Karim H. Shalaby

Selective control of SIRP-α-positive airway dendritic cell trafficking through CD47 is critical for the development of TH2-mediated allergic inflammation

BACKGROUND Dendritic cells (DCs) are essential for the initiation and maintenance of T(H)2 responses to inhaled antigen that lead to the establishment of allergic diseases. Two subpopulations of nonplasmacytoid DCs (ie, CD11b(low)CD103+ and CD11b(high)CD103(-)) are found in lung/airway tissues. Yet the identification and migratory properties of the DC subset that contributes to T(H)2-mediated responses remain to be clarified. CD47, a signal regulatory protein (SIRP)-alpha partner, reportedly governed skin DC migration. OBJECTIVE We here thought to investigate the role of CD47/SIRP-alpha interactions in airway DC trafficking and the development of allergic airway inflammation. METHODS We characterized the DC influx into lungs and mediastinal lymph nodes in CD47(-/-) and CD47(+/+) BALB/c mice by using experimental models of allergic asthma. Mice were systemically (intraperitoneal ovalbumin/alum) or locally (intratracheal ovalbumin-loaded bone marrow-derived DCs) immunized and challenged by ovalbumin aerosol. We also evaluated the consequences of SIRP-alpha-Fc fusion molecule administration on the induction of airway disease in BALB/c mice. RESULTS SIRP-alpha selectively identified the CD11b(high)CD103(-) DC subset that predominantly accumulated in mediastinal lymph nodes during airway inflammation. However, CD103(-)SIRP-alpha+ DC trafficking, T(H)2 responses, and airway disease were impaired in CD47(-/-) mice. Importantly, the adoptive transfer of CD103(-) SIRP-alpha+CD47(+/+) but not CD47(-/-) DCs elicited a strong T(H)2 response in CD47(-/-) mice. Finally, the administration of SIRP-alpha-Fc molecule protected BALB/c mice from allergic airway inflammation. CONCLUSION Lung CD11b(high)CD103(-)SIRP-alpha+ DC migration is governed by self-CD47 expression, and manipulation of the CD47/SIRP-alpha pathway suppresses CD103(-)SIRP-alpha(+) DC-driven pathogenic T(H)2 responses and airway inflammation.

Combined forced oscillation and forced expiration measurements in mice for the assessment of airway hyperresponsiveness

BackgroundPulmonary function has been reported in mice using negative pressure-driven forced expiratory manoeuvres (NPFE) and the forced oscillation technique (FOT). However, both techniques have always been studied using separate cohorts of animals or systems. The objective of this study was to obtain NPFE and FOT measurements at baseline and following bronchoconstriction from a single cohort of mice using a combined system in order to assess both techniques through a refined approach.MethodsGroups of allergen- or sham-challenged ovalbumin-sensitized mice that were either vehicle (saline) or drug (dexamethasone 1 mg/kg ip)-treated were studied. Surgically prepared animals were connected to an extended flexiVent system (SCIREQ Inc., Montreal, Canada) permitting NPFE and FOT measurements. Lung function was assessed concomitantly by both techniques at baseline and following doubling concentrations of aerosolized methacholine (MCh; 31.25 - 250 mg/ml). The effect of the NPFE manoeuvre on respiratory mechanics was also studied.ResultsThe expected exaggerated MCh airway response of allergic mice and its inhibition by dexamethasone were detected by both techniques. We observed significant changes in FOT parameters at either the highest (Ers, H) or the two highest (Rrs, RN, G) MCh concentrations. The flow-volume (F-V) curves obtained following NPFE manoeuvres demonstrated similar MCh concentration-dependent changes. A dexamethasone-sensitive decrease in the area under the flow-volume curve at the highest MCh concentration was observed in the allergic mice. Two of the four NPFE parameters calculated from the F-V curves, FEV0.1 and FEF50, also captured the expected changes but only at the highest MCh concentration. Normalization to baseline improved the sensitivity of NPFE parameters at detecting the exaggerated MCh airway response of allergic mice but had minimal impact on FOT responses. Finally, the combination with FOT allowed us to demonstrate that NPFE induced persistent airway closure that was reversible by deep lung inflation.ConclusionsWe conclude that FOT and NPFE can be concurrently assessed in the same cohort of animals to determine airway mechanics and expiratory flow limitation during methacholine responses, and that the combination of the two techniques offers a refined control and an improved reproducibility of the NPFE.

