Treatment with a sphingosine analog after the inception of house dust mite-induced airway inflammation alleviates key features of experimental asthma
© Gendron et al.; licensee BioMed Central. 2015
Received: 6 August 2014
Accepted: 21 January 2015
Published: 3 February 2015
In 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.
BALB/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.
AAL-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.
Airway 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.
KeywordsFTY720 Fingolimod Gilenya Dermatophagoides pteronyssinus Apoptosis Dendritic cells CD4+ T cells Asthma S1P AAL-R AAL-S Sphingosine
Asthma is a chronic lung disorder with no cure that affects 235 million people worldwide . Asthma decreases the quality of life and its yearly cost per country ranges from 10 to 266 billion dollars . Although a myriad of interventions exists to alleviate symptoms, current practices only ensure adequate control of asthma in less than 50% of patients . Furthermore, up to 10 percent of patients suffer from severe forms of asthma that are largely refractory to current therapies  and too many still succumb . Given the health and the economic burden of asthma, identification of new mechanisms and targets for intervention is required.
Sphingosine analogs emerge as potent immunomodulators with therapeutic efficacy proven in humans. For instance, the drug FTY720 (Fingolimod, Gilenya) is currently used for patients with multiple sclerosis . The compound AAL-R is an analog of FTY720 that has been broadly used to study the mechanisms of action of this class of compounds [6-9]. Sphingosine analogs such as FTY720 and AAL-R are cell-permeant and become phosphorylated intracellularly to become a sphingosine-1-phosphate (S1P) analog. They are then actively exported from the cells. Owing to their phosphorylation, the secreted forms of the drugs are cell-impermeant and their mechanisms of action become then restricted to extracellular effects, which are mediated by binding on four of the five cell surface S1P receptors (S1P1,3–5) . The archetypal mechanism of action of sphingosine analogs is sequestration of lymphocytes in secondary lymphoid organs after activation of S1P1 . However, it is increasingly recognized that activation of S1P receptors following the intracellular phosphorylation and secretion of the analogs is not the only mechanism underlying their bioactivity. These compounds can indeed modulate cellular functions independently of S1P receptor engagement, including among others cell fate and the activity of both phospholipase and phosphatase subsets [6,11,12]. The mechanisms of action of sphingosine analogs are thus complex, misunderstood, and might vary under different pathophysiological conditions.
When administered in a prophylactic fashion, phosphorylatable sphingosine analogs interfere with the development of experimental allergic airway inflammation. This was demonstrated in the classical model induced by systemic sensitization with ovalbumin (OVA) emulsified in aluminum (Alum), followed by airway challenges with OVA (OVA-Alum model) . The mechanism of action under these conditions is potent inhibition of dendritic cell (DC) accumulation in draining lymph nodes, interference with antigen presentation, and failure to launch an efficient antigen-specific T cell response [13,14]. However, whether the phosphorylatable sphingosine analogs are salutary when administered in a therapeutic fashion, i.e. after the onset of allergic airway inflammation, is undefined. Additionally, the efficacy of phosphorylatable sphingosine analogs to interfere with airway inflammation induced by a natural allergen has never been studied. In contrast with the classical OVA-Alum model, natural allergens have specific modi operandi – including proteolytic activity and endogenous adjuvant activity . Importantly, the natural allergens evoke a classical allergic response by local mechanisms involving pulmonary DC when delivered through natural routes of exposure, such as the airways . They thus recapitulate more genuinely the mechanisms of allergic airway inflammation in humans.
Under these premises, we aimed to determine if a sphingosine analog, namely AAL-R, alleviates experimental asthma elicited by subchronic airway inflammation induced by exposure to a natural allergen, namely Dermatophagoides pteronyssinus (house dust mite extract; HDM). We hypothesized that treatment with AAL-R after the onset of HDM-induced airway inflammation interferes with mechanisms leading to key features of asthma in a murine model.
