Sepsis, a life-threatening response to systemic infection, is a worldwide health-care problem and sepsis-associated encephalopathy (SAE), a diffuse brain dysfunction secondary to sepsis without an obvious brain infection, is one of the most common complications. Sepsis pathophysiology is a complex combination of immune homeostasis, poor tissue perfusion, and reactive oxygen species leading to the multi-organ injury (1) with high mortality rate (2). Up to 70% of patients with sepsis demonstrate some symptoms of SAE ranging from mild (confusion, inattention, and concentration deficits) to severe symptoms (deep coma) (1, 3). Due to the variation of clinical manifestations of SAE and the similarity between the characteristics of SAE and encephalopathy from other causes (uremia, hepatic encephalopathy, electrolyte imbalance, and glycemic coma), SAE diagnosis depends on the consideration of other possible causes without definite clinical criteria (3). In addition, SAE pathophysiology is still unclear with multifactorial factors such as endothelial injury, blood–brain barrier (BBB) defect, inflammation, neuron signaling interference, and cell apoptosis. Indeed, BBB is a highly selective semipermeable membrane that consists of a physical barrier (endothelial tight junctions) and a transport barrier (receptor-mediated transcytosis) (4, 5) that protects the brain from several toxins and pathogens in blood circulation during sepsis. Sepsis-induced BBB defects are considered a major cause of SAE (6) that allows the influx-transmigration of circulating molecules into the brain and, conversely, the translocation of several brain-specific molecules into blood circulation.
As such, the detection, in blood, of several brain-specific proteins such as s100-β (a glial-specific protein of astrocytes) and neuron-specific enolase (an enzyme of the mature neurons) are proposed as SAE biomarkers (7), but unfortunately are not reliable enough for clinical use. Because microRNA (miR), a small endogenous non-coding RNA, is smaller than protein, miR might be an easier molecule that passes through BBB and is possibly a more sensitive biomarker of sepsis-induced BBB injury and/or encephalopathy. In addition, miRs are stable enough in blood to use as proposed biomarkers in several diseases including brain disorders (8–11). Here, transcriptomic profiling of brains from sepsis mice was explored for the discovery of SAE biomarker and candidate miRs were validated on mouse models and patient samples.
MATERIALS AND METHODS
Animal, animal models, and encephalopathy determination
Animal protocols were approved by the Institutional Animal Care and Use Committee of the Faculty of Medicine, Chulalongkorn University following the US National Institutes of Health animal care and use protocol. Male, 8-week-old C57BL/6 mice (National Laboratory Animal Center, Nakhornpathom, Thailand) weighing approximately 20 g to 22 g each were used. Mice, housed in standard clear plastic cages (three–five mice per cage), had free access to water and food (SmartHeart Rodent; Perfect companion pet care, Bangkok, Thailand) with a light/dark cycle of 12: 12 h in 22 ± 2°C with 50 ± 10% relative humidity.
Encephalopathy was induced by cecal ligation and puncture (CLP) and bilateral nephrectomy (BiNx) for SAE and uremic encephalopathy, respectively. CLP (ligation at 10 mm from cecal tip and punctured twice with a 21-gauge needle) or BiNx surgery were performed through an abdominal incision under isoflurane anesthesia following the previous publications (12, 13). In sham operation, kidney and cecum were identified before closing the abdomen. Fentanyl at 0.03 mg/kg in 0.5 mL of normal saline solution was administered subcutaneously for analgesia and fluid replacement postoperatively. The severity of encephalopathy was determined by SHIRPA score (SmithKline Beecham, Harwell, Imperial College, Royal London Hospital, phenotype assessment) that was designed for the evaluation of behavior, neurological aspects, and physiological features in mouse based on mouse position, respiration, activity, reflex responses, body tone, etc. (14). Mice were sacrificed at the specific time-points by cardiac puncture under isoflurane anesthesia. The internal organs were collected in RNAlater (Thermo Fisher Scientific, Waltham, Mass) and homogenized with Qiazol lysis (QIAGEN, Hilden, Germany) for miR evaluation. Blood was put in ethylene diamine tetraacetic acid (EDTA) before plasma separation and was kept at −80°C until analyzed. Kidney and liver injury were determined by QuantiChrom Creatinine Assay (DICT-500; BioAssay, Hayward, Calif) and EnzyChrom ALT assay (EALT-100, BioAssay), respectively. Serum cytokines (TNF-α, IL-6, and IL-10) were measured with ELISA (eBioscience, San Diego, Calif). For the detection of apoptosis in brain, 10% formalin-fixed, paraffin-embedded sections were stained with anti-active caspase-3 antibody (Cell Signaling Technology, Beverly, Mass), examined in whole sections in ×200 fields and expressed as positive cells per high-power field. For cytokine detection in brain, samples were weighed, sonicated thoroughly and the supernatant from homogenous tissue preparation was evaluated by ELISA assay (eBioscience) and demonstrated in pg/g of tissue weight.
