MicroRNAs (miRNAs) are short noncoding single-stranded RNA species found in a wide variety of organisms. MicroRNAs mostly regulate gene expression by forming imperfect base pairings with target mRNA and subsequently guiding mRNA cleavage or translational repression (1, 2). In mammals, miRNAs have been associated with diverse physical and biological processes (3, 4). Altered miRNA expression levels were correlated with disease occurrence and progression.
Recently, studies have shown that miRNAs play a role in the innate immune response, which is the first line of defense that relies on phagocytes such as granulocytes and macrophages (5, 6). Increased levels of miR-146 in human monocytic cell lines and miR-155 expression in murine macrophages in response to endotoxin stimulation have been reported (7, 8). After lipopolysaccharide (LPS) injection, a specific whole-blood–derived miRNA signature was observed in mice (9). A relatively small number of miRNAs (miR-143, miR-146b, miR-150, miR-342, and let-7 g) in leukocytes of human volunteers are subject to differential expression on treatment with LPS (10).
Bacterial LPS is an effective trigger of the inflammatory response during infection with gram-negative bacilli (GNB), which implicates the pathogenesis of sepsis and septic shock (11, 12). Lipopolysaccharide-induced toll-like receptor (TLR) 4 signal transduction activates well-characterized pathways against pathogens, including those involving nuclear factor-κB (NF-κB) and activator protein 1 (AP-1), leading to the production of down stream proinflammatory cytokines, chemokines, or leukocyte adhesion molecules (13). Uncontrolled activation of LPS-induced mechanisms results in sepsis. Despite much effort in the development of antimicrobial therapies, more than 200,000 deaths still occur from sepsis in the United States each year (14). Urinary tract infection (UTI) accounts for a significant number of emergency department visits. In the elderly, UTI is a major cause of GNB sepsis with significant mortality (15).
MicroRNAs have been found to be involved in the fine-tuning of TLR-mediated inflammatory response. Fine-tuning TLR/NF-κB signaling dynamics may involve both negative and positive feedback regulators, which function in concert, to ensure finely controlled immunity against microbial infection (16, 17). MiR-146a, miR155, miR-125b, miR-21, and miR-98 were shown to inhibit the TLR-triggered inflammatory cytokines (7, 18, 19); miR-98 and let-7 were reported to regulate cytokine-inducible Src homology 2–containing protein (CIS) expression via translational suppression in human cholangiocytes (20).
In this study, we profiled the miRNA expression levels in peripheral leukocytes of GNB urosepsis patients and compared them with those of healthy controls. We explored the regulatory mechanism of endotoxin-responsive miRNAs in the TLR and cytokine signaling by using a human monocytic cell line (THP-1 cells) treated with LPS antigen stimulation to mimic GNB sepsis in vitro.
This prospective noninterventional study was conducted in the emergency department (ED) of a tertiary care medical center located in Taipei City, Taiwan. The institutional review board approved the study, and patients or their next of kin provided written informed consent before enrollment. Convenience sampling was used in this study. Patients aged 18 years or older with a diagnosis of urosepsis between March 2010 and October 2010 were eligible for inclusion in the study. The diagnosis of urosepsis was based on UTI in combination with two or more of the following criteria of systemic inflammatory response syndrome: temperature more than 38°C or less than 36°C; pulse rate more than 90 beats/min; respiratory rate more than 20 breaths/min or hyperventilation with PaCO2 less than 32 mmHg; white blood cell count more than 12,000/μL or less than 4,000/μL or more than 10% immature cells (21). A UTI is defined as the presence of nitrites and leukocytes in a spot midstream urine sample and later confirmed by a urine culture. Given that the purpose of this study was to examine the profile of miRNA expressions in peripheral neutrophils of the patients with GNB urosepsis, only patients with GNB as the definite causative pathogens after microbiological workup were enrolled. Patients were excluded if they were pregnant, under corticosteroids, bone marrow or organ transplant recipients, leukopenic (white blood cell count <1,000/μL), and neutropenic (polymorphonuclear granulocyte count <500/μL) or if diagnosed with acquired immune deficiency syndrome. In addition, 20 healthy volunteers were included as healthy controls for further analysis.
At the ED, the following items were recorded for each patient: age, vital signs, comorbid diseases, routine blood test values, and microbiological culture results. The attending physician ordered microbiological tests and antimicrobial therapy according to the usual practice of the ED without interference by the research team.
