Study On The Effect And Mechanism Of Cell-free DNA Scavengers In Sepsis

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection.It is a major public health concern due to its high mortality rate and lack of effective treatment drugs.Excessive activation of Toll-like receptors?TLRs?can initiate and drive inflammatory dysregulation.TLRs recognize pathogen-associated molecular patterns?PAMPs?or damage-associated molecular patterns?DAMPs?.Among them,cell-free DNAs?cfDNA?,have been demonstrated to not only represent prognostic,predictive biomarkers of sepsis,but also contribute to magnitude and duration of inflammatory response through TLR activation in immune cells.In this context,neutralization of cfDNA may modulate the overwhelming immune response and ameliorate organ injury caused by severe sepsis.Therefore,it is necessary to develop a strategy and material that can efficiently and safely combine and remove cfDNA.Positive polymers such as 3rd generation polyamidoamine dendrimer?PAMAM-G3?and polyethyleneimine?PEI?are widely used in gene delivery due to their charge interaction with nucleic acids.This kind of nucleic acid binding polymer?NABPs?is expected to be used as a scavenger of cfDNA for the regulation of inflammatory diseases.At present,it has been proved that PAMAM-G3,a classic NABP,could prevent TLR activation in the target immune cells through exogenous scavenging of pro-inflammatory nucleic acids and nucleic acid-protein complexes.It is reported that nucleic acid-binding nanoparticles?NABNs?may produce better scavenging effects compared with soluble NABPs.Here,we first examined the effect of PAMAM-G3 on both the cecal ligation and puncture?CLP?-induced severe sepsis model and the CpG-induced SIRS model.Then we synthesized two biodegradable mesoporous silica nanoparticles functionalized with polyethylenimine?MSN-PEI?with controllable charge density.We compared the scavenging capabilities and anti-inflammatory effects between PEI and MSN-PEI with different charge densities in vitro and in vivo.The dissertation consists of two parts.The first part:to investigate the role of scavenging cfDNA strategy in sepsis.?1?Anti-inflammatory effect of PAMAM-G3 in vitroWe first investigated the binding affinity of PAMAM-G3 with DNA and anti-inflammatory effect in vitro.The results showed that PAMAM-G3 exhibited high binding affinity with calf thymus DNA,and formed stable scavenger-DNA complexes.The serum cfDNA level in septic patients was significantly higher than that in healthy volunteers.PAMAM-G3 not only inhibited CpG ODN and septic serum-mediated TLR9 activation in engineered HEK-TLR9 reporter cells,but also reduced the pro-inflammatory cytokine tumor necrosis factor-??TNF-??secreted by RAW264.7macrophages.These findings indicate that the cfDNA in septic serum induced TLR9activation and pro-inflammatory cytokine secretion,while this pathological phenotype could be abrogated by PAMAM-G3.?2?Therapeutic effect of PAMAM-G3 in CpG ODN-induced fatal SIRS modelIn order to further verify the anti-inflammatory effect of PAMAM-G3 on DNA-mediated inflammation,we established the CpG ODN-induced fatal SIRS model.PAMMA-G3?20 mg/kg,i.p.?,XBJ?4 mL/kg?were intraperitoneally injected30 min after CpG administration.Mice were monitored for survival rate as described above,and serum was collected 8 hours after CpG ODN injection for analysis of TNF-?and IL-6 via ELISA.TLR9⁺peritoneal cells were analyzed.The results showed that all the mice died within 48 hours after CpG ODN injection,while PAMAM-G3 remarkably reduced mortality by 90%.In addition to the improvement on survival,we also found significant reduction in other inflammatory markers,such as fewer TLR9⁺peritoneal cells and reduced serum TNF-?and IL-6 level.These findings clearly verified that the protective effect of PAMAM-G3 and its downstream anti-inflammatory responses in CpG ODN-induced SIRS model were from cfDNA scavenging.?3?Therapeutic effect of PAMAM-G3 in CLP-induced sepsis modelWe developed the CLP septic model with different grades by varying the distance of cecum ligation in wide-type BALB/c mice.The highest clinical score and the highest serum and peritoneal fluid?PLF?cfDNA level were designated as the severe grade.This severe model was chosen to demonstrate the protective potential of PAMAM-G3.PAMAM-G3?20 mg/kg,i.p.?,XBJ?4 mL/kg?were administered 12 hours prior to CLP and 1 and 12 hours after CLP.Mice were monitored for survival rate and clinical score.24 hours after CLP,serum and peritoneal fluids were collected for analysis of cfDNA,TNF-?,IL-6,MCP-1 level,and tissue enzyme level.The lung,kidney,liver,spleen,and heart were collected and fixed with 4%paraformaldehyde for histopathological examination.The results showed that all CLP-induced animals without any treatments died within 72 