Inflammatory sub-phenotypes in sepsis: relationship to outcomes, treatment effect and transcriptomic sub-phenotypes

Rationale Heterogeneity of sepsis limits discovery and targeting of treatments. Clustering approaches in critical illness have identified patient groups who may respond differently to therapies. These include in acute respiratory distress syndrome (ARDS) two inflammatory sub-phenotypes, using latent class analysis (LCA), and in sepsis two Sepsis Response Signatures (SRS), based on transcriptome profiling. It is unknown if inflammatory sub-phenotypes such as those identified in ARDS are present in sepsis and how sub-phenotypes defined with different techniques compare. Objectives To identify inflammatory sub-phenotypes in sepsis using LCA and assess if these show differential treatment responses. These sub-phenotypes were compared to hierarchical clusters based on inflammatory mediators and to SRS sub-phenotypes. Methods LCA was applied to clinical and biomarker data from two septic shock randomized trials. VANISH compared norepinephrine to vasopressin and hydrocortisone to placebo and LeoPARDS compared levosimendan to placebo. Hierarchical cluster analysis (HCA) was applied to 65, 21 and 11 inflammatory mediators measured in patients from the GAinS (n=124), VANISH (n=155) and LeoPARDS (n=484) studies. Measurements and Main Results LCA and HCA identified a sub-phenotype of patients with high cytokine levels and worse organ dysfunction and survival, with no interaction between LCA classes and trial treatment responses. Comparison of inflammatory and transcriptomic sub-phenotypes revealed some similarities but without sufficient overlap that they are interchangeable. Conclusions A sub-phenotype with high levels of inflammation and increased disease severity is consistently identifiable in sepsis, with similarities to that described in ARDS. There was limited overlap with the transcriptomic sub-phenotypes. ### Competing Interest Statement ACG reports that he has received speaker fees from Orion Corporation Orion Pharma and Amomed Pharma. He has consulted for Ferring Pharmaceuticals, Tenax Therapeutics, and received grant support from Orion Corporation Orion Pharma, and Tenax Therapeutics with funds paid to his institution. DFM reports grants to his institution from the MRC/NIHR EME programme for the conduct of this work. Outside the submitted work, DFM reports personal fees for consultancy from Bayer, GSK, Boehringer Ingelheim, Eli Lilly, Novartis and SOBI and for being a member of the data monitoring and ethics committee for Vir Biotechnology and Faron studies and as an educational seminar speaker for GSK. DFM has received funding to his institution from the NIHR, MRC, Wellcome Trust, Innovate UK, Northern Ireland Health and Social Care Research and Development Office, Novavax and Randox. In addition, DFM is a named inventor on a patent US8962032 covering the use of sialic acid-bearing nanoparticles as anti-inflammatory agents issued to his institution. DFM was a Director of Research for the Intensive Care Society and is the Director for the MRC/NIHR EME Programme. MSH is a Director of Research for the Intensive Care Society, Member of the MRC/NIHR EME Programme, UK Representative of the ESICM, and on the Board of International Sepsis Forum. MSH declares that he has done advisory board activity either directly or indirectly through International Sepsis Forum for Biotest, Endpoint Health, Janssen, Pfizer, and Santersus, with payments going into the unrestricted institutional research funds. CO reports grants to her institution from the MRC/NIHR EME programme for the conduct of this work. Outside the submitted work, CO reports personal fees for consultancy from INSMED and for committee membership for the California Institute of Regenerative Medicine. Outside of the submitted work CO has received research funding from MRC, Wellcome Trust, NIHSC R&D and Innovate UK. CO's spouse has received personal fees for consultancy outside of the submitted work from Bayer, GSK, Boehringer Ingelheim, Eli Lilly, Novartis and SOBI and fees for being a member of the data monitoring and ethics committee for Vir Biotechnology and Faron studies and as an educational seminar speaker for GSK. CO's spouse is a named inventor on a patent US8962032 covering the use of sialic acid-bearing nanoparticles as anti-inflammatory agents issued to his institution. All other authors declare no conflict of interest directly applicable to this research. ### Funding Statement This paper presents independent research funded by the UK National Institute for Health and Care Research (NIHR) under its Research for Patient Benefit program, an NIHR Clinician Scientist Award (NIHR/CS/009/007), and an NIHR Research Professor award held by Prof Gordon and the U.K. Efficacy and Mechanism Evaluation (EME) Programme, a Medical Research Council (MRC) and NIHR partnership (16/33/01; 08/99/08; 11/14/08). It was also supported by the NIHR Imperial Biomedical Research Centre, and also by the UK Intensive Care Foundation. Further support was received from Wellcome Trust Grant (090532/Z/09/Z) to core facilities at the Wellcome Centre for Human Genetics, a Wellcome Trust Investigator Award (204969/Z/16/Z) to Prof Knight, the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China, the NIHR Oxford Biomedical Research Centre, and from a China Scholarship Council, University of Oxford Scholarship held by Mi. Drs Davenport and Burnham are supported by Wellcome Sanger Institute Core Funding from the Wellcome Trust [206194 and 108413/A/15/D]. For the purpose of Open Access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. The authors Dr Antcliffe and Prof Gordon are affiliated with the Department of Health and Social Care, Centre for Antimicrobial Optimization at Imperial College, London. The views expressed in this article are those of the authors and not necessarily those of the MRC, National Health Service, NIHR, or Department of Health. The funders of the study had no role in design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript, or the decision to submit for publication. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethical approval was obtained from the following ethics committee for aspects of this work: Oxford A research ethics committee ref 12/SC/0014 (VANISH) London-Harrow Research and Ethics Committee ref 13/LO/0365 (LeoPARDS) Scotland A Research Ethics Committee ref 05/MRE00/38 and Berkshire Research Ethics Committee ref 08/H0505/78 (GAinS) all gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Gene-expression data is available on ArrayExpress. Individual participant data that underlie the results in this article, after de-identification (text, table, and figures) and inflammatory mediator data will be made available from the corresponding author on submission of a data request application.