Murine model of asthma
Pathogen-free BALB/c female mice (8 weeks old, Charles River) were anesthetized with isoflurane and instilled intranasally (i.n.) with 50 μl of saline or with saline containing 50 or 75 μg HDM extract (Greer), once daily, from day 1 to 10. Unless otherwise indicated, all measurements were taken at day 11. The mice were either euthanized with ketamine-xylazine overdose for collection of the bronchoalveolar lavage fluid (BALF) and for tissue sampling; or tested for airway responsiveness to methacholine (MCh) using documented methods . Experimental, housing and care procedures were approved by the Committee of Animal Care of Laval University in accordance with the guidelines of the Canadian Council on Animal Care (protocols 2013037, 2009128).
Plates were coated with 50 μg/mL HDM and incubated with serial dilution of serum (1:200 to 1:145800). Titers were determined by a colorimetric reaction as reported .
Naïve or HDM-instilled mice were treated intratracheally (i.t.) with either 50 μl of distilled water (dH2O) containing 2.5 μg of AAL-R, or the vehicle (dH2O) from day 7 to day 10. This dose, which corresponds to 0.1 mg/kg, was chosen because it does not induce histological alterations per se, and because it was shown to potently induce immunomodulation in the airways [13,14]. Treatments were delivered under isoflurane anesthesia and animals were thereafter placed in standard housing conditions for 1 h until re-anesthesized with isoflurane prior to HDM instillation. In a second series of experiments, an additional group was treated with a molar equivalent dose (3.5 μg) of the phosphorylated sphingosine analog, namely AFD-R. Compounds were either provided by Dr Hugh Rosen (TSRI) or synthetized using a modification of published methods (NuChem Therapeutics Inc.) [19,20].
Differential counts, flow cytometric analyses and quantification of cytokines
BALF as well as single-cell suspensions from both the right and central lobes and from draining mediastinal lymph nodes (MLN) were obtained as previously reported . Antibodies are anti- CD4 (RM4-5) (eBioscience); CD8a (53–6.7), CD11c (N418), CD19 (6D5), CD90.2 (30-H12), Ki-67 (16A8) (Biolegend); MHC-II (2G9) (BD Biosciences). Viability was assessed using annexin V (Biolegend) and LIVE/DEAD (Life Technologies). Fluorescence and autofluorescence (AF) were acquired using a Diva-driven LSRFortessa (Becton Dickinson) and analyzed with the FlowJo software (Tree Star inc). To compute the total numbers of cell subsets, absolute cell counts obtained with a hemocytometer were multiplied by cell frequencies obtained either by flow cytometry or following differential cell counts as previously reported [17,21]. For cytokine quantification, cell-free BALF were concentrated with Amicon 3 K (Millipore) and incubated with cytometric bead array Flex Set for interleukin (IL)-5, IL-13 and CCL5 according to the manufacturer’s instructions (BD Biosciences).
After BALF were performed, the left caudal lobes were processed as previously described  and stained with either hematoxylin and eosin (H&E) for the evaluation of inflammatory cell infiltrate; or with Periodic Acid-Schiff (PAS) stain and hematoxylin for the evaluation of mucus-producing cells. Histological evaluation was performed by an American College of Veterinary Pathologists Board-certified pathologist (CT). The nomenclature used to describe the lesion and the severity of the lesions (5 grades scale) was based on the guidelines provided by the Society of Toxicological Pathology [22,23].
Measurement of airway responsiveness
Methacholine (MCh) challenge test was performed on tracheotomized mice connected to a flexiVent apparatus (SCIREQ) as previously reported .
Unless otherwise stated, the data shown are means ± SEM. Data were analyzed using one-way ANOVAs. To fulfill the normality and variance assumptions, variables were log-transformed. An arc sinus transformation on the square root was applied on BALF measurements that were expressed in percentage. Homogeneity of covariance parameter assumption among groups was verified. A posteriori comparisons were performed with Tukey’s comparison technique. All reported P values are based on these transformations. In addition, the nonparametric Kruskal-Wallis test was performed on histology data. The results were considered significant with p values ≤ 0.05, unless otherwise stated. All analyses were conducted using the statistical package SAS v9.3 (SAS Institute Inc).
