The circulatory small non‐coding RNA landscape in community‐acquired pneumonia on intensive care unit admission

Abstract Community‐acquired pneumonia (CAP) is a major cause of sepsis. Despite several clinical trials targeting components of the inflammatory response, no specific treatment other than antimicrobial therapy has been approved. This argued for a deeper understanding of sepsis immunopathology, in particular factors that can modulate the host response. Small non‐coding RNA, for example, micro (mi)RNA, have been established as important modifiers of cellular phenotypes. Notably, miRNAs are not exclusive to the intracellular milieu but have also been detected extracellular in the circulation with functional consequences. Here, we sought to determine shifts in circulatory small RNA levels of critically ill patients with CAP‐associated sepsis and to determine the influence of clinical severity and causal pathogens on small RNA levels. Blood plasma was collected from 13 critically ill patients with sepsis caused by CAP on intensive care unit admission and from 5 non‐infectious control participants. Plasma small RNA‐sequencing identified significantly altered levels of primarily mature miRNAs in CAP relative to controls. Pathways analysis of high or low abundance miRNA identified various over‐represented cellular biological pathways. Analysis of small RNA levels against common clinical severity and inflammatory parameters indices showed direct and indirect correlations. Additionally, variance of plasma small RNA levels in CAP patients may be explained, at least in part, by differences in causal pathogens. Small nuclear RNA levels were specifically altered in CAP due to Influenza infection in contrast to Streptococcus pneumoniae infection. Pathway analysis of plasma miRNA signatures unique to Influenza or Streptococcus pneumoniae infections showed enrichment for specific proteoglycan, cell cycle, and immunometabolic pathways.


| INTRODUC TI ON
Community-acquired pneumonia (CAP) is a major determinant of sepsis, which accounts for substantial morbidity and mortality worldwide. 1 Sepsis is understood to be mainly initiated by a severe, and often protracted, reaction to infection leading to organ failure. 2 The aetiology of sepsis is multifaceted, involving various microbiological and host response variables. 3 The early oversimplified model of an overwhelming inflammatory reaction failed to capture the complex pathophysiology of the sepsis syndrome, exemplified by unsuccessful clinical trials with a variety of anti-inflammatory agents. 4 New insights have suggested that while inflammation is responsible for 'collateral' tissue damage, immune suppression that accompanies sepsis also contributes to adverse clinical outcome. 5  CNAs are segments of a host's or pathogen's genome present in biological fluids and have been known to exist for decades. 9 These molecules are understood to be released in the host's circulation via either passive secretion, for example, cell death, or active secretion mechanisms. 7 CNAs have been found in the circulation of noninfectious control participants and also shown to be significantly altered in disease conditions, such as cancer, 10 trauma 11 and autoimmune disorders. 12 Such observations have suggested that CNAs may represent promising, non-invasive, tools for early detection of several diseases.
In this study, we sought to determine changes in CNAs, specifically small non-coding RNAs, measured in blood plasma samples from patients with CAP-associated sepsis on intensive care unit (ICU) admission. Levels of plasma small RNAs in CAP were firstly compared to non-infectious control participants, as well as patients with blood culture positive Streptococcus (S.) pneumoniae compared to patients with polymerase chain reaction (PCR) positive Influenza (A or B) infections. Significantly different plasma miRNA levels were analysed for biological pathway enrichment, revealing putative functional targets of paracrine and/or autocrine signalling.

| Study design
The study was performed within the context of the Molecular Diagnosis and Risk Stratification of Sepsis (MARS) project, a prospective observational cohort study in the two tertiary referral centres in the Netherlands (ClinicalTrials.gov identifier NCT01905033) (Academic Medical center, Amsterdam and University Medical Center, Utrecht). 13,14 The current study comprised a total of 13 ICU-admitted CAP patients with positive blood cultures for S pneumoniae and PCR positive Influenza A or B infection. Sepsis diagnosis was described in detail previously. 13,15 This cohort was enrolled between January 2011 and July 2012. The Medical Ethics committees approved an opt-out consent method (IRB no. 10-056C). For each patient, demographics, comorbidities, severity indices and outcomes were gathered. Severity was assessed by APACHE IV 16 and mSOFA excluding the central nervous system component. 17 Septic shock was defined by the use of noradrenaline for hypotension in a dose of more than 0.1µg/kg/min during at least 50% of the ICU day. Five non-infectious control participants (age and gender-matched) were also included in the study. From all noninfectious control participants' written informed consent was obtained. Procedures were performed in accordance with the Helsinki declaration of 1975 (revised 1983). Using 13 sepsis patient samples and 5 non-infectious control participants, and estimated effect size of 0.8, we had 80% power to reject the null hypothesis (5% false discovery rate).

