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. 2023 Jul 18:14:1220028.
doi: 10.3389/fimmu.2023.1220028. eCollection 2023.

Transcriptomic profiles of multiple organ dysfunction syndrome phenotypes in pediatric critical influenza

Tanya Novak  1   2   3 , Jeremy Chase Crawford  1   4   5 , Georg Hahn  6 , Mark W Hall  7 , Simone A Thair  1   8 , Margaret M Newhams  1   3 , Janet Chou  9   10 , Peter M Mourani  11 , Keiko M Tarquinio  12 , Barry Markovitz  13 , Laura L Loftis  14 , Scott L Weiss  15 , Renee Higgerson  16 , Adam J Schwarz  17 , Neethi P Pinto  18 , Neal J Thomas  19 , Rainer G Gedeit  20 , Ronald C Sanders Jr  21 , Sidharth Mahapatra  22 , Bria M Coates  23 , Natalie Z Cvijanovich  24 , Kate G Ackerman  25 , David W Tellez  26 , Patrick McQuillen  27 , Stephen C Kurachek  28 , Steven L Shein  29 , Christoph Lange  30 , Paul G Thomas  4   5 , Adrienne G Randolph  1   2   3   10
Affiliations

Transcriptomic profiles of multiple organ dysfunction syndrome phenotypes in pediatric critical influenza

Tanya Novak et al. Front Immunol. .

Abstract

Background: Influenza virus is responsible for a large global burden of disease, especially in children. Multiple Organ Dysfunction Syndrome (MODS) is a life-threatening and fatal complication of severe influenza infection.

Methods: We measured RNA expression of 469 biologically plausible candidate genes in children admitted to North American pediatric intensive care units with severe influenza virus infection with and without MODS. Whole blood samples from 191 influenza-infected children (median age 6.4 years, IQR: 2.2, 11) were collected a median of 27 hours following admission; for 45 children a second blood sample was collected approximately seven days later. Extracted RNA was hybridized to NanoString mRNA probes, counts normalized, and analyzed using linear models controlling for age and bacterial co-infections (FDR q<0.05).

Results: Comparing pediatric samples collected near admission, children with Prolonged MODS for ≥7 days (n=38; 9 deaths) had significant upregulation of nine mRNA transcripts associated with neutrophil degranulation (RETN, TCN1, OLFM4, MMP8, LCN2, BPI, LTF, S100A12, GUSB) compared to those who recovered more rapidly from MODS (n=27). These neutrophil transcripts present in early samples predicted Prolonged MODS or death when compared to patients who recovered, however in paired longitudinal samples, they were not differentially expressed over time. Instead, five genes involved in protein metabolism and/or adaptive immunity signaling pathways (RPL3, MRPL3, HLA-DMB, EEF1G, CD8A) were associated with MODS recovery within a week.

Conclusion: Thus, early increased expression of neutrophil degranulation genes indicated worse clinical outcomes in children with influenza infection, consistent with reports in adult cohorts with influenza, sepsis, and acute respiratory distress syndrome.

Keywords: MODS; critical care; influenza; neutrophil degranulation; neutrophil transcripts; organ failure; pediatric intensive care; sepsis.

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Conflict of interest statement