NLRX1 prevents mitochondrial induced apoptosis and enhances macrophage antiviral immunity by interacting with influenza virus PB1-F2 protein

Significance Apoptosis refers to the ability of a cell to undergo programmed cell death under normal physiological conditions or in response to stress signals. During infection, influenza A viruses have the capacity to induce early apoptosis of immune cells, thereby preventing them from performing their antiviral function. In this study, we identified that a host innate immune sensor [NLRX1 (nucleotide-binding oligomerization domain-like receptor X1)] has the capacity to bind a small death-inducing protein from influenza A virus [PB1-F2 (polymerase basic protein 1-frame 2)] and defend immune cells against virus-driven apoptosis. This phenomenon allows the immune cells to survive longer and effectively restrict viral replication, protecting the host against the detrimental consequences of influenza. To subvert host immunity, influenza A virus (IAV) induces early apoptosis in innate immune cells by disrupting mitochondria membrane potential via its polymerase basic protein 1-frame 2 (PB1-F2) accessory protein. Whether immune cells have mechanisms to counteract PB1-F2–mediated apoptosis is currently unknown. Herein, we define that the host mitochondrial protein nucleotide-binding oligomerization domain-like receptor (NLR)X1 binds to viral protein PB1-F2, preventing IAV-induced macrophage apoptosis and promoting both macrophage survival and type I IFN signaling. We initially observed that Nlrx1-deficient mice infected with IAV exhibited increased pulmonary viral replication, as well as enhanced inflammatory-associated pulmonary dysfunction and morbidity. Analysis of the lungs of IAV-infected mice revealed markedly enhanced leukocyte recruitment but impaired production of type I IFN in Nlrx1−/− mice. Impaired type I IFN production and enhanced viral replication was recapitulated in Nlrx1−/− macrophages and was associated with increased mitochondrial mediated apoptosis. Through gain- and loss-of-function strategies for protein interaction, we identified that NLRX1 directly bound PB1-F2 in the mitochondria of macrophages. Using a recombinant virus lacking PB1-F2, we confirmed that deletion of PB1-F2 abrogated NLRX1-dependent macrophage type I IFN production and apoptosis. Thus, our results demonstrate that NLRX1 acts as a mitochondrial sentinel protecting macrophages from PB1-F2–induced apoptosis and preserving their antiviral function. We further propose that NLRX1 is critical for macrophage immunity against IAV infection by sensing the extent of viral replication and maintaining a protective balance between antiviral immunity and excessive inflammation within the lungs.

Intravenous immunoglobulin attenuates airway inflammation through induction of forkhead box protein 3–positive regulatory T cells

BACKGROUND Intravenous immunoglobulin (IVIG) is a frequently used disease-modifying therapy for a large spectrum of autoimmune and inflammatory conditions, yet its mechanisms of action are incompletely understood. Using a robust murine model of antigen-driven allergic airways disease, we have demonstrated that IVIG markedly improves ovalbumin (OVA)-induced airway hyperresponsiveness characterized by 4- to 6-fold enhancement in regulatory T (Treg) cells in pulmonary and associated lymphoid tissues. OBJECTIVE We sought to determine whether IVIG induces antigen-specific Treg cells and to address cellular interactions that lead to induction of Treg cells by IVIG. METHODS C57Bl/6 mice were sensitized and challenged by means of intranasal OVA exposure. IVIG or albumin control was administered 24 hours before challenge. Treg cells were tracked by using green fluorescent protein (GFP)-forkhead box protein 3 (Foxp3) knock-in reporter mice (Foxp3(GFP)), and Treg cell and dendritic cell (DC) phenotypes and activities were elucidated by using coculture and flow cytometry. RESULTS IVIG therapy of OVA-sensitized and OVA-challenged mice induced antigen-specific forkhead box protein 3 (Foxp3)-positive Treg cells from non-Treg cell precursors. The induced Treg cells home specifically to the lungs and draining lymph nodes and have greatly potentiated suppressive activity compared with that seen in Treg cells purified from control mice. Induction of Treg cells is mediated by tolerogenic DCs generated after IVIG exposure. Compared with albumin-treated, OVA-exposed mice, IVIG-primed DCs express altered Notch ligands, including increased Delta-4 and reduced Jagged-1 levels, reflecting decreased T(H)2 polarization. Furthermore, IVIG-primed DCs can stimulate Treg cell differentiation from uncommitted Foxp3(-)CD4(+) T cells ex vivo, and adoptive transfer of IVIG-primed DCs abrogates airway hyperresponsiveness and induces Treg cells. CONCLUSION The anti-inflammatory effects of IVIG therapy can be mediated by the immunomodulation of DCs, creating a bridge that induces antigen-specific, highly suppressive Treg cells.

