TNF Superfamily Member 14 Drives Post-Influenza Depletion of Alveolar Macrophages Enabling Secondary Pneumococcal Pneumonia

Secondary bacterial infection, often caused by Streptococcus pneumoniae (Spn), is one of the most frequent and severe complications of influenza A virus (IAV)-induced pneumonia. Phenotyping of the pulmonary innate immune landscape after IAV infection revealed a significant depletion of the tissue-resident alveolar macrophage (TR-AM) population at day 7, which was associated with increased susceptibility to Spn outgrowth. To elucidate the molecular mechanisms underlying TR-AM depletion, and to define putative targets for treatment, we combined single-cell transcriptomics and cell-specific PCR profiling in an unbiased manner, using in vivo models of IAV infection and IAV/Spn co-infection. The TNF superfamily 14 (TNFSF14) ligand-receptor axis was revealed as the driving force behind post-influenza TR-AM death during the early infection phase, enabling the transition to pneumococcal pneumonia, while intrapulmonary transfer of genetically modified TR-AMs and antibody-mediated neutralization of specific pathway components alleviated disease severity. With a mainly neutrophilic expression and a high abundance in the bronchoalveolar fluid (BALF) of patients with severe virus-induced ARDS, TNFSF14 emerged as a novel determinant of virus-driven lung injury. Targeting the TNFSF14-mediated intercellular communication network in the virus-infected lung can, therefore, improve host defense, minimizing the risk of subsequent bacterial pneumonia, and ameliorating disease outcome. To identify the main leukocyte source of the TNFSF14 ligand upon IAV pneumonia, 500,000 live leukocytes (gated as Sytox- CD45+) from the homogenized single-cell lung suspensions of IAV-infected mice on day 3 (ext303) and 7 post infection (pi) (ext294) were flow-sorted into FACS tubes containing 0.35μL 0.04% BSA/PBS-/-. Following viability control, 10,000 cells were loaded onto Chromium Chip B (10X Genomics). Gel beads in emulsion (GEM) generation, cDNA synthesis and amplification, and library preparation were performed with the Chromium Single Cell 3’ Reagent Kit v3.1 (10X Genomics), as per the manufacturer’s protocol. Indexed libraries were sequenced on an Illumina NextSeq2000. Prior to analysis, reads were aligned against the mouse genome (GRCm38.p6) and quantified using StarSolo (https://github.com/alexdobin/STAR). Analysis was conducted with the Scanpy software (https://github.com/theislab/scanpy). After quality filtering, raw cell counts were normalized to the median count over all cells and transformed into log space for variance stabilization. Principal component analysis (PCA) identified 14 and 11 components on days 3 and 7 pi, respectively. Uniform Manifold Approximation and 609 Projection (UMAP) embedding was created to identify cell type clusters through Leiden clustering. Doublet analysis was conducted on day 7 using Scrublet (https://github.com/swolock/scrublet), leading to the removal of a doublet cluster (118 cells).