Sequencing data analysis
MircoRNA profiling from miR-arrays of mouse brains with severe sepsis (at 24 h post-CLP) versus sepsis survivors (at 120 h post-CLP) was performed for the discovery of miR that was associated with SAE. Brains were prepared by QIAGEN miRNeasy kit (QIAGEN) and the libraries starting with 1 μg total RNA for each sample. Then, the samples were processed with the miRNA array of BGISEQ-500 platform. Data were subsequently processed in R-bioconductor (library package EdgeR) and normalized by trimmed mean of M-value between each pair of samples. Pairwise analysis was conducted for each group. The P value was corrected with Benjamini method and values less than 0.05 were considered statistically significant. The predicted mRNA targets were obtained from DIANA tool micro-T-CDS (15, 16) and TargetScan version 7.2 (17) with high confidence scores (≥ 0.7) and high aggregated probability of conserved target (8mer ≥ 0.8; 7mer-m8 ≥ 1.3; 7mer-1A ≥ 1.6). The overlapping mRNA targets were collected for biological pathway analysis by Reactome Database (18) which corrected P values [False discovery rate technique] of less than 0.05 were shown as interaction mapping (Cytoscape version 3.4.0) (19). MGI-gene names were used according to ENSEMBL database gene identification data (ID).
Total RNAs of plasma and tissue samples were extracted using QIAGEN miR-Neasy serum/plasma kit (QIAGEN) according to the manufacturer's instruction. Briefly, miRs were converted to cDNA by TaqMan MicroRNA Assays kit (Applied Biosystems, Waltham, Mass) in RT-PCR (reverse transcription polymerase chain reaction) machine (SimpliAmp Thermal Cycler systems, Applied Biosystems, Foster city, CA). All primers (mmu-miR-370–3p, ID 002275; mmu-miR-137-5p, ID 001129; mmu-let-7a-1-3p, ID 002478) were purchased from Thermo Fisher Scientific (Waltham, Mass) and cDNA samples for quantitative real-time PCR through TaqMan Universal PCR Master Mix on a qRT-PCR (real-time polymerase chain reaction) machine were used (Applied Biosystems). Relative expression was calculated using the ΔΔCT method and normalized to the expression of snoRNA-202 and cel-miR-39 for tissue and plasma samples, respectively (Applied Biosystems).
Blood–brain barrier permeability analysis
The analysis of blood–brain barrier permeability was performed by plasma S100β and the Evans blue dye (EB) assay for the detection of microvascular leakage in the same mice. At 45 min before sacrifice, blood was collected through facial artery for the detection of S100β by ELISA assay (SEA567Mu, Cloud-Clone Corp, Tex). After that, 1% EB (Sigma-Aldrich, Mo) at 2 mL/kg in 0.9% sodium chloride was administered via tail vein at 30 min before sacrifice following a published procedure (20). At sacrifice, phosphate buffer solution (PBS) was perfused through left ventricle until the blue color in blood was eliminated and brains were weighed, snap-frozen in liquid nitrogen. Then the brains were homogenized in formamide (Sigma-Aldrich) in a ratio of brain: formanide at 0.4 mg: 1 mL at 55°C for 18 h before centrifugation and EB in the supernatant was measured with the absorbance of 620 nm in comparison with the EB standard curve for the quantitative values.