Overview of asthma; the place of the T cell.

Asthma is an inflammatory disease which is associated with activated T cells in the airway wall. The contribution of the T lymphocyte to inflammation in asthma has been extensively studied through descriptions of T cell subsets in the airway wall of asthmatic patients and from animal and cellular models. Allergy-driven airway disease is mediated primarily by the T helper (Th)2 cell subset. Other subsets, such as Th1, Th17, invariant natural killer T and CD8+ T cells likely contribute to the development, and possibly the progression of established disease. Resolution of inflammation is controlled in part by regulatory T cells. Therapies directed at T cells and their cytokines have been disappointing in asthma despite, in some instances, promising results on allergen challenge. This suggests that the induction of asthma may be T-cell-mediated and allergen-triggered, whereas disease may be sustained and exacerbated by other mechanisms.

Sites of allergic airway smooth muscle remodeling and hyperresponsiveness are not associated in the rat

The cause-and-effect relationship between airway smooth muscle (ASM) remodeling and airway hyperresponsiveness (AHR) following allergen challenge is not well established. Using a rat model of allergen-induced ASM remodeling we explored the relationship between the site of ASM remodeling and AHR. Brown Norway rats, sensitized and challenged (3 times at 5-day intervals) with ovalbumin, were intranasally administered 0.1 mg/kg budesonide 24 and 1 h before challenge. Airway responses to aerosolized methacholine were assessed 48 h or 1 wk after three challenges. Airways were stained and analyzed for total airway wall area, area of smooth muscle-specific α-actin, and goblet cell hyperplasia, and the constant-phase model was used to resolve the changes in respiratory system mechanics into large airway and peripheral lung responses. After three ovalbumin challenges, there was a significant increase in ASM area and in the total wall area in all sized airways as well as an increase in goblet cells in the central airways. Budesonide inhibited ASM growth and central airway goblet cell hyperplasia following ovalbumin challenges. Budesonide also inhibited small but not large airway total wall area. AHR was attributable to excessive responses of the small airways, whereas responsiveness of the large airways was unchanged. Budesonide did not inhibit AHR after repeated challenge. We conclude that ASM remodeling induced by repeated allergen challenges involves the entire bronchial tree, whereas AHR reflects alterations in the lung periphery. Prevention of ASM remodeling by corticosteroid does not abrogate AHR.

EGF receptor activation during allergic sensitization affects IL‐6‐induced T‐cell influx to airways in a rat model of asthma

EGF receptor (EGFR) is involved in cell differentiation and proliferation in airways and may trigger cytokine production by T cells. We hypothesized that EGFR inhibition at the time of allergic sensitization may affect subsequent immune reactions. Brown Norway rats were sensitized with OVA, received the EGFR tyrosine kinase inhibitor, AG1478 from days 0 to 7 and OVA challenge on day 14. OVA‐specific IgE in serum and cytokines and chemokines in BAL were measured 24 h after challenge. To evaluate effects on airway hyperresponsiveness (AHR), rats were sensitized, treated with AG1478, intranasally challenged, and then AHR was assessed. Furthermore chemotactic activity of BALF for CD4+ T cells was examined. The eosinophils, neutrophils and lymphocytes in BAL were increased by OVA and only the lymphocytes were reduced by AG1478. OVA significantly enhanced IL‐6 concentration in BAL, which was inhibited by AG1478. However AHR, OVA‐specific IgE and IL‐4 mRNA expression in CD4+ T cells were not affected by AG1478. BALF from OVA‐sensitized/challenged rats induced CD4+ T‐cell migration, which was inhibited by both AG1478 treatment in vivo and neutralization of IL‐6 in vitro. EGFR activation during sensitization may affect the subsequent influx of CD4+ T cells to airways, mainly mediated through IL‐6.