hours.In contrast,repetitive i.p.administration of PAMAM-G3 produced notable protection against the lethal condition,which triggered death starting at 24 hours post CLP-induction.The survival prolongation conferred by PAMAM-G3 corresponded with a significant reduction in the cfDNA level and pro-inflammatory cytokines in serum and PLF.In addition,PAMAM-G3ameliorated lung,kidney,liver and heart tissue injury.Consistently,biochemical analyses confirmed the protective effects of PAMAM-G3 in preventing the failure of these organs.Importantly,PAMAM-G3 exhibited an efficacy similar to that provided by XBJ.These data suggest that PAMAM-G3 is able to reduce septic death and ameliorate multiple organ injury in a clinically relevant severe sepsis model.?4?Investigating the mechanism of PAMAM-G3 in CLP-induced sepsis model8 hours after CLP,peritoneal fluids were collected to determine the number of TLR9⁺cells and macrophages and peritoneal macrophages were isolated.NF-?B p65?phospho S536?,NF-?B p65,MyD88 and TLR9 were detected via western blot analysis.TNF-?,iNOS and Arg-1 mRNA were detected via quantitative real-time PCR assay.The results showed that the amount of TLR9⁺peritoneal cells in the PAMAM-G3-treated septic mice were remarkably lower than that in the untreated CLP mice.We identified that a higher portion of peritoneal macrophages were CD11c⁺?M1-polarized macrophages?in the septic mice,while the PAMAM-G3treatment reduced the population of those cells.In addition,the M1-polarized markers,TNF-?and iNOS,which were upregulated in CLP-challenged peritoneal macrophages,were significantly repressed by PAMAM-G3,and expression of the M2-polarized marker,Arg-1,was partly restored.The expression of TLR9 and MyD88 were dramatically upregulated in the peritoneal macrophages of model mice,and the level of phosphorylated p65 increased substantially as well.In contrast,treatment with PAMAM-G3 significantly limited the activation of TLR9-MyD88-NF-?B signaling pathway.These data supported our hypothesis and suggested that PAMAM-G3reversed the M1 polarization of peritoneal macrophages through TLR9-MyD88-NF-?B signaling pathway during severe sepsis progression.The second part:to investigate the therapeutic potential of MSN-PEI in sepsis.?1?Synthesis and characterization of MSN-PEIWe synthesized MSN as a model for developing NABNs.Its GSH responsive degradation capability also motivated us to select this biomaterial.To evaluate the surface charge density effect,we designed three NABNs with different charge densities through functionalizing the MSNs with or without PEIs?Mn 25 KDa or 800Da?.MSN-PEI 25K and MSN-PEI 800 have similar size,zeta potential,and degradable behavior,and the former held a stronger charge density than the latter one.Additionally,the cationic MSNs?MSN-NH₂?with the lowest charge density was used for further comparison.?2?Anti-inflammatory effect of MSN-PEIWe investigated the DNA binding ability and anti-inflammatory effect of MSN-PEI in vitro.The results showed that both types of MSN-PEI,but not MSN-NH₂,complexed with cfDNA in a dose-depending manner and formed stable scavenger-DNA complexes.All NABNs and NABPs significantly inhibited both CpG ODN and sepsis serum-induced TLR9 activation under the same dosage.MSN-PEI25K exhibited comparable TLR9 inhibition with PEI 25K on a mass-based comparison although the MSN-PEI 25K has a lower N content.Among all the nanoparticles,the NABN with the higher charge density was more efficient in blocking the TLR9 activation.We saw similar trends in TNF-?secretion from those danger signals-activated macrophages.As a result,we could confirm that charge density played an important role in NABN-mediated cfDNA scavenging and inflammation inhibition.?3?Therapeutic effect of MSN-PEI in CLP-induced sepsis modelMSN-PEI?20 mg/kg,i.p.?,XBJ?4 mL/kg?were administered 12 hours prior to CLP and 1 and 12 hours after CLP.Mice were monitored for survival rate and clinical score.24 hours after CLP,serum and peritoneal fluids were collected for analysis of cfDNA,TNF-?,IL-6,MCP-1 level,and tissue enzyme level.The lung,kidney,liver,spleen,and heart were collected and fixed with 4%paraformaldehyde for histopathological examination.8 hours after CLP,peritoneal fluids were collected to determine the number of TLR9⁺cells.The results showed that both NABNs conferred protection against CLP-induced death,which was consistent with transient clinical scores from the 24 to 48 hours'time points.The MSN-PEI 25K group outperformed the MSN-PEI 800 group in agreement with the in vitro results.Neither PEI 25K nor PEI 800,the soluble cationic counterparts,could improve the survival of CLP-induced mice compared with the control group.Consistent with the decreased mortality,the serum and peritoneal cfDNA level in the MSN-PEI 25K-treated mice were