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Interleukin-21 sustains inflammatory signals that contribute to sporadic colon tumorigenesis.

De Simone V, Ronchetti G, Franzè E, Colantoni A, Ortenzi A, Fantini MC, Rizzo A, Sica GS, Sileri P, Rossi P, MacDonald TT, Pallone F, Monteleone G, Stolfi C - Oncotarget (2015)

Bottom Line: Stimulation of human CRC cell lines with IL-21 did not directly activate the oncogenic transcription factors STAT3 and NF-kB and did not affect CRC cell proliferation and survival.IL-21 deficiency was associated with reduced STAT3/NF-kB activation in both immune cells and neoplastic cells, diminished synthesis of protumorigenic cytokines (that is, IL-17A, IL-22, TNF-α and IL-6), downregulation of COX-2/PGE2 pathway and decreased angiogenesis in the lesions of Apc(min/+) mice.Altogether, data suggest that IL-21 promotes a protumorigenic inflammatory circuit that ultimately sustains the development of sporadic CRC.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Medicine, University of Rome "Tor Vergata", Rome, Italy.

ABSTRACT
Interleukin (IL)-21 triggers inflammatory signals that contribute to the growth of neoplastic cells in mouse models of colitis-associated colorectal cancer (CRC). Because most CRCs are sporadic and arise in the absence of overt inflammation we have investigated the role of IL-21 in these tumors in mouse and man. IL-21 was highly expressed in human sporadic CRC and produced mostly by IFN-γ-expressing T-bet/RORγt double-positive CD3+CD8- cells. Stimulation of human CRC cell lines with IL-21 did not directly activate the oncogenic transcription factors STAT3 and NF-kB and did not affect CRC cell proliferation and survival. In contrast, IL-21 modulated the production of protumorigenic factors by human tumor infiltrating T cells. IL-21 was upregulated in the neoplastic areas, as compared with non-tumor mucosa, of Apc(min/+) mice, and genetic ablation of IL-21 in such mice resulted in a marked decrease of both tumor incidence and size. IL-21 deficiency was associated with reduced STAT3/NF-kB activation in both immune cells and neoplastic cells, diminished synthesis of protumorigenic cytokines (that is, IL-17A, IL-22, TNF-α and IL-6), downregulation of COX-2/PGE2 pathway and decreased angiogenesis in the lesions of Apc(min/+) mice. Altogether, data suggest that IL-21 promotes a protumorigenic inflammatory circuit that ultimately sustains the development of sporadic CRC.

No MeSH data available.


