The contribution of p53 to kidney dysfunction, inflammation, and tubular cell death, hallmark features of ischemic renal injury (IRI), remains undefined. Here, we studied the role of proximal tubule cell (PTC)-specific p53 activation on the short-and long-term consequences of renal ischemia/reperfusion injury in mice. After IRI, mice with PTC-specific deletion of p53 (p53 knockout [KO]) had diminished whole-kidney expression levels of p53 and its target genes, improved renal function, which was shown by decreased plasma levels of creatinine and BUN, and attenuated renal histologic damage, oxidative stress, and infiltration of neutrophils and macrophages compared with wild-type mice. Notably, necrotic cell death was attenuated in p53 KO ischemic kidneys as well as oxidant-injured p53-deficient primary PTCs and pifithrin-a-treated PTC lines. Reduced oxidative stress and diminished expression of PARP1 and Bax in p53 KO ischemic kidneys may account for the decreased necrosis. Apoptosis and expression of proapoptotic p53 targets, including Bid and Siva, were also significantly reduced, and cell cycle arrest at the G2/M phase was attenuated in p53 KO ischemic kidneys. Furthermore, IRI-induced activation of TGF-b and the long-term development of inflammation and interstitial fibrosis were significantly reduced in p53 KO mice. In conclusion, specific deletion of p53 in the PTC protects kidneys from functional and histologic deterioration after IRI by decreasing necrosis, apoptosis, and inflammation and modulates the long-term sequelae of IRI by preventing interstitial fibrogenesis.
Female mice transgenic for the rat proto-oncogene c-erb-B2, under control of the mouse mammary tumor virus (MMTV) promoter (neuN), spontaneously develop metastatic mammary carcinomas. The development of these mammary tumors is associated with increased number of GR-1(+)CD11b(+) myeloid derived suppressor cells (MDSCs) in the peripheral blood (PB), spleen and tumor. We report a complex relationship between tumor growth, MDSCs and immune regulatory molecules in non-mutated neu transgenic mice on a FVB background (FVB-neuN). The first and second tumors in FVB-neuN mice develop at a median of 265 (147-579) and 329 (161-523) days, respectively, resulting in a median survival time (MST) of 432 (201 to >500) days. During tumor growth, significantly increased number of MDSCs is observed in the PB and spleen, as well as, in infiltrating the mammary tumors. Our results demonstrate a direct correlation between tumor size and the number of MDSCs infiltrating the tumor and an inverse relationship between the frequency of CD4(+) T-cells and MDSCs in the spleen. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assessment of enzyme and cytokine transcript levels in the spleen, tumor, tumor-infiltrating non-parenchymal cells (NPCs) and mammary glands revealed a significant increase in transcript levels from grossly normal mammary glands and tumor-infiltrating NPCs during tumor progression. Tumor NPCs, as compared to spleen cells from wild-type (w/t) mice, expressed significantly higher levels of arginase-1 (ARG-1), nitric oxide synthase (NOS-2), vascular endothelial growth factor (VEGF-A) and significantly lower levels of interferon (IFN)-gamma, interleukin (IL)-2 and fms-like tyrosine kinase-3 ligand (Flt3L) transcript levels. Transcript levels in the spleens of tumor-bearing (TB) mice also differed from normal mice, although to a lesser extent than transcript levels from tumor-infiltrating NPCs. Furthermore, both spleen cells and NPCs from TB mice, but not control mice, suppressed alloantigen responses by syngeneic control spleen cells. Correlative studies revealed that the number of MDSCs in the spleen was directly associated with granulocyte colony stimulating factor (G-CSF) transcript levels in the spleen; while the number of MDSCs in the tumors was directly correlated with splenic granulocyte macrophage stimulating factor (GM-CSF) transcript levels, tumor volume and tumor cell number. Together our results support a role for MDSCs in tumor initiation and progressive, T-cell depression and loss of function provide evidence which support multiple mechanisms of MDSC expansion in a site-dependent manner.