Blood sampling and RNA isolation
Venous blood samples (5 mL) were drawn within 6 h after study enrollment. The blood was collected into EDTA tubes and immediately processed following the LeukoLock Filter System protocol (Ambion Europe Ltd., Huntingdon, UK). All blood samples were fractionated by binding leukocytes to a filter while removing erythrocytes, reticulocytes, and platelets by washing with phosphate-buffered saline. The filter-bound leukocyte fraction was then stabilized by adding 2.5 mL of RNALater (Ambion Europe Ltd.). We modified the protocol from the LeukoLock RNA Isolation (Ambion Europe Ltd.) system and subjected the leukocyte fraction to lysis with TRI reagent (Sigma-Aldrich, St. Louis, Mo) to purify the total RNA retained in the small RNA fraction. Total RNA was recovered by phenol-chloroform extraction and isopropanol precipitation and purified by ethanol–sodium acetate precipitation. All RNA samples were quality controlled by measuring the optical density at 260 and 280 nm and by analyzing an aliquot of the RNA preparation on an Agilent 2100 Bioanalyzer using RNA 6000 Nano chips (Agilent Technologies, Santa Clara, Calif). RNA samples were stored at −80°C until further processing.
MiRNA microarray screening
A total of 14 RNA samples from peripheral leukocytes (seven urosepsis patients and seven healthy controls) were analyzed using a Geniom real-time analyzer (GRTA, febit GmbH, Heidelberg, Germany) with the Geniom biochip miRNA homo sapiens. Each array contains seven replicates of 1,048 miRNAs and miRNA star sequences as annotated in the Sanger miRBase 16.0 (22). Sample labeling with biotin was carried out by microfluidic-based enzymatic on-chip labeling of miRNAs as described before (23).
After hybridization for 16 h at 42°C, the biochip was washed automatically. A program for signal enhancement was processed with the GRTA. The resulting detection pictures were evaluated using the Geniom Wizard Software. For each array, the median signal intensity was extracted from the raw data file such that, for each miRNA, seven intensity values were calculated according to each replicate copy of miRBase on the array. After background correction, the seven replicate intensity values of each miRNA were summarized using their median value. Quantile normalization was applied to normalize the data across different arrays. All further analyses were carried out using the normalized and background-subtracted intensity values.
Quantification of miRNA expression
MicroRNA expression was quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) experiments to validate the findings from the initial array-based screen. Approximately 5 ng of total RNA was subjected to complementary DNA synthesis using miRNA-specific stem-loop reverse transcription primers (Applied Biosystems). Real-time qRT-PCR used the TaqMan miRNA assay kits (Applied Biosystems) together with TaqMan Universal PCR Master Mix (Applied Biosystems) according to the manufacturer’s instruction. The qRT-PCR used an ABI PRISM 7300 detection system. Each sample was measured thrice in a 96-well plate, and U6 snRNA was used as an internal control. Calculations were normalized against U6 snRNA levels.
The human monocytic cell line THP-1 was obtained from the American Tissue Culture Collection and maintained in HEPES-buffered RPMI 1640 medium (Sigma-Aldrich) supplemented with 2 mM of L-glutamine, 1 mM of sodium pyruvate, and 10% fetal bovine serum (Invitrogen, Carlsbad, Calif).
LPS antigen stimulation, endotoxin-responsive miRNAs, and cytokine quantification
For stimulation with LPS (Escherichia coli serotype 0111:B4, Sigma-Aldrich), THP-1 cells were seeded at 1 × 106 per well on a six-well plate 2 days before exposure. Lipopolysaccharide was directly added to the cell culture medium at the indicated concentration. After 6 or 12 h of LPS (1 μg/mL) antigen stimulation, total RNA was extracted from the THP-1 cells by using TRI reagent (Sigma-Aldrich). Endotoxin-responsive miRNAs and cytokine mRNA quantification were determined using real-time RT-PCR TaqMan assays (Applied Biosystems). We used U6 snRNA as internal controls to normalize the expression levels of miRNAs. Calculations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 cytokine mRNA expression levels were performed using the comparative △△Ct method and normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). TaqMan probes used for the detection of specific cytokines included the assays ID for TNF-α, Hs00174128_ml; for IL-1β, Hs01555410_ml; for IL-6, Hs00985641_ml; and for GAPDH, Hs99999905_ml.