AGR, TN, and MMN: NIH/NIAID as declared in the funding statement - grant support paid to Boston Children’s Hospital. Also current CDC grant for COVID-19 work unrelated to current manuscript also paid to Boston Children’s Hospital under PI AGR. AGR also receives Royalties from UpToDate, Inc. as Section editor of Pediatric Critical Care Medicine. Honoraria: Grand Rounds presentations on MIS-C not related to current manuscript. Participation on a Data Safety Monitoring/Advisory Board: NIH Grace Study. Leadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid: Medical Advisory Board for Families Fight Flu and Chair of International Sepsis Forum. JCC: additionally declares support from ALSAC for the current work. Other entities not related to the current work: U01AI150747, R01AI136514, U01AI144616; American Association of Immunologists Abstract award 2021, Patent: Methods for Treating or Reducing the Severity of a Viral Infection Publication no. WO 2021/226174 A1. MWH declared Kiadis, Licensing income, unrelated to the content of this manuscript, American Board of Pediatrics Payment, Abbive payment. Partner Therapeutics Provision of study drug, unrelated to the current manuscript, Sobi Provision of study drug, unrelated to the current manuscript. JC: NIH, paid to institution not related to this work; GLG Group payment to her, Elsevier payment made to her for work done as Associate Editor; Patent pending for Methods and compositions for treating and preventing T cell-driven diseases. BMC: Not related to current work: receives grants paid to institution from American Lung Association. Sobi Participation on a Data Safety Monitoring Board/Advisory Board. SLW: Receives royalties from UpToDate, Inc. Has grants/contracts with Pennsylvania Dept of Health not related to current manuscript. PGT: declares support from ALSAC and NIAID R01 AI154470 for the current work. Has several grants not associated with current work: NIAID 5R01AI128805-05; 1R01AI154470; NIAID 75N93019C00052; 5R01AI136514; 5R01AI35025; NIAID 5U01AI144616; NIAID U01AI150747. Royalties/licenses paid to both PGT and his institution: TCR cloning technology—Miltenyi Biotec & TCR amplification technology—Shennon Bio. Consulting fees paid to him: Johnson and Johnson, Cytoagents, Immunoscape, Shennon Bio. Payment or honoraria for lectures, presentations, or other events: Illumina—Future Genomic Advances, Yale University, CZ Biohub, PACT Pharma, UCSD, Tufts, University of Arizona, Mt. Sinai, Umass, OSU, Korean Association of Immunology, SISMID, University of Washington, MSKCC, Washington University, University of Missouri, Fred Hutch, UNM Illumina Single Cell, Cincinnati Childrens, University of Toronto, Purdue University, Iowa State University, University of Georgia. Patent: Methods for Treating or Reducing the Severity of a Viral Infection Publication no. WO 2021/226174 A1. (continued disclosures for PGT:) Support for attending meetings and/or travel: NIH Study Section, Keystone Viral Immunity, NIAID CEIRR, CEIRS and CIVIC meetings, GRC, 10X Users Symposium, Illumina Symposia, ImmunOktoberfest, Options for the Control of Influenza XI, Carghese Workshop, FOCIS, Max Planck for Complex Systems, Santa Fe Institute, AAI, Weizmann Institute, APS Society, Banff Institute, ISIRV meeting, University of Melbourne, British Society of Immunology. Stock or stock options: Shennon Bio Scientific advisory board payments. (Additional patents:) US20170304293A1 Coordinated metabolic reprogramming in response to productive viral infections; Cloning and expression system for t-cell receptors; License payments made to me and institution; Method for detecting SARS-CoV-2 infection. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Children with influenza critical illness were stratified by their multiple organ dysfunction syndrome (MODS) status during blood collection and final outcome. Grey boxes indicate that patient groups were excluded from analysis. Longitudinal blood samples were collected from a subgroup of Prolonged and Recovered MODS patients in which a second blood sample was collected approximately 7 days after the first while still in hospital. PICU, Pediatric Intensive Care Unit. ECMO, Extracorporeal Membrane Support.
Figure 2
Figure 2
Whole blood collected during MODS revealed significantly increased mRNA expression of nine genes associated with neutrophil degranulation in pediatric patients who had Prolonged MODS or Died (n=38) compared to patients that Recovered from MODS (n=27). Never MODS was included as an influenza positive PICU control (n=126). After adjustment for multiple comparisons, transcription levels between Recovered vs Never MODS did not have any statistically differences. Note the different y-axis for H and I. ****q<0.0001, ***q ≤ 0.001, **q ≤ 0.01, *q<0.05. All Tukey box plots represent the median (center line of the box), 25% IQR (Q1, bottom line of the box), 75% IQR (Q3, top line of the box), error bars are the minimum and maximum values within 1.5x the Q1 and Q3 quadrants, outliers indicated by circles. MODS, Multiple Organ Dysfunction Syndrome. (A) RETN, Resistin. (B) TCN1, Haptocorin. (C) OLFM4, Olfactomedin 4. (D) LCN2, Lipocalin 2. (E) BPI, Bactericidal Permeability Increasing Protein. (F) MMP8, Matrix Metallopeptidase 8. (G) LTF, Lactotransferrin. (H) S100A12, S100 Calcium Binding Protein A12. (I) GUSB, Glucuronidase Beta.
Figure 3
Figure 3
Whole blood collected during MODS showed significantly increased mRNA expression of eight genes not directly associated with neutrophil degranulation in patients that had Prolonged MODS or Died (n=38) compared to those that who recovered from MODS (n=27). Never MODS was included as an influenza positive PICU control (n=126). After adjustment for multiple comparisons, transcription levels between Recovered vs Never MODS did not have any statistically differences. ****q<0.0001, **q ≤ 0.01, *q<0.05. MODS, Multiple Organ Dysfunction Syndrome. (A) MS4A4A, Membrane Spanning 4-Domains A4A. (B) GAPDH, Glyceraldehyde-3-Phosphate Dehydrogenase. (C) LGALS1, Galectin 1. (D) CLEC1B, C-Type Lectin Domain Family 1 Member B. (E) ESPL1, Separin. (F) GNA15, G protein subunit alpha 15. (G) DUSP4, Dual specificity phosphatase 4. (H) TWISTNB, RNA polymerase I subunit F.
Figure 4
Figure 4
Longitudinal, paired blood samples from Prolonged MODS/Died versus Recovered MODS patients: Five genes whose mRNA expression significantly differed between their first and second samples obtained approximately 7 days apart. Median normalized mRNA levels are plotted for each subgroup with 95% confidence intervals of the median. These genes showed a differential change over time between the first, early blood collection, Sample 1, and Sample 2 collected approximately 7 days later from Prolonged MODS/Died patients (n=23, staggered line, ◊ median expression) and Recovered MODS patients (n=22, solid line, ● median expression), FDR q<0.05. Note: for each graph, y-axis linear scales vary. RPL3, Ribosomal protein L3. EEF1G, Eukaryotic Translation Elongation Factor 1 Gamma protein. HLA-DMB, Major histocompatibility complex, class II, DM beta. MRPL3, Mitochondrial Ribosomal Protein L3. CD8A, T-cell surface glycoprotein CD8 alpha chain.
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