Dr Kelly Asquith

Virus-induced asthma exacerbation is a health burden worldwide and lacks effective treatment. To better understand the disease pathogenesis and find novel therapeutic targets, we established a mouse model of steroid (dexamethasone (DEX)) resistant asthma exacerbation using ovalbumin (OVA) and influenza virus (FLU) infection. Using liquid chromatography-tandem mass spectrometry (LC-MC/MS), we performed a shotgun proteomics assay coupled with label-free quantification to define all dysregulated proteins in the lung proteome of asthmatic mice. Compared to control, 71, 89, and 30 proteins were found significantly upregulated by at least two-fold (p-value = 0.05) in OVA-, OVA/FLU-, and OVA/FLU/DEX-treated mice, respectively. We then applied a Z-score transformed hierarchical clustering analysis and Ingenuity Pathway Analysis (IPA) to highlight the key inflammation pathways underlying the disease. Within all these upregulated proteins, 64 proteins were uniquely highly expressed in OVA/FLU mice compared to OVA mice; and 11 proteins were DEX-refractory. IPA assay revealed two of the most enriched pathways associated with these over-expressed protein clusters were those associated with MHC class I (MHC-I) antigen-presentation and interferon (IFN) signaling. Within these pathways, signal-transducer-and-activator-of-transcription-1 (STAT1) protein was identified as the most significantly changed protein contributing to the pathogenesis of exacerbation and the underlying steroid resistance based on the label-free quantification; and this was further confirmed by both Parallel Reaction Monitoring (PRM) proteomics assay and western blots. Further, the pharmacological drug Fludarabine decreased STAT1 expression, restored the responsiveness of OVA/FLU mice to DEX and markedly suppressed disease severity. Taken together, this study describes the proteomic profile underpinning molecular mechanisms of FLU-induced asthma exacerbation and identifies STAT1 as a potential therapeutic target, more importantly, we provided a novel therapeutic strategy that may be clinically translated into practice.

Gene variants that disrupt TCR signaling can cause severe immune deficiency, yet less disruptive variants are sometimes associated with immune pathology. Null mutations of the gene encoding the scaffold protein Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76), for example, cause an arrest of T cell positive selection, whereas a synthetic membrane-targeted allele allows limited positive selection but is associated with proinflammatory cytokine production and autoantibodies. Whether these and other enigmatic outcomes are due to a biochemical uncoupling of tolerogenic signaling, or simply a quantitative reduction of protein activity, remains to be determined. In this study we describe a splice variant of Lcp2 that reduced the amount of wild-type SLP-76 protein by ~90%, disrupting immunogenic and tolerogenic pathways to different degrees. Mutant mice produced excessive amounts of proinflammatory cytokines, autoantibodies, and IgE, revealing that simple quantitative reductions of SLP-76 were sufficient to trigger immune dysregulation. This allele reveals a dose-sensitive threshold for SLP-76 in the balance of immunity and immune dysregulation, a common disturbance of atypical clinical immune deficiencies.