Western blot analysis
After LPS (1 μg/mL) antigen stimulation for 24 h, 25 μg of THP-1 whole-cell lysate was separated on 10% acrylamide gels and electrotransferred to nitrocellulose membrane in Tris-glycine buffer with 20% ethanol. Membranes were blocked with 5% milk in 1× Tris-buffered saline and 0.1% Tween-20 for 1 h at room temperature. Blots were then probed with primary antibodies and tubulin (Santa Cruz Biotechnology, Santa Cruz, Calif), followed by a horseradish peroxidase–conjugated secondary antibody (Bio-Rad, Hercules, Calif). Primary antibody was incubated overnight at 4°C with rocking. After washing, incubations with secondary antibody were performed at room temperature for 45 min. Blots were visualized using supersignal chemiluminescence substrate (Pierce, Rockford, Ill) exposed to film (Kodak, Rochester, NY). Target protein level was expressed as its ratio to tubulin.
Transduction of miRNA
For miRNA transduction studies, THP-1 cells were seeded at 1 × 104 per well in six-well culture dishes and were transduced with 50 nM of endotoxin-responsive miRNA through pLV-miRNA lentiviral transduction protocols (Biosettia, San Diego, Calif). Transduced cells were collected for comparison of cytokine production before and after 24 h of LPS (1 μg/mL) antigen stimulation. The amount of TNF-α and IL-1β present in culture supernatants was measured using an enzyme-linked immunosorbent assay according to the manufacturer’s protocol (R&D Systems, Minneapolis, Minn).
Descriptive results were reported as mean ± SD. Variables were evaluated to determine their association with the diagnosis by using Pearson χ2 test for categorical data and Mann-Whitney U test for numerical data. The groups were compared with the use of the Mann-Whitney U test (or the nonparametric Kruskal-Wallis test when appropriate) for numerical data and the Pearson χ2 test for categorical data. All statistical analyses were completed with SPSS 18.0 version software, and a two-tailed P < 0.05 was considered significant.
Characteristics of the study sample
A total of 22 patients with urosepsis were eligible in the study. The basic demographic data are shown in Table 1. The mean age of patients was 60.6 ± 20.3 years. A total of five patients (22.7%) fit the criteria for septic shock, and seven patients (31.8%) had bacteremia. The etiology distribution of causative organism is summarized in Table 1. Tumor necrosis factor-α and IL-1β mRNA expression ratio had no missing or indeterminate values. Baseline TNF-α and IL-1β mRNA expressions on peripheral leukocytes were significantly higher among urosepsis patients than among healthy controls. The TNF-α mRNA expression ratios at admission were 3.94 ± 2.73 in urosepsis patients and 1.61 ± 0.42 in healthy controls (P = 0.005). The IL-1β mRNA expression ratios at admission were 117.54 ± 85.29 in urosepsis patients and 2.53 ± 1.18 in healthy controls (P < 0.001) (Fig. 1).
Differentially expressed miRNAs detected by microarray in GNB urosepsis patients and healthy controls
We used microarray to detect miRNAs in peripheral leukocytes that were differentially expressed by comparing samples from seven urosepsis patients and seven healthy controls. Gram-negative bacilli infection altered the expression level of 30 miRNAs (15 had upregulated and 15 had downregulated expressions; Fig. 2A). A selection of miRNAs was chosen for validation based on statistically significant high levels of logarithmized fold changes seen on the microarray. Two upregulated (miR-1249 and miR-199b-5p) and two downregulated (let-7a and miR-150) miRNAs were chosen for further qRT-PCR validation.
Let-7a and miR-150 expression levels in peripheral leukocytes are significantly reduced in GNB urosepsis patients compared with healthy controls
To confirm the microarray results, we used TaqMan assays to verify the expression level changes from urosepsis patients (n = 22) and healthy controls (n = 20) of four candidate miRNAs. The primer sequences used in real-time PCR are summarized in Table 2 (TaqMan miRNA assay ID for let-7a, 000377; for miR-150, 000473; for miR-1249, 002868; for miR-199b-5p, 000500; for U6 snRNA, 001093). These four candidate miRNAs altered the expression patterns in the validation set. The results are consistent with the results from the microarray set. However, only let-7a (P < 0.001) and miR-150 (P < 0.001) were significantly downregulated in urosepsis patients compared with healthy controls (Fig. 2, B and C); miR-1249 (P = 0.298) and miR-199b-5p (P = 0.089) did not significantly change between both groups (data not shown).
