Sputum alarmin levels delineate distinct T2 cytokine pathways and patient subgroups in asthma

Rationale Asthma is a chronic airway disease driven by multiple immunologic pathways that determine the clinical response to therapy. Current diagnostic methods are incapable of discriminating subtypes of asthma and guiding targeted treatment. We hypothesized that sputum cytokine profiles could help to identify immunologically-defined disease subtypes and individualize therapy in patients with severe asthma. Objectives Define asthma subtypes associated with sputum alarmin and cytokine levels. Methods Cross-sectional analysis of clinical features and sputum from 200 asthmatic patients was performed. 10 cytokines belonging to alarmin, T2, and non-T2 pathways were measured. Pearson correlation was used to identify cytokine modules. Latent class analysis was used to cluster patients by cytokine expression. Measurements and Main Results Three modules of highly correlated cytokines were identified including a non-T2 module, the IL-1βmod (IL-1β, IL-6, GCSF), and two distinct T2 modules: TSLPmod (TSLP, IL-4, IL-5, IL-9) and IL-33mod (IL-33, IL-13, IL-21). The TSLPmod was associated with asthma severity, airway obstruction, eosinophilia, and elevated FeNO. Patient clustering revealed three subgroups; two different subgroups showed expression of T2 modules. Conclusions Analysis of sputum cytokines revealed three discrete signaling modules in patients with asthma. Unexpectedly, the inclusion of alarmins led to separation of canonical T2 cytokines into two unique modules; IL-5 grouped with TSLP, while IL-13 grouped with IL-33. In addition, patient clustering revealed two distinct endotypes associated with T2 immune signaling. These findings indicate a new layer of immunologic heterogeneity within the T2 paradigm, and suggest that sputum cytokine profiling may hold diagnostic utility for patients with asthma. ### Competing Interest Statement All authors have completed the ICMJE uniform disclosure form at [www.icmje.org/coi_disclosure.pdf][1] and declare: no support from any organization for the submitted work; SG has received payment for his Advisory Board role for AstraZeneca, GLC has received payments for his Speakers Bureau / Advisory Board roles for AstraZeneca, Glaxo Smith-Kline, Boehringer Ingelheim, Sanofi, Regeneron, Genentech. ### Funding Statement This study was funded by: F32 (HL154641), K08 (HL159422), CFF Fellowship (GAUTAM20D0), PBF Fellowship, Patterson Award, YCCI Scholar Award, and Doris Duke FRCS at Yale Award (#2021266) to SG. R01 (HL153604) to JG. R21 (LM012884) to XY. UM1 (AI114271), U23 (HL138998), and R01 (HL153604) to GLC. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Institutional Review Board of Yale University gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors. * ACT : Asthma control test BDR : Bronchodilator response BMI – : Body mass index FeNO : Fractional exhaled nitric oxide IgE : Immunoglobulin E LCA : Latent class analysis PFT : Pulmonary function test T2 : Type 2 TSLP : Thymic stromal lymphopoietin YCAAD : Yale Center for Asthma and Airway Diseases [1]: http://www.icmje.org/coi_disclosure.pdf