Silencing microRNA155 transforms the properties of endotoxin tolerant dendritic cells and enhance its therapeutic effect on sepsis murine model .

Sepsis refers to the body's dysfunctional response to infection and leads to life-threatening organ dysfunction. Dentritic cells (DCs), a kind of powerful functional antigen-presenting cell, plays a critical role in immune response and has been used in the field of immunotherapy for multiple diseases. Endotoxin tolerance is a phenomenon that believed to prevent the organism from producing persistent and excessive inflammatory reaction and numerous studies indicated that endotoxin tolerant dendritic cells (ETDCs) have therapeutic effects on experimental models of several diseases. MicroRNA155(miR155), which plays a pivotal role in various physiological and pathological processes in immunity, was reported associated with the formation of endotoxin tolerance. In this paper, we explored the change of properties after silencing miR155 in ETDCs then attempted to study its effects on the murine model of sepsis. Endotoxin tolerant dendritic cells were transfected with adenovirus silencing miR155 and negative control adenovirus, thereafter collected for reverse transcription polymerase chain reaction of miR155, costimulatory molecules analysis, allogeneic mixed lymphocyte reaction and cytokine concentration evaluation. 42 balb/c mice were randomly divided into control group, sham operation group, sepsis group, ETDCs treatment group and miR155 -/- ETDCs treatment group. Two treatment groups respectively received tail vein injection of ETDCs and miR155 -/- ETDCs 24h earlier than cacal ligation puncture. Our results revealed that silencing miR155 could deepen the immature status of ETDCs and both ETDCs and miR155 -/- ETDCs performed a protective effect in murine sepsis models while miR155 -/- ETDCs got a better protection. We reasoned that miR155 participates the formation of endotoxin tolerance and the protective effects of ETDCs and miR155 -/- ETDCs in the early stage of sepsis may achieved by altering the concentration of mouse cytokines to affect T cell differentiation and exert negative immune regulation.

Sepsis refers to a syndrome of physiologic, pathologic, and biochemical abnormalities induced by infection, leads to life-threatening organ dysfunction 1 .
Characterised by a dysfunctional host immune response comprising both proinflammatory and anti-inflammatory or immunosuppressing components, sepsis affects all types of immune cells and their compartments 2 . Despite a variety of clinical treatment measures, sepsis remains a major health problem worldwide, associated with high mortality rates in all countries 3 . Compelling evidence indicate that the derailment from immune homeostasis underlies sepsis-associated acute and long-term mortality 4,5 .
Therefore, there is an emergency to seek new strategies to target immune deficiency in sepsis to improve the prognosis. Considering sepsis involving in complex network of proinflammatory and anti-inflammatory cytokines 6 , specific targeting a single mediator maybe not a ideal choice. Recent years, adoptive cellular immunotherapy gradually has become a research hotspot.
Dentritic cells (DCs), a kind of powerful functional antigen-presenting cell, plays a critical role in mediating innate immune response and inducing adaptive immune response 7 , has been used in the field of immunotherapy for diseases such as autoimmune diseases, malignant tumors, and parasitic infections 8 .Endotoxin tolerance is a phenomenon that prior exposure of innate immune cells like monocytes/macrophages to minute amounts of endotoxin cause them to become refractory to subsequent endotoxin challenge 9 .The formation of endotoxin tolerance prevents the organism from producing persistent and excessive inflammatory reaction.
Our previous study indicated that the utilization of endotoxin tolerance could attenuate experimental acute liver failure 10,11 .Similarly, ample studies has proved endotoxin tolerant dendritic cells (ETDCs) have therapeutic effects on experimental models of several diseases [12][13][14] . Although endotoxin tolerance has attracted lots of attention these years, the biological machanism remains elusive.
MicroRNAs(miRNAs) is evolutionarily conserved non-coding small RNAs that range from 18 to 25 nucleotides (nt) in length 15 , participates in the fine-tuning of many fundamental biological processes 16 . As one of the best characterized miRNAs, microRNA155(miR155) plays a pivotal role in various physiological and pathological processes such as immunity, inflammation, viral infections, cancer, cardiovascular disease, hematopoietic lineage differentiation, and Down syndrome 17 . miR155's uniquely expressed in activated cells of the immune system while its deficiency can cause serious defects in immune function 18  In this study,we investigated the change of properties after silencing miR155 in ETDCs then attempted to explore its effects on the murine model of sepsis.

Animals
Male balb/c and c57bl/6 mice aged 6-8 weeks were purchased from the Shanghai Laboratory Animal Center (Shanghai,China) and housed under specific pathogen-free conditions in the Experimental Animal Laboratory of Wenzhou Medical University (Wenzhou, Zhejiang, China).All mice had free access to standard laboratory water and chow and were treated in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The research protocol was approved by the Animal Ethics Committee of Wenzhou Medical University.

