Background Airway instillation of bleomycin (BLM) in mice is a widely used, yet challenging, model for acute lung injury (ALI) with high variability in treatment scheme and animal outcomes among investigators. Whether the gut microbiota plays any role in the outcome of BLM-induced lung injury is currently unknown. Methods Intratracheal instillation of BLM into C57BL/6 mice was performed. Fecal microbiomes were analyzed by 16s rRNA amplicon and metagenomic sequencing. Germ-free mice conventionalization and fecal microbiota transfer between SPF mice were performed to determine dominant commensal species that are associated with more severe BLM response. Further, lungs and gut draining lymph nodes of the mice were analyzed by flow cytometry to define immunophenotypes associated with the BLM-sensitive microbiome. Results Mice from two SPF barrier facilities at the University of Chicago exhibited significantly different mortality and weight loss during BLM-induced lung injury. Conventionalizing germ-free mice with SPF microbiota from two different housing facilities recapitulated the respective donors’ response to BLM. Fecal microbiota transfer from the facility where the mice had worse mortality into the mice in the facility with more survival rendered recipient mice more susceptible to BLM-induced weight loss in a dominant negative manner. BLM-sensitive phenotype was associated with the presence of Helicobacter and Desulfovibrio in the gut, decreased Th17-neutrophil axis during steady state, and augmented lung neutrophil accumulation during the acute phase of the injury response. Conclusion The composition of gut microbiota has significant impact on BLM-induced wasting and death suggesting a role of the lung-gut axis in lung injury.

Expression of the transcription factor Interferon Regulatory Factor 4 (IRF4) is required for the development of lung conventional dendritic cells type 2 (cDC2s) that elicit Th2 responses, yet how IRF4 functions in lung cDC2s throughout the acute and memory allergic response is not clear. Here, we use a novel mouse model that loses IRF4 expression after lung cDC2 development to demonstrate that mice with IRF4-deficient DCs display impaired memory responses to allergen. This defect in the memory response is a direct result of ineffective Th2 induction and impaired recruitment of activated effector T cells to the lung after sensitization. IRF4-deficient DCs demonstrate defects in their migration to the draining lymph node and in T cell priming. Finally, T cells primed by IRF4-competent DCs mediate potent memory responses independently of IRF4-expressing DCs, demonstrating that IRF4-expressing DCs are not necessary during the memory response. Thus, IRF4 controls a program in mature DCs governing Th2 priming and effector responses, but IRF4-expressing DCs are dispensable during tissue resident-memory T cell (TRM cell)-dependent memory responses.

Interstitial lung diseases (ILDs) are a heterogeneous group of disorders that can develop in patients with connective tissue diseases (CTD). Establishing autoimmunity in ILD impacts prognosis and treatment. ILD patients are screened for autoimmunity by assaying for anti-nuclear autoantibodies, rheumatoid factors and other non-specific tests. However, this approach has not been rigorously validated and may miss autoimmunity that manifests as autoantibodies to tissue antigens not previously defined in ILD. Here, we use Phage Immunoprecipitation-Sequencing (PhIP-Seq) to conduct a large, multi-center unbiased autoantibody discovery screen of ILD patients and controls. PhIP-Seq identified 17 novel autoreactive targets, and machine learning classifiers derived from these targets discriminated ILD serum from controls. Among these 17 candidates, we validated Cadherin Related Family Member 5 (CDHR5) as an autoantigen and found CDHR5 autoantibodies in patients with rheumatologic disorders and importantly, subjects not previously diagnosed with autoimmunity. Lung tissue of CDHR5 autoreactive patients showed transcriptional profiles consistent with activation of NFκB signaling and upregulation of chitotriosidase (CHIT1), a molecular pathway linked to fibrosis. Our study shows PhIP-Seq uncovers novel autoantibodies in ILD patients not revealed by standard clinical tests. Furthermore, CDHR5 autoantibodies may define a novel molecular endotype of ILD characterized by inflammation and fibrosis.

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