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TNF Neutralization Results in the Delay of Transplantable Tumor Growth and Reduced MDSC Accumulation.

Atretkhany KS, Nosenko MA, Gogoleva VS, Zvartsev RV, Qin Z, Nedospasov SA, Drutskaya MS - Front Immunol (2016)

Bottom Line: Under the influence of inflammatory cytokines, these cells become MDSCs, acquire immunosuppressive phenotype, and accumulate in the affected tissue, as well as in the periphery.TNF is a critical factor for the induction, expansion, and suppressive activity of MDSCs.Both etanercept and infliximab treatments resulted in a delayed growth of MCA 205 fibrosarcoma in hTNF KI mice, significantly reduced tumor volume, and also resulted in less accumulated MDSCs in the blood 3 weeks after tumor cell inoculation.

View Article: PubMed Central - PubMed

Affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia; Immunology Department, Faculty of Biology, Beloszersky Institue of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia.

ABSTRACT
Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of immature myeloid cells (IMCs) that, under normal conditions, may differentiate into mature macrophages, granulocytes, and dendritic cells. However, under pathological conditions associated with inflammation, cancer, or infection, such differentiation is inhibited leading to IMC expansion. Under the influence of inflammatory cytokines, these cells become MDSCs, acquire immunosuppressive phenotype, and accumulate in the affected tissue, as well as in the periphery. Immune suppressive activity of MDSCs is partly due to upregulation of arginase 1, inducible nitric oxide synthase, and anti-inflammatory cytokines, such as IL-10 and TGF-β. These suppressive factors can enhance tumor growth by repressing T-cell-mediated anti-tumor responses. TNF is a critical factor for the induction, expansion, and suppressive activity of MDSCs. In this study, we evaluated the effects of systemic TNF ablation on tumor-induced expansion of MDSCs in vivo using TNF humanized (hTNF KI) mice. Both etanercept and infliximab treatments resulted in a delayed growth of MCA 205 fibrosarcoma in hTNF KI mice, significantly reduced tumor volume, and also resulted in less accumulated MDSCs in the blood 3 weeks after tumor cell inoculation. Thus, our study uncovers anti-tumor effects of systemic TNF ablation in vivo.

No MeSH data available.


Related in: MedlinePlus

Infliximab or etanercept efficiently reduce tumor growth and MDSC accumulation in hTNF KI mice. (A) Tumor growth in hTNF KI mice undergoing treatment with PBS (black), etanercept (red), or infliximab (blue). Each line represents the growth curve of mean tumor volume ±SD. (B) MDSC accumulation in the blood of tumor-bearing hTNF KI mice undergoing treatment with PBS (black), etanercept (red), or infliximab (blue). Each bar represents mean relative MDSC level in the blood of tumor-bearing mice normalized to tumor-free mice ±SD. (C) Representative dot plots for MDSC staining in the blood from mice without tumors (left), anti-TNF (middle), or PBS-treated (right) mice with tumors on day 16 after tumor cells inoculation. Cells were first gated on VD−CD45+. MDSCs were defined as CD11b+Gr-1+ cells (with Gr-1 antibody clone RB6-8C5 recognizing both Ly6G and Ly6C epitopes). (D) MDSC accumulation in the spleens and peripheral lymph nodes of tumor-bearing mice three weeks after tumor inoculation. Graph shows relative numbers of Ly6C+Ly6G+cells among CD11b+ cells. Cells were gated as VD-CD45+B220–. Data are representative of more than three independent experiments with five to six mice per each group; *p < 0.5 and **p < 0.01.
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Figure 3: Infliximab or etanercept efficiently reduce tumor growth and MDSC accumulation in hTNF KI mice. (A) Tumor growth in hTNF KI mice undergoing treatment with PBS (black), etanercept (red), or infliximab (blue). Each line represents the growth curve of mean tumor volume ±SD. (B) MDSC accumulation in the blood of tumor-bearing hTNF KI mice undergoing treatment with PBS (black), etanercept (red), or infliximab (blue). Each bar represents mean relative MDSC level in the blood of tumor-bearing mice normalized to tumor-free mice ±SD. (C) Representative dot plots for MDSC staining in the blood from mice without tumors (left), anti-TNF (middle), or PBS-treated (right) mice with tumors on day 16 after tumor cells inoculation. Cells were first gated on VD−CD45+. MDSCs were defined as CD11b+Gr-1+ cells (with Gr-1 antibody clone RB6-8C5 recognizing both Ly6G and Ly6C epitopes). (D) MDSC accumulation in the spleens and peripheral lymph nodes of tumor-bearing mice three weeks after tumor inoculation. Graph shows relative numbers of Ly6C+Ly6G+cells among CD11b+ cells. Cells were gated as VD-CD45+B220–. Data are representative of more than three independent experiments with five to six mice per each group; *p < 0.5 and **p < 0.01.

