Cloning, Expression and Characterization of a Peptibody To Deplete Myeloid Derived Suppressor Cells in a Murine Mammary Carcinoma Model

Myeloid derived suppressor cells (MDSCs) are an immature heterogeneous population of myeloid lineage that attenuate the anti-tumor immune responses. Depletion of MDSCs has been shown to improve efficacy of cancer immunotherapeutic approaches. Here, we produced and characterized a recombinant peptibody capable of recognizing and depleting murine MDSCs. Using SOE-PCR, the coding sequence of the MDSC binding peptide and linker were synthesized and then ligated into a home-made expression plasmid containing mouse IgG2a Fc. The peptibody construct was transfected into CHO-K1 cells by lipofectamine 3000 reagent and the resulting fusion protein was purified with protein G column and subsequently characterized by ELISA, SDS-PAGE and immunoblotting. The binding profile of the peptibody to splenic MDSCs and its MDSC depletion ability were then tested by flow cytometry. The purified peptibody appeared as a 70 kDa band in Western blot. It could bind to 98.8% of splenic CD11b+/Gr-1+ MDSCs. In addition, the intratumoral MDSCs were significantly depleted after peptibody treatment compared to their PBS-treated negative control counterparts (P <0.05). In this study, a peptibody capable of depleting intratumoral MDSCs, was produced. Our results imply that it could be considered as a potential drug effective for immunotherapy of cancers.

Changes in Functional Glucocorticoid Sensitivity of Isolated Splenocytes Induced by Chronic Psychosocial Stress – A Time Course Study

Chronic psychosocial stress is a risk factor for the development of numerous disorders, of which most are associated with chronic low-grade inflammation. Given the immunosuppressive effects of glucocorticoids (GC), one underlying mechanism might be the development of stress-induced GC resistance in certain immune cell subpopulations. In line with this hypothesis, male mice exposed to the chronic subordinate colony housing (CSC, 19 days) model develop GC resistance of in vitro lipopolysaccharide (LPS)-stimulated splenocytes, splenomegaly and an increased percentage of splenic CD11b+ cells. Here male C57BL/6N mice were euthanized at different days during CSC, and following 30 days of single housing after stressor termination to assess when CSC-induced splenic GC resistance starts to develop and whether this is a transient effect. Moreover, splenic CD11b, GC receptor (GR) and/or macrophage migration inhibiting factor (MIF) protein levels were quantified at respective days. While mild forms of CSC-induced GC resistance, increased splenic CD11b expression and/or splenomegaly were detectable on days 8 and 9 of CSC, more severe forms took until days 15 and 16 to develop, but normalized almost completely within 30 days following stressor termination (day 51). In contrast, splenic GR expression was decreased in CSC versus single-housed control (SHC) mice at all days assessed. While MIF expression was increased on days 15 and 16 of CSC, it was decreased in CSC versus SHC mice on day 20 despite persisting splenomegaly, increased CD11b expression and functional GC resistance. In summary, our data indicate that GC resistance and CD11b+ cell-mediated splenomegaly develop gradually and in parallel over time during CSC exposure and are transient in nature. Moreover, while we can exclude that CSC-induced reduction in splenic GR expression is sufficient to induce functional GC resistance, the role of MIF in CD11b+ cell-mediated splenomegaly and GC resistance requires further investigation.

Role of spleen-derived CD11b+Gr-1+ cells in sepsis-induced acute kidney injury

Purpose: CD11b+Gr-1+ cells play a key role in inflammation and the purpose of this study was to determine whether splenic CD11b+Gr-1+ cells are mobilized to the kidney and lead to acute kidney injury during sepsis. Methods: The sepsis model was generated via cecum ligation and puncture (CLP). The mice were randomly distributed into control, sham operated, CLP and CLP+splenectomy (CLPS) groups (n=5–10/group). The percentage of CD11b+Gr-1+ cells in circulating, bone marrow and spleen were determined. Plasma concentrations of interleukin-6, interleukin-1β, creatinine (Cr) and neutrophil gelatinase-associated lipocalin were measured. CD11b+Gr-1+ cells were detected by immunofluorescence and qRT-PCR. Hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) were performed. Expression of mammalian target of rapamycin (mTOR), hypoxia-inducible factor-1α (HIF-1α) and cleaved caspase-3 was measured. Results: The percentage of CD11b+Gr-1+ cells in blood was significantly higher in the CLP group and lower in CLPS group. CD11b+Gr-1+ cells in the spleen were significantly lower in the CLP group. In the CLP group, the plasma concentrations of interleukin-6, interleukin-1β, Cr and neutrophil gelatinaseassociated lipocalin were higher. The expression of Gr-1 and CD11b were higher in CLP. The CD11b+Gr-1+ cells were detected in the kidneys of the CLP group. HE, PAS and TUNEL showed inflammatory cell infiltration and cell apoptosis in CLP. Western blot indicated dephosphorylation of mTOR, down-expression of HIF-1α and increased expression of cleaved caspase-3 in sepsis kidney. Conclusion: Splenic CD11b+Gr-1+ cells migrated to the kidney in sepsis, which led to acute kidney injury via the inhibition of mTOR/HIF-1α.

Ferumoxytol Attenuates the Function of MDSCs to Ameliorate LPS-Induced Immunosuppression in Sepsis

Sepsis-induced immunosuppression is recognized as one of the main features responsible for therapeutic failures. Myeloid-derived suppressor cells (MDSCs), which are mainly characterized by their suppressive properties, have been reported to be expanded in sepsis. Ferumoxytol (FMT), an FDA-approved iron supplement, has been shown to possess immune-modulatory properties in tumors. However, it is unclear whether FMT alters the functions of MDSCs to reduce late-sepsis immunosuppression. Here, we showed an immunomodulatory effect of FMT on MDSCs to ameliorate lipopolysaccharide (LPS)–induced immunosuppression in the late stage of sepsis. Separation of cells with internalized FMT and detection of the intracellular iron content showed that MDSCs could uptake FMT. Low doses of FMT had no effects on the cell viability of MDSCs, but FMT inhibited the expansion of MDSCs in vitro. Moreover, FMT significantly downregulated the expression levels of Arg-1, S100A8, S100A9, and p47phox as well as ROS production in MDSCs. FMT decreased the percentage of granulocytic MDSCs (G-MDSCs) and promoted the differentiation of MDSCs into macrophages. Furthermore, FMT reduced white blood cell recruitment and alveolar wall thickening in the lungs and areas of necrosis in the liver as well as some biochemical markers of liver dysfunction. FMT decreased the percentage of G-MDSCs and monocytic MDSCs (M-MDSCs) in the spleens of LPS-induced septic mice. Of note, FMT reduced the T cell immunosuppressive functions of both G-MDSCs and M-MDSCs. Expectedly, FMT also significantly reduced Arg-1 and p47phox gene expression in splenic CD11b+Gr-1+ cells isolated from LPS-challenged mice. These data indicate that FMT decreased the immunosuppressive functions of MDSCs by decreasing Arg-1 and ROS production, suggesting that FMT may reduce long-term immunosuppression in the late stage of sepsis.