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. 2015 Sep 22;12(11):1902-14.
doi: 10.1016/j.celrep.2015.08.033. Epub 2015 Sep 10.

TNF Counterbalances the Emergence of M2 Tumor Macrophages

Franz Kratochvill  1 , Geoffrey Neale  2 , Jessica M Haverkamp  1 , Lee-Ann Van de Velde  1 , Amber M Smith  1 , Daisuke Kawauchi  3 , Justina McEvoy  4 , Martine F Roussel  3 , Michael A Dyer  4 , Joseph E Qualls  5 , Peter J Murray  6
Affiliations

TNF Counterbalances the Emergence of M2 Tumor Macrophages

Franz Kratochvill et al. Cell Rep. .

Abstract

Cancer can involve non-resolving, persistent inflammation where varying numbers of tumor-associated macrophages (TAMs) infiltrate and adopt different activation states between anti-tumor M1 and pro-tumor M2 phenotypes. Here, we resolve a cascade causing differential macrophage phenotypes in the tumor microenvironment. Reduction in TNF mRNA production or loss of type I TNF receptor signaling resulted in a striking pattern of enhanced M2 mRNA expression. M2 gene expression was driven in part by IL-13 from eosinophils co-recruited with inflammatory monocytes, a pathway that was suppressed by TNF. Our data define regulatory nodes within the tumor microenvironment that balance M1 and M2 populations. Our results show macrophage polarization in cancer is dynamic and dependent on the balance between TNF and IL-13, thus providing a strategy for manipulating TAMs.

