Tropical urban environments reveal a strong association of CD45RBloCD161+ Th2 subset to allergic rhinitis To the Editor:Allergic airway diseases such as allergic rhinitis (AR) affects more than 400 million individuals worldwide and afflicts substantial health and economic morbidity. [1] AR is strongly associated with a type 2 response, characterized by the cytokines IL-5, IL-4 and IL-13. However, the key drivers behind AR immunopathogenesis remained to be elucidated. This study aims to identify critical pathogenic cell populations associated with AR using the Singapore System Immunology Cohort (SSIC) [2] and a clinician-diagnosed paediatric cohort with active AR manifestation (Supplementary Table 1 ). In both cohorts, the eosinophilic nature of AR was confirmed by higher blood eosinophil numbers (Supplementary Figure 1 ).Whole blood gene expression analysis revealed a total of 23 probes representing 20 unique genes were associated with AR in the SSIC (Table 1A ). To account for ethnicity and environmental influences we validated our findings in BAMSE population-based cohort comprising of Swedish adolescents. Table 1B shows 11 DEGs which was also associated with AR, confirming the transferability of our findings to other populations. For the top DEGs that reached nominal significance in the SSIC we performed an Ingenuity Pathway Analysis (IPA). Supplementary Table 2 revealed important pathways related to hypersensitivity and inflammation and also functional enrichment for eosinophils, basophils and mast cells. In particular, functional activation of Th2 was highlighted as a key pathway for AR pathogenesis. As CRTH2 was reported to be expressed by cell types involved in the eosinophilic response, [3] an unsupervised cluster analysis was performed on the CRTH2+ subset in PBMC of individuals from SSIC (Figure 1 A and B ) to determine CRTH2+ subsets associated with AR. We found that CD161+Th2 subsets in particularly to be strongly associated with AR (Figure 1C and D ) (Supplementary Figure 2 ). Further characterization found that the marker CD45RB to be significantly downregulated on CD161+Th2 cells of AR individuals (Figure 1E ). Low CD45RB expression on T cells is indicative of a mature phenotype. Interestingly, significantly higher circulatory plasma IL-5 levels (Figure 1F) . Furthermore we could also demonstrate AR individuals produced significantly higher IL-5 in an in vitro PMA-stimulation assay (Figure 1G ).While we noted a small population of IL-5 secreting conventional CD161-Th2 (cTh2), IL-5 secretion was significantly elevated in CD161+Th2 cells (Figure 1H ). Strikingly, IL-5 was found to be predominantly secreted by CD45RBlo subset in both cTh2 and CD161+Th2 (Figure 1H and I ). There was also a significant increase in the IL-5 producing CD45RBloCD161+Th2 population from the AR individuals (Figure 1I ). These findings confirm CD45RBloCD161+Th2 as the main producers of IL-5.We further validated our findings in a second paediatric cohort with clinically diagnosed active AR manifestations. To further refine CD161+Th2 subset that is associated with AR, we performed unsupervised PhenoGraph and UMAP clustering on CD161+Th2 (Supplementary Figure 3A and B ). Amongst the UMAP clusters, “cluster 3” was found to be significantly associated with active AR (Supplementary Figure 3C and D ). Deep characterization reveals “cluster 3” to be an IL-5 secreting CD45RBlopopulation, confirming our earlier observation (Supplementary Figure 3E ). Furthermore, this cluster appeared to be a highly differentiated population of mature CD161+Th2 cells with an activated phenotype secreting IL-2, IL-3, IL-4, IL-9 and IL-13 concomitantly (Supplementary Figure 3E and F ). Thus, the severity of eosinophilic airway allergies such as AR seems to be driven by an activated terminally differentiated CD161+Th2 subset that is able to secrete a complex set of inflammatory cytokines.The presence of CD45RBloCD161+Th2 population in both cohorts shows the persistence and pertinence of this population in the pathogenesis of AR. Both cohorts described in this study were collected in Singapore, whereby majority of the individuals are sensitized against HDM. HDM is a perennial allergen in tropical nations such as Singapore, thus T cells in atopic individuals undergo constant stimulation. This could explain the strong association observed between CD45RB expression on CD161+Th2 cells and atopy markers despite the fact that not all subjects demonstrated active AR symptoms during the collection of SSIC cohort. Taken together, our current study unifies the markers previously reported for allergic-specific Th2 subsets and provides clarity for the pathogenic Th2 subset previously reported in different allergic diseases.[4-6] Neutralizing the CD45RBloCD161+Th2 subset should disrupt the allergic response pathway, thus providing a target for lasting therapeutic interventions. Moreover, these cells may also be leveraged as a biomarker for the effectiveness of immunotherapy as well as a potential biomarker of public health surveillance of allergic individuals.