Concomitant exposure to ovalbumin and endotoxin augments airway inflammation but not airway hyperresponsiveness in a murine model of asthma.

Varying concentrations of lipopolysaccharide (LPS) in ovalbumin (OVA) may influence the airway response to allergic sensitization and challenge. We assessed the contribution of LPS to allergic airway inflammatory responses following challenge with LPS-rich and LPS-free commercial OVA. BALB/c mice were sensitized with LPS-rich OVA and alum and then underwent challenge with the same OVA (10 µg intranasally) or an LPS-free OVA. Following challenge, bronchoalveolar lavage (BAL), airway responsiveness to methacholine and the lung regulatory T cell population (Treg) were assessed. Both OVA preparations induced BAL eosinophilia but LPS-rich OVA also evoked BAL neutrophilia. LPS-free OVA increased interleukin (IL)-2, IL-4 and IL-5 whereas LPS-rich OVA additionally increased IL-1β, IL-12, IFN-γ, TNF-α and KC. Both OVA-challenged groups developed airway hyperresponsiveness. TLR4-deficient mice challenged with either OVA preparation showed eosinophilia but not neutrophilia and had increased IL-5. Only LPS-rich OVA challenged mice had increased lung Tregs and LPS-rich OVA also induced in vitro Treg differentiation. LPS-rich OVA also induced a Th1 cytokine response in human peripheral blood mononuclear cells.We conclude that LPS-rich OVA evokes mixed Th1, Th2 and innate immune responses through the TLR-4 pathway, whereas LPS-free OVA evokes only a Th2 response. Contaminating LPS is not required for induction of airway hyperresponsiveness but amplifies the Th2 inflammatory response and is a critical mediator of the neutrophil, Th1 and T regulatory cell responses to OVA.

Pattern Recognition Receptors and Aging

Pattern recognition receptors (PRRs), such as the toll-like receptors (TLRs), C-type lectin receptors (CLRs), nucleotide-binding oligomerization domain and leucine-rich repeat-containing receptors (NLRs), and the more recently discovered cytosolic RNA- and DNA-sensing receptors, are a crucial element of the innate immune system owing to their function of enabling immune cells to detect macromolecules of foreign and potentially harmful origin, such as those of infectious agents, and instructing appropriate protective inflammatory and adaptive immune responses. However, PRRs and their signaling components are expressed on not only innate immune cells but various other immune and structural cells of the body including endothelial, epithelial, muscle, and stromal cells, as well as fibroblasts, adipocytes, and cells of the nervous system, and are increasingly being shown to play an active and diverse role in the regulation of cellular senescence and tissue homeostasis. For instance, PRRs maintain balanced immunity towards nonpathogenic commensals at host-environment interfaces such as the gut and skin, detect “danger signals” released during cell stress or injury, and interact with physiological processes such as autophagy, DNA repair, and cellular apoptosis. The regulation of many of these processes and PRR families is altered in the course of ageing, and the mechanisms underlying this dysregulation are discussed in this chapter. We also review the extensive evidence from gene association or functional studies indicating that loss or gain of PRR expression or activity is pertinent to the increased susceptibility to infection and poor responsiveness to vaccination of aged individuals and, furthermore, is implicated in numerous age-related diseases such as metabolic syndrome, cardiovascular and neurodegenerative diseases, and cancer. The hyperactivation of PRR pathways, to which both infectious agents and endogenous sources of ligands have been shown to contribute, may promote a state of persistent low-grade inflammation in elderly individuals, referred to as “inflammageing,” which in turn may also facilitate the emergence of chronic age-related diseases. Most notably, deficiency in TLR activity may be responsible for the impaired immunity to infection observed in ageing, whereas hyperactivation of TLRs and inflammasomes has been frequently associated with chronic age-related diseases, suggesting that these PRR families may be particularly amenable to therapies aimed at enhancing immunity in the elderly or treating age-related disorders.