Effect Of Ferumoxytol On The Immunosuppressive Status Of The Late Sepsis By Regulating MDSCs

Myeloid-derived suppressor cells(MDSCs)are a heterogeneous population of immature myeloid cells with a potent immune suppressive capacity.Most knowledge of MDSCs stems from cancer studies,in recent years,an expansion of MDSCs has also been described in acute and chronic inflammatory diseases including inflammation,burns and autoimmune diseases.MDSCs consist of two large groups of cells:granulocytic MDSCs(G-MDSCs)and monocytic MDSCs(M-MDSCs),both populations have immune suppressive functions through the production of iNOS,ROS and arginase 1.Sepsis,a dysregulated host inflammatory response to severe infection with the presence of life-threatening organ dysfunction,is the most common cause of death in the intensive care unit.Sepsis initiates an overwhelming pro-inflammatory response,and patients with late sepsis will quickly exhibit a long-term immunosuppressive state.The expansion of MDSCs has been described in the spleens and lymph nodes from septic animal models,as well as in patients with sepsis.Increased numbers of MDSCs correlate with suppression of lymphocyte proliferation,it has been reported that MDSCs acquire their phenotype in the bone marrow and then migrate to secondary lymphoid organs to inhibit T cells responses and suppress their proliferation in LPS-immunosuppressed mice.Activation of immunosuppressive cells such as MDSCs is critical for appropriate control of hyper-responsiveness of the innate immune system,but their excess expansion may lead to hyper-immunosuppressive that is the predominant driving force for secondary infection and mortality in late sepsis.Most patients with protracted sepsis exhibit an immunosuppressive status and most sepsis mortality is due to late-sepsis immunosuppression.Therefore,how to improve the immune status of late sepsis has become an urgent problem to be solved.So eliminating MDSCs may have beneficial effects in late sepsis by restoring the immune response.Ferumoxytol,FDA-approved iron supplement,has been widely used as drug carriers and contrast agents in MRI scanning.It has been showed that ferumoxytol has immune-modulatory properties such as induced a phenotypic shift in M2 macrophages towards a high CD86+,TNFa positive M1 macrophages subtype.Previous studies of ferumoxytol have focused on their effects on macrophages in cancer,however,the effects of ferumoxytol on other immune cells have not been examined.Therefore,this study analyzed the effect of ferumoxytol on the quantity and functions of MDSCs,and discussed the mechanism of ferumoxytol through MDSCs to improve the symptoms of LPS-induced late sepsis in mice.We find that ferumoxytol decrease the MDSCs population and attenuate their immunosuppressive function to ameliorate LPS-induced the later stages of sepsis in mice.Detailed results are as follows:1、Ferumoxytol attenuate the immunosuppressive function of MDSCs via down regulation expression of Arg-1 and ROS.It has been found that ferumoxytol inhibited cancer growth by inducing a pro-inflammatory immune response with M1 macrophages polarization,indicating that ferumoxytol has immune-modulatory functions.In this study,we first assessed the phagocytosis of MDSCs on ferumoxytol using MACS;to determine if ferumoxytol would alter the functions of MDSCs,we obtained BM-derived MDSCs and audited their transcriptomes for expression of ferumoxytol-treated MDSCs versus untreated controls mRNA via RT-PCR and evaluated ROS levels and compared the change in subpopulations of MDSCs among ferumoxytol-treated and no treated;to assess the effect of ferumoxytol on MDSCs differentiation,we treated cells with ferumoxytol on day 0 and performed flow cytometry staining for macrophage-associated maker CD11b and F4/80 on days 1,3 and 5.Our study showed that MDSCs are able to take up magnetic nanoparticles-ferumoxytol,and our data further demonstrated that ferumoxytol attenuate the immunosuppressive function of MDSCs via down-regulation of Arg-1 and ROS,and decreased the percentage of G-MDSCs but increased the percentage of M-MDSCs;Our data also indicated that ferumoxytol has a direct effect of MDSCs differentiation,in vitro experiments,about fifth cells differentiated evenly into macrophages at 5d.Although we did not investigate the phenotype of macrophages exposed to ferumoxytol,we hypothesized that they were pro-inflammatory M1 subtype.2、Ferumoxytol alleviate the symptoms of late sepsis in mice by reducing the percentages and attenuate the functions of MDSCs to restore the amount of T cells.It had previously shown MDSCs expand massively in the bone marrow,spleens and lymph nodes of mice with ongoing septic processes and contribute to sepsis-induced T cells suppression;additionally,previous study reported that M-MDSCs and G-MDSCs strongly contribute to T cells dysfunction in patients with sepsis.In ferumoxytol an iron core is wrapped in a carbohydrate shell,which leads to low toxicity and lysosomal uptake and degradation.It has been reported that ferumoxytol does not cause liver toxicity in patients or animal models and is typically metabolized within two months.To evaluate the effect of ferumoxytol on the late sepsis in mice,we performed in vivo assay using a murine sepsis model,AST and ALT levels in serum of mice were measured,and livers and lungs were taken for H&E staining;current models of sepsis associate adverse outcomes with elevated production of inflammatory cytokines,so we assessed changes in the serum levels of pro-inflammatory cytokines;to determine whether ferumoxytol would modulate MDSCs populations in bone morrow and spleens of mice,we measured the proportions of MDSCs in bone marrow and spleens and T cells in the spleens;to further confirm the in vivo role that MDSCs play in suppressing T cells proliferation,MDSCs that isolated from spleens of mice were co-cultured with T cells.A histopathological examination indicated that ferumoxytol ameliorated alveolar wall thickening and decreased areas of necrosis in the livers;our data also showed and ferumoxytol does not increase the production of inflammatory factors in early sepsis,suggesting that it will not negatively affect the occurrence of sepsis;moreover,our data demonstrated that ferumoxytol significantly decreased the percentages of MDSCs and attenuate functions of MDSCs to restore the amount of T cells in the late sepsis of mice.In conclusion,this study demonstrates a novel immune-modulatory property of ferumoxytol,which ferumoxytol attenuate the immunosuppressive function of MDSCs via down-regulation of Arg-1 and ROS;furthermore,we provide an attractive therapeutic approach for the treatment of sepsis immunosuppression,targeting MDSCs may provide a promise for restoring the immune response during sepsis.