Late inflammatory monocytes define circulatory immune dysregulation observed in skin microbiome-stratified atopic dermatitisTo the Editor,Atopic dermatitis (AD) is a skin inflammatory disorder well described for significant disease heterogeneity. (1) Previously, we defined steady-state microbial configurations dermotypes A and B that robustly reflected heterogeneity in AD clinical severity, cutaneous barrier properties and skin microbiome composition. (2) Here, we explored circulating immune dysregulation underlying dermotype-stratified AD.We performed single-cell RNA sequencing (scRNA-seq) of PBMCs from healthy subjects with dermotype A (n = 4) and AD patients with dermotypes A (n = 4) and B (n = 6).  UMAP reduction distinguished 13 major immune clusters using transcriptional profile differences, identified using DISCO CELLiD (3) and top differentially expressed genes (DEGs; Supplementary Figure 1A ). Major clusters corroborated well across all groups as a testimony to limited sample distortion by inter-individual or batch effects (Supplementary Figure 1B ). Critically, AD patients harboured a marked enrichment in monocyte population cluster 1, whereby dermotype stratification saw a further enrichment in dermotype B (Supplementary Figure 1B ).To reveal finer expression details, we sub-clustered myeloid cells, obtaining 11 sub-populations whose identities were similarly assigned following DISCO CELLiD and top DEGs (Supplementary Figure 1C ). Clusters with low cell counts and not of myeloid origin were removed prior to down sampling for normalisation, yielding 7 myeloid clusters of interest. Majority of clusters represent CD14+ monocytes except for clusters 5 and 7 corresponding to CD16+ monocytes and conventional dendritic cells (cDC) respectively (Figure 1A ). CD14+ monocytes segregated into 4 transcriptionally distinct states (Figure 1A and B ): early activation monocytes expressing proinflammatory alarmins (S100A8 and S100A12; clusters 0 and 1), transitional monocytes concomitantly expressing alarmins and chemokines (S100A12 and CCL3; cluster 2), intermediate monocytes expressing antigen presentation genes (HLA-DPB1 andHLA-DPA1 ; cluster 3), and late inflammatory monocytes expressing proinflammatory cytokines (IL1B, CCL3 and TNF ; cluster 4,Supplementary Figure 1D ) (4, 5). Late inflammatory monocytes (cluster 4) were notably enriched in AD dermotype B (Figure 1A and C ) and exhibited upregulation in MHC class II (HLA-DQA2, HLA-DQB1 ), monocyte-related (ITGAM, FGD2 ) and proinflammatory genes (CCL4L2, CCL3L1 and CCL20 ), suggestive of inflammatory dysregulation (Figure 1D and Supplementary Table 1 ). Taken together, dermotype stratification addressed part of the cellular heterogeneity observed in circulating immune profiles, characterised particularly by late inflammatory monocytes enrichment in dermotype B.Complimenting scRNA-seq findings with CytoFLEX (Supplementary Figure 2A ), unstimulated PBMCs were stained and secretion of inflammatory cytokines CCL3, CCL4, TNF-α, IL-1β, and IL-8 was monitored (Supplementary Table 2 ). Cytokine secretion by classical (CD14+ CD16-) monocytes were significantly elevated in AD compared to healthy controls (Figures 2A, B and Supplementary Figure 2B ). Atopy-relevant cell types were examined, revealing similar levels between AD dermotypes A and B but elevated presence of Th2 and Th2a in AD dermotype B as opposed to healthy controls (Figures 2C and D ). The reverse was observed for NK and ILC3s, where AD subjects with dermotype B registered cell deficiencies (Figures 2E and F ). Comparing between AD dermotypes, IL3Cs were significantly diminished in dermotype B (Figure 2F ). Healthy and AD subjects harbouring dermotype A shared similar expression of aforementioned cells, suggesting that disease alone could not differentiate immune dysregulation observed. Next, we quantified 92 inflammatory biomarkers in plasma using Olink Proteomics Proximity Extension Assay (PEA) Target 96 Inflammation Panel. AD subjects recorded significantly higher expression of TNF-associated cytokines TRANCE and TRAIL, monocyte chemoattractant MCP-4, IL-18, and IL-2 than healthy controls (Figures 2G-J and Supplementary Figure 2C ). Noticeably, higher basal abundance of TRANCE and MCP-4 was observed in AD dermotype B than dermotype A (Figures 2G and I ), lending additional support to circulatory immune dysregulation previously observed in inflammatory monocytes.Our findings provide unique insights into immune heterogeneity underlying AD patients stratified by defined skin