Blood was collected in EDTA for plasma separation from patients with sepsis in the medical ward and medical intensive care unit at the King Chulalongkorn Memorial Hospital following approval from the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University (IRB 113/60). Inclusion criteria were adults (>18 years old) with the diagnosis of bacterial sepsis at admission as determined by fever (body temperature > 38°C) with a possible source of bacterial infection. The exclusion criteria were metabolic encephalopathy such as electrolyte imbalance, hypo-/hyper-glycemia, severe liver injury (alanine transaminase > 500 U/L), and drug-induced encephalopathy, intracranial causes of encephalopathy such as head trauma, intracerebral lesions, epilepsy, malignancy, and unstable vital signs (mean arterial pressure less than 65 mm Hg, hypoxia and post-cardiopulmonary resuscitation). The examination for Glasgow Coma Scale (GCS) and sepsis-related organ failure assessment (SOFA) scores were determined at the time of enrollment. Patients with GCS less than 13 were determined as patients with SAE and samples from the volunteers were used as a control group. As a cross-sectional analysis, there were 17 patients with SAE and 12 patients as sepsis without encephalopathy. In addition, plasma from patients with severe uremia with an indication of renal replacement therapy (normal GCS) was used as a representative of uremic encephalopathy due to the very low incidence of uremic encephalopathy. Epidemiology of patients is presented in Table 1.
Induction of miR expression in a neuron cell-line
Since sepsis consists of several factors that possibly induce miR expression in neuron including inflammatory cytokines, starvation injury, endotoxin, and uremic toxin, these factors were challenged upon the neuron cell line of PC-12 (American Type Culture Collection; ATCC CRL-1721, Manassas, Va). PC-12 cell was incubated in complete media (RPMI1640) with 10% v/v horse serum, 5% v/v fetal bovine serum, and 1% v/v Penicillin-Streptomycin (Thermo-Fisher Scientific) for 24 h before starting the experiment. Then PC-12 cell at 1×105 cells/wells was incubated for 24 h with each of the following factors; murine inflammatory cytokine (Bio legend, San Diego, Calif) including TNF-α (100 ng/mL) (21), IL-6 (20 ng/mL), or IL-10 (20 ng/mL) Earle balance salt solution (Thermo Fisher Scientific) to explore the influence of cell-starvation, endotoxin (Sigma-Aldrich) at dose 1 mg/well, uremic mouse serum from mice with 48 h post-BiNx with BUN and Scr at 112 ± 11 mg/dL and 2.7 ± 0.2 mg/dL, respectively, or PBS control, prior to cell retrieval for miR evaluation by qRT-PCR as mentioned above. Of note, mouse serum was heated at 56°C for 1 h to eliminate several cytotoxic factors (e.g., complement and cytokines) in mouse serum before incubation into the cell following the previous publications.
Transfection of miR 370-3p and flow cytometry analysis
To explore the influence of miR-370-3p in neuron, PC-12 cells were transfected before activating with several conditions as mentioned above. Transfection efficacy was tested in PC-12 at 5 × 105 cells/well after incubation with plasmid containing green fluorescent protein at 1 μL in of OptiMEM I (100 μL) with lipofectamine 2,000 (100 μL) (Invitrogen, Carlsbad, Calif) in 5% CO2 at 37°C for 48 h before direct microscopic visualization (Olympus IX81). In parallel, the transfection efficacy of PC-12 (5 × 105 cells/well) after incubation with hsa-miR-370-3p (Ambion Inc, Thermo Fisher Scientific) (5 μL) in OptiMEM I (final concentration of 100 nM in 50 μL RNAimax) or miR negative control (Ambion) at 5% CO2, 37°C for 24 to 72 h before retrieving cells for miR evaluation by qRT-PCR as mentioned above. Transfection protocol with 72 h incubation was used for further experiments due to the highest transfection efficacy. Because of sepsis-induced brain apoptosis and the possible association of miR-370-3p with apoptosis cell apoptosis of the miR-370-3p transfected cells after several activations was determined by standard protocol of flow cytometry. In short, the stimulated cells at 5 × 105 cell/well were washed in Dulbecco buffer, centrifuged and stained with fluorochrome-conjugated antibodies against annexin V and propidium iodide (PI) (BioLegend, San Diego, Calif). All stained cells were analyzed by flow cytometry analysis BD LSR-II (BD Biosciences) with FlowJo software. Cells stained with only annexin V (but not PI) and annexin V together with PI represented early and late apoptotic cells, respectively.