| Sampling, small RNA isolation and sequencing
Whole blood (EDTA) collected from non-infectious control participants and sepsis patients were centrifuged at 1200rpm for 20 minutes to obtain 300ul plasma, and stored in −80C. Plasma from sepsis patients was obtained within 6 hours of ICU admission. Prior to nucleic acid isolation, plasma samples were thawed over ice and centrifuged at 12,000rpm for 10 minutes. Small noncoding RNA was isolated from plasma samples using the miRNeasy mini kit (Qiagen) and MinElute PCR purification kit (Qiagen) as described by the manufacturer. Next generation sequencing libraries of small RNA were prepared using the Illumina TruSeq Small RNA-Seq sample preparation kit (Illumina). Small non-coding RNA was isolated from plasma samples using the miRNeasy mini kit

| Statistics
Statistical analysis was performed in the R statistical environment (v 3.5.0; R Core Team (2019). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-proje ct.org/.). For correlation studies, we used the Spearman correlation method. The Hmisc R package was used to compute the significance levels for spearman correlations and corrplot R package was used to create the correlogram.
Heat maps were generated using the pheatmap R method and principal component analysis (PCA) was done using the FactoMinerR R package. Venn diagrams were made using the VennDiagram R package.
Pathway analysis of the high abundance miRNA revealed significant over-representation for proteoglycans in cancer, hippo signalling pathway, protein processing in endoplasmic reticulum, endocytosis as well as known metabolic pathways, for example, p53 signalling and hypoxia-inducible factor (HIF)-1 signalling ( Figure 1E). Low abundance circulatory microRNAs were associated with ECM-receptor interaction, bacterial invasion of epithelial cells and vasopressin-regulated water reabsorption. Considering adjusted p-values and highest enrichment ratios, the hippo signalling pathway was uncovered as top overrepresented pathway ( Figure 1F). RNAs relative to controls ( Figure 3A and Table S2). MiRNAs were the predominant species (79%) followed by misc_RNA (13%), snoRNA (2%) and rRNA (1%) ( Figure 3B). CAP patients with Influenza infection had 32 significantly altered circulatory small RNAs as compared to non-infectious control participants ( Figure 3C and Table S2).

F I G U R E 2
Of note, 5 snRNA species (16%) were altered solely in patients with influenza infection ( Figure 3D), in contrast to patients with S pneumoniae infection ( Figure 3B). These circulating snRNA species included RNU5A-1, RNU2-7P and RNU2-33P. 10 small non-coding RNAs were common to both S pneumoniae and Influenza virus infections ( Figure 3E and Table S3). Unique transcriptional alterations were also identified, with 28 and 22 significantly altered small RNAs unique to S pneumoniae or Influenza virus infected patients, respectively ( Figure 3E and Table S3). Pathway analysis of elevated plasma miRNAs unique to S pneumoniae infected patients revealed an overrepresentation for adherens junction, lysine degradation, bacterial invasion of the epithelial cells and endocytosis pathways ( Figure 3F).
Low abundance miRNAs were associated with focal adhesion and translational processing including RNA transport and mRNA surveillance pathways ( Figure 3F). Pathway analysis of elevated miRNAs unique to Influenza infected patients revealed significant associations to canonical signalling pathways that included p53 signalling pathway, hippo signalling, TGF-beta signalling pathways and MAPK signalling pathway ( Figure 3G).  various canonical signalling pathways, notably HIF-1α signalling, hippo signalling and proteoglycan reactions. HIF-1α signalling has been shown to influence cytokine responses both in vitro and in vivo, which is understood to be a major cog in immunometabolism wherein defects can precipitate to a state of immune paralysis (immunosuppression). 56,57 Hippo signalling is an evolutionary conserved cell developmental pathway that controls organ size by primarily regulating cell differentiation, proliferation and apoptosis. 58 Proteoglycans represent important composite molecules of the extracellular matrix that contribute to cell adhesion, angiogenesis and in the context of cancer, metastasis and tumour progression. 59 In particular, endocan, a proteoglycan secreted by vascular endothelium, 60 has been reported as a candidate predictor of poor prognosis in acute respiratory distress syndrome, hospital-acquired pneumonia, as well as in all-cause sepsis. [61][62][63] Our study has limitations. This interpretation of the study is hindered by its small sample size. Despite this limitation, genomewide significant differences in small non-coding RNA were detected. The choice of source material (plasma) precluded identification of circulating small RNA cells-of-origin, which would enhance the functional interpretability of the small RNA profiles herein delineated.

| D ISCUSS I ON
In conclusion, we provide a comprehensive map of circulatory small non-coding RNAs in sepsis caused by CAP relative to controls.
Significantly altered circulating miRNA in patients were predicted to target various cellular signalling cascades, particularly immunometabolic, cell proliferation and survival pathways. Our findings prioritize specific circulating small RNAs for functional validation studies as well as potential biomarker studies to discriminate bacterial and viral infections in larger cohorts.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
All RNA-sequencing data used in this study are accessible through the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database with accession code GSE137294 (https://www.ncbi.nlm.nih.gov/geo/query/ acc. cgi?acc=GSE13 7294).