Let-7a and miR-150 are downregulated in THP-1 cells after LPS stimulation
To investigate whether downregulated let-7a and miR-150 are the direct result of TLR4 signaling or events secondary to systemic TLR4 activation, we established THP-1 cell culture treated with LPS antigen stimulation to mimic GNB sepsis in vitro. After 6 or 12 h of LPS antigen stimulation, we identified increased TNF-α, IL-1β, and mRNA expression in THP-1 cells (Fig. 3A). Furthermore, we tested how the stimulation of monocytic cells with bacterial LPS affects intracellular let-7a and miR-150 levels in cell culture. As shown in Figure 3, B and C, stimulation of cultured THP-1 cells with 1 μg/mL LPS led to a significant downregulation of let-7a and miR-150 in these cells.
TLR2 or TLR3 signaling pathways did not similarly affect the expression levels of let-7a and miR-150
To investigate whether TLR2 or TLR3 signaling pathways similarly affect the expression levels of let-7a and miR-150, we stimulated THP-1 cells with 1 μg/mL lipoteichoic acid (LTA) from Staphylococcus aureus or polyinosinic-polycytidylic acid (poly [I:C]) double-stranded RNA (InvivoGen, San Diego, Calif). After 6 or 12 h of antigen stimulation, LTA or poly (I:C) did not downregulate the expression of let-7a and miR-150 in THP-1 cells (Fig. 4, A–D). The results showed that TLR2 or TLR3 signaling pathways did not have a similar effect on let-7a and miR-150 in THP-1 cells.
Downregulated let-7a is correlated with induced expression of CIS protein without change in CIS mRNA levels in THP-1 cells by activating the TLR signaling pathway
MiR-150 was proven to be a biomarker of sepsis. These results are in line with previous reports that downregulation of miR-150 in both leukocytes and plasma had been identified in sepsis patients (10, 24); let-7a has not been well studied in this area. We sought to investigate let-7a–mediated posttranscriptional control in the fine-tuning of TLR signaling. Previous study had demonstrated that let-7 targets CIS 3′-UTR result in translational repression (20). The CIS and suppressors of cytokine signaling proteins are key physiological regulators of both innate and adaptive immunity (25). Forced expression of CIS could promote LPS-induced IκBα degradation and enhance NF-κB activity (20). The CIS mRNA levels were detected by real-time PCR analysis after 12 h of LPS stimulation (TaqMan probe assay ID for CIS, Hs00367082_g1). The CIS protein content was evaluated by Western blot after 24 h of LPS stimulation. Coupled with downregulation of let-7a after LPS stimulation, we found that LPS induces a significant increase of CIS protein without change in CIS mRNA levels in THP-1 cells (Fig. 5, A and B).
Let-7a plays a functional role in the suppression of LPS-induced TNF-α and IL-1β expression in THP-1 cells
To investigate whether the expression of let-7a was functionally involved in the production of cytokines by LPS, we overexpressed let-7a in THP-1 cells by pLV-miRNA lentiviral transduction system with let-7a or miR-control as control. Transduced THP-1 cells were stimulated with LPS (1 μg/mL) for 24 h, and the levels of TNF-α and IL-1β production were analyzed in the cell-free supernatants. Overexpressed let-7a significantly decreased TNF-α and IL-1β production in THP-1 cells in response to LPS (Fig. 6, A and B).
In this study, the expressions of two miRNAs, namely, let-7a and miR-150, were confirmed to be significantly downregulated in GNB urosepsis patients compared with those of healthy controls. Vasilescu et al. (24) proposed that miR-150 is a biomarker of sepsis. To the best of our knowledge, let-7a is the first identified biomarker of GNB sepsis. By using in vitro models with human monocytic cell lines, we demonstrated that LPS stimulation downregulated THP-1 cell expression of let-7a. The downregulation of let-7a is correlated with induced expression of CIS protein without a change in CIS mRNA levels in THP-1 cells via TLR signaling pathway activation. Moreover, gain of function by overexpression of let-7a revealed that let-7a significantly decreased TNF-α and IL-1β production in response to LPS. Let-7a may be relevant to the regulation of TLR-mediated innate immune response.
The miRNAs in body fluids are candidate diagnostics for a variety of conditions and diseases. Circulating miRNAs have been reported to be biomarkers for sepsis diagnosis and prognosis (24, 26–29). We studied peripheral leukocyte rather than circulating miRNA expression in urosepsis patients. Innate immune response constitutes the first line of defense against invading microbial pathogens and relies on phagocytes, such as granulocytes and macrophages. In mice, miRNA profiles from whole blood may be detectable at a very early stage after exposure to LPS, even as early as 2 h after treatment (9). The extracellular and cellular miRNA profiles are different. Expression of circulating miRNAs is considered to reflect the extrusion of miRNAs from relevant remote tissues or organs or disease processes (30). However, no studies show whether these observed dysregulated circulating miRNAs are the direct result of TLR4 signaling or events secondary to systemic TLR4 activation. Peripheral leukocyte miRNAs probably do not reflect miRNAs expressed in remote tissues. A previous study has demonstrated that approximately 30% of the released miRNAs in vitro and in vivo do not reflect the cellular profile (31).
