Understanding the impact of opposing signals on human neutrophils in Atopic Dermatitis

Mel Diedro1, Selina Huber1, Patrick Westerman 2,3, Teresa Zinsli2,3, Milad Noori4, Christina Ravens4, Marie-Charlotte Brüggen5, CK-CARE Study Group, Christoph Messner2,3, Laurent Falquet6, Pierre-Yves Mantel1.7, Paola Martinez Murillo1

  1. Christine Kühne – Center for Allergy Research and Education, Davos, Switzerland
  2. Swiss Institute of Allergy and Asthma Research (SIAF), University of Zurich, Davos, Switzerland
  3. Precision Proteomics Center, Swiss Institute of Allergy and Asthma Research, University of Zurich, Davos, Switzerland
  4. Hochgebirgsklinik Davos, Switzerland
  5. Faculty of Medicine, University of Zurich, Zurich, Switzerland
  6. Department of Biology, University of Fribourg, Fribourg, Switzerland
  7. Department of Oncology, Microbiology, and Immunology, University of Fribourg, Fribourg, Switzerland

Introduction
Neutrophils, once considered short-lived and terminally differentiated, are now recognized as transcriptionally active and functionally plastic cells. In type 2 dominant inflammatory skin diseases such as atopic dermatitis (AD), their contribution remains insufficiently defined. Despite frequent bacterial colonization of AD lesions, neutrophil accumulation is limited. IL-4Rα signaling has been shown to suppress neutrophil effector functions in murine models and human samples, yet its mechanistic impact on human neutrophils under opposing cytokine cues is not fully understood.

Methods
Primary neutrophils were isolated from healthy human donors and stimulated in vitro with IL-4, IL-13, IFN-gamma or their combinations. Phospho-flow cytometry was used to monitor STAT6 and STAT1 activation. NET formation was quantified by immunofluorescence, and transcriptomic profiling was performed using bulk RNA-seq to assess cytokine-driven transcriptional reprogramming. In parallel, sample collection is ongoing for proteomic and ATAC-seq analyses, leveraging validated pipelines to further explore IL-4Rα–mediated regulation at the protein and chromatin accessibility levels.

Results

Phospho-flow cytometry confirmed canonical STAT6 and STAT1 activation, validating the mechanistic engagement of IL-4Ralpha and IFN-gamma pathways. Functionally, IL-4Rα signaling appeared to reduced NET formation, while IFN-γ promoted it. Co-stimulation partially reversed the suppressive effects of IL-4/IL-13, highlighting a dynamic balance between opposing cytokine signals. Transcriptomic profiling further revealed that the addition of IFN-gamma to IL-4 or IL-13 significantly modulated genes associated with neutrophil migration, chemotaxis, and cellular activation, underscoring the antagonistic interplay between Th2 and Th1 environments.

Conclusions
These results reveal that IL-4Ralpha signaling dampens neutrophil antimicrobial functions, while IFN-gamma seems to override this suppression and restore key effector programs. Ongoing proteomic and ATAC-seq studies are expected to extend these insights by uncovering additional regulatory layers involved in neutrophil plasticity. Ex vivo validation studies in AD patients are underway to confirm these findings in a disease-relevant context.

To overview