microbiome configurations. Definingly, dermotype B is characterised by microbial dysbiosis with reduced species richness, fewer commensal species and increased presence of S. aureus virulence genes. (2) Rather than atopy-relevant Type 2 cells, late inflammatory monocytes demonstrated a striking enrichment in AD dermotype B, particularly in the upregulation of proinflammatory cytokine transcripts. Coupled with skin microbial dysbiosis that increases cutaneous exposure to potent allergic inflammation inducers such as δ-toxin (2, 6), immune dysregulation directed by monocytes could predispose AD skin towards flare predisposition and clinical severity as observed in dermotype B. Basal secretion levels of proinflammatory cytokines by classical monocytes are elevated in AD regardless of dermotype, suggesting higher baseline inflammation driven by disease. While no stimulation was conducted here, earlier reports have demonstrated that LPS activation of monocytes upregulates transcriptional expression of proinflammatory cytokines IL-1β, CCL3 and IL-8. (4) Similarly, heightened proinflammatory cytokines and monocyte-related proteins were detected in plasma of AD dermotype B compared to dermotype A subjects, validating scRNA-seq findings of monocyte-driven immune dysregulation and inflammation.Collectively, circulatory immune profiling of dermotypes, defined by alterations in skin microbiota compositions, suggest a pathophysiological role for inflammatory monocytes. Future studies are needed to ascertain the functional and mechanistic roles of late inflammatory monocytes in dermotype-stratified AD. Importantly, our study highlights the potential clinical utility of dermotype stratification to indicate the cellular basis for increased inflammation and exacerbated immune dysregulation, setting precedence for better understanding of disease prognosis.

Background: Allergic rhinitis (AR) is strongly associated with a type 2 response, characterized by the cytokines IL-5, IL-4 and IL-13. Several studies have implicated ILC2 and TH2A (CD161+ TH2) but it is not yet entirely clear which subsets are driving the common allergic reactions underlying AR. The objective of this study aims to identify critical pathogenic cell populations associated with AR and to determine their phenotype and functional contribution to disease progression. Methods: We identified integral allergic-specific cell types by transcriptomic sequencing. Whole blood, PBMCs and plasma from a cross-sectional cohort of 216 individuals were analysed by 9-colour flow cytometry and ultra-sensitive cytokine bead arrays using unsupervised clustering algorithms. Clinically active AR cases were further analysed by functional mass cytometry to define phenotype and cytokine secretion (IL-2, IL-3, IL-4, IL-5, IL-9, IL13, IL-17A and IL-22) as well as the expression of the hematopoietic prostaglandin D synthetase (HPGDS). Results: The unbiased analysis revealed that atopy and AR manifestation corelated only with eosinophils, plasma IL-5 and CD161+ TH2 cells. In-depth characterization further revealed that the CD45RB lo CD161+ TH2 subset were most closely associated with severity. This subset is able to concomitantly secrete multiple cytokines including IL-5, IL-13 and IL-4 and has been previously reported to be associated with other eosinophilic allergies. Conclusion: CD45RB lo CD161+ TH2 have key roles in driving the allergic response in AR. Neutralizing the CD45RB lo CD161+ TH2 subset should disrupt the allergic response pathway, thus providing a target for lasting therapeutic interventions.

Article type: Letter to the Editor

Disrupted epithelial barrier in nasal polyps characterizes aspirin exacerbated respiratory disease Anand Kumar Andiappan1*, Mohammad Asad2, Celine Chua3, Esha Sehanobish2 , Zhen Ren4, Xue Ying Chan1, Josephine Lum1, Nicholas Ang1, Duan Kaibo1, Adam Gersten2, Waleed M. Abuzeid5, Nadeem Akbar2, Marc Gibber2, Shanshan Howland1, Bernett Lee1, Olaf Rotzschke1, Steven A. Porcelli2, Elina Jerschow2*1 Singapore Immunology Network, Agency for Science, Technology and Research2 Albert Einstein College of Medicine/Montefiore Medical Center, Bronx, NY, USA3 Department of Biological Science, National University of Singapore4 Division of Allergy and Immunology, Department of Medicine, Washington University Schoolf of Medicine, St Louis, MO, USA5Rhinology and Endoscopic Skull Base Surgery, Department of Otolaryngology – Head and Neck Surgery, University of Washington, Seattle, WA, USA*Correspondence: [email protected]; [email protected]