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4T1 Murine Mammary Carcinoma Cells Enhance Macrophage-Mediated Innate Inflammatory Responses.

Madera L, Greenshields A, Coombs MR, Hoskin DW - PLoS ONE (2015)

Bottom Line: BMDMs exposed to 4T1 mammary carcinoma-conditioned medium demonstrated enhanced production of pro-inflammatory cytokines tumor necrosis factor α, interleukin-6, and CCL2 in response to lipopolysaccharide (LPS) while production of interleukin-10 remained unchanged.The increased LPS-induced production of pro-inflammatory cytokines was transient and correlated with enhanced cytokine production in response to other Toll-like receptor agonists, including peptidoglycan and flagellin.Furthermore, peritoneal macrophages obtained from 4T1 tumor-bearing mice displayed enhanced pro-inflammatory cytokine production in response to LPS.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology & Immunology, Dalhousie University, Halifax, Nova Scotia, Canada.

ABSTRACT
Tumor progression and the immune response are intricately linked. While it is known that cancers alter macrophage inflammatory responses to promote tumor progression, little is known regarding how cancers affect macrophage-dependent innate host defense. In this study, murine bone-marrow-derived macrophages (BMDM) were exposed to murine carcinoma-conditioned media prior to assessment of the macrophage inflammatory response. BMDMs exposed to 4T1 mammary carcinoma-conditioned medium demonstrated enhanced production of pro-inflammatory cytokines tumor necrosis factor α, interleukin-6, and CCL2 in response to lipopolysaccharide (LPS) while production of interleukin-10 remained unchanged. The increased LPS-induced production of pro-inflammatory cytokines was transient and correlated with enhanced cytokine production in response to other Toll-like receptor agonists, including peptidoglycan and flagellin. In addition, 4T1-conditioned BMDMs exhibited strengthened LPS-induced nitric oxide production and enhanced phagocytosis of Escherichia coli. 4T1-mediated augmentation of macrophage responses to LPS was partially dependent on the NFκB pathway, macrophage-colony stimulating factor, and actin polymerization, as well as the presence of 4T1-secreted extracellular vesicles. Furthermore, peritoneal macrophages obtained from 4T1 tumor-bearing mice displayed enhanced pro-inflammatory cytokine production in response to LPS. These results suggest that uptake of 4T1-secreted factors and actin-mediated ingestion of 4T1-secreted exosomes by macrophages cause a transient enhancement of innate inflammatory responses. Mammary carcinoma-mediated regulation of innate immunity may have significant implications for our understanding of host defense and cancer progression.

No MeSH data available.


Related in: MedlinePlus

4T1- and LLC-conditioned macrophages exhibit increased pro-inflammatory cytokine production in response to LPS.(A) C57BL/6 or (B) BALB/c BMDMs were cultured in DMEM alone or in the presence of culture medium conditioned by 4T1 mammary carcinoma, E0771 mammary carcinoma, ID8 ovarian carcinoma, or LLC cells for 24 h. Medium conditioned by HC11 normal mouse epithelial cells was used as a negative control. BMDMs were then washed with PBS and stimulated with 100 ng/ml LPS for 4 h, then culture supernatants were collected and protein levels of cytokines TNFα, IL-6, CCL2, and IL-10 were determined by ELISA. Results are shown as the mean (± SEM) of at least 5 independent experiments. Statistical analyses were done by ANOVA, followed by Tukey’s multiple comparison tests; * p < 0.05, ** p < 0.01, *** p < 0.001.
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pone.0133385.g001: 4T1- and LLC-conditioned macrophages exhibit increased pro-inflammatory cytokine production in response to LPS.(A) C57BL/6 or (B) BALB/c BMDMs were cultured in DMEM alone or in the presence of culture medium conditioned by 4T1 mammary carcinoma, E0771 mammary carcinoma, ID8 ovarian carcinoma, or LLC cells for 24 h. Medium conditioned by HC11 normal mouse epithelial cells was used as a negative control. BMDMs were then washed with PBS and stimulated with 100 ng/ml LPS for 4 h, then culture supernatants were collected and protein levels of cytokines TNFα, IL-6, CCL2, and IL-10 were determined by ELISA. Results are shown as the mean (± SEM) of at least 5 independent experiments. Statistical analyses were done by ANOVA, followed by Tukey’s multiple comparison tests; * p < 0.05, ** p < 0.01, *** p < 0.001.