significantly lower than those in other groups.Similarly,MSN-PEI 25K administration significantly reduced CLP-induced TNF-?,IL-6 and MCP-1 release in both serum and the peritoneal fluid while the serum cfDNA and pro-inflammatory level of the mice treated with its counterpart?PEI-25K?were somewhat higher,suggesting toxicity,although the peritoneal cfDNA level was decreased.MSN-PEI25K significantly attenuated the multiple organ injury.Consistent with these changes in organ histology,MSN-PEI 25K markedly reversed the increased serum tissue enzyme level.Similarly,the PEI 25K group held comparable score for each organ,and significantly increased the level of serum biochemical indicators.These in vivo data agreed with the in vitro results that NABNs were better than NABPs and the NABN with the higher charge density was more effective.?4?The biodistribution of MSN-PEI in CLP-induced sepsis modelTo check the biodistribution of MSN-PEI,at 12 hours prior to CLP and 1 and 12hours post-CLP procedure,NABNs?20 mg/kg?were injected into the mice?i.p.?.24hours after surgery,the main organs?intestines,spleen,heart,liver,lung and kidney?and blood were disrupted with concentrated nitric acid.The concentrations of Si ion in the solutions were determined by ICP-OES.We next studied the biodistribution of the Cy7-labeled NABNs and NABPs in abdominal organs through ex vivo near-infrared fluorescence?NIRF?imaging.At 1 hour post-surgery,the cationic materials were intraperitoneally injected into the mice at a dose of 20 mg/kg.At 2,12and 24 hours post-surgery,mice were sacrificed and the images of the heart,lung,liver,spleen,kidney and intestines were collected using an NIR Imaging System.In the sham mice,the MSN-PEI were mainly observed in the liver,with little difference of retention in the cecum.However,in septic mice,both MSN-PEI preferentially localized in the cecum,with NABNs showing longer retention in the colonic tissue than NABPs.MSN-PEI 25K exhibited higher retention in the colonic tissue than MSN-PEI 800.Correspondingly,its counterpart PEI-25K had much higher accumulation in the liver and kidney.This differential accumulation was confirmed by quantitative measurement of Si in various tissues,reinforcing the NIRF imaging results that higher charge density correlated with higher accumulation in the inflamed site.?5?The safety of MSN-PEIWe used MTT method to detect the cytotoxicity of RAW264.7 cells and HUVEC cells.For the subsequent repeated-dose toxicity study,mice were i.p.injected with different MSN-PEI doses at 0,13 and 24 hours.The mouse mortality was recorded for14 days.Then,the mice treated with a 20 mg/kg dose were sacrificed at 24 hours and7 days after the last injection.The blood samples were collected at 24 hours,and the tissue enzyme level were measured.The number of each type of blood cell?RBCs,WBCs and PLTs?was quantified.The serum TNF-?,IL-6 and MCP-1 level were measured.The heart,liver,spleen,kidney and lung tissues were collected at 24 hours and on the 7th day after the last injection and fixed with 4%paraformaldehyde.The results show that MSN-PEI 25K reduced toxicity compared with the soluble PEI 25K in macrophages,and as expected,the one with higher charge density was more toxic in both RAW 264.7 and HUVEC cells.In line with the repetitive administration procedure,we evaluated the acute toxicity of these scavengers with thrice-repeated dosing within 24 hours.All the PEI-treated mice?both 25K and 800?died at the dose of 40 mg/kg.In contrast,all the mice in both MSN-PEI 25K and MSN-PEI 800groups behaved normally under the same dosage,showing no mortality at 320 mg/kg but suffering a 50%and 20%mortality,respectively,at the dose of 640 mg/kg.We further probed the possible mechanism of NABP-induced toxicity and observed that PEI 25K increased the level of biochemical parameters in comparison with those of the control group.Correspondingly,markedly higher level of cfDNA and pro-inflammatory cytokines,abnormal number of blood cells,and damaged tissue of liver,kidney and lung in the PEI 25K group were also observed,implying that the toxicity originated from the systematic proinflammatory response,resulting in thrombosis.Importantly,both MSN-PEI 25K and MSN-PEI 800 did not significantly change the level of any serum biochemical indicators,blood cell number or pro-inflammatory cytokine level,consistent with negligible pathological changes in the major organs described earlier.To sum up,this paper draws the following conclusions:?1?We have revealed a role of cfDNA in stimulating the TLR9-mediated proinflammatory response and provided a new target for sepsis management.?2?Scavenging cfDNA strategy is an effective treatment strategy in ameliorating systemic inflammation in severe sepsis.?3?The NABNs MSN-PEI 25K showed better therapeutic efficacy in vivo.