Treatment with a sphingosine analog after the inception of house dust mite-induced airway inflammation alleviates key features of experimental asthma

Abstract

BackgroundIn vivo phosphorylation of sphingosine analogs with their ensuing binding and activation of their cell-surface sphingosine-1-phosphate receptors is regarded as the main immunomodulatory mechanism of this new class of drugs. Prophylactic treatment with sphingosine analogs interferes with experimental asthma by impeding the migration of dendritic cells to draining lymph nodes. However, whether these drugs can also alleviate allergic airway inflammation after its onset remains to be determined. Herein, we investigated to which extent and by which mechanisms the sphingosine analog AAL-R interferes with key features of asthma in a murine model during ongoing allergic inflammation induced by Dermatophagoides pteronyssinus.MethodsBALB/c mice were exposed to either D. pteronyssinus or saline, intranasally, once-daily for 10 consecutive days. Mice were treated intratracheally with either AAL-R, its pre-phosphorylated form AFD-R, or the vehicle before every allergen challenge over the last four days, i.e. after the onset of allergic airway inflammation. On day 11, airway responsiveness to methacholine was measured; inflammatory cells and cytokines were quantified in the airways; and the numbers and/or viability of T cells, B cells and dendritic cells were assessed in the lungs and draining lymph nodes.ResultsAAL-R decreased airway hyperresponsiveness induced by D. pteronyssinus by nearly 70%. This was associated with a strong reduction of IL-5 and IL-13 levels in the airways and with a decreased eosinophilic response. Notably, the lung CD4+ T cells were almost entirely eliminated by AAL-R, which concurred with enhanced apoptosis/necrosis in that cell population. This inhibition occurred in the absence of dendritic cell number modulation in draining lymph nodes. On the other hand, the pre-phosphorylated form AFD-R, which preferentially acts on cell-surface sphingosine-1-phosphate receptors, was relatively impotent at enhancing cell death, which led to a less efficient control of T cell and eosinophil responses in the lungs.ConclusionAirway delivery of the non-phosphorylated sphingosine analog, but not its pre-phosphorylated counterpart, is highly efficient at controlling the local T cell response after the onset of allergic airway inflammation. The mechanism appears to involve local induction of lymphocyte apoptosis/necrosis, while mildly affecting dendritic cell and T cell accumulation in draining lymph nodes.

Lipid Mediators of Allergic Disease: Pathways, Treatments, and Emerging Therapeutic Targets

“…In murine asthma models, S1P has a central role in mediating AHR (via smooth muscle S1P1 activation) and enhancing inflammation via effects on MCs, eosinophils, and DCs [140, 177]. Nebulized delivery of FTY720 (figolimod), an S1P1 functional antagonist, and multiple SphK1 inhibitors (SK1-I, N,N-dimethylsphingosine (DMS), AAL-R) have demonstrated that S1P mediates both the sensitization and effector phases of murine allergic asthma [35, 39, 40, 178]. …”

A Phosphorylatable Sphingosine Analog Induces Airway Smooth Muscle Cytostasis and Reverses Airway Hyperresponsiveness in Experimental Asthma

“…Contrary to AAL-R, corticosteroid intervention (dexamethasone; 1 mg/kg/day) failed to reverse AHR, furthering the concept of usefulness for sphingosine analogs in the context of asthma. On that note, we previously showed that AAL-R efficiently interfered with the inflammatory cascade in response to HDM soon after its instatement in the airways ( Gendron et al, 2015 ). In face of differing immune mechanisms occurring in the remodeled airways ( Mushaben et al, 2013 ; Sordillo and Raby, 2014 ), and since alleviation of eosinophilic inflammation can interfere with pathogenic mechanisms of asthma, we also confirmed that AAL-R blunted the antigen rechallenge-induced lymphocyte accumulation in the bronchoalveolar lavage fluid, which was accompanied by a profound 54% decrease of eosinophil numbers (Supplementary Figure 1 ).…”

“…Mice were then left untouched during week 6 ( Figure 2 ). Sphingosine analogs (AAL-S or AAL-R; see Gendron et al, 2015 ) or dexamethasone (Sigma Aldrich, Oakville, ON, Canada) were injected i.p. at a dose of 1 mg/kg once daily from day 7 to day 14 following the last HDM instillation, and compared with a group receiving an equal volume of the vehicle.…”

“…Heart rate was monitored until euthanasia in order to ensure proper anesthesia. Bronchoalveolar lavage fluid was harvested as described ( Gendron et al, 2015 ) and tissues were then fixed in phosphate buffered saline containing 4% paraformaldehyde for histological analyses. Experiments, housing and care procedures were approved by the Committee of Animal Care of Université Laval in accordance with the guidelines of the Canadian Council on Animal Care (protocol 2013037).…”

“…Human primary ASM cells (CC-2576; Lonza, Walkersville, MD, USA) were plated at a density of 10 4 cells per cm 2 in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum (complete medium) for 24 h, and then incubated with sphingosine analogs, the S1P lyase inhibitor (SM4; Zhao et al, 2015 ) or the dual S1P receptor (S1P 1-3 ) antagonist (VPC23019; Tocris, Burlington, ON, Canada) for 24–72 h. Cells were either incubated with tetrazolium dye (MTT assay; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma Aldrich, Oakville, ON, Canada) to determine the viable cell numbers; or incubated with bromodeoxyuridine (BrdU; 5-Bromo-2′-deoxyuridine; Roche, Laval, QC, Canada) overnight to assess proliferation. Flow cytometric analyses were performed as described ( Gendron et al, 2015 ).…”