Early diagnosis of GNB bacterial infection is critical for preventing further complications. The results of our study are from patients diagnosed as having GNB infection. We found reduced expression levels of let-7a and miR-150 in peripheral leukocytes in such cases. These potential biomarkers could have a crucial influence on the initial antimicrobial therapy. Innate immunity preserves host integrity by recognizing the pathogen-associated molecular patterns of invading microorganisms. Different immune responses may underlie various microorganisms. Signaling and sensing differ significantly in gram-negative and gram-positive bacteria. These findings are consistent with our results, which demonstrate that the expression profiles of miRNAs from GNB urosepsis patients were not similar to those of previous studies from a heterogeneous infected population (24, 26, 28, 29).
In this study, we identified two differentially expressed miRNAs, namely, let-7a and miR-150, by microarrays in our discovery set. MiR-150 had been proven to be a biomarker of sepsis. MiR-150 was suggested to regulate the differentiation and the activation state of immune cells by alteration of c-Myb signaling and expression levels of CXCR4, both representing direct targets of miR-150 (32, 33). A previous study has shown that decreased miR-150 expression was identified in peripheral blood leukocytes from sepsis patients, as detected by microarrays (24). Downregulation of miR-150 was observed in the leukocytes of human volunteers on treatment with LPS (10). Our results are in line with these previous studies. However, the present study is the first to identify let-7a as a biomarker of GNB sepsis.
Toll-like receptor/NF-κB signaling initiates a series of host cell defense reactions against pathogens. The activation of macrophages by LPS initiates negative feedback signals that aim to establish tolerance to a subsequent LPS stimulation. Positive feedback regulators are activated to encounter the negative regulator for a quick restoration of TLR/NF-κB pathway susceptibility. Fine-tuning of TLR/NF-κB signaling dynamics may involve both negative and positive feedback regulators, which function in concert, to ensure finely controlled immunity against microbial infection (16, 17). MicroRNAs have been found to be involved in the fine-tuning of TLR-mediated inflammatory response (23). In this study, we demonstrated that LPS stimulation downregulated THP-1 cell expression of let-7a. Hu et al. (20) reported that LPS stimulation decreased the expression of let-7 in a TLR4/MyD88-dependent manner, resulting in the relief of translational suppression of CIS in human cholangiocytes. The CIS promotes LPS-induced IκBα degradation and enhances NF-κB activity in cholangiocytes (20). Our results showed that LPS stimulation induces the upregulation of CIS protein without a change in CIS mRNA levels in THP-1 cells by activating the TLR signaling pathway. Moreover, gain of function by overexpression of let-7a revealed that let-7a significantly decreased TNF-α and IL-1β production in response to LPS. These data suggested that let-7a is relevant to the regulation of TLR-mediated innate immune response.
This study has several limitations. First, because the arrays were performed on total leukocytes, blood leukocyte counts and different leukocyte subpopulations will obviously affect greatly the outcome of gene array in each sample. In the validation phase, we used the same volume (5 ng) of extracted RNA for RT-PCR and normalized the values to the constitutive expression of the U6 snRNA to address the concern in blood cell counts. In addition, a retrospective analysis was undertaken to correct the presentation of the miRNA data for different leukocyte subpopulations. Briefly, the formula was as follows: (normalized miRNAs data)/(total percentages of polymorphonuclear neutrophils and monocytes) = final miRNAs expression ratio. The results and conclusions of our study did not change after correction. Second, no case of gram-positive bacterial, viral, or fungal infection was included in this study. The miRNA expression profile is unknown in such cases. Third, the sample size in the validation phase is limited. Some small sample bias may be present. Regardless, a statistically significant difference was found, which implied a large differential power.
In conclusion, reduced let-7a and miR-150 levels in peripheral leukocytes correlate with GNB sepsis. Let-7a is relevant to the regulation of TLR-mediated innate immune response.
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Keywords:© 2015 by the Shock Society
Hsa-let-7a; hsa-miR-150; microarrays; gram-negative bacteria; toll-like receptor