Background Early-life human rhinovirus (RV) infection has been linked to asthma development in high risk infants and children. Nevertheless, the role of RV infection in the initiation of asthma remains unclear. Objective We hypothesized that, in contrast to infection of mature BALB/c mice, neonatal infection with RV promotes an IL-25-driven type 2 response which causes persistent mucous metaplasia and airway hyperresponsiveness. Methods Six day-old and eight week-old BALB/c mice were inoculated with sham HeLa cell lysate or RV. Airway responses from 1 to 28 days after infection were assessed by qPCR, ELISA, histology, immunofluorescence microscopy, flow cytometry and methacholine responsiveness. Selected mice were treated with a neutralizing antibody to IL-25. Results Compared to mature mice, RV infection in neonatal mice increased lung IL-13 and IL-25 production whereas IFN-γ, IL-12p40 and TNF-α expression were suppressed. In addition, the population of IL-13-secreting type 2 innate lymphoid cells (ILC2s) was expanded with RV infection in neonatal but not in mature mice. ILC2 cells were the major cell type secreting IL-13 in neonates. Finally, anti-IL-25 neutralizing antibody attenuated ILC2 expansion, mucous hypersecretion and airways responsiveness. Conclusions These findings suggest that early-life viral infection could contribute to asthma development by provoking age-dependent, IL-25-driven type 2 immune responses.

Borrelia burgdorferi (Bb) is a tick-borne spirochete that is the causative agent for Lyme disease. Our previous studies indicate that virulent Bb can potently enhance IL-10 production by macrophages (MØs) and that blocking IL-10 production significantly enhances bacterial clearance. We hypothesize that skin-associated APC types, such as MØs and dendritic cells (DCs) are potent producers of IL-10 in response to Bb, which may act in autocrine fashion to suppress APC responses critical for efficient Bb clearance. Our goal is to delineate which APC immune functions are dysregulated by Bb-elicited IL-10 using a murine model of Lyme disease. Our in vitro studies indicated that both APCs rapidly produce IL-10 upon exposure to Bb, that these levels inversely correlate with the production of many Lyme-relevant proinflammatory cytokines and chemokines, and that APCs derived from IL-10-/- mice produced greater amounts of these proinflammatory mediators than wild-type APCs. Phagocytosis assays determined that Bb-elicited IL-10 levels can diminish Bb uptake and trafficking by MØs, suppresses ROS production, but does not affect NO production; Bb-elicited IL-10 had little effect on phagocytosis, ROS, and NO production by DCs. In general, Bb exposure caused little-to-no upregulation of several critical surface co-stimulatory markers by MØs and DCs, however eliminating Bb-elicited IL-10 allowed a significant upregulation in many of these co-stimulatory receptors. These data indicate that IL-10 elicited from Bb-stimulated MØs and DCs results in decreased production of proinflammatory mediators and co-stimulatory molecules, and suppress phagocytosis-associated events that are important for mediating both innate and adaptive immune responses by APCs.

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.

Background We have shown that rhinovirus (RV), a cause of asthma exacerbations, colocalizes with CD68- and CD11b-positive airway macrophages following experimental infection in humans. We have also shown that RV-induced cytokine expression is abolished in TLR2−/− bone marrow-derived macrophages. Objective We hypothesize that TLR2+ macrophages are required and sufficient for RV-induced airway inflammation in vivo. Methods To determine the requirement and sufficiency of TLR2 for RV-induced airway responses, naïve and ovalbumin-sensitized and challenged C57BL/6 wild-type and TLR2−/− mice were infected with RV1B followed by IgG or anti-TLR2. Bone marrow chimera experiments using OVA-treated C57BL/6 and TLR2−/− mice were also performed. Finally, naïve TLR2−/− mice underwent intranasal transfer of bone marrow-derived wild type macrophages. Results RV1B infection of naïve wild-type mice induced an influx of airway neutrophils and CD11b+ exudative macrophages which was reduced in TLR2−/− mice. In allergen-exposed mice, RV-induced neutrophilic and eosinophilic airway inflammation and hyperresponsiveness were reduced in TLR2−/− and anti-TLR2-treated mice. Transfer of TLR2−/− bone marrow into wild type ovalbumin-treated, C57BL/6 mice blocked RV-induced airway responses, whereas transfer of wild type marrow to TLR2−/− mice restored them. Finally, transfer of wild-type macrophages to naïve TLR2−/− mice was sufficient for neutrophilic inflammation after RV infection, whereas macrophages treated with IL-4 (to induce M2 polarization) were sufficient for eosinophilic inflammation, mucous metaplasia and airways hyperresponsiveness. Conclusions TLR2 is required for early inflammatory responses induced by RV, and TLR2+ macrophages are sufficient to confer airway inflammation to TLR2−/− mice, with the pattern of inflammation depending on macrophage activation state.

