Study On The Function And Mechanism Of Lnc-309 In Negatively Regulating The Activation Of Inflammatory Macrophages Induced By HTNV Infection

BackgroundHantaan virus（HTNV）is one of the most widely spread hemorrhagic fever viruses,which belongs to Bunyavirales and Hantaviridae.Apodemus agrarius,the main natural reservoirs,carry viruses without disease,while HTNV can cause hemorrhagic fever with renal syndrome（HFRS）in human beings.People are infected through inhaling of aerosols containing virus,ingesting contaminated food/water or contacting animal hair/excreta with virus particles.Research shows that HFRS is characterized by systemic inflammation and vascular leakage,and its pathogenesis is closely related to the dysfunction of intrinsic immune cells such as monocytes/macrophages.Our previously research showed that there were significant differences in the activation patterns between human and murine macrophages post HTNV infection.Macrophages from HFRS patients show persistent inflammatory activation（M1）,while murine macrophages inhibit M1 activation by expressing a series of species-specific long noncoding RNA（Lnc RNA）,such as Lnc-309.However,the specific molecular mechanism of Lnc-309 is still unclear.ObjectiveThe purpose of this study is to clarify the mechanism of murine-specific Lnc-309 in negatively regulating the activation of inflammatory macrophages post HTNV infection,and to preliminarily evaluate its protective effect on HTNV infection in vivo,so as to provide a potential target for constructing a new clinical treatment strategy for HFRS.Methods and Results1.Clarify the role of Lnc-309 in negatively regulating inflammatory macrophage activation post HTNV infection1.1 Sequence analysis and expression level of Lnc-309Based on RNA-seq and si RNA screening,our laboratory initially determined that murine-specific Lnc-309 had an anti-inflammatory effect of inhibiting TNF-αproduction.To further clarify the biological characteristics of Lnc-309 and its relationship with viral infection,we firstly performed systematic analysis of the non-coding characteristics and secondary structure of Lnc-309 using bioinformatics means such as LGC,UCSC and RNAfold.Lnc-309 does not contain a coding region and its sequence species are less conserved with a pocket-like structure for protein binding.Secondly,the expression levels of Lnc-309 in murine macrophages post viral infection（HTNV,vesicular stomatitis virus（VSV）and sendai virus（Se V））was detected by quantitative real-time PCR（qRT-PCR）.The results showed that HTNV infection could induce upregulation of Lnc-309 both in BMDMs and RAW264.7,and the trend of Lnc-309 expression was time dependent（48hpi,p<0.001）.Thirdly,the level of Lnc-309 was detected in organs by qRT-PCR,and the results suggested that Lnc-309 was upregulated in the heart,liver,spleen,lung and kidney of mice 3 dpi,with the most significant in the liver（p<0.0001）.1.2 Explore the function of Lnc-309 on HTNV replication in macrophagesUsing recombinant lentivirus,Lnc-309 was over-expressed in BMDMs（murine macrophages）and THP-1（PMA-induced differentiation,human macrophages）respectively.Then,HTNV was infected with MOI=1.Immunoblotting and qRT-PCR were used to detect HTNV replication at the protein and nucleic acid levels.The results hint that Lnc-309 over-expression promoted viral protein（NP）expression（p<0.05）and viral nucleic acid（S）replication（p<0.01）at 24 hpi.1.3 Clarify the role of Lnc-309 in negatively regulating the activation of inflammatory macrophages by inhibiting the production of various inflammatory factorsTo further clarify the function of Lnc-309 on macrophages,Lnc-309 was over-expressed in BMDMs and THP-1 using recombinant lentivirus,and HTNV was incubated with MOI=1.Flow cytometry,enzyme-linked immunosorbent assay（ELISA）and qRT-PCR were performed to assess the immune-related functions such as antigen presentation and inflammatory regulation.The results showed that Lnc-309 over-expression induced CD86and CX3CR1 expression and inhibited the expression of TNF-α,IL-6 and i NOS.To sum up,Lnc-309 mainly played a negative role in regulating the activation of inflammatory macrophages.2.The molecular mechanism of Lnc-309-RIPK3-STAT1 axis in negatively regulating inflammatory macrophage activation2.1 Exploring the effect of Lnc-309 on signaling pathways related to inflammatory macrophage activationLnc-309 was over-expressed in BMDMs and THP-1 respectively,and then infected with HTNV（MOI=1）.Immunoblotting was conducted to assess the activation of inflammation-related signaling pathways,such as Janus kinase-Signal transducers and activators of transcription（JAK/STAT）and nuclear factor kappa B（NF-κB）.The results showed that Lnc-309 significantly suppressed the phosphorylation of