Onset of allergic airway inflammation in response to daily exposure to HDM
AAL-R alleviates airway responsiveness induced by HDM
Histopathological alterations of mice lungs exposed to HDM
Perivascular infiltration of mononuclear cells
Peribronchial/bronchiolar infiltration of mononuclear cells
Perivascular infiltration of granulocytes
Hypertrophy/hyperplasia of the bronchial epithelium
AAL-R interferes with lung accumulation of cells that drive inflammation induced by HDM
AAL-R enhances apoptosis/necrosis of lymphocytes in the lung
Effect of intratracheal delivery of AAL-R on lymphocytes and DC in the draining lymph nodes
Sphingosine analogs are new therapeutic molecules with potent immunomodulatory properties. Yet, their potential in the treatment of asthma is misunderstood. The main finding of this study is the efficacy of a phosphorylatable sphingosine analog to interfere with asthma in a murine model after the onset of allergic inflammation induced by a natural allergen. We also reveal that lymphocyte and DC numbers in draining lymph nodes are not modulated by AAL-R, as it was previously observed in the OVA-Alum model of asthma . Conversely, AAL-R attenuates the accumulation of eosinophils, DC, CD4+ T cells and CD19+ B cells in the lungs. This was associated with significant decreases of IL-5 and IL-13 levels in the BALF. We also identify the induction of T cell apoptosis as a significant mechanism contributing to the alleviation of allergic airway inflammation by AAL-R; and this phenomenon was not observed using the pre-phosphorylated cell impermeant form of the drug.
Previous studies have addressed the roles of S1P pathway modifiers in the phase of intra peritoneal sensitization with OVA-Alum . In addition, administration of S1P pathway modifiers prior to, or concomitantly with, repetitive OVA challenges in peripherally sensitized animals was shown to be effective in preventing cardinal features of experimental asthma . However, it was heretofore unknown whether the drugs were able to attenuate key features of established allergic airway inflammation. The findings of the present study represent the first evidence that a phosphorylatable sphingosine analog can alleviate experimental asthma after the onset of allergic airway inflammation.
It is becoming clear that the airway mucosa can be a critical site for amplification and maintenance of allergic disease . Consequently, models using natural allergens that cause signs of experimental asthma when delivered through natural routes are currently preferred to assess the preclinical potential of experimental treatments . In the model used herein, airway delivery of HDM was sufficient to trigger functional impairment of the airways, accumulation of T cells, B cells and eosinophils. Remarkably, all of which were inhibited by AAL-R. In accordance with our current finding, FTY720 dampens eosinophil accumulation within the mucosa in a model of ongoing allergic rhinitis . Unfortunately, the number of DC in draining lymphoid tissues was not assessed in that latter study . In our study, both local DC and T cells were severely altered by local treatment with AAL-R, which is a major interest owing to the predominant role played by these cell subsets in pulmonary inflammation. This alteration of the local T cell response was also associated with a strong decrease of IL-5 and IL-13 levels in the airways. Of potential importance is the fact that these interleukins can by themselves increase the contractile capacity of airway smooth muscle . It is thus likely that AAL-R interfered with airway hyperresponsiveness, at least in part, by reducing the levels of soluble factors that potentiate the contractile capacity of airway smooth muscle. On note, airway smooth muscle enlargement is not observed in this acute model; so impairment of this feature may not be related to the beneficial effect of AAL-R on airway hyperresponsiveness. The location of T cells has also recently emerged as a critical mechanism influencing the contractile capacity of airway smooth muscle. Indeed, T cells in the vicinity of airway muscle cells modify their contractile capacity [30,31]. It is tempting to speculate that the profound reduction of T cell numbers by AAL-R might have contributed to alleviate hyperresponsiveness by reducing the number of T cells located proximally to the contractile apparatus of the airways.
