Rationale: The association between immune-cell-specific transcriptomic profiles and Idiopathic Pulmonary Fibrosis (IPF) mortality is unknown. Objectives: To determine immune-cell-specific transcriptomic profiles associated with IPF mortality. Methods: We profiled peripheral blood mononuclear cells (PBMC) in 18 participants [University of South Florida: IPF, COVID-19, post-COVID-19 Interstitial Lung Disease (Post-COVID-19 ILD), controls] by single-cell RNA sequencing (scRNA-seq) and identified 16 immune-cell-specific transcriptomic profiles. The Scoring Algorithm of Molecular Subphenotypes (SAMS) was used to calculate Up-scores based on these 16 gene profiles. Their association with outcomes was investigated in peripheral blood, Bronchoalveolar Lavage (BAL) and lung tissue of N=416 IPF patients from six cohorts. Findings were validated in an independent IPF, PBMC scRNA-seq dataset (N=38). Measurements and main results: Cox-regression models demonstrated that 230 genes from CD14+CD163-HLA-DRlow circulating monocytes predicted IPF mortality [Pittsburgh (p=0.02), Chicago (p=0.003)]. PBMC proportions of CD14+CD163-HLA-DRlow monocytes were higher in progressive versus stable IPF (Yale, 0.13±0.05 versus 0.09±0.05, p=0.034). Receiving operating characteristic identified a 230 gene, Up-score >41.84 (Pittsburgh) predictive of mortality in Chicago (HR: 6.58, 95%CI: 2.15-20.13, p=0.001) and in pooled analysis of BAL cohorts (HR: 2.20, 95%CI: 1.44-3.37, p=0.0003). High-risk patients had decreased expression of the T-cell co-stimulatory genes CD28, ICOS, ITK and LCK (Pittsburgh and Chicago, p<0.01). 230 gene-up-scores negatively correlated with Forced Vital Capacity (FVC) in IPF lung tissues (LGRC, rho=-0.2, p=0.02). Results were replicated using a subset of 13 genes from the 230-gene signature (pooled PBMC cohorts - HR: 5.34, 95%CI: 2.83-10.06, p<0.0001). Conclusions: The transcriptome of CD14+CD163-HLA-DRlowmonocytes is associated with increased IPF mortality.

Competing interests: NK is a scientific founder at Thyron, served as a consultant to Boehringer Ingelheim, Pliant, GSK, Merck, Pliant, Three Lake Partners, Astra Zeneca, RohBar, Veracyte, CSL Behring, Gilead, Galapagos, Chiesi, Arrowhead, Fibrogen, Sofinnova and Thyron over the last 3 years, reports Equity in Pliant and Thyron, and grants from Boehringer Ingelheim, Three Lakes Foundation, BMS and non-financial support from MiRagen and Astra Zeneca. IN reports grant for research to institution from Veracyte and personal honoraria from Boehringer Ingelheim and Sanofi. The rest authors have nothing to disclose.

This study was funded by NIH grants including R21HL161723, R01HL127349, R01HL141852, U01HL145567, UH2HL123886 (NK), R01HL127349 and R01HL159805 (PVB), UG3HL145266, R01HL171918, R01HL162659 (IN and institution) and the USF Foundation, Ubben Family Fund (JHM).

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Ethical approval for this study was given by the Institutional Review Boards / Research Integrity & Compliance of University of South Florida (Identity: Pro00032158_MODCR000003, Title: University of South Florida-Tampa General Hospital Lung Transplant Biorepository, Principal Investigator: Jose D. Herazo-Maya).

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Data analyzed are available in GEO under the following GEO accession numbers: University of South Florida: GSE264196, Yale: GSE233844, Pittsburgh: GSE28221, Chicago: GSE27957, Siena, Leuven and Freiburg: GSE70867, LGRC: GSE47460.