Although spontaneous protein crystallization is a rare event in vivo, Charcot-Leyden crystals (CLCs) consisting of galectin-10 (Gal10) protein are frequently observed in eosinophilic diseases, such as asthma. We found that CLCs derived from patients showed crystal packing and Gal10 structure identical to those of Gal10 crystals grown in vitro. When administered to the airways, crystalline Gal10 stimulated innate and adaptive immunity and acted as a type 2 adjuvant. By contrast, a soluble Gal10 mutein was inert. Antibodies directed against key epitopes of the CLC crystallization interface dissolved preexisting CLCs in patient-derived mucus within hours and reversed crystal-driven inflammation, goblet-cell metaplasia, immunoglobulin E (IgE) synthesis, and bronchial hyperreactivity (BHR) in a humanized mouse model of asthma. Thus, protein crystals may promote hallmark features of asthma and are targetable by crystal-dissolving antibodies.

The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) is pivotal to the pathophysiology of widespread allergic diseases mediated by type 2 helper T cell (Th2) responses, including asthma and atopic dermatitis. The emergence of human TSLP as a clinical target against asthma calls for maximally harnessing its therapeutic potential via structural and mechanistic considerations. Here we employ an integrative experimental approach focusing on productive and antagonized TSLP complexes and free cytokine. We reveal how cognate receptor TSLPR allosterically activates TSLP to potentiate the recruitment of the shared interleukin 7 receptor α-chain (IL-7Rα) by leveraging the flexibility, conformational heterogeneity and electrostatics of the cytokine. We further show that the monoclonal antibody Tezepelumab partly exploits these principles to neutralize TSLP activity. Finally, we introduce a fusion protein comprising a tandem of the TSLPR and IL-7Rα extracellular domains, which harnesses the mechanistic intricacies of the TSLP-driven receptor complex to manifest high antagonistic potency.

Glutathione (GSH) is a vital intracellular cysteine-containing tripeptide across all kingdoms of life and assumes a plethora of cellular roles. Such pleiotropic behavior relies on a finely tuned spatiotemporal distribution of glutathione and its conjugates, which is not only controlled by synthesis and breakdown, but also by transport. Here, we show that import of glutathione in the obligate human pathogen Haemophilus influenzae , a glutathione auxotrophe, is mediated by the ATP-binding cassette (ABC)-like dipeptide transporter DppBCDF, which is primed for glutathione transport by a dedicated periplasmic-binding protein (PBP). We have identified the periplasmic lipoprotein HbpA, a protein hitherto implicated in heme acquisition, as the cognate PBP that specifically binds reduced (GSH) and oxidized glutathione (GSSG) forms of glutathione with physiologically relevant affinity, while it exhibits marginal binding to hemin. Dissection of the ligand preferences of HbpA showed that HbpA does not recognize bulky glutathione S conjugates or glutathione derivatives with C-terminal modifications, consistent with the need for selective import of useful forms of glutathione and the concomitant exclusion of potentially toxic glutathione adducts. Structural studies of the highly homologous HbpA from Haemophilus parasuis in complex with GSSG have revealed the structural basis of the proposed novel function for HbpA-like proteins, thus allowing a delineation of highly conserved structure-sequence fingerprints for the entire family of HbpA proteins. Taken together, our studies unmask the main physiological role of HbpA and establish a paradigm for glutathione import in bacteria. Accordingly, we propose a name change for HbpA to glutathione-binding protein A.