All data was analyzed by the Statistical Package for Social Sciences software (SPSS 22.0, SPSS Inc, Ill) and Graph Pad Prism version 7.0 software (La Jolla, Calif). The results were presented as mean ± standard error. The differences between groups were examined for statistical significance by one-way analysis of variance followed by Tukey analysis or Student t test for comparisons of multiple or two groups, respectively, and a P value < 0.05 was statistically significant.
Transcriptomic analysis from mouse brains of sepsis encephalopathy versus sepsis recovery
Transcriptomic analyses from mouse brains of SAE (24 h post-CLP) versus recovery mice (120 h post-CLP) demonstrated an elevation of miR-497, −144, -let7a, −137, −21a, −7b, −1249, −486b, and −370 expression (Fig. 1, A and B). Among these miRs, only miR-370-3p, miR-137-5p, and let-7A-1-3p were associated with neurological disorders from previous publications (22–24). Thus these three miRs were further explored in two mouse models of encephalopathy caused by sepsis and uremia to determine the disease specificity of the candidate biomarkers. The encephalopathy in mice, as determined by SHIRPA score, in CLP sepsis and BiNx uremia was demonstrated as early as 6 h post-surgery. Although the progression of encephalopathy was more rapid in CLP compared with BiNx, the encephalopathy score was similar between 24 h post-CLP and 48 h post-BiNx (Fig. 1C). Hence, the further comparison of encephalopathy parameters was performed between 24 h post-CLP versus 48 h post-BiNx. Because all tested parameters between 24 and 48 h-post-sham operation (data not shown) were similar, only the data of 48 h post-sham operation (but not 24 h post-sham) was demonstrated.
A brain-specific miR of sepsis encephalopathy, miR-370-3p
Organ specificity of these miRs in sepsis and uremia was tested and all miRs demonstrated the highest expression in sepsis brain in comparison with the brain of sham mice (Fig. 2, A–C). In parallel, miR let-7a-1-3p was not only expressed in brain but also in liver, heart, and lung of sham and in lung of BiNx (Fig. 2A). Similarly, in addition to the miR-137-5p expression in brain, it was also expressed in liver and heart of sham and BiNx (Fig. 2B). In contrast, miR-370-3p was expressed only in brain of sepsis mice but not in other organs (Fig. 3C). In addition, the expression of these miRs in several organs within each mouse model (relative to the brain expression) was demonstrated (Fig. 2, D–F). In sham mice, the expression of miR-137-5p and miR-let-7a-1-3p in brain was lower than several internal organs (liver, heart, and lung), while miR-370-3p was only expressed only in brain (Fig. 2D). In the sepsis CLP model, the expression of these three miRs was very high in brain implying the major influence of miRs against neuron in sepsis (Fig. 2E). Likewise, in BiNx mice, miR-370-3p expressed only in brain, while miR-let-7a-1-3p and miR-137-5p expressed more in lung and heart, respectively (Fig. 2F). Moreover, all three miRs in brain of SAE mice (24 h post-CLP) were higher than BiNx and sepsis at the recovery phase (120 h post-CLP) as calculated with the relative expression to sham brains (Fig. 2G). Of note, miR-370-3p expression was also demonstrated in brains of BiNx mice but in a lower level than the sepsis brain (Fig. 2G). These data imply prominent miR-370-3p expression in brain of mice with SAE and the specificity of miR-370-3p toward brain tissue.