Among the prerequisites to be effective, immunotherapy must work in the presence of the host immunosuppression. Myeloid-derived suppressor cells (MDSCs) are one of the cellular mediators that have a role in limiting the induction of adaptive immune response and host responses to immunotherapy. MDSCs are an immature, heterogeneous cellular population, including hematopoietic precursors for granulocytes, macrophages, dendritic cells, and endothelial cells. They are infrequent in healthy hosts, but increase during tumor progression as a result of dysregulated myeloid cell expansion and differentiation. Tumor growth is frequently associated with an increased frequency of MDSCs that results in a loss in T-cell number and function through increased ROS and peroxynitrites production and arginase 1 (ARG1) and nitric oxide synthase 2 (NOS-2) upregulation. A better understanding of MDSCs expansion and the regulation of biodistribution and arrest would improve interventional strategies and cancer immunotherapy outcomes. Using five mammary tumor models, we have examined tumor-associated expansion of MDSCs and observed that this occurs in an organ-specific manner. Murine MDSCs can be subdivided into phenotypically and functionally distinct sub-populations according to Gr-1 expression levels; the CD11b+Gr-1 dim MDSCs with monocytic nuclei and strong T-cell suppressive activity, and CD11b+Gr-1bright having ring-shaped nuclei (polymorphonuclear cells) and a lower T-cell suppression profile. These cells can also be subset based on the expression of Ly-6C which is highly expressed on immature, bone marrow-derived cells and combined this Gr-1 expression Our studies revealed a significant increase in the Ly-6Cbright expression on the CD11b+Gr-1dim MDSCs in all organs studied in tumor-bearing mice as compared to non-tumor-bearing mice. In association with the increase in CD11b+Gr-1dimLy-6Cbright cells in the tumor non-parenchymal cells of 4T1 tumor bearing mice, increased transcripts levels of cytokines associated with MDSCs expansion (granulocyte-colony stimulating factor [G-CSF]) and MDSCs-associated immunosuppression (ARG-1, NOS-2, and cyclooxygenase 2 [COX-2]) while lower levels of these cytokines were observed in tumor NPCs from Cl66 tumor-bearing mice and non-tumor-bearing mice. In association with the increase in MDSCs and loss of T-cells in the 4T1 tumor-bearing mice there was a significant decrease in cytokine transcript levels that can enhance immune response (interferon gamma [IFN-γ] and interleukin 12 [IL-12]) from tumor non-parenchymal cells (NPCs) in 4T1 tumor bearing mice as compared to Cl66 tumor bearing mice and non-tumor bearing mice. Studies to assess where the MDSCs were expanding revealed that in 4T1 tumor bearing mice, frequency of the proliferating MDSCs is increased only in the spleen and liver compared to the non-tumor-bearing mice. Our studies also examined the affect of tumor burden on the trafficking of MDSCs. We observed that, after adoptive transfer of MDSCs isolated from 4T1 tumor-bearing mice, they arrest initially within the lungs in non-tumor-bearing mice. While in 4T1 tumor-bearing mice they rapidly transverse the lungs with arrest within the tumor tissue. We and others reported that the increase in MDSCs frequencies is associated with a decrease in CD3+ cells function and numbers. We observed a decrease in frequency of all T-cell subsets in the thymus of tumor bearing mice tested compared to the non-tumor-bearing mice suggesting defects in T-cell maturation associated with tumor burden. We investigated the effect of tumor burden on the phenotype of the CD3+ cells using the Annexin V staining and found that 4T1 tumor bearing mice have increased frequency of Annexin V positive CD3+ T-cells that are infiltrating tumor tissue compared to those in other organs. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B52.