Mentions: One important difference between etanercept and antibody-based anti-TNF drugs (such as infliximab, adalimumab, and others) is that the former may also bind and neutralize soluble LTα3 (45). Because several non-redundant functions of sLTα3 were reported, for instance, the role of LTα3 produced by innate lymphoid cells in the gut (46), we wanted to make sure that the effects of etanercept in our tumor model (including results presented in Figure 2) were due to neutralization of TNF and not to soluble LTα3. To this end, we compared the effects of anti-TNF therapy on tumor growth using humanized knock-in mice, which produce human TNF instead of murine TNF (further referred to as hTNF KI mice) (41, 42). This model allowed us to compare therapeutical effects of various human TNF blockers in vivo, in our case – etanercept and infliximab. First of all, tumor growth (Figures 2A and 3A) and the accumulation of MDSCs (Figures 2B and 3B) in tumor-bearing hTNF KI mice were comparable to those in wild-type mice, in both cases MDSCs reached 30–40% in the blood 3 weeks after MCA 205 tumor cells inoculation. We then compared the effects of TNF blockade with either etanercept or infliximab on MCA 205 growth and found that both drugs efficiently and comparably inhibited tumor growth (Figure 3A), indicating that anti-tumor effects were most likely due to TNF and not to sLTa neutralization. Three weeks after inoculation, the tumor volume in mice undergoing anti-TNF treatment reached only 200 mm3, as compared to 500–600 mm3 in the control group. Histological analysis revealed infiltration of the tumor tissue by myeloid cells in all three experimental groups (Figure S3 in Supplementary Material). We also observed a significant reduction in MDSC levels in the blood of mice treated with either of the two blockers (Figure 3B). MDSC accumulation in control mice reached 30–40% of total leukocytes in the blood, while in mice treated with etanercept or infliximab it only reached 15–20% (Figure 3C). Tumor-free mice in each group, despite receiving TNF blockers, showed the same frequency of CD11b+Gr-1+ myeloid cells, at approximately 8–10% of total blood leukocytes (Figure 3C). We also followed accumulation of MDSCs in the periphery: in lymph nodes and in the spleen, and found that the frequency of MDSCs was higher in PBS-treated group (Figure 3D). These data indicated that TNF plays a crucial role in MCA205 tumor growth and MDSC accumulation.


TNF Neutralization Results in the Delay of Transplantable Tumor Growth and Reduced MDSC Accumulation.

Atretkhany KS, Nosenko MA, Gogoleva VS, Zvartsev RV, Qin Z, Nedospasov SA, Drutskaya MS - Front Immunol (2016)