Plasma miR-370-3p outperformed s100β as a biomarker of sepsis encephalopathy in mouse
Biomarkers from blood are easier to use in clinical practice and plasma miRs are reported as biomarkers (7). Interestingly, plasma miR-370-3p increased only in mice with SAE (24 h post-CLP) while miR-137-5p and miR-let-7a-1-3p in plasma were elevated both in SAE and in uremia (48 h post-BiNx) (Fig. 2H). In addition, plasma miR-370-3p increased as early as 6 h after CLP (Fig. 3A), an onset of SAE according to the SHIRPA score (Fig. 1C), but non-detectable in BiNx mice even at the peak of uremic encephalopathy at 48 h post-BiNx (Figs. 3A, 1C) indicating the specificity toward SAE. In parallel, plasma s100β (a current candidate of SAE biomarker) and BBB defect (EB dye assay) were detectable at 24 h post-CLP, but not at 6 h post-CLP (Fig. 3, B and C), while plasma miR-370-3p was demonstrated at 6 h post-CLP (Fig. 3A). Moreover, plasma s100β also increased at 48 h post-BiNx (Fig. 3B), implying the less specificity toward SAE. Plasma miR-370-3p in sepsis mice increased together with serum cytokines and liver enzyme, but not serum creatinine, (Fig. 3, D–H) suggesting the influence of systemic inflammation upon miR-370-3p expression. However, only plasma miR-370-3p, but not s100β, correlated with the encephalopathy score, without the correlation between both biomarkers (Fig. 3, I–K). In parallel, miR-370-3p was not correlated with plasma creatinine (Fig. 3L).
Plasma miR-370-3p as a biomarker of sepsis encephalopathy in patients
Interesting miR-370-3p data in sepsis mice prompted a pilot study to test patient serum. Interestingly, plasma miR-370-3p was approximately 40 times higher in patients with SAE compared with healthy volunteers and did not increase in sepsis without encephalopathy (Fig. 4A). In contrast, plasma s100β increased in sepsis (regardless of encephalopathy), but not uremia (Fig. 4B), implying the limitation of s100β as a SAE biomarker as mentioned previously (25). However, the severity of sepsis in patients with SAE (lower GCS; Fig. 4D) was more severe than patients without SAE as evaluated by plasma creatinine and SOFA score (Fig. 4, E and F). Additionally, miR-370-3p was associated with GCS and SOFA score but was less correlated with plasma creatinine (Fig. 4, G–I) implying the lesser impact of renal injury against this miR. On the other hand, s100β was correlated only with GCS but not SOFA or plasma creatinine (Fig. 4, J–L). Due to the low incidence of uremic encephalopathy, plasma of patients with severe uremia from chronic kidney disease who need renal replacement therapy initiation was used instead of uremic encephalopathy. None of these patients demonstrated high plasma miR-370-3p compared with healthy volunteers (Fig. 4A).
The influence of miR-370-3p in apoptosis of neuron cell
Sepsis-induced brain apoptosis is, in part, responsible for BBB damage in SAE. To understand the role of miR-370-3p, the enrichment analysis of miR-370-3p overlapped the predicted targets (n = 402 genes from TargetScan and microT tool) indicating the association between this miR and the small ubiquitin like modification (SUMOylation) which controls protein translocation (nucleus and cytoplasm) (26) and apoptosis (27) (Fig. 5). As a result, increased apoptosis in mouse brain at 24 h post-CLP was also demonstrated (Fig. 6,A and B) supporting previous publications (28–31). In addition, prominent inflammation in septic brains was also demonstrated by the increased inflammatory cytokines (TNF-α and IL-6) in brain tissue of CLP compared with sham (Fig. 6C). Additionally, miR-370-3p was inducible in PC-12 cell only by TNF-α and starvation, but not other factors (Fig. 6D), while miR-137-5p and miR-let-7a-1-3p were inducible only by LPS (Fig. 6, E and F) implying the impact of TNF-α in enhancing miR-370-3p in the brain during sepsis. Further, mimic-miR-370-3p was transfected into PC-12 neuron cell line (Fig. 6G) before activation by several factors. Interestingly, apoptosis was inducible in the over-expressed mimic-miR-370-3p cells after the activation with cytokines (TNF-α, IL-6, and IL-10) and LPS but not starvation and uremic serum (Fig. 6, Hand I and supplement Fig. 1, Supplemental Digital Content, https://links.lww.com/SHK/A976).
The transcriptomic profiling from SAE brains implies increased expression of miR-370-3p and suggests a candidate of SAE biomarker. Indeed, the elevation of plasma miR-370-3p was demonstrated in SAE, but not in uremic encephalopathy, in mouse models and in patients. Perhaps the prominent cytokine storm (especially TNF-α) and severe hypoxia (cell starvation) in sepsis enhanced miR-370-3p expression in neuron and facilitated the translocation of this miR from brain into blood circulation through sepsis-induced BBB damage.
