Serotonin 5-HT2 receptor activation prevents allergic asthma in a mouse model

Serotonin (5-HT) acts through multiple receptor subtypes and is implicated in various physiological and immune processes. Prior work has shown that the 5-HT2A receptor, best known for roles in the central nervous system and as the target of classic hallucinogens, is also expressed in immune-related tissues and certain pulmonary cell types. Nau and colleagues previously reported potent anti-inflammatory effects of 5-HT2A receptor agonists in vitro and in vivo, particularly with the high-affinity agonist (R)-DOI, which blocks expression of multiple proinflammatory markers and pathways. This study set out to test whether activation of 5-HT2 receptors by inhaled (R)-DOI can prevent key features of allergic airways disease. Using the well-established ovalbumin (OVA) mouse model of allergic asthma, the investigators examined whether pulmonary delivery of (R)-DOI blocks airways hyperresponsiveness (AHR), mucus hyperproduction, pulmonary inflammation and eosinophil recruitment, and they profiled relevant cytokine and chemokine gene expression in the lung.

The study used the OVA-induced allergic asthma model in male mice aged 6–8 weeks. Mice were sensitised by intraperitoneal injection of 20 μg OVA emulsified in alum on days 0 and 14, then challenged with a 1% (wt/vol) OVA aerosol for 20 minutes on days 24–26. Thirty minutes before each aerosol challenge mice received nose-only inhalation of (R)-DOI at either 0.01 mg/kg or 1.0 mg/kg, or vehicle control, delivered with an ultrasonic nebuliser. Pulmonary physiology was assessed on day 28. Two complementary methods were used: the forced oscillation technique in anaesthetised, mechanically ventilated animals (FlexiVent) with tidal volume 10 ml/kg and frequency 2.5 Hz, and whole-body plethysmography in awake mice recording peak enhanced pause (PenH). Bronchoconstrictor responses were evoked by serial aerosolised methacholine (MeCh) challenges; MeCh concentrations differed slightly between methods (forced oscillation up to 50 mg/ml, whole-body plethysmography up to 100 mg/ml). Bronchoalveolar lavage fluid (BALF) was collected after pulmonary testing on day 28 for total and differential cell counts; two blinded observers classified cells and at least 200 cells were counted per animal. Lung histopathology was performed on paraffin sections stained with Periodic acid–Schiff (PAS) for mucus and haematoxylin and eosin for morphology and inflammation. BALF total protein was measured by BCA assay. Pulmonary gene expression of a panel of cytokines and chemokines (Il-4, Il-5, Il-6, Il-10, Il-13, Tnfα, Mcp-1, and Gm-csf) was quantified by quantitative PCR using Roche LightCycler instrumentation and analysed by the ΔΔCT method normalised to Gapdh. Total and OVA-specific serum IgE were measured, although assay details are not clearly reported in the extracted text. Statistical analyses were performed using GraphPad Prism. The extracted text does not clearly report group sizes or specific statistical tests used for individual comparisons.

In both measures of airway function, OVA-sensitised and challenged mice developed significant AHR as expected. Pulmonary administration of (R)-DOI prior to each OVA challenge effectively prevented this increase in airway responsiveness. Mice treated with either 0.01 mg/kg or 1.0 mg/kg (R)-DOI showed airway responses that were not significantly different from naïve controls by both forced oscillation and whole-body plethysmography. Histological examination revealed pronounced peribronchial inflammation and mucus hyperproduction in OVA-only mice. Pretreatment with 1.0 mg/kg (R)-DOI produced very little peribronchial inflammation or mucus, whereas the low dose of 0.01 mg/kg led to a significant reduction in inflammation and mucus compared to OVA alone but did not fully normalise histology. Cellular analysis of BALF showed that OVA provoked a marked increase in total recovered cells driven largely by eosinophils. Both (R)-DOI doses significantly reduced total BALF cell numbers and eosinophil counts relative to OVA-only mice. Eosinophil counts in the (R)-DOI groups were higher than in naïve mice but not significantly different from naïve. A non-significant trend toward reduced neutrophils was observed in (R)-DOI treated mice. Measures of lung vascular/epithelial leak and systemic humoral responses were not altered by (R)-DOI. BALF total protein was elevated in OVA mice versus naïve controls, and (R)-DOI did not reduce BALF protein compared with OVA-only. Similarly, OVA increased total and OVA-specific serum IgE, and these increases were not affected by (R)-DOI treatment. At the transcriptional level, OVA increased lung mRNA for several Th2 and innate cytokines and chemokines (Il-4, Il-5, Il-10, Il-13, Mcp-1, and Gm-csf), with Il-6 and Tnfα showing non-significant trends. (R)-DOI did not alter the OVA-induced increases in Il-4 or Il-10. In contrast, (R)-DOI significantly repressed OVA-induced Mcp-1, Il-13 and Il-5 expression and completely blocked the increase in Gm-csf. Although Il-6 was not significantly upregulated by OVA in this dataset, (R)-DOI significantly reduced Il-6 expression in OVA-treated mice.

Nau and colleagues interpret their findings as evidence that selective activation of serotonin 5-HT2 receptors with inhaled (R)-DOI has potent anti-inflammatory effects in the lung and prevents the development of allergic airways disease in the OVA mouse model. They emphasise that previous work indicates the anti-inflammatory actions of (R)-DOI are mediated via the 5-HT2A receptor subtype, and the authors detected 5-HT2A receptor mRNA in whole lung tissue. Two doses were examined: 1.0 mg/kg, comparable to doses used in behavioural studies, and a much lower 0.01 mg/kg dose chosen to test the compound's predicted super-potency; both doses produced anti-inflammatory effects. The investigators propose that the therapeutic site of action is pulmonary tissue, acting on resident activated T cells and/or innate immune cells. Support for this mechanism includes repression by (R)-DOI of key mediators of eosinophil recruitment and Th2 responses, including Il-5, Gm-csf and Mcp-1, and the observed blockade of eosinophil recruitment, mucus hyperproduction and AHR. Because (R)-DOI did not reduce BALF protein or modulate total or OVA-specific IgE, the authors suggest the drug acts on activated rather than naïve immune populations and that its protective effects are likely non-mast-cell dependent. The discussion contrasts these results with earlier reports that implicated 5-HT2A receptors in promoting AHR; the authors note those studies used the relatively non-selective antagonist ketanserin, which complicates interpretation. Two explanations are offered for the apparent discrepancy: selective activation of 5-HT2 receptors may avoid engagement of other serotonin receptor subtypes that drive inflammation, and (R)-DOI may act as a functionally selective ligand at 5-HT2A receptors, recruiting anti-inflammatory signalling pathways that endogenous serotonin does not. The authors acknowledge several uncertainties: they were unable in this study to directly validate the receptor subtype mediating the effect, systemic administration of (R)-DOI was not tested, and the precise downstream signalling pathways remain to be elucidated. Concluding, the study identifies a previously unrecognised functional role for 5-HT2 receptor activation in the lung and proposes that selective 5-HT2A agonists such as (R)-DOI might represent a novel small-molecule therapeutic approach for asthma, effective at doses well below those required to cause behavioural or cardiovascular adverse effects.