bioRxivImmunologyPreprint
Therapeutic signature mapping of paired direct and indirect LPS injury in an ex vivo human lung perfusion platform reveals injury-specific druggable programs
Ahad Abdalla, David A. DorwardiD, Antonella PellicoroiD, Tom Michael QuinniD, Stuart Dickson, Adam D. L. MarshalliD, Annya M. Bruce, John J ColeiD, Keith FinlaysoniD, Richard A. O’ConnoriD, Chris Haslett, Manu Shankar‐HariiD, Kevin DhaliwaliD
Abstract
Acute Respiratory Distress Syndrome (ARDS) remains highly morbid and lacks approved disease-modifying pharmacotherapies. Direct (pulmonary) and indirect (extrapulmonary) insults may initiate biologically distinct early injury programs, but human tissue-level evidence from the first hours is scarce. Here we establish a paired, acellular ex vivo lung perfusion (EVLP) platform using human donor lungs unsuitable for transplantation to model direct (endobronchial) and indirect (perfusate) lipopolysaccharide (LPS) injury within the same donor. We profiled lung tissue proteomes at 4 h post-insult and performed therapeutic nomination by querying proteomics-derived injury signatures against the CLUE L1000 perturbational compendium with independent cross-platform validation. Both models developed histological injury and robust cytokine release. Direct injury preferentially enriched neutrophil degranulation, extracellular matrix remodelling and metabolic reprogramming modules, whereas indirect injury showed prominent complement/coagulation perturbation with greater endothelial activation markers in perfusate. Cross-platform prioritisation converged on tractable signalling and epigenetic axes, including JAK/STAT, PI3K/AKT/mTOR, SYK, CDK and HDAC inhibitor classes - yielding a tiered shortlist for EVLP intervention testing. This intact human lung perturbation platform enables injury-stratified mechanistic inference and therapeutic prioritisation in early lung injury relevant to ARDS.
- organism: human
- assay: proteomics
- research focus: differential expression
- research focus: drug response / drug discovery
- approach: experimental assay
In-depth summary
Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (18 Aug 2026) · CC BY 4.0
Direct and indirect LPS injury in human donor lungs produce distinct 4 h proteomic programs; signature reversal favours SYK, CDK, HDAC, JAK/STAT and PI3K/AKT/mTOR pathway modulation.
ARDS has no approved disease-modifying drugs and is usually treated as one entity despite direct versus indirect aetiologies, and human lung tissue from the first hours of injury is rarely accessible. This work uses paired ex vivo perfused donor lungs with tissue proteomics to resolve route-specific early programs and link them to drug-signature reversal.
Key results
ShownBoth the endobronchial and perfusate LPS models produced histological injury, neutrophil (MPO+) infiltration and perfusate cytokine release by 4 h. Soluble endothelial adhesion markers rose more in the indirect model.
Lung injury score increased from baseline in both models (p < 0.01); MPO+ cell increase in both (p < 0.05); IL-8, IL-6, IL-1beta and TNFalpha rose over time; ICAM-1 and VCAM-1 rose more in indirect injury. Paired t-tests. · Distinct pathological features and mediator…; Figure legends
ShownProteomic profiling separated injured tissue from non-injured controls across three contrasts that include the EVLP effect. Once EVLP-associated changes were removed, proteins common to both injury types were enriched for neutrophil degranulation, and each type kept distinct pathways.
5,650 proteins quantified (paired subset of n = 5 donors); 874 differentially expressed across three contrasts (MS-DAP, q < 0.01); after excluding EVLP effects, 116 proteins shared between direct and indirect injury. · Establishment of a paired human EVLP model of…; Global tissue proteomics resolves EVLP effects…
ShownDirect injury was dominated by neutrophil degranulation with extracellular matrix and metabolic remodelling. Azurocidin, neutrophil elastase and myeloperoxidase were increased in injured tissue.