Mentions: To assess the inflammatory phenotype of tumor-conditioned macrophages, BMDMs were exposed to tumor-conditioned media for 24 h and then stimulated with 100 ng/ml LPS from Escherichia coli for 4 h. As expected, LPS stimulation of non-conditioned BMDMs from C57BL/6 or BALB/c mice resulted in the production of pro-inflammatory cytokines tumor necrosis factor α (TNFα) and IL-6, macrophage chemokine CCL2, and anti-inflammatory cytokine IL-10 (Fig 1A and 1B). Surprisingly, C57BL/6 BMDMs cultured with 4T1 mouse mammary carcinoma-conditioned medium exhibited a 2–3 fold increase in the production of TNFα, IL-6, and CCL2 in the presence of LPS (Fig 1A). Lewis lung carcinoma (LLC)-conditioned BMDMs also showed a significant increase in TNFα and CCL2 levels in response to LPS. ID8 ovarian carcinoma-conditioned BMDMs demonstrated a modest increase in TNFα and CCL2 following LPS stimulation, although this effect was not statistically significant. In contrast, none of these tumor-conditioned media affected LPS-stimulated IL-10 production by BMDMs when compared to non-conditioned BMDMs. Neither E0771 mouse mammary carcinoma-conditioned medium nor control medium conditioned with HC11 mouse mammary epithelial cells affected the LPS response of BMDMs. Similar trends were observed in macrophages from BALB/c mice; 4T1-conditioned Balb/c BMDMs showed a striking increase in TNFα, IL-6, and CCL2 production in response to LPS, while IL-10 levels remained unaffected (Fig 1B), demonstrating that this effect was not mouse strain-specific. TNFα and IL-6 were not detected in the tumor-conditioned media nor in the supernatants of conditioned BMDMs, prior to LPS stimulation, indicating that the tumor-conditioning step itself did not elicit an inflammatory response (data not shown). These results indicate that secreted factors of 4T1 murine mammary carcinomas and LLC can potentiate macrophage inflammatory cytokine production in response to LPS. Furthermore, this increased sensitivity is selective since TNFα, IL-6, and CCL2 production was increased while anti-inflammatory cytokine IL-10 production was unaffected. Subsequent experiments employed 4T1-conditioned C56BL/6 BMDMs because the effect was most striking in these cells.


4T1 Murine Mammary Carcinoma Cells Enhance Macrophage-Mediated Innate Inflammatory Responses.

Madera L, Greenshields A, Coombs MR, Hoskin DW - PLoS ONE (2015)

