Neutrophilic asthma is a vexing disease, but mechanistic and therapeutic advancements will require better models of allergy-induced airway neutrophilia. Here, we find that periodic ovalbumin (OVA) inhalation in sensitized mice elicits rapid allergic airway inflammation and pathophysiology mimicking neutrophilic asthma. OVA-experienced murine lungs harbor diverse clusters of CD4+ resident memory T (TRM) cells, including unconventional RORgtnegative/lowT helper 17 (TH17) cells. Acute OVA challenge instigates interleukin (IL)-17A secretion from these TRM cells, driving CXCL5 production from Muc5achigh airway secretory cells, leading to destructive airway neutrophilia. The TRM and epithelial cell signals discovered herein are also observed in adult human asthmatic airways. Epithelial antigen presentation regulates this biology by skewing TRM cells toward TH2 and TH1 fates so that TH1-related interferon (IFN)-g suppresses IL-17A-driven, CXCL5mediated airway neutrophilia. Concordantly, in vivo IFN-g supplementation improves disease outcomes. Thus, using our model of neutrophilic asthma, we identify lung epithelial-CD4+ TRM cell crosstalk as a key rheostat of allergic airway neutrophilia.

RATIONALE: Neutrophilic asthma is an aggravating disease diagnosed predominantly in adult humans. Though good models for eosinophilic asthma exist, few models for neutrophilic asthma are available. Here, we hypothesized that repeated and intermittent exposures of sensitized mice to inhaled ovalbumin (OVA) over extended durations would better model adult human experience leading to neutrophilic asthma. As such, our model differs from the conventional OVA-based model of eosinophilic asthma by introducing intermittent and recurrent inhalation of aerosolized OVA in pre-sensitized mice. METHODS: We used diverse transgenic mice, flow cytometry, and an array of standard analytic assays for evaluating disease outcomes in our model. Human relevance of our murine findings were confirmed by mining publicly available scRNA-Seq datasets. RESULTS: Acute allergic challenge in mice with inhaled OVA experience presented with accelerated, steroid-resistant neutrophilia accompanied by airway hyperreactivity and unrestrained lung injury. We observed that OVA-experienced lungs exhibited rapid IL-17A production which augmented CXCL5 secretion by lung epithelial cells leading to robust airway neutrophilia on allergic exacerbation. Consistent with its extensive experience, we found that such murine lungs possessed diverse subsets of CD4+ tissue-resident memory (TRM) cells including TH1, TH2, and Treg TRM cells; a feature lacking in naïve lungs. Interestingly, despite rapid IL-17A accumulation, experienced lungs were starkly devoid of conventional IL-17A-producing TH17 cells (identified by their RORγt positivity). Instead, we discovered that a novel unconventional RORγtnegative/low TH17 subset was the predominant producer of IL-17A that kickstarted the allergic neutrophilia. Among the lung intrinsic regulators, we found that epithelial MHC-II was key. Specifically, MHC-II expressed by lung epithelia enhanced TH1 TRM cell abundance which negatively correlated with CXCL5 accumulation, neutrophilia, and airway edema. As a result of this serendipitous observation, we found that IFN-γ muted IL-17A-induced CXCL5 production by lung epithelial cells in vitro and by airways in vivo to mediate this immunoregulatory effect. Indeed, prophylactic or therapeutic delivery of IFN-γ was sufficient to prevent neutrophilia, and mice lacking IFN-γ displayed worse disease outcomes in our model. Of note, all the CD4+ TRM and epithelial signatures discovered in our mouse model were conserved in adult asthmatic human airways via scRNA-Seq datasets. CONCLUSIONS: Thus, using a tractable murine model for allergic airway neutrophilic disease that mimics adult human airway biology, we identify CD4+ TRM – epithelial crosstalk as an important regulator of this disease. Our findings suggest that IFN-γ and the downstream pathways it activates may represent promising avenues to mitigate allergic airway neutrophilic disease.