The current work also highlights that the immunomodulatory mechanisms of sphingosine analogs after the onset of allergic inflammation likely differ from the ones observed in the context of a prophylactic treatment. When administered prophylactically, i.e. prior to airway OVA challenge, sphingosine analogs strongly inhibit antigen presentation in draining lymph nodes, which is responsible for the alleviation of experimental allergic airway disease [13,14]. In agreement with these results, airway delivery of a single dose of AAL-R soon after influenza virus infection inhibits both DC accumulation in draining lymph nodes and the ensuing immunopathological responses in the airways [9,32]. Conversely, our results show that AAL-R strongly inhibits DC accumulation in the lungs while having mild effects on DC numbers in MLNs over the course of a 4-day treatment. These results are in line with the ability of sphingosine analogs to interfere with de novo DC recruitment from blood to tissues , and to blunt the release of chemotactic factors in the lung [9,13]. While we cannot fully exclude the possibility that antigen presentation was modulated, our results rather suggest that once DCs have migrated to the lymph node, sphingosine analogs are relatively impotent at interfering with the chain of events leading to the antigen-triggered amplification of lymphocytes.
Although several studies used local delivery of sphingosine analogs [9,13,14,21,34,35], and sphingosine analogs are well known inducers of cell death , this is the first evidence supporting the idea that cell death could contribute to alleviate allergic airway disease. Indeed, aminoalcohols like AAL-R readily penetrate the plasma membrane and become phosphorylated intracellularly to interfere with cell survival . Accordingly, we show that AAL-R, but not AFD-R, potently induces lymphocyte apoptosis/necrosis in the lung. This is consistent with AAL-R’s rapid phosphorylation, low turnover, and potent induction of apoptosis in murine splenocytes through intracellular accumulation of AFD-R . Interestingly, AAL-R-induced apoptosis/necrosis appears to affect CD4+ T cells and B cells, which are crucially involved in allergic airway disease, while having no effect on CD8+ T cells, DCs and the CD90−CD19−AF−SSCmed-hi granulocyte-enriched fraction. These results certainly argue against a non-specific toxic effect. Of note, FTY720 was associated with preferential apoptosis of activated T cells in another model , while not potently affecting DC apoptosis in vitro  and myeloid leukocyte apoptosis in vivo . In a preclinical perspective, drugs that are customarily used to treat asthma can induce apoptosis in cells from the airways, supporting the viability of this drug-related mechanism of action in humans [39,40].
Lymphopenia has long been associated with the immunomodulatory effects of sphingosine analogs. Yet, a number of facts challenge the idea that systemic lymphopenia per se is central to immunomodulation. Indeed, local treatments with sphingosine analogs induce more potent immunosuppression than systemic delivery . In addition, naïve T cells are more sensitive to sphingosine analogs-induced lymphopenia than activated T cells . Our results reveal that distinct mechanisms of action could be associated with the non-phosphorylated and the phosphorylated forms of the compounds. Specifically, our results demonstrated that AAL-R induces a more severe local T cell inhibition than AFD-R, which correlates with the ability of AAL-R to induce local death of lymphocytes. The idea that lymphopenia-independent mechanisms could explain, at least in part, the local immunosuppression induced by sphingosine analogs is also supported by the observation that prophylactic treatment with the non-phosphorylatable AAL-S enantiomer, yet at a somewhat superior dosage than in the current study, inhibits cytokine release as well as T cell and DC accumulation in the airways in a model of experimental asthma . This effect was associated with activation of protein phosphatase 2A, but it remains unknown if such mechanism is viable under the current conditions.
We conclude that a phosphorylatable sphingosine analog can interfere with allergic airway inflammation caused by a natural allergen, resulting in improved respiratory function. The mechanism of action appears to differ from that previously observed in a prophylactic context. Our results also support that the phosphorylation status of the drug can influence the mechanisms by which the immune response is modulated, with enhancement of T cell apoptosis being restricted to the non-phosphorylated form.
We thank Serge Simard for statistical analyses, Marc Veillette and Marie-Josée Beaulieu for their help and expertise. We thank Hugh Rosen for providing AAL-R and Daniel Guay for support with synthesis of sphingosine analogs.
Supported by CIHR grant # 274357 and funding from the Respiratory Health Network of the FRQS. DG receives a scholarship from the Training program in respiratory health of Quebec – IRSC. DM, MRB, NF, EB are members of the FRQS Respiratory Health Network and DM, NF, MRB are FRQS Junior 1 Scholars. This work was also supported by the Canadian Foundation for Innovation.
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