STAT1（p-STAT1ser727）,but not that of p65（p-p65 ser276/ser529）.To further clarify the mechanism by which Lnc-309 regulates STAT1 activation,we performed reciprocal analysis using RPIseq and predicted that Lnc-309 has low binding potential with STAT1（RF classifier score=0.5）.Subsequently,we over-expressed both STAT1-Flag and Lnc-309 in HEK293T.The RIP assay suggested that STAT1 did not enrich Lnc-309.In summary,Lnc-309 could block STAT1-mediated inflammatory macrophage activation post HTNV infection,which was independent on the direct interaction,and the mechanism needs to be further explored.2.2 RIPK3 directly bound and blocked STAT1-mediated activation of inflammatory macrophages after HTNV infectionRNA-Seq revealed that death-related signal pathways such as necroptosis were significantly activated post HTNV infection.Whereas HTNV infection did not cause cytopathic effect,suggesting that death-related molecules might exert a non-classical role.qRT-PCR and immunoblotting showed that RIPK3 increased significantly post HTNV infection,and its total protein expression was negatively correlated with the phosphorylation of STAT1.We isolated BMDMs from RIPK3^-/-mice,and infected with HTNV（MOI=1）.The results illustrated that the phosphorylation of STAT1 in RIPK3^-/-BMDMs was significantly increased（at 24/48 hpi）compared with WT.Consistently,RIPK3 over-expression could block the phosphorylation of STAT1 at 24/48 hpi.These results indicated that RIPK3 could inhibit inflammatory macrophage activation by blocking STAT1 signaling.In order to clarify the molecular mechanism of RIPK3 in regulating the phosphorylation of STAT1,we used confocal fluorescence microscope to examine the subcellular localization of the above molecules,it showed that RIPK3 and STAT1 co-localized and aggregated in a punctate manner during HTNV infection.Additionally,RIPK3over-expression could significantly hinder the nuclear translocation of STAT1 in the exogenous system.The results of co-immunoprecipitation（Co-IP）also confirmed that RIPK3 interacted with STAT1,and the interaction became tighter post HTNV infection.We concluded that RIPK3 could directly bind and inhibit STAT1 phosphorylation post HTNV infection.2.3 Lnc-309 directly bound RIPK3 and enforced its interaction with STAT1 to exert anti-inflammatory effectsRPIseq analysis showed that Lnc-309 had the potential to combine with RIPK3（RF classifier score=0.65）.RIPK3-Flag and Lnc-309 were over-expressed in HEK293T,and anti-Flag antibody was used for RIP.The results showed that RIPK3 could significantly enrich Lnc-309,suggesting an interaction between Lnc-309 and RIPK3.More importantly,their interaction strengthened under over-expressing Lnc-309.Lnc-309 might bind RIPK3to promote its interaction with STAT1 and suppress the activation of STAT1,which ultimately regulated the activation of inflammatory macrophages.3.Evaluation of the protective effect of Lnc-309 in vivoTo explore the protective effect of Lnc-309 in vivo,we firstly constructed pc DNA6.0-CMV-309 based on RNA delivery technology to over-express Lnc-309 in eukaryotic cells and validated the expression effect in HEK293T cells.Secondly,we injected 100μg of the plasmid through caudal vein every 48 h into the HTNV-infected nude mice（control group was injected with empty vector and experimental group was injected with over-expression plasmid）.qRT-PCR was performed to detect the expression effect of each organ,and the results showed that the expression of Lnc-309 in liver,lung and kidney of the experimental nude mice was elevated compared with the control group,suggesting successful in vivo overexpression.Thirdly,in the in vivo protection assay,the overall index changes such as body temperature and body weight of mice were monitored to assess the overall efficacy of Lnc-309.The results showed that there was no statistical difference in body temperature and body weight in the experimental group,but the survival time was significantly longer compared with the control group.Finally,HE staining and qRT-PCR was performed to analyze the immunoinflammatory damage of organs.The results suggested that the expression of various inflammatory cytokines,including TNF-αand IL-6,was significantly decreased,while the expression of IL-10 was up-regulated in the lungs of the experimental mice.The above findings revealed that Lnc-309 exerted a protective effect by inhibiting the inflammatory pathological damage caused by HTNV infection.ConclusionHTNV-infected macrophages could induce the up-regulation of murine-specific Lnc-309.Lnc-309 bound RIPK3 to promote its interaction with STAT1 and blocked STAT1-mediated activation of inflammatory macrophages,which played an important anti-inflammatory protective role during HTNV infection.