Related in: MedlinePlus

Characterization of IL-21-producing cells in human sporadic colorectal cancer (CRC)A. Total proteins extracted from both tumor (T) and non-tumor (NT) areas of two patients with sporadic CRC were evaluated for IL-21 expression by western blotting. β-actin was used as loading control. The figure is representative of five separate experiments. Right inset. Quantitative analysis of IL-21/β-actin protein ratio. Values are expressed in arbitrary units (a.u.) and indicate the mean ± SEM of all experiments (n=10). B. IL-21 is preferentially made by CD3+CD8− cells in tumor infiltrating leukocytes (TILs). Left panel. Representative histograms showing the percentage of CD45+IL-21+ cells in TILs. The example is representative of five experiments in which cells isolated from five patients with sporadic CRC were analyzed. Numbers above lines indicate the percentages of CD45+IL-21+ cells. Staining of cells with PE-conjugated control isotype IgG is also shown. Central panels. Representative dot-plots showing the fraction of IL-21+ cells in TILs. CD45+IL-21+ and CD45+IL-21- cells were gated and then analyzed for the indicated markers. The numbers indicate the percentage of cells in the designated quadrants. The example is representative of five experiments in which cells isolated from five patients with sporadic CRC were analyzed. Right panel. Representative histograms showing the fraction of IL-21+ cells in TILs. CD45+IL-21+ cells were gated and then analyzed for the indicated markers. Data are expressed as mean ± SEM of five experiments in which cells isolated from five patients with sporadic CRC were analyzed. C-D. IL-21 is preferentially made by IFN-γ-expressing T-bet/RORγt double-positive CD3+CD8− cells in human sporadic CRC. C. Representative histograms showing the percentages of IL-21-producing CD3+CD8− cells expressing the indicated transcription factors. Data are expressed as mean ± SEM of five experiments in which TILs isolated from five patients with sporadic CRC were analyzed. D. IL-17A and IFN-γ expression was analyzed in T-bet-RORγt+ and T-bet/RORγt double-positive IL-21-producing CD3+CD8− cells by flow cytometry. The numbers indicate the percentage of cells in the designated quadrants. Right panels. Percentages of T-bet-RORγt+ and T-bet/RORγt double-positive IL-21-producing CD3+CD8− cells co-expressing IL-17A and/or IFN-γ. Data are expressed as mean ± SEM of five experiments in which TILs isolated from five patients with sporadic CRC were analyzed. E. TILs isolated from four patients with sporadic CRC were cultured either with a control IgG or anti-IL-21 antibody (both used at 10 μg/ml) for 48 h. Each point represents the percentage of IL-17A-, IL-22-, IL-6- and IFN-γ-producing CD45+CD3+CD8− cells, assessed by flow cytometry, in TILs isolated from a single patient.
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Figure 1: Characterization of IL-21-producing cells in human sporadic colorectal cancer (CRC)A. Total proteins extracted from both tumor (T) and non-tumor (NT) areas of two patients with sporadic CRC were evaluated for IL-21 expression by western blotting. β-actin was used as loading control. The figure is representative of five separate experiments. Right inset. Quantitative analysis of IL-21/β-actin protein ratio. Values are expressed in arbitrary units (a.u.) and indicate the mean ± SEM of all experiments (n=10). B. IL-21 is preferentially made by CD3+CD8− cells in tumor infiltrating leukocytes (TILs). Left panel. Representative histograms showing the percentage of CD45+IL-21+ cells in TILs. The example is representative of five experiments in which cells isolated from five patients with sporadic CRC were analyzed. Numbers above lines indicate the percentages of CD45+IL-21+ cells. Staining of cells with PE-conjugated control isotype IgG is also shown. Central panels. Representative dot-plots showing the fraction of IL-21+ cells in TILs. CD45+IL-21+ and CD45+IL-21- cells were gated and then analyzed for the indicated markers. The numbers indicate the percentage of cells in the designated quadrants. The example is representative of five experiments in which cells isolated from five patients with sporadic CRC were analyzed. Right panel. Representative histograms showing the fraction of IL-21+ cells in TILs. CD45+IL-21+ cells were gated and then analyzed for the indicated markers. Data are expressed as mean ± SEM of five experiments in which cells isolated from five patients with sporadic CRC were analyzed. C-D. IL-21 is preferentially made by IFN-γ-expressing T-bet/RORγt double-positive CD3+CD8− cells in human sporadic CRC. C. Representative histograms showing the percentages of IL-21-producing CD3+CD8− cells expressing the indicated transcription factors. Data are expressed as mean ± SEM of five experiments in which TILs isolated from five patients with sporadic CRC were analyzed. D. IL-17A and IFN-γ expression was analyzed in T-bet-RORγt+ and T-bet/RORγt double-positive IL-21-producing CD3+CD8− cells by flow cytometry. The numbers indicate the percentage of cells in the designated quadrants. Right panels. Percentages of T-bet-RORγt+ and T-bet/RORγt double-positive IL-21-producing CD3+CD8− cells co-expressing IL-17A and/or IFN-γ. Data are expressed as mean ± SEM of five experiments in which TILs isolated from five patients with sporadic CRC were analyzed. E. TILs isolated from four patients with sporadic CRC were cultured either with a control IgG or anti-IL-21 antibody (both used at 10 μg/ml) for 48 h. Each point represents the percentage of IL-17A-, IL-22-, IL-6- and IFN-γ-producing CD45+CD3+CD8− cells, assessed by flow cytometry, in TILs isolated from a single patient.

Mentions: IL-21 was significantly increased in CRC samples as compared to non-tumor mucosa (Figure 1A). To assess the cellular source of IL-21 in human sporadic CRC, we isolated tumor infiltrating leukocytes (TILs) from patients who underwent surgical resection. Flow cytometry analysis revealed that IL-21 was mostly produced by CD3+CD8− T cells (Figure 1B) and minimally expressed by CD3+CD8+ T cells and natural killer (NK) T cells (CD3+CD56+ cells) (2.54±0.4 and 0.96±0.28 respectively); no IL-21 staining was seen in NK cells (CD3-CD56+ cells), macrophages (CD68+ cells) and B cells (CD19+ cells) thus ruling out such cells as sources of IL-21 in human CRC (Figure 1B). Next, we evaluated whether IL-21 was co-expressed with Th1/Th17 signature cytokines (that is, IFN-γ and IL-17A respectively) within CD3+CD8− and CD3+CD8+ populations. Nearly 5% of CD3+CD8− cells produced IL-21 either alone or in combination with IFN-γ (5.15±0.66 and 4.45±0.67 respectively), whereas less then 2% (1.65±0.46) of such cells co-expressed IL-21 and IL-17A (Suppl. Figure 1A and B). Most of the CD3+CD8+ cells synthesized IFN-γ (77.1±8.25), whereas less than 3% of such cells expressed either IL-21 alone (0.3±0.11) or in combination with IFN-γ or IL-17A (2.8±1.17 and 0.42±0.14 respectively) (Suppl. Figure 1C and D).


