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Increased expression of surface CD44 in hypoxia-DCs skews helper T cells toward a Th2 polarization.

Yang M, Liu Y, Ren G, Shao Q, Gao W, Sun J, Wang H, Ji C, Li X, Zhang Y, Qu X - Sci Rep (2015)

Bottom Line: However, the underlying mechanisms still remain largely unknown.In this study, we found the over-expression of surface CD44 in DCs was involved in this process via ligand binding.Moreover, KIF2A expression was found negatively regulated by HIF-1α in hypoxic microenvironment.

View Article: PubMed Central - PubMed

Affiliation: Institute of Basic Medical Sciences, Qilu Hospital, Shandong University, Jinan, 250012, Shandong, China.

ABSTRACT
A low partial oxygen pressure (hypoxia) occurs in many pathological environments, such as solid tumors and inflammatory lesions. Understanding the cellular response to hypoxic stress has broad implications for human diseases. As we previously reported, hypoxia significantly altered dendritic cells (DCs) to a DC2 phenotype and promoted a Th2 polarization of naïve T cells with increased IL-4 production. However, the underlying mechanisms still remain largely unknown. In this study, we found the over-expression of surface CD44 in DCs was involved in this process via ligand binding. Further investigation showed hypoxia could reduce the surface expression of membrane type 1 metalloprotease (MT1-MMP) via down-regulating the kinesin-like protein KIF2A, which subsequently alleviated the shedding of CD44 from DCs. Moreover, KIF2A expression was found negatively regulated by HIF-1α in hypoxic microenvironment. These results suggest a previously uncharacterized mechanism by which hypoxia regulates the function of DCs via KIF2A/MT1-MMP/CD44 axis, providing critical information to understand the immune response under hypoxia.

No MeSH data available.


Related in: MedlinePlus

MT1-MMP was involved in regulating the surface expression of CD44 in LPS induced mature DCs.(a) MT1-MMP mRNA expression in mDCs cultured under normoxia (N) or hypoxia (H) was evaluated by qRT-PCR. Results are shown as fold changes relative to CD44 mRNA levels in normoxic mDCs and represent the average of three independent experiments. (b) Micrographs of mature DCs stained for endogenous MT1-MMP using specific primary and FITC-labeled secondary antibody, and stained for nucleus using DAPI. The location of MT1-MMP was detected using immunofluorescence microscopy (×400). (c) Relative fluorescence intensity of MT1-MMP in mDCs under normoxia (N) and hypoxia (H) was measured by Image J software and results are expressed as fold relative to MT1-MMP expression in normoxic mDCs (equal to 1). Data are expressed as the mean ± SD of three independent experiments. (d) Expression of MT1-MMP protein in mDCs was detected by western blotting. Total proteins prepared from normoxic (N) or hypoxic (H) mDCs were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-MT1-MMP mAb or by parallel with β-actin antibody as a loading control. (e,f) imDCs were matured with LPS in the absence or presence of anti-MT1-MMP mAb for 48 hours. (e) Cell samples were immunostained and analyzed by flow cytometry. The fluorescence mean of surface stained CD44 on control IgG treated mDCs or anti-MT1-MMP treated mDCs are presented. (f) Cell culture supernatants were harvested from each group and the shed CD44 protein was measured using the ELISA kit. Data are expressed as the mean ± SD of four independent experiments in Fig. 2e,f. *P < 0.05.
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f2: MT1-MMP was involved in regulating the surface expression of CD44 in LPS induced mature DCs.(a) MT1-MMP mRNA expression in mDCs cultured under normoxia (N) or hypoxia (H) was evaluated by qRT-PCR. Results are shown as fold changes relative to CD44 mRNA levels in normoxic mDCs and represent the average of three independent experiments. (b) Micrographs of mature DCs stained for endogenous MT1-MMP using specific primary and FITC-labeled secondary antibody, and stained for nucleus using DAPI. The location of MT1-MMP was detected using immunofluorescence microscopy (×400). (c) Relative fluorescence intensity of MT1-MMP in mDCs under normoxia (N) and hypoxia (H) was measured by Image J software and results are expressed as fold relative to MT1-MMP expression in normoxic mDCs (equal to 1). Data are expressed as the mean ± SD of three independent experiments. (d) Expression of MT1-MMP protein in mDCs was detected by western blotting. Total proteins prepared from normoxic (N) or hypoxic (H) mDCs were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-MT1-MMP mAb or by parallel with β-actin antibody as a loading control. (e,f) imDCs were matured with LPS in the absence or presence of anti-MT1-MMP mAb for 48 hours. (e) Cell samples were immunostained and analyzed by flow cytometry. The fluorescence mean of surface stained CD44 on control IgG treated mDCs or anti-MT1-MMP treated mDCs are presented. (f) Cell culture supernatants were harvested from each group and the shed CD44 protein was measured using the ELISA kit. Data are expressed as the mean ± SD of four independent experiments in Fig. 2e,f. *P < 0.05.

Mentions: Shedding of CD44 from cell surface is closely controlled by MT1-MMP32, we therefore explored whether hypoxia regulated CD44 expression in DCs through MT1-MMP. Firstly, the effect of hypoxia on MT1-MMP expression was investigated. As expected, hypoxic mDCs expressed a lower level of MT1-MMP compared with the normoxic mDCs validated by real-time PCR (Fig. 2a). Subsequently, we confirmed the appearance of MT1-MMP throughout the cytoplasm with high concentrations in the periphery or the leading edge of spontaneously migrating cells by immunofluorescence microscopy and observed relatively weaker expression of MT1-MMP in hypoxic mDCs (Fig. 2b,c). Also further confirmation was acquired by western blotting results (Fig. 2d).