BMDCs preparation and the induce of ETDCs
Bone marrow-derived DC cells (BMDCs) were isolated from femurs and tibias of male balb/c mice, resuspended in RPMI 1640 medium (Gibco, USA) containing 10% fetal bovine serum(Gibco) and 1% penicillinstreptomycin mixture, seeded in 6-well tissue culture plates and left to adhere for 8 h at 37°C in a humidified 5% CO2 atmosphere. Then BMDCs were cultured with RPMI 1640 medium supplemented with 10% FBS in the presence of 20ng/ml rmGM-CSF and 10ng/ml IL-4 (PeproTech, USA).
All of the culture supernatants were replaced with new medium mixed with cytokines in day2. We changed half medium and add cytokines every two days. BMDCs were plated in a 6-well plate and add LPS(100ng/ml) into culture supernatants to induce ETDCs at day4.

adenovirus transfection
According to different treatments, all cells were separated into five groups named imDCs,mDCs(DCs+large dose LPS), ETDCs(ETDCs+ large dose LPS), ETDCs-con(ETDCs+ negative adenovirus + large dose LPS), miR155 -/-ETDCs (miR155 -/-ETDCs + high dose LPS). At day5,the ETDCs were transfected with aden-miR155 -/and aden-con at an appropriate multiplicity of infection(MOI) for 6 hours in RPMI1640 with 10%FBS.Subsequently the culture supernatants containing virus suspension was replaced by new medium mixed with IL-4 and rmGM-CSF. After stimulated by large doses of LPS(10mg/ml) at day7 for 24h,all groups of cells were collected for subsequent experiments.

RNA isolation and qRT-PCR analysis
After endotoxin tolerance induction,total RNA was extracted from culture cells using TRIzol regent (Life technologies) according to the manufacturer's instructions.
Total microRNA was isolated from DCs using a miRcute miRNA isolation kit (TiangenBiotech,Beijing China) under manufatrer's guidance. Whereafter, we used a RevertAidFirstStand cDNA Kit(Thermo Fisher) to reverse-transcribe total RNA to cDNA. The cDNAs were subjected to realtime PCR analysis using a ABI7000 prism

Cytokine analysis
The cell supernatant of each group and serum of each group mice were collected. The concentrations of IL-10 and TNF-α of each group were examined using ELISA kit by manufactrer's instruction.

Flow cytometry analysis
The cells of five groups were harvested and stained with PE-, PEcy7-or APCconjugated anti-mouse MHC-II, CD86 and CD11c Abs. The spleen T lymphocytes (1×10 7 /ml)of each group mice were isolated after execution. 100μl cell suspension was stained with CD4,FITC and CD25,APC followed by incubation of Foxp3,PE, while another 100μl stained with CD4,PerCP-Cy5.5 and IL-17A, PE. All cells were fixed with 1% paraformaldehyde and analyzed by flow cytometry.

Histopathological analysis
After sacrificing all the mice 24 hours after the operation,the liver, kidney and ileal tissue of each group were taken and made into pathological section. Hematoxylin-eosin (HE) staining was performed to visualize the pathological changes.

Statistical analysis
All data are presented as means ± standard deviation (SD). One-way analysis of variance (ANOVA) and the least significant difference (LSD) test in SPSS19.0 software were used to analyze the statistical differences among multiple groups. A value of P less than 0.05 was considered statistically significant (P<0.05), each result was repeated at least three times.

successful transduction of aden-miR155 -/inhibited miR155 expression
Previous studies have confirmed that recombinant adenovirus is a efficient vehicle for DC gene transfer 20 . Adenovirus-miR155 -/and control adenovirus were transfected into ETDCs respectively, thereafter we observed the cells with inverted fluorescence microscope and photographed the fluorescent images, confirming that the transfection rate exceeded 85%. (Fig.1) The expression of miR155 in ETDCs transducted with aden-miR155 -/were significantly decreased, approved that the aden-miR155 -/inhibit the expression of miR155 successfully (Fig.2).

Discussion
Sepsis can lead to immune system dysfunction, and eventually result in multiple organ failure and septic shock 24,25 . Due to the specific mechanism of sepsis has not been fully elucidated, a effective treatment of sepsis is still lacking. It is believed that the activation of mass immune cells and the inflammatory factor storm formed by the massive secretion of cytokines play an important role in the early onset of sepsis 24 . DC, the only professional antigen presenting cell that can activate the initial T lymphocyte activation, not only play a pivotal role in regulating immune response or immune tolerance but also exhibit important application value in cell adoptive immunotherapy 8,26 . Predictably, the research of DC targeting immunomodulatory therapy is emerging as a new direction for the treatment of sepsis and hence has a wide prospect. Endotoxin tolerance, as a self-protective mechanism against the inflammatory response induced by lipopolysaccharides, can be utilized as a preventive treatment against endotoxemia 27 .Our previous studies confirmed that endotoxin tolerance manifested a protective role in mice with acute liver failure, which can improve survival rate and alleviate liver pathological injuries 10 .It is reported that ETDCs have a therapeutic effect on autoimmune cerebrospinal myelitis 12 and tracheitis 13 , but there is little research on its application in sepsis.
As the initiating factor of inflammatory response, the concentration of TNF-α in the blood of sepsis patients was remarkblely increased 28,29  DCreg can reduce pro-inflammatory cytokines such as TNF-α, negatively regulate the immune response, and alleviate the damages caused by excessive immunity.
In conclusion, according to our experimental results, we speculate that miR155 。 to negatively regulate immune response than ETDCs. The protective effects of ETDCs and miR155 -/-ETDCs in the early stage of sepsis may achieved by altering the concentration of mouse cytokines to affect T cell differentiation and exert negative immune regulation.The regulation of miR155 may provides a novel approach of adoptive immunotherapy for sepsis.

Ethics approval and consent to participate：
All protocols were authorized by the Animal Ethics Committee of Wenzhou Medical University.