Interleukin-21 sustains inflammatory signals that contribute to sporadic colon tumorigenesis.

De Simone V, Ronchetti G, Franzè E, Colantoni A, Ortenzi A, Fantini MC, Rizzo A, Sica GS, Sileri P, Rossi P, MacDonald TT, Pallone F, Monteleone G, Stolfi C - Oncotarget (2015)

Characterization of IL-21-producing cells in human sporadic colorectal cancer (CRC)A. Total proteins extracted from both tumor (T) and non-tumor (NT) areas of two patients with sporadic CRC were evaluated for IL-21 expression by western blotting. β-actin was used as loading control. The figure is representative of five separate experiments. Right inset. Quantitative analysis of IL-21/β-actin protein ratio. Values are expressed in arbitrary units (a.u.) and indicate the mean ± SEM of all experiments (n=10). B. IL-21 is preferentially made by CD3+CD8− cells in tumor infiltrating leukocytes (TILs). Left panel. Representative histograms showing the percentage of CD45+IL-21+ cells in TILs. The example is representative of five experiments in which cells isolated from five patients with sporadic CRC were analyzed. Numbers above lines indicate the percentages of CD45+IL-21+ cells. Staining of cells with PE-conjugated control isotype IgG is also shown. Central panels. Representative dot-plots showing the fraction of IL-21+ cells in TILs. CD45+IL-21+ and CD45+IL-21- cells were gated and then analyzed for the indicated markers. The numbers indicate the percentage of cells in the designated quadrants. The example is representative of five experiments in which cells isolated from five patients with sporadic CRC were analyzed. Right panel. Representative histograms showing the fraction of IL-21+ cells in TILs. CD45+IL-21+ cells were gated and then analyzed for the indicated markers. Data are expressed as mean ± SEM of five experiments in which cells isolated from five patients with sporadic CRC were analyzed. C-D. IL-21 is preferentially made by IFN-γ-expressing T-bet/RORγt double-positive CD3+CD8− cells in human sporadic CRC. C. Representative histograms showing the percentages of IL-21-producing CD3+CD8− cells expressing the indicated transcription factors. Data are expressed as mean ± SEM of five experiments in which TILs isolated from five patients with sporadic CRC were analyzed. D. IL-17A and IFN-γ expression was analyzed in T-bet-RORγt+ and T-bet/RORγt double-positive IL-21-producing CD3+CD8− cells by flow cytometry. The numbers indicate the percentage of cells in the designated quadrants. Right panels. Percentages of T-bet-RORγt+ and T-bet/RORγt double-positive IL-21-producing CD3+CD8− cells co-expressing IL-17A and/or IFN-γ. Data are expressed as mean ± SEM of five experiments in which TILs isolated from five patients with sporadic CRC were analyzed. E. TILs isolated from four patients with sporadic CRC were cultured either with a control IgG or anti-IL-21 antibody (both used at 10 μg/ml) for 48 h. Each point represents the percentage of IL-17A-, IL-22-, IL-6- and IFN-γ-producing CD45+CD3+CD8− cells, assessed by flow cytometry, in TILs isolated from a single patient.
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Related In: Results  -  Collection