Infliximab or etanercept efficiently reduce tumor growth and MDSC accumulation in hTNF KI mice. (A) Tumor growth in hTNF KI mice undergoing treatment with PBS (black), etanercept (red), or infliximab (blue). Each line represents the growth curve of mean tumor volume ±SD. (B) MDSC accumulation in the blood of tumor-bearing hTNF KI mice undergoing treatment with PBS (black), etanercept (red), or infliximab (blue). Each bar represents mean relative MDSC level in the blood of tumor-bearing mice normalized to tumor-free mice ±SD. (C) Representative dot plots for MDSC staining in the blood from mice without tumors (left), anti-TNF (middle), or PBS-treated (right) mice with tumors on day 16 after tumor cells inoculation. Cells were first gated on VD−CD45+. MDSCs were defined as CD11b+Gr-1+ cells (with Gr-1 antibody clone RB6-8C5 recognizing both Ly6G and Ly6C epitopes). (D) MDSC accumulation in the spleens and peripheral lymph nodes of tumor-bearing mice three weeks after tumor inoculation. Graph shows relative numbers of Ly6C+Ly6G+cells among CD11b+ cells. Cells were gated as VD-CD45+B220–. Data are representative of more than three independent experiments with five to six mice per each group; *p < 0.5 and **p < 0.01.
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Figure 3: Infliximab or etanercept efficiently reduce tumor growth and MDSC accumulation in hTNF KI mice. (A) Tumor growth in hTNF KI mice undergoing treatment with PBS (black), etanercept (red), or infliximab (blue). Each line represents the growth curve of mean tumor volume ±SD. (B) MDSC accumulation in the blood of tumor-bearing hTNF KI mice undergoing treatment with PBS (black), etanercept (red), or infliximab (blue). Each bar represents mean relative MDSC level in the blood of tumor-bearing mice normalized to tumor-free mice ±SD. (C) Representative dot plots for MDSC staining in the blood from mice without tumors (left), anti-TNF (middle), or PBS-treated (right) mice with tumors on day 16 after tumor cells inoculation. Cells were first gated on VD−CD45+. MDSCs were defined as CD11b+Gr-1+ cells (with Gr-1 antibody clone RB6-8C5 recognizing both Ly6G and Ly6C epitopes). (D) MDSC accumulation in the spleens and peripheral lymph nodes of tumor-bearing mice three weeks after tumor inoculation. Graph shows relative numbers of Ly6C+Ly6G+cells among CD11b+ cells. Cells were gated as VD-CD45+B220–. Data are representative of more than three independent experiments with five to six mice per each group; *p < 0.5 and **p < 0.01.
Mentions: One important difference between etanercept and antibody-based anti-TNF drugs (such as infliximab, adalimumab, and others) is that the former may also bind and neutralize soluble LTα3 (45). Because several non-redundant functions of sLTα3 were reported, for instance, the role of LTα3 produced by innate lymphoid cells in the gut (46), we wanted to make sure that the effects of etanercept in our tumor model (including results presented in Figure 2) were due to neutralization of TNF and not to soluble LTα3. To this end, we compared the effects of anti-TNF therapy on tumor growth using humanized knock-in mice, which produce human TNF instead of murine TNF (further referred to as hTNF KI mice) (41, 42). This model allowed us to compare therapeutical effects of various human TNF blockers in vivo, in our case – etanercept and infliximab. First of all, tumor growth (Figures 2A and 3A) and the accumulation of MDSCs (Figures 2B and 3B) in tumor-bearing hTNF KI mice were comparable to those in wild-type mice, in both cases MDSCs reached 30–40% in the blood 3 weeks after MCA 205 tumor cells inoculation. We then compared the effects of TNF blockade with either etanercept or infliximab on MCA 205 growth and found that both drugs efficiently and comparably inhibited tumor growth (Figure 3A), indicating that anti-tumor effects were most likely due to TNF and not to sLTa neutralization. Three weeks after inoculation, the tumor volume in mice undergoing anti-TNF treatment reached only 200 mm3, as compared to 500–600 mm3 in the control group. Histological analysis revealed infiltration of the tumor tissue by myeloid cells in all three experimental groups (Figure S3 in Supplementary Material). We also observed a significant reduction in MDSC levels in the blood of mice treated with either of the two blockers (Figure 3B). MDSC accumulation in control mice reached 30–40% of total leukocytes in the blood, while in mice treated with etanercept or infliximab it only reached 15–20% (Figure 3C). Tumor-free mice in each group, despite receiving TNF blockers, showed the same frequency of CD11b+Gr-1+ myeloid cells, at approximately 8–10% of total blood leukocytes (Figure 3C). We also followed accumulation of MDSCs in the periphery: in lymph nodes and in the spleen, and found that the frequency of MDSCs was higher in PBS-treated group (Figure 3D). These data indicated that TNF plays a crucial role in MCA205 tumor growth and MDSC accumulation.

Bottom Line: Under the influence of inflammatory cytokines, these cells become MDSCs, acquire immunosuppressive phenotype, and accumulate in the affected tissue, as well as in the periphery.TNF is a critical factor for the induction, expansion, and suppressive activity of MDSCs.Both etanercept and infliximab treatments resulted in a delayed growth of MCA 205 fibrosarcoma in hTNF KI mice, significantly reduced tumor volume, and also resulted in less accumulated MDSCs in the blood 3 weeks after tumor cell inoculation.

View Article: PubMed Central - PubMed

Affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia; Immunology Department, Faculty of Biology, Beloszersky Institue of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia.

ABSTRACT
Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of immature myeloid cells (IMCs) that, under normal conditions, may differentiate into mature macrophages, granulocytes, and dendritic cells. However, under pathological conditions associated with inflammation, cancer, or infection, such differentiation is inhibited leading to IMC expansion. Under the influence of inflammatory cytokines, these cells become MDSCs, acquire immunosuppressive phenotype, and accumulate in the affected tissue, as well as in the periphery. Immune suppressive activity of MDSCs is partly due to upregulation of arginase 1, inducible nitric oxide synthase, and anti-inflammatory cytokines, such as IL-10 and TGF-β. These suppressive factors can enhance tumor growth by repressing T-cell-mediated anti-tumor responses. TNF is a critical factor for the induction, expansion, and suppressive activity of MDSCs. In this study, we evaluated the effects of systemic TNF ablation on tumor-induced expansion of MDSCs in vivo using TNF humanized (hTNF KI) mice. Both etanercept and infliximab treatments resulted in a delayed growth of MCA 205 fibrosarcoma in hTNF KI mice, significantly reduced tumor volume, and also resulted in less accumulated MDSCs in the blood 3 weeks after tumor cell inoculation. Thus, our study uncovers anti-tumor effects of systemic TNF ablation in vivo.

No MeSH data available.


Related in: MedlinePlus