Plasma miR-370-3p, a candidate biomarker of sepsis-associated encephalopathy from CLP sepsis model
Despite the expression of these three miRs in sepsis mouse brains, only miR-370-3p was solely demonstrated in brains, not other organs. Although miR-370-3p was also increased in BiNx brains, the expression was 20 to 30 times lower than SAE brain implying miR-370-3p as the most specific-miR for sepsis brain determination. While miR-137-5p and miR-let-7a-1-3p in plasma were expressed in both CLP sepsis and BiNx mice, plasma miR-370-3p was expressed only in SAE (24 h post-CLP). Because BBB damage (Evan blue assay and plasma s100β) between 24 h post-CLP versus 48 h post-BiNx was similar (Fig. 3, B and C), TNF-α, but not uremic serum, induced miR-370-3p in PC-12 cell (Fig. 6D), and very prominent serum TNF-α in sepsis in comparison with uremia, high plasma miR-370-3p in sepsis was possibly a result of increased miR expression in neurons from sepsis-induced brain inflammation and transferred into blood due to BBB damage. Further, miR-370-3p increased as early as 6 h post-CLP, an onset of encephalopathy as determined by SHIRPA score, but not in BiNx even at 48 h post-surgery which supports the specificity of this miR in sepsis. Interestingly, miR-370-3p was detectable at 6 h post-CLP while s100β and Evan blue dye assay were not different from sham mice, suggesting the higher sensitivity of miR-370-3p for SAE detection. Perhaps, the smaller molecular size of miRs, in comparison with protein, makes it easier for the translocation from brain into blood resulting in the earlier detection of miR-370-3p than s100β in plasma. Nevertheless, uremic toxin could not induce miR-370-3p as demonstrated by very low plasma miR-370-3p in BiNx despite the more severe uremia (plasma creatinine) than CLP sepsis, and uremic serum fails to induce miR-370-3p expression in PC-12 cell. Moreover, increased miR-370-3p was demonstrated in a pilot study of patients with SAE (GCS less than 13), demonstrating a proof of concept to use as a biomarker for SAE. Plasma miR-370-3p in patients with uremia was very low and was not correlated with serum creatinine, supporting the less impact of renal injury against plasma miR-370-3p level. However, sepsis severity in our patients with SAE was more severe than patients without SAE and the healthy controls were younger than study patients. Hence, further studies on patients with similar sepsis severity with and without SAE are warranted.
Sepsis-induced brain apoptosis and miR-370-3p
Although the actual mechanism of SAE has not been completely characterized, brain apoptosis is frequently mentioned as one of the contributing factors toward SAE (32). Our results from miR-370-3p predicted targets suggested the association with SUMOylation pathway, a post-translational modification process of several cell mechanisms including apoptosis. As such, the over-expression miR-370-3p in glioblastoma cells enhances inflammation from Japanese encephalitis virus (33) and in hepatocellular carcinoma cell limits cancer metastasis (34) perhaps through apoptosis induction. Therefore, miR-370-3p might be another regulator of inflammation and apoptosis in SAE of the brain. Indeed, miR-370-3p over-expression in PC-12 neuron cell line enhanced susceptibility toward apoptosis after the activation with cytokine or LPS (but not with starvation or uremia). Because both cytokine and LPS in serum are prominent in sepsis over uremia, these data highlight the influence of miR-370-3p in sepsis. Moreover, miR-370-3p was inducible in PC-12 only by TNF-α (but not by other cytokines) suggesting that TNF-α enhanced miR-370-3p expression in neurons, increasing cell vulnerability toward apoptosis (by second-hit injury). Although TNF-α has been mentioned as a key mediator of SAE through the activation of TNF-receptor-1 (35), TNF-α pathway is non-specific for SAE and not a proper SAE biomarker. Hence, miR-370-3p (a down-stream molecule of TNF-α) should be a better candidate of the SAE biomarker. Moreover, miR-370-3p manipulation for the attenuation of brain apoptosis in SAE is also an interesting future topic.
Transcriptomic analyses from brains of sepsis mice and the validation in two mouse models of encephalopathy (sepsis and uremia) identified miR-370-3p as a more specific biomarker for SAE because it was inducible by sepsis molecules (LPS and cytokines), but not by uremic toxin. We propose plasma miR-370-3p as an SAE biomarker for future studies.
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