumin-treated mice, and confocal fluorescence microscopy revealed co-localization of rhinovirus, eotaxin-1 and IL-4 in CD68-positive cells. RV inoculation of lung macrophages from ovalbumin-treated, but not PBS-treated, mice induced expression of eotaxin-1, IL-4, and IL-13 ex vivo. Macrophages from ovalbumin-treated mice showed increased expression of arginase-1, Ym-1, Mgl-2 and IL-10, indicating a shift in macrophage activation status. Depletion of macrophages from ovalbumin-sensitized and -challenged mice reduced eosinophilic inflammation and airway hyperreactivity following RV infection. We conclude that augmented airway eosinophilic inflammation and hyperresponsiveness in RV-infected mice with allergic airways disease is directed in part by eotaxin-1. Airway macrophages from mice with allergic airways disease demonstrate a change in activation state characterized in part by altered eotaxin and IL-4 production in response to RV infection. These data provide a new paradigm to explain RV-induced asthma exacerbations.

Rhinovirus (RV), a single-stranded RNA picornavirus, is the most frequent cause of asthma exacerbations. We previously demonstrated in human bronchial epithelial cells that melanoma differentiation-associated gene (MDA)-5 and the adaptor protein for Toll-like receptor (TLR)-3 are each required for maximal RV1B-induced interferon (IFN) responses. However, in vivo, the overall airway response to viral infection likely represents a coordinated response integrating both antiviral and pro-inflammatory pathways. We examined the airway responses of MDA5- and TLR3-deficient mice to infection with RV1B, a minor group virus which replicates in mouse lungs. MDA5 null mice showed a delayed type I IFN and attenuated type III IFN response to RV1B infection, leading to a transient increase in viral titer. TLR3 null mice showed normal IFN responses and unchanged viral titers. Further, RV-infected MDA5 and TLR3 null mice showed reduced lung inflammatory responses and reduced airways responsiveness. Finally, RV-infected MDA5 null mice with allergic airways disease showed lower viral titers despite deficient IFN responses, and allergic MDA5 and TLR3 null mice each showed decreased RV-induced airway inflammatory and contractile responses. These results suggest that, in the context of RV infection, binding of viral dsRNA to MDA5 and TLR3 initiates pro-inflammatory signaling pathways leading to airways inflammation and hyperresponsiveness.

Recent studies link early rhinovirus (RV) infections to later asthma development. We hypothesized that neonatal RV infection leads to an IL-13-driven asthma-like phenotype in mice. BALB/c mice were inoculated with RV1B or sham on day 7 of life. Viral RNA persisted in the neonatal lung up to 7 days after infection. Within this time frame, IFNs-α, -β and -γ peaked 1 day after infection, whereas IFN-λ levels persisted. Next, we examined mice on day 35 of life, 28 days after initial infection. Compared to sham-treated controls, virus-inoculated mice demonstrated airways hyperresponsiveness. Lungs from RV-infected mice showed increases in several immune cell populations, as well as the percentages of CD4-positive T cells expressing IFN-γ and of NKp46/CD335+, TCR-β+ cells expressing IL-13. Periodic acid-Schiff and immunohistochemical staining revealed mucous cell metaplasia and muc5AC expression in RV1B- but not sham-inoculated lungs. Mucous metaplasia was accompanied by induction of gob-5, MUC5AC, MUC5B and IL-13 mRNA. By comparison, adult mice infected with RV1B showed no change in IL-13 expression, mucus production or airways responsiveness 28 days after infection. Intraperitoneal administration of anti-IL13 neutralizing antibody attenuated RV-induced mucous metaplasia and methacholine responses, and IL-4R null mice failed to show RV-induced mucous metaplasia. Finally, neonatal RV increased the inflammatory response to subsequent allergic sensitization and challenge. We conclude that neonatal RV1B infection leads to persistent airways inflammation, mucous metaplasia and hyperresponsiveness which are mediated, at least in part, by IL-13.