457 proteins differed (injured vs uninjured segment): 340 differentially expressed (87 increased; 253 decreased) and 117 differentially detected; STRING modules for neutrophil degranulation, ECM, metabolism and MAPK signalling. · Direct injury is dominated by neutrophil granule…
ShownIndirect injury mainly perturbed complement and coagulation, ECM and acute-phase programs. Its interaction network hubs were predominantly ribosomal proteins, consistent with translation-linked regulation.
394 proteins differed (MS_EmpiRe): 252 differentially expressed (87 increased; 165 decreased) and 142 differentially detected; STRING network of 394 nodes and 661 edges; top ten cytoHubba hubs mostly ribosomal. · Indirect injury features complement/coagulation…
SuggestedSignature reversal queries and cross-resource checks converged on a few druggable axes, giving a tiered shortlist for later EVLP testing. This is computational only; no compound was tested.
CLUE L1000 negative connectivity at FDR < 0.05 (all cell lines, then A549 and HCC515); concordance with Enrichr, L1000CDS² and Open Targets; signatures Direct UP (99), DOWN (358), Indirect UP (91), DOWN (303), truncated to 150 proteins. · Therapeutic discovery; Cross-platform validation of therapeutic…; Cross-platform drug repurposing validation; Therapeutic connectivity analysis (CLUE/L1000)
Methods, data and reproducibility
Paired endobronchial or perfusate LPS was applied to human donor lungs under acellular EVLP, and lung tissue was profiled by LC-MS/MS proteomics at 4 h, with MS-DAP differential testing, enrichment and STRING networks. Injury signatures were queried against CLUE L1000 and cross-checked in Enrichr, L1000CDS² and Open Targets.
- Human donor lungs declined for transplantation, used in single-lung EVLP experiments with direct or indirect LPS injury · human · n = Ten donors, sixteen single lungs
- Discovery tissue proteomics in a paired subset (within-donor comparisons) · human · n = 5 donors; 5,650 proteins passed filtering
- Histology, MPO staining and perfusate cytokine/adhesion molecule measurements across the broader experimental set · human
- Human reference proteome used to map precursors · human · UP000005640_9606
- Input signatures for drug repurposing queries (direct and indirect, up and down) · human · n = Direct UP 99, DOWN 358; Indirect UP 91, DOWN 303
- Methods & tools
- Spectronaut, DIA-NN, MS-DAP v1.0.6 with MS-EmpiRe, clusterProfiler v4.4.4 with MSigDB v2023.2.Hs, STRING v12.0 in Cytoscape v3.10.1, cytoHubba v0.1, WebCSEA, CLUE L1000, Enrichr, L1000CDS², Open Targets Platform, timsTOF FleX LC-MS/MS
- Reagents & environment
- MS-DAP v1.0.6 under R 4.2.1; PCAtools v2.8.0; clusterProfiler v4.4.4; org.Hs.eg.db v3.15.0; STRING v12.0 (stringApp v2.0.3); Cytoscape v3.10.1; cytoHubba v0.1.
- Reproducibility gaps
- Raw and processed proteomics data and analysis scripts are to be deposited in a public repository only upon publication; no accession or URL is given yet.
Limitations (6)
- EVLP lacks whole-body immune and neurohumoral interactions, which may especially affect indirect injury. · stated by the authors
- Only the first 4 h after insult were sampled, so later-phase transitions in injury cannot be resolved. · stated by the authors
- CLUE L1000 predictions derive from cancer cell line perturbations (A549 and HCC515) and are hypothesis-generating, not proof of efficacy. · stated by the authors
- The acellular perfusate lacks circulating blood cells, limiting modelling of later cell recruitment and immunothrombotic amplification. · stated by the authors
- Discovery proteomics used a paired subset of 5 donors, and no nominated compound was tested in EVLP, so drug tiers rest on database concordance alone. · noted in this summary
- In the direct model, uninjured controls came from the right upper lobe while injured and baseline samples came from the right lower lobe, so lobe differences may confound the injury contrast. · noted in this summary
Read this if you want a human whole-organ proteomic comparison of direct versus indirect early lung injury and a cross-database drug-reversal shortlist for ARDS.
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