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Figure 1: Characterization of IL-21-producing cells in human sporadic colorectal cancer (CRC)A. Total proteins extracted from both tumor (T) and non-tumor (NT) areas of two patients with sporadic CRC were evaluated for IL-21 expression by western blotting. β-actin was used as loading control. The figure is representative of five separate experiments. Right inset. Quantitative analysis of IL-21/β-actin protein ratio. Values are expressed in arbitrary units (a.u.) and indicate the mean ± SEM of all experiments (n=10). B. IL-21 is preferentially made by CD3+CD8− cells in tumor infiltrating leukocytes (TILs). Left panel. Representative histograms showing the percentage of CD45+IL-21+ cells in TILs. The example is representative of five experiments in which cells isolated from five patients with sporadic CRC were analyzed. Numbers above lines indicate the percentages of CD45+IL-21+ cells. Staining of cells with PE-conjugated control isotype IgG is also shown. Central panels. Representative dot-plots showing the fraction of IL-21+ cells in TILs. CD45+IL-21+ and CD45+IL-21- cells were gated and then analyzed for the indicated markers. The numbers indicate the percentage of cells in the designated quadrants. The example is representative of five experiments in which cells isolated from five patients with sporadic CRC were analyzed. Right panel. Representative histograms showing the fraction of IL-21+ cells in TILs. CD45+IL-21+ cells were gated and then analyzed for the indicated markers. Data are expressed as mean ± SEM of five experiments in which cells isolated from five patients with sporadic CRC were analyzed. C-D. IL-21 is preferentially made by IFN-γ-expressing T-bet/RORγt double-positive CD3+CD8− cells in human sporadic CRC. C. Representative histograms showing the percentages of IL-21-producing CD3+CD8− cells expressing the indicated transcription factors. Data are expressed as mean ± SEM of five experiments in which TILs isolated from five patients with sporadic CRC were analyzed. D. IL-17A and IFN-γ expression was analyzed in T-bet-RORγt+ and T-bet/RORγt double-positive IL-21-producing CD3+CD8− cells by flow cytometry. The numbers indicate the percentage of cells in the designated quadrants. Right panels. Percentages of T-bet-RORγt+ and T-bet/RORγt double-positive IL-21-producing CD3+CD8− cells co-expressing IL-17A and/or IFN-γ. Data are expressed as mean ± SEM of five experiments in which TILs isolated from five patients with sporadic CRC were analyzed. E. TILs isolated from four patients with sporadic CRC were cultured either with a control IgG or anti-IL-21 antibody (both used at 10 μg/ml) for 48 h. Each point represents the percentage of IL-17A-, IL-22-, IL-6- and IFN-γ-producing CD45+CD3+CD8− cells, assessed by flow cytometry, in TILs isolated from a single patient.
Mentions: IL-21 was significantly increased in CRC samples as compared to non-tumor mucosa (Figure 1A). To assess the cellular source of IL-21 in human sporadic CRC, we isolated tumor infiltrating leukocytes (TILs) from patients who underwent surgical resection. Flow cytometry analysis revealed that IL-21 was mostly produced by CD3+CD8− T cells (Figure 1B) and minimally expressed by CD3+CD8+ T cells and natural killer (NK) T cells (CD3+CD56+ cells) (2.54±0.4 and 0.96±0.28 respectively); no IL-21 staining was seen in NK cells (CD3-CD56+ cells), macrophages (CD68+ cells) and B cells (CD19+ cells) thus ruling out such cells as sources of IL-21 in human CRC (Figure 1B). Next, we evaluated whether IL-21 was co-expressed with Th1/Th17 signature cytokines (that is, IFN-γ and IL-17A respectively) within CD3+CD8− and CD3+CD8+ populations. Nearly 5% of CD3+CD8− cells produced IL-21 either alone or in combination with IFN-γ (5.15±0.66 and 4.45±0.67 respectively), whereas less then 2% (1.65±0.46) of such cells co-expressed IL-21 and IL-17A (Suppl. Figure 1A and B). Most of the CD3+CD8+ cells synthesized IFN-γ (77.1±8.25), whereas less than 3% of such cells expressed either IL-21 alone (0.3±0.11) or in combination with IFN-γ or IL-17A (2.8±1.17 and 0.42±0.14 respectively) (Suppl. Figure 1C and D).

Bottom Line: Stimulation of human CRC cell lines with IL-21 did not directly activate the oncogenic transcription factors STAT3 and NF-kB and did not affect CRC cell proliferation and survival.IL-21 deficiency was associated with reduced STAT3/NF-kB activation in both immune cells and neoplastic cells, diminished synthesis of protumorigenic cytokines (that is, IL-17A, IL-22, TNF-α and IL-6), downregulation of COX-2/PGE2 pathway and decreased angiogenesis in the lesions of Apc(min/+) mice.Altogether, data suggest that IL-21 promotes a protumorigenic inflammatory circuit that ultimately sustains the development of sporadic CRC.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Medicine, University of Rome "Tor Vergata", Rome, Italy.

ABSTRACT
Interleukin (IL)-21 triggers inflammatory signals that contribute to the growth of neoplastic cells in mouse models of colitis-associated colorectal cancer (CRC). Because most CRCs are sporadic and arise in the absence of overt inflammation we have investigated the role of IL-21 in these tumors in mouse and man. IL-21 was highly expressed in human sporadic CRC and produced mostly by IFN-γ-expressing T-bet/RORγt double-positive CD3+CD8- cells. Stimulation of human CRC cell lines with IL-21 did not directly activate the oncogenic transcription factors STAT3 and NF-kB and did not affect CRC cell proliferation and survival. In contrast, IL-21 modulated the production of protumorigenic factors by human tumor infiltrating T cells. IL-21 was upregulated in the neoplastic areas, as compared with non-tumor mucosa, of Apc(min/+) mice, and genetic ablation of IL-21 in such mice resulted in a marked decrease of both tumor incidence and size. IL-21 deficiency was associated with reduced STAT3/NF-kB activation in both immune cells and neoplastic cells, diminished synthesis of protumorigenic cytokines (that is, IL-17A, IL-22, TNF-α and IL-6), downregulation of COX-2/PGE2 pathway and decreased angiogenesis in the lesions of Apc(min/+) mice. Altogether, data suggest that IL-21 promotes a protumorigenic inflammatory circuit that ultimately sustains the development of sporadic CRC.

No MeSH data available.


Related in: MedlinePlus