Background.Human immunodeficiency virus (HIV)-infected individuals have increased risk for vascular thrombosis, potentially driven by interactions between activated leukocytes and the endothelium.Methods.Monocyte subsets (CD14+CD16−, CD14+CD16+, CD14DimCD16+) from HIV negative (HIV−) and antiretroviral therapy-treated HIV positive (HIV+) participants (N = 19 and 49) were analyzed by flow cytometry for adhesion molecule expression (lymphocyte function-associated antigen 1 [LFA-1], macrophage-1 antigen [Mac-1], CD11c/CD18, very late antigen [VLA]-4) and the fractalkine receptor (CX3CR1); these receptors recognize ligands (intercellular adhesion molecules [ICAMs], vascular cell adhesion molecule [VCAM]-1, fractalkine) on activated endothelial cells (ECs) and promote vascular migration. Plasma markers of monocyte (soluble [s]CD14, sCD163) and EC (VCAM-1, ICAM-1,2, fractalkine) activation and systemic (tumor necrosis factor receptor [TNFR-I], TNFR-II) and vascular (lipoprotein-associated phospholipase A2 [Lp-PLA2]) inflammation were measured by enzyme-linked immunosorbent assay.Results.Proportions of CD16+ monocyte subsets were increased in HIV+ participants. Among all monocyte subsets, levels of LFA-1 were increased and CX3CR1 levels were decreased in HIV+ participants (P < .01). Levels of sCD163, sCD14, fractalkine, ICAM-1, VCAM-1, TNFR-II, and Lp-PLA2 were also increased in HIV+ participants (P < .05), and levels of sCD14, TNFR-I, and TNFR-II were directly related to ICAM-1 and VCAM-1 levels in HIV+ participants. Expression of CX3CR1 on monocyte subsets was inversely related to plasma Lp-PLA2 (P < .05 for all).Conclusions.Increased proportions of CD16+ monocytes, cells with altered adhesion molecule expression, combined with elevated levels of their ligands, may promote vascular inflammation in HIV infection.

Human rhinovirus (HRV) infections lead to exacerbations of lower airways disease in asthmatic patients but not in healthy individuals. However, underlying mechanisms remain to be completely elucidated. We hypothesized that the Th2-driven allergic environment enhances HRV-induced CC chemokine production, leading to asthma exacerbations. Ovalbumin (OVA)-sensitized and -challenged mice inoculated with HRV showed significant increases in the expression of lung CC chemokine ligand (CCL)-2/monocyte chemotactic protein (MCP)-1, CCL4/macrophage inflammatory protein (MIP)-1β, CCL7/MCP-3, CCL19/MIP-3β, and CCL20/MIP3α compared with mice treated with OVA alone. Inhibition of CCL2 with neutralizing antibody significantly attenuated HRV-induced airways inflammation and hyperresponsiveness in OVA-treated mice. Immunohistochemical stains showed colocalization of CCL2 with HRV in epithelial cells and CD68-positive macrophages, and flow cytometry showed increased CCL2(+), CD11b(+) cells in the lungs of OVA-treated, HRV-infected mice. Compared with lung macrophages from naïve mice, macrophages from OVA-exposed mice expressed significantly more CCL2 in response to HRV infection ex vivo. Pretreatment of mouse lung macrophages and BEAS-2B human bronchial epithelial cells with interleukin (IL)-4 and IL-13 increased HRV-induced CCL2 expression, and mouse lung macrophages from IL-4 receptor knockout mice showed reduced CCL2 expression in response to HRV, suggesting that exposure to these Th2 cytokines plays a role in the altered HRV response. Finally, bronchoalveolar macrophages from children with asthma elaborated more CCL2 upon ex vivo exposure to HRV than cells from nonasthmatic patients. We conclude that CCL2 production by epithelial cells and macrophages contributes to HRV-induced airway hyperresponsiveness and inflammation in a mouse model of allergic airways disease and may play a role in HRV-induced asthma exacerbations.

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Background Lipid profiles are altered by HIV infection and antiretroviral therapy (ART). Among HIV-uninfected (HIV−) populations the concentrations of various lipid classes (ie, lyso-phosphatidylcholine, LPC) and their saturated (SaFA), mono-unsaturated (MUFA), and polyunsaturated fatty acid (PUFA) composition are related to cardiometabolic disease risk. Associations between changes in the lipidome and immune activation in HIV-infected (HIV+) individuals beginning ART have not been described. Methods Plasma lipid concentrations and their fatty acid composition were measured by differential mobility spectroscopy in samples from 35 treatment-naive HIV+ participants beginning raltegravir (RAL)-based ART and from HIV- individuals (n = 13) matched for age and sex. Results The levels of SaFA, including palmitic (16:0) and stearic (18:0) acid were enriched in HIV+ participants (pre- and post-ART), and SaFA levels were often positively correlated with levels of immune activation (ie, IL-6, sCD14, and TNFR1) at baseline and week 48. Levels of PUFAs (including 18:3, 20:4, and 20:5) were lower in HIV+ participants at baseline compared to levels in HIV- participants (P < 0.01), and levels of these PUFAs were increased following 48 weeks of ART. Levels of PUFAs were often inversely related to immune activation. Levels of LPC were increased in HIV+ participants, both pre- and post-ART vs HIV- participants, and the composition of LPC was enriched for SaFAs among HIV+ individuals. At week 48, several LPC molecules containing SaFAs were positively correlated with levels of sCD14, D-dimer, and TNFR1 (P < 0.01), and levels of PUFA-containing LPC (18:3, 20:5, 22:5, 22:6) were positively correlated with CD4+ T cell counts and inversely correlated with sCD14 and IL-6 (P < 0.01). Conclusions The composition of the lipidome is altered in HIV infection and changes when ART is administered. Alterations in SaFAs were generally associated with inflammatory markers and may contribute to comorbid disease pathogenesis.