Immune manipulation can control tumor initiation and growth while tumor growth may suppress host immunity. In our studies of tumor–host interactions, we observed a tumor associated expansion of IMSCs and suppression of T cell number and tumor infiltration. Further, the cyclooxygenase-2 (COX-2) inhibitor, Celecoxib had chemopreventative and as shown for the first time, therapeutic activity for mammary tumors in HER-2/neu transgenic BALB/c mice. Tumor growth was associated with a significant decrease in T cells and a significant increase in IMSCs (CD11b+Gr1+). In addition type 1 cytokine transcripts were depressed, while transcription levels of COX-2, arginase-1 (ARG1), nitric oxide synthetase-2 (NOS2), granulocyte (G) and G-monocyte colony stimulating factor, and vascular endothelial growth factor-alpha (VEGF-a) were increased in the preneoplastic lesions, tumors, and spleens and to a greater extent tumor infiltrating leukocytes. Celecoxib chemoprevention reduced the changes in IMSC and T cell numbers and cytokine and enzyme transcription levels. We posit that the increase in IMSCs may be due to the increased levels of COX-2, VEGF-a and growth factor(s), while the loss of T cell numbers and function maybe due to the heightened levels of ARG1 and NOS2. Further, as Celecoxib administration does not increase T cell infiltration of tumors or decrease IMSC infiltration, the changes in cytokine and growth factor levels maybe critical to the improved clinical outcomes. Together, these studies suggest an important role for IMSCs and its regulation by Celecoxib in cancer induction, and therapeutic intervention. Supported in part by funding from NIH grant 5 RO1 AT001739, Eppley Cancer Center and Nebraska DHHS LB506.
We have previously examined the effect of transplantable, murine mammary tumor growth on the expansion of myeloid‐derived suppressor cells (MDSCs) and their tumor infiltration, as well as the relationship of MDSC frequency to T cells. We report an extension of these studies using mouse mammary tumor virus (MMTV) promoter driven HER2/Neu proto‐oncogene transgenic, FBV mice (neu transgenic mice). Studies in this model also included an assessment of the tumor chemopreventative activity by the cyclooxygenase‐2 inhibitor, celecoxib (80 mg/kg in drinking water). We report that celecoxib administration delayed tumor induction, increased the incidence of apoptotic tumor cells in the treated mice, and prolonged survival. In addition, immune surrogates in the spleens and tumors were studied by flow cytometry and quantitative reverse transcriptase polymerase chain reaction analysis. These studies revealed that tumor development expanded MDSCs numbers and depressed the T cell frequency in the spleen. Celecoxib administration reduced the MDSC expansion and T cell depression in the spleen and tumors. The tumor associated increase in transcript levels of COX‐2, G‐CSF, GM‐CSF TGF‐β, NOS‐2, ARG‐1 and VEGF‐α were reduced by celecoxib administration. The greatest increase in cytokine and enzyme levels were observed within tumor infiltrating leukocytes relative to spleen cells, suggesting a tumor‐immune cell interaction.Supported in part by funding from NIH grant 5 RO1 AT001739, Eppley Cancer Center and Nebraska DHHS LB506.
MDSCs are increased in the marrow (BM), lungs, liver, and tumors of TB hosts as compared to non-TB (NTB) mice. In vivo, BrdU labeling of leukocytes from 4T1 mammary TB mice revealed that MDSCs had an increased rate of proliferation in BM, spleen, lungs, and liver. MDSCs are observed in the highest frequency in the blood and spleen, suggesting hematopoietic progenitors mobilized from BM and proliferated following arrest. We assessed the biodistribution of circulating cells using CFSE-labeled spleen cells from TB mice, adoptively transferred to normal mice by iv injection and rapidly arrested in the lungs. In contrast, CFSE-labeled MDSCs injected into 4T1 TB mice transversed the lungs, briefly arrested in the liver, and were retained in BM and tumors. We posit that in 4T1 TB mice, the normal arrest in the pulmonary capillary bed is prevented by increased levels of nitric oxide from activated MDSCs resulting in capillary vasodilation and reduced arrest. In 4T1 TB mice, CD3+ T-cells are decreased, although the MFI of CFSE suggests proliferation. Annexin V staining revealed a high frequency of apoptotic CD3+ T-cells, associated with MDSC accumulation. In summary, MDSCs proliferate in lymphoid and parenchymal organs resulting in T-cell apoptosis possibly associated with activation-induced cell death. Further, although MDSCs proliferate in many organs, they rapidly circulate preferentially homing to the spleen and liver.
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