4T1- and LLC-conditioned macrophages exhibit increased pro-inflammatory cytokine production in response to LPS.(A) C57BL/6 or (B) BALB/c BMDMs were cultured in DMEM alone or in the presence of culture medium conditioned by 4T1 mammary carcinoma, E0771 mammary carcinoma, ID8 ovarian carcinoma, or LLC cells for 24 h. Medium conditioned by HC11 normal mouse epithelial cells was used as a negative control. BMDMs were then washed with PBS and stimulated with 100 ng/ml LPS for 4 h, then culture supernatants were collected and protein levels of cytokines TNFα, IL-6, CCL2, and IL-10 were determined by ELISA. Results are shown as the mean (± SEM) of at least 5 independent experiments. Statistical analyses were done by ANOVA, followed by Tukey’s multiple comparison tests; * p < 0.05, ** p < 0.01, *** p < 0.001.
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pone.0133385.g001: 4T1- and LLC-conditioned macrophages exhibit increased pro-inflammatory cytokine production in response to LPS.(A) C57BL/6 or (B) BALB/c BMDMs were cultured in DMEM alone or in the presence of culture medium conditioned by 4T1 mammary carcinoma, E0771 mammary carcinoma, ID8 ovarian carcinoma, or LLC cells for 24 h. Medium conditioned by HC11 normal mouse epithelial cells was used as a negative control. BMDMs were then washed with PBS and stimulated with 100 ng/ml LPS for 4 h, then culture supernatants were collected and protein levels of cytokines TNFα, IL-6, CCL2, and IL-10 were determined by ELISA. Results are shown as the mean (± SEM) of at least 5 independent experiments. Statistical analyses were done by ANOVA, followed by Tukey’s multiple comparison tests; * p < 0.05, ** p < 0.01, *** p < 0.001.
Mentions: To assess the inflammatory phenotype of tumor-conditioned macrophages, BMDMs were exposed to tumor-conditioned media for 24 h and then stimulated with 100 ng/ml LPS from Escherichia coli for 4 h. As expected, LPS stimulation of non-conditioned BMDMs from C57BL/6 or BALB/c mice resulted in the production of pro-inflammatory cytokines tumor necrosis factor α (TNFα) and IL-6, macrophage chemokine CCL2, and anti-inflammatory cytokine IL-10 (Fig 1A and 1B). Surprisingly, C57BL/6 BMDMs cultured with 4T1 mouse mammary carcinoma-conditioned medium exhibited a 2–3 fold increase in the production of TNFα, IL-6, and CCL2 in the presence of LPS (Fig 1A). Lewis lung carcinoma (LLC)-conditioned BMDMs also showed a significant increase in TNFα and CCL2 levels in response to LPS. ID8 ovarian carcinoma-conditioned BMDMs demonstrated a modest increase in TNFα and CCL2 following LPS stimulation, although this effect was not statistically significant. In contrast, none of these tumor-conditioned media affected LPS-stimulated IL-10 production by BMDMs when compared to non-conditioned BMDMs. Neither E0771 mouse mammary carcinoma-conditioned medium nor control medium conditioned with HC11 mouse mammary epithelial cells affected the LPS response of BMDMs. Similar trends were observed in macrophages from BALB/c mice; 4T1-conditioned Balb/c BMDMs showed a striking increase in TNFα, IL-6, and CCL2 production in response to LPS, while IL-10 levels remained unaffected (Fig 1B), demonstrating that this effect was not mouse strain-specific. TNFα and IL-6 were not detected in the tumor-conditioned media nor in the supernatants of conditioned BMDMs, prior to LPS stimulation, indicating that the tumor-conditioning step itself did not elicit an inflammatory response (data not shown). These results indicate that secreted factors of 4T1 murine mammary carcinomas and LLC can potentiate macrophage inflammatory cytokine production in response to LPS. Furthermore, this increased sensitivity is selective since TNFα, IL-6, and CCL2 production was increased while anti-inflammatory cytokine IL-10 production was unaffected. Subsequent experiments employed 4T1-conditioned C56BL/6 BMDMs because the effect was most striking in these cells.

Bottom Line: BMDMs exposed to 4T1 mammary carcinoma-conditioned medium demonstrated enhanced production of pro-inflammatory cytokines tumor necrosis factor α, interleukin-6, and CCL2 in response to lipopolysaccharide (LPS) while production of interleukin-10 remained unchanged.The increased LPS-induced production of pro-inflammatory cytokines was transient and correlated with enhanced cytokine production in response to other Toll-like receptor agonists, including peptidoglycan and flagellin.Furthermore, peritoneal macrophages obtained from 4T1 tumor-bearing mice displayed enhanced pro-inflammatory cytokine production in response to LPS.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology & Immunology, Dalhousie University, Halifax, Nova Scotia, Canada.

ABSTRACT
Tumor progression and the immune response are intricately linked. While it is known that cancers alter macrophage inflammatory responses to promote tumor progression, little is known regarding how cancers affect macrophage-dependent innate host defense. In this study, murine bone-marrow-derived macrophages (BMDM) were exposed to murine carcinoma-conditioned media prior to assessment of the macrophage inflammatory response. BMDMs exposed to 4T1 mammary carcinoma-conditioned medium demonstrated enhanced production of pro-inflammatory cytokines tumor necrosis factor α, interleukin-6, and CCL2 in response to lipopolysaccharide (LPS) while production of interleukin-10 remained unchanged. The increased LPS-induced production of pro-inflammatory cytokines was transient and correlated with enhanced cytokine production in response to other Toll-like receptor agonists, including peptidoglycan and flagellin. In addition, 4T1-conditioned BMDMs exhibited strengthened LPS-induced nitric oxide production and enhanced phagocytosis of Escherichia coli. 4T1-mediated augmentation of macrophage responses to LPS was partially dependent on the NFκB pathway, macrophage-colony stimulating factor, and actin polymerization, as well as the presence of 4T1-secreted extracellular vesicles. Furthermore, peritoneal macrophages obtained from 4T1 tumor-bearing mice displayed enhanced pro-inflammatory cytokine production in response to LPS. These results suggest that uptake of 4T1-secreted factors and actin-mediated ingestion of 4T1-secreted exosomes by macrophages cause a transient enhancement of innate inflammatory responses. Mammary carcinoma-mediated regulation of innate immunity may have significant implications for our understanding of host defense and cancer progression.

No MeSH data available.


Related in: MedlinePlus