BackgroundThe mechanisms by which viruses cause asthma exacerbations are not precisely known. Previously, we showed that, in ovalbumin (OVA)-sensitized and -challenged mice with allergic airway inflammation, rhinovirus (RV) infection increases type 2 cytokine production from alternatively-activated (M2) airway macrophages, enhancing eosinophilic inflammation and airways hyperresponsiveness. In this paper, we tested the hypothesis that IL-4 signaling determines the state of macrophage activation and pattern of RV-induced exacerbation in mice with allergic airways disease.MethodsEight week-old wild type or IL-4 receptor knockout (IL-4R KO) mice were sensitized and challenged with OVA and inoculated with RV1B or sham HeLa cell lysate.ResultsIn contrast to OVA-treated wild-type mice with both neutrophilic and eosinophilic airway inflammation, OVA-treated IL-4R KO mice showed increased neutrophilic inflammation with few eosinophils in the airways. Like wild-type mice, IL-4R KO mice showed OVA-induced airway hyperreactivity which was further exacerbated by RV. There was a shift in lung cytokines from a type 2-predominant response to a type 1 response, including production of IL-12p40 and TNF-α. IL-17A was also increased. RV infection of OVA-treated IL-4R KO mice further increased neutrophilic inflammation. Bronchoalveolar macrophages showed an M1 polarization pattern and ex vivo RV infection increased macrophage production of TNF-α, IFN-γ and IL-12p40. Finally, lung cells from OVA-treated IL-4R KO mice showed reduced CD206+ CD301+ M2 macrophages, decreased IL-13 and increased TNF-α and IL-17A production by F4/80+, CD11b+ macrophages.ConclusionsOVA-treated IL-4R KO mice show neutrophilic airway inflammation constituting a model of allergic, type 1 cytokine-driven neutrophilic asthma. In the absence of IL-4/IL-13 signaling, RV infection of OVA-treated mice increased type 1 cytokine and IL-17A production from conventionally-activated macrophages, augmenting neutrophilic rather than eosinophilic inflammation. In mice with allergic airways inflammation, IL-4R signaling determines macrophage activation state and the response to subsequent RV infection.

Rhinovirus (RV) causes asthma exacerbations. Previously, we showed that adherent bronchoalveolar cells from allergen-treated mice produce IL-13 when stimulated with RV ex vivo, implicating cells of the monocyte/macrophage lineage in viral-induced airway inflammation. In this study, we hypothesized that RV infection of allergen-treated mice results in IL-13 production by CD11b1 exudative macrophages in vivo. We sensitized and challenged BALB/c mice with ovalbumin (OVA), after which mice were inoculated with RV or sham HeLa cell lysate. After 1 day, lungs were harvested, and cell suspensions were analyzed by flow cytometry. We repeated this process in IL-13 reporter mice, CD11b-DTR mice in which diphtheria toxin selectively depletes CD11b1 cells, and chemokine receptor 2 (CCR2) null mice. We found that lungs of mice infected with RV alone showed increases in CD451, CD681, F4/801, Ly6C1, and CD11b high cells, indicating an influx of inflammatory monocytes and exudative macrophages. The combination of OVA and RV had synergistic effects on the exudative macrophage number. However, CD11b1 cells from OVA-treated, RV-infected mice showed M2 polarization, including expression of CD206 and CD301 and production of IL-13. Similar results were obtained in IL-13 reporter mice. Diphtheria toxin depleted CD11b1, IL-13-producing cells in OVA-treated, RV-infected, CD11b-DTR mice, decreasing airway inflammation and responsiveness. CD11b1, Ly6C1 cells were reduced in CCR2 knockout mice. We conclude that, in contrast to naive mice, RV infection of mice with allergic airways disease induces an influx of IL-13-producing CD11b1 exudative macrophages bearing M2 macrophage markers. This finding further implicates alternatively activated macrophages in RV-induced asthma exacerbations.

Rhinovirus (RV) is responsible for the majority of virus-induced asthma exacerbations. We showed previously that RV infection of ovalbumin-sensitized and -challenged BALB/c mice induces production of type 2 cytokines from M2-polarized macrophages. In the present study, we sought to determine the mechanism of RV-induced cytokine expression. We infected bone marrow-derived macrophages (BMMs) from BALB/c mice with RV serotype 1B, a minor group virus that infects mouse cells. Selected cultures were pretreated with IL-4, a type 2 cytokine increased in allergic asthma. RV infection of untreated cells increased messenger RNA and protein expression of the M1 cytokines TNF-a, CXCL1, and IL-6 but failed to induce expression of the M2 cytokines CCL22 and CCL24. Cells pretreated with IL-4 showed decreased expression of M1 cytokines but increased expression of Ym-1, Arg-1 (M2 markers), CCL22, and CCL24. Infection with ultraviolet (UV)-irradiated, replication-deficient RV elicited similar cytokine responses, suggesting that the outcome is replication independent. Consistent with this, viral RNA copy number did not increase in RV-treated BMMs or bronchoalveolar macrophages. RVinduced cytokine expression was not affected when cells were pretreated with cytochalasin D, suggesting that viral endocytosis is not required for the response. Finally, RV-induced cytokine expression and viral attachment were abolished in BMMs from myeloid differentiation factor 88 and Toll-like receptor (TLR)2 KO mice, suggesting a specific requirement of TLR2. We conclude that RV elicits a proinflammatory cytokine response in BMMs through a cellsurface-mediated, TLR2-dependent mechanism that does not require viral endocytosis or replication.

Immune cells have important roles during disease and are known to contribute to secondary, inflammation-induced injury after traumatic brain injury. To delineate the functional role of macrophages during traumatic brain injury, we depleted macrophages using transgenic CD11b-DTR mice and subjected them to controlled cortical impact. We found that macrophage depletion had no effect on lesion size assessed by T2-weighted MRI scans twenty-eight days after injury. Macrophage depletion resulted in a robust increase in proinflammatory gene expression in both the ipsilateral and contralateral hemispheres after controlled cortical impact. Interestingly, this sizeable increase in inflammation did not affect lesion development. We also showed that macrophage depletion resulted in increased proinflammatory gene expression in the brain and kidney in the absence of injury. These data demonstrate that depletion of macrophages in CD11b-DTR mice can significantly modulate the inflammatory response during brain injury without affecting lesion formation. These data also reveal a potentially confounding inflammatory effect in CD11b-DTR mice that must be considered when analyzing the effects of macrophage depletion in disease models.