Aldose Reductase-deficiency Inhibits Endotoxin-induced Innate Immune Response In Macrophages By Autophagy

BackgroundAldose reductase (AR), a rate-limiting enzyme of the polyol pathway that reduces glucose intosorbitol in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH), hasemerged as the molecular target that mediates various infammatory diseases. Ravindranath et al.have demonstrated that transgenic mice over-expressing AR showed a greater inflammatoryresponse in cecal ligation and puncture model. In addition, AR inhibit ion suppresses infammationin several other models.It has been reported that AR regulates the infammatory signals via nuclear factor-κB (NF-κB)activation. NF-κB was considered as a key regulator of inducible gene expression in the immunesystem. The canonical NF-κB pathway is triggered by many inflammatory stimuli to induce theIKKβ that specifically phosphorylates the IκB proteins, thereby targeting them for ubiquitinationand proteolysis.Autophagy is an essential cell-intrinsic protection to starvation as well as a quality-control system that can deliver damaged organelles, long-lived proteins, misfolded proteins and invadingmicroorganisms from the cytoplasm to lysosomes for degradation. Macroautophagy is a process inwhich cells form double-membrane vesicles called autophagosomes around a portion of thecytoplasm. There is a growing body of evidence, suggesting that the specifcity factor for selectiveautophagy is determined by ubiquitination and binding of this ubiquitin signal by autophagicadaptor proteins. The protein p62, one of those adaptors, recognizes the polyubiquitinated targetsand binds the ubiquitin-like autophagosome membrane light-chain3(LC3) in the autophagicdegradation pathway, and functions as a scaffold protein for several signal transductions throughinteraction with various signaling proteins. Eventually, these autophagosomes fuse with lysosomes,resulting in the degradation of their contents.Accumulating data suggest that autophagy is not only an innate immune effector but also acrucial innate immune regulator. It has been reported that blocking autophagy by genetic deletionof the autophagy regulator Atg16L1enhances LPS-induced IL-1β production. Recently, Shi et al.have demonstrated that activation of autophagy by inflammatory signals limits IL-1β productionby targeting ubiquitinated inflammasomes for destruction. In addition, Baginska et al.demonstrated that hypoxia impairs breast cancer cell susceptibility to NK-mediated lysis in vitrovia Granzyme B degradation by the activation of autophagy. We found that LPS stimuli elicitedmore autophagosomes, but less iNOS in AR-deficient macrophages. These results prompted us toinvestigate the inherent connections among those things.ObjectiveThe present study was aimed to invetigate the mechanisms involved in AR and innateimmune responseMethods1) The RAW264.7murine macrophage cell line transfected with negative control siRNAor ARsiRNA andbone marrow-derived macrophages (BMDMs) from WT or AR KO micewere applied in this study.2) Using western blot to detect those important adaptors in the classical NF-κB pathway, suchas TLR4, IKK complex, IκBα,andp65.The result showed thatthe IKKβ andIKKγ proteinswere lower in LPS-induced AR-deficient cells (RAW264.7cell transfected with ARsiRNA or BMDMs from AR KO mice.3) Using qPCR to test the mRNA levels of IKKβ and IKKγ. The results showed that their mRNAexpressions were not impaired inLPS-induced AR-deficient cells.These data indicated thatIKK-complex proteins were decreased at the post-transcriptional level in LPS-inducedAR-deficient macrophages.4) Applying immunocytochemistry to examine LC3(Autophagosome marker) and thelocalization of LC3and LAMP1(Lysosome marker); Using western blot to examine the ratioof LC3II/LC3I and LAMP1; Employing transmission electron microscopy to observeautophagic vacuoles and autolysosomes.5) Using immunocytochemistry to detect the localization of IKK complex and LC3, and thelocalization of IKK complex and LAMP1.6) Using immunocytochemistry to detect the localization of IKK complex and ubiquitin, and thelocalization of IKK complex andp62.7) Employing western blot to detect the changes of LC3, IKKβ, IKKγ, and iNOS after blockingautophagy by3-MA.ResultsOur study has shown that AR knockout led to the suppression of innate immune response,which acted to limit inflammatory activity by a defect of the IKK complex. Knockout of ARtriggered autophagosome formation, which was initially independent of LPS stimulation. But theexposure of AR KO macrophages to stimuli that markedly enhanced autophagosome formationand maturation. Autophagy was able to capture and degrade the IKK complex via IKKβ and IKK ubiquitination, which led to the recruitment of p62and LC3. Autophagosome formation in thesetting of AR knockout was functionally important, pharmacological blockade of autophagyenhanced the production of iNOS and the level of the IKK complex. These data suggested anintimate relationship among AR-deficiency, innate immune response and autophagosomebiogenesis.