While childhood-onset asthma is extensively modeled in labs and includes TH2 cell driven-, steroid-responsive, eosinophilic etiology, little is understood about late-onset asthma which presents as severe, steroid-resistant, and destructive neutrophilic disease with poor outcomes. Lack of appropriate disease models widens this knowledge gap leading to fewer therapies and poorer quality of life for neutrophilic asthma patients. Here, we discover that while conventional models of allergic asthma (relying on naïve sensitized mice acutely challenged with aerosolized ovalbumin) induces eosinophilic asthma, transient and recurrent aeroallergenic exposure over extended durations (as would occur in humans progressing into adulthood) reprograms the lung myeloid and lymphoid landscape to instigate neutrophilic asthma. Mice with such lung history harbor diverse clusters of lung-resident CD4+ TRM cells including a novel RORγt-negative-IL17A+ TH17 subset; the latter detectable in asthmatic adult human lungs. On allergen reencounter, these RORγt-negative TH17 cells, rapidly secrete IL-17A which signals into lung epithelial and stromal cells to express CXCL5 and induce neutrophilic asthma including peribronchial neutrophilia, vascular leakage and lung damage. We find that lung epithelial antigen presentation is crucial to regulate disease severity by skewing CD4+ TRM phenotypes in asthmatic lungs. Specifically, epithelial MHC-II supports TH1 TRM cells and IFNγ secretion; the latter being a potent suppressor of IL-17A-induced CXCL5 and airway neutrophilia. Thus, using a relevant model of the disease, we identify an ‘epithelium-lymphocyte-neutrophil’ circuitry as a critical regulator of late-onset neutrophilic asthma. Supported by NIH grants including HL147461 to F.T.K., HL142199 to K.A.B., HL136725 to M.R.J., GM120060 and HL111449 to L.J.Q., AI115053, HL135756, and HL137081 to J.P.M. and T32 HL007035 for support of trainees.

Barrier tissues are populated by functionally plastic CD4+ resident memory T (TRM) cells. Whether the barrier epithelium regulates CD4+ TRM cell locations, plasticity and activities remains unclear. Here we report that lung epithelial cells, including distinct surfactant protein C (SPC)lowMHChigh epithelial cells, function as anatomically-segregated and temporally-dynamic antigen presenting cells. In vivo ablation of lung epithelial MHC-II results in altered localization of CD4+ TRM cells. Recurrent encounters with cognate antigen in the absence of epithelial MHC-II leads CD4+ TRM cells to co-express several classically antagonistic lineage-defining transcription factors, changes their cytokine profiles, and results in dysregulated barrier immunity. In addition, lung epithelial MHC-II is needed for surface expression of PD-L1, which engages its ligand PD-1 to constrain lung CD4+ TRM cell phenotypes. Thus, we establish epithelial antigen presentation as a critical regulator of CD4+ TRM cell function and identify epithelial-CD4+ TRM cell immune interactions as core elements of barrier immunity.

Barrier tissues are sentinelled by CD4+ TRM cells with potent anti-microbial activities and considerable lineage plasticity. We hypothesized that local antigen presentation by lung epithelial cells (LECs) instruct CD4+ TRM cell activities. Pneumococcal infections in transgenic mice, flow- and spectral-cytometry, computational biology, and immunofluorescence were used to study this biology. All LECs including a novel alveolar surfactant protein C (SPC)low LEC were adept at antigen presentation. Temporal analysis of LECs for MHC-II and costimulatory/coinhibitory molecules revealed that airway club cells were T-cell stimulatory via CD40 while alveolar LECs expressed T-cell inhibitory PD-L1. This anatomical segregation of LEC antigen presentation correlated with deposition of CD4+ TRM cells around airways such that ablation of LEC MHC-II disrupted CD4+ TRM niches and blockade of CD40 signals prevented accumulation of CD4+ TRM cells. Recurrent memory recalls in absence of LEC MHC-II led to expansion of unconventional CD4+ TRM cells co-expressing classically incompatible lineage-defining transcription factors, changing their cytokine repertoire and leading to dysregulated immunity that phenocopied clinical features of checkpoint blockade therapy. Consequently, a tight correlation between MHC-II and PD-L1 was confirmed in mouse and human LECs. We discovered that LEC MHC-II functions in post-translational trafficking lockstep with PD-L1 to exert its restraints on TRM cell activities. Our results identify epithelial antigen presentation as critical instructors of CD4+ TRM cell locations, phenotypes and activities and establish epithelial-CD4+ TRM cell immunological synapses as key components of barrier immunity.