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LRRK1 is critical in the regulation of B-cell responses and CARMA1-dependent NF-κB activation.

Morimoto K, Baba Y, Shinohara H, Kang S, Nojima S, Kimura T, Ito D, Yoshida Y, Maeda Y, Sarashina-Kida H, Nishide M, Hosokawa T, Kato Y, Hayama Y, Kinehara Y, Okuno T, Takamatsu H, Hirano T, Shima Y, Narazaki M, Kurosaki T, Toyofuku T, Kumanogoh A - Sci Rep (2016)

Bottom Line: Concomitantly, B cells lacking LRRK1 exhibited a profound defect in proliferation and survival upon BCR stimulation, which correlated with impaired BCR-mediated NF-κB activation and reduced expression of NF-κB target genes including Bcl-xL, cyclin D2, and NFATc1/αA.Furthermore, LRRK1 physically interacted and potently synergized with CARMA1 to enhance NF-κB activation.Our results reveal a critical role of LRRK1 in NF-κB signaling in B cells and the humoral immune response.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunopathology, World Premier International (WPI) Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan.

ABSTRACT
B-cell receptor (BCR) signaling plays a critical role in B-cell activation and humoral immunity. In this study, we discovered a critical function of leucine-rich repeat kinase 1 (LRRK1) in BCR-mediated immune responses. Lrrk1(-/-) mice exhibited altered B1a-cell development and basal immunoglobulin production. In addition, these mice failed to produce IgG3 antibody in response to T cell-independent type 2 antigen due to defects in IgG3 class-switch recombination. Concomitantly, B cells lacking LRRK1 exhibited a profound defect in proliferation and survival upon BCR stimulation, which correlated with impaired BCR-mediated NF-κB activation and reduced expression of NF-κB target genes including Bcl-xL, cyclin D2, and NFATc1/αA. Furthermore, LRRK1 physically interacted and potently synergized with CARMA1 to enhance NF-κB activation. Our results reveal a critical role of LRRK1 in NF-κB signaling in B cells and the humoral immune response.

No MeSH data available.


Related in: MedlinePlus

LRRK1 mediates BCR-dependent NF-κB activation.(a) BCR endocytosis of wild-type and Lrrk1−/− mice, monitored by flow cytometry at the indicated times after staining with biotinylated anti-IgM. Cells were incubated for 30 min on ice in the presence of biotinylated anti-IgM, and then incubated at 37 °C for 0, 5, 15, 30, or 60 min. The level of BCR remaining on the cell surface is presented as mean fluorescence intensity relative to that of the 0 min sample (defined as 100%). Results are representative of three similar experiments. (b) Phosphorylation status of Syk and PLCγ2 of wild-type and Lrrk1−/− B cells assessed by immunoblotting of whole-cell lysates obtained from B cells stimulated with anti-IgM. Data are representative of at least two independent experiments. (c) Ca2+ mobilization in response to stimulation with 10 μg/ml anti-IgM in wild-type and Lrrk1−/− splenocytes stained with anti-B220 (total B cell), anti-CD21, and anti-CD23 (FO, B220+ CD21loCD23hi; MZ, B220+ CD21hiCD23lo). Ca2+ flux was monitored by Indo-1AM imaging in the presence of 2 mM extracellular Ca2+, and all values are plotted as the FL5/FL4 fluorescence ratio (FL4 = 500–520 nm; FL5 = 400–420 nm). Data are representative of three independent experiments. (d,f) Activation status of NF-κB, NFAT, and MAPK signaling or induction of Bcl-xL, cyclin D2, and NFATc1/αA in wild-type and Lrrk1−/− B cells stimulated with anti-IgM, as determined by immunoblot analysis. Data are representative of three independent experiments. (e) Nuclear translocation of p65 after stimulation with anti-IgM in wild-type and Lrrk1−/− B cells assessed by immunoblot analysis. Lamin B and GAPDH serve as nuclear and cytosolic markers, respectively, and as loading controls. Data are representative of three independent experiments. (g) Quantitative RT-PCR analysis of Bcl-xL and cyclin D2 expression in B cells 6 h after stimulation with anti-IgM. GAPDH and β-actin were used as