Increased expression of surface CD44 in hypoxia-DCs skews helper T cells toward a Th2 polarization.

Yang M, Liu Y, Ren G, Shao Q, Gao W, Sun J, Wang H, Ji C, Li X, Zhang Y, Qu X - Sci Rep (2015)

MT1-MMP was involved in regulating the surface expression of CD44 in LPS induced mature DCs.(a) MT1-MMP mRNA expression in mDCs cultured under normoxia (N) or hypoxia (H) was evaluated by qRT-PCR. Results are shown as fold changes relative to CD44 mRNA levels in normoxic mDCs and represent the average of three independent experiments. (b) Micrographs of mature DCs stained for endogenous MT1-MMP using specific primary and FITC-labeled secondary antibody, and stained for nucleus using DAPI. The location of MT1-MMP was detected using immunofluorescence microscopy (×400). (c) Relative fluorescence intensity of MT1-MMP in mDCs under normoxia (N) and hypoxia (H) was measured by Image J software and results are expressed as fold relative to MT1-MMP expression in normoxic mDCs (equal to 1). Data are expressed as the mean ± SD of three independent experiments. (d) Expression of MT1-MMP protein in mDCs was detected by western blotting. Total proteins prepared from normoxic (N) or hypoxic (H) mDCs were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-MT1-MMP mAb or by parallel with β-actin antibody as a loading control. (e,f) imDCs were matured with LPS in the absence or presence of anti-MT1-MMP mAb for 48 hours. (e) Cell samples were immunostained and analyzed by flow cytometry. The fluorescence mean of surface stained CD44 on control IgG treated mDCs or anti-MT1-MMP treated mDCs are presented. (f) Cell culture supernatants were harvested from each group and the shed CD44 protein was measured using the ELISA kit. Data are expressed as the mean ± SD of four independent experiments in Fig. 2e,f. *P < 0.05.
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Related In: Results  -  Collection

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f2: MT1-MMP was involved in regulating the surface expression of CD44 in LPS induced mature DCs.(a) MT1-MMP mRNA expression in mDCs cultured under normoxia (N) or hypoxia (H) was evaluated by qRT-PCR. Results are shown as fold changes relative to CD44 mRNA levels in normoxic mDCs and represent the average of three independent experiments. (b) Micrographs of mature DCs stained for endogenous MT1-MMP using specific primary and FITC-labeled secondary antibody, and stained for nucleus using DAPI. The location of MT1-MMP was detected using immunofluorescence microscopy (×400). (c) Relative fluorescence intensity of MT1-MMP in mDCs under normoxia (N) and hypoxia (H) was measured by Image J software and results are expressed as fold relative to MT1-MMP expression in normoxic mDCs (equal to 1). Data are expressed as the mean ± SD of three independent experiments. (d) Expression of MT1-MMP protein in mDCs was detected by western blotting. Total proteins prepared from normoxic (N) or hypoxic (H) mDCs were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-MT1-MMP mAb or by parallel with β-actin antibody as a loading control. (e,f) imDCs were matured with LPS in the absence or presence of anti-MT1-MMP mAb for 48 hours. (e) Cell samples were immunostained and analyzed by flow cytometry. The fluorescence mean of surface stained CD44 on control IgG treated mDCs or anti-MT1-MMP treated mDCs are presented. (f) Cell culture supernatants were harvested from each group and the shed CD44 protein was measured using the ELISA kit. Data are expressed as the mean ± SD of four independent experiments in Fig. 2e,f. *P < 0.05.
Mentions: Shedding of CD44 from cell surface is closely controlled by MT1-MMP32, we therefore explored whether hypoxia regulated CD44 expression in DCs through MT1-MMP. Firstly, the effect of hypoxia on MT1-MMP expression was investigated. As expected, hypoxic mDCs expressed a lower level of MT1-MMP compared with the normoxic mDCs validated by real-time PCR (Fig. 2a). Subsequently, we confirmed the appearance of MT1-MMP throughout the cytoplasm with high concentrations in the periphery or the leading edge of spontaneously migrating cells by immunofluorescence microscopy and observed relatively weaker expression of MT1-MMP in hypoxic mDCs (Fig. 2b,c). Also further confirmation was acquired by western blotting results (Fig. 2d).

Bottom Line: However, the underlying mechanisms still remain largely unknown.In this study, we found the over-expression of surface CD44 in DCs was involved in this process via ligand binding.Moreover, KIF2A expression was found negatively regulated by HIF-1α in hypoxic microenvironment.

View Article: PubMed Central - PubMed

Affiliation: Institute of Basic Medical Sciences, Qilu Hospital, Shandong University, Jinan, 250012, Shandong, China.

ABSTRACT
A low partial oxygen pressure (hypoxia) occurs in many pathological environments, such as solid tumors and inflammatory lesions. Understanding the cellular response to hypoxic stress has broad implications for human diseases. As we previously reported, hypoxia significantly altered dendritic cells (DCs) to a DC2 phenotype and promoted a Th2 polarization of naïve T cells with increased IL-4 production. However, the underlying mechanisms still remain largely unknown. In this study, we found the over-expression of surface CD44 in DCs was involved in this process via ligand binding. Further investigation showed hypoxia could reduce the surface expression of membrane type 1 metalloprotease (MT1-MMP) via down-regulating the kinesin-like protein KIF2A, which subsequently alleviated the shedding of CD44 from DCs. Moreover, KIF2A expression was found negatively regulated by HIF-1α in hypoxic microenvironment. These results suggest a previously uncharacterized mechanism by which hypoxia regulates the function of DCs via KIF2A/MT1-MMP/CD44 axis, providing critical information to understand the immune response under hypoxia.

No MeSH data available.


Related in: MedlinePlus