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Figures

Figure 1
Figure 1. Cooperative MyD88-TNF signaling drives expression of M1/M2 genes in TAMs
(A) M1 and M2 associated gene expression in TAMs isolated from 3 tumor models (Neuroblastoma: Neuro; Tymoma: Thymo; Glioma: Glio) and analyzed by microarray. Signal intensities are shown with the rank among 45037 probe sets (in N-TAMs). Color code represents high (yellow) to low (blue) intensities. Data are representative of the log2 signal intensities (n = 3 per TAM type). (B) Immunoblot analysis of ARG1 expression in CD11b+ and CD11b− fractions isolated from EG7 thymomas or gliomas grown in Arg1flox/flox;Tie2-cre (Arg1KO) and WT (Arg1flox/flox littermates) mice. (C, D and E) qRT-PCR of EG7 TAM RNA isolated from WT, TNFRKO, Myd88ΔH or DKO mice. Each dot represents the expression values from individual mice from at least 2 experiments with the black line representing the mean. Error bars, SEM. Statistical significance was calculated using two-tailed Student's t test and is indicated by *p< 0.05, **p< 0.01, ***p< 0.001.
Figure 2
Figure 2. MyD88 and TNFR1 signaling suppress M2 gene expression in TAMs
(A) Gating strategy of TAM populations A (red ellipse), B (orange ellipse) and C (green ellipse) as well as tumor-infiltrating eosinophils (Eos, purple square) and double negative (DN) cells for sorting experiments comparing EG7 TAMs from WT and Ccr2-/- mice. (B) Schematic representation of TAM development from TAM-A to TAM-C. (C) F4/80 and CX3CR1-GFP expression in EG7 TAMs isolated from WT and CX3CR1-GFP mice in TAM fractions as shown in A and B. Data are representative histograms of 3 (F4/80; n = 10) or 1 (CX3CR1-GFP; n = 3) experiments. (D) TAM fractions in macrophages gated as shown in A isolated from different transplantable tumor models. Blots are representative for at least 2 experiments where each experiment used at least 5 mice per tumor type. (E) Transcriptome analysis of EG7 TAM populations A and C derived from WT, TNFRKO, Myd88ΔH or DKO mice. Depicted values of M1 and M2 associated genes are heat maps arranged by log2 signal intensities (n = 2 per genotype).
Figure 3
Figure 3. TNF signaling blocks IL-13 derived M2 polarization
(A-C) qRT-PCR analysis of BMDMs left unstimulated or treated with a cocktail of IL-4 and IL-13 in combination with or without TNF for the times indicated. Data are the mean expression values (n = 2) and are representative for 1 out of 2 experiments. (D) Flow histograms of IL-4Rα and IL-13Rα expression in EG7 TAM populations A and C, representative of 2 experiments (n = 10). Unstained control is shown in grey. (E) qRT-PCR of M2 genes EG7 TAM populations A and C isolated from WT and Stat6KO mice. Data are the expression values from individual mice (n ≥ 7) from 2 experiments with the black line representing the mean. (F) The expression of the M2 marker PD-L2 in EG7 TAM populations A and C was analyzed by flow cytometry isolated from WT and Stat6KO mice. Data are shown as Median Fluorescence Intensity (MFI) (n = 3) and representative flow analysis in TAM-C. (G) IL-4Rα and IL-13Rα expression in EG7 TAM populations A and C analyzed by flow cytometry as shown in (D) isolated from WT, TNFRKO and Stat6KO mice. Data are shown as Median Fluorescence Intensity (MFI) (WT, Stat6KO: n = 4; TNFRKO: n = 3) and represent 1 out of 3 experiments. Statistical significance was calculated using a two-tailed Student's t test and is indicated by *p< 0.05, **p< 0.01, ***p< 0.001. Error bars, SEM.
Figure 4
Figure 4. Tumor infiltrating eosinophil derived IL-13 drives M2 expression in TAMs
(A and B) qRT-PCR analysis of IL-13 production in EG7 TAM unsorted (A) or sorted for TAM subpopulations as depicted in Figure 2A and B. Values from individual mice (A: n ≥ 21; B: n ≥ 10) from at least 2 experiments are shown. Data in B were normalized to eosinophils (Eos). Error bars, SEM. (C) SiglecF expression analyzed by flow cytometry in TAM subpopulations gated as shown in Figure 2. Plots are representative for 3 experiments. (D) H&E stained cytospin slides of EG7 TAM populations (40× magnification). Pictures shown are representative of 2 experiments (n = 4). (E) qRT-PCR analysis of CD11b+ EG7 TAMs derived from control IgG1 or anti-IL-13 antibody treated TNFRKO mice (n ≥ 5). Mean expression values from 1 of 2 experiments are shown. Error bars, SEM. Statistical significance was calculated using a One-way Anova (B) or two-tailed Student's t test (A and E) and is indicated by *p< 0.05, **p< 0.01, ***p< 0.001.
Figure 5
Figure 5. M2 gene expression in TNFRKO TAMs correlates with tumor infiltrating eosinophils
(A) Frequency of tumor infiltrating eosinophils in 3 different tumor models (EG7, LLC and B16) in WT and TNFRKO mice gated as in Figure 2A. Data are values from individual mice (n ≥ 6) from at least 2 independent experiments per tumor model. Cell frequencies are shown as % Ly6C+/− MHCII− cells of all CD11b+Ly6G− cells. Error bars, SEM. (B and C) qRT-PCR analysis of CD11b+ TAMs isolated from different tumor models in WT and TNFRKO mice as in A (n ≥ 5). Data are the mean expression values normalized to the corresponding WT TAMs in each tumor model and represent values from individual mice from at least 2 independent experiments per tumor model. Error bars, SEM.
Figure 6
Figure 6. Treatment with the anti-TNF drug Etanercept enhances IL-13 and M2-associated gene expression in TAMs
(A, B and C) qRT-PCR analysis of CD11b+ EG7 TAMs isolated from PBS (n ≥ 17) or Etanercept (n ≥ 18) treated WT mice compared to TAMs from TNFRKO mice (n ≥ 11). Data represent individual mice from 3 experiments normalized to the corresponding mean PBS treated WT TAMs per experiment. Error bars, SEM. Statistical significance was calculated using a two-tailed Student's t test and is indicated by *p< 0.05, **p< 0.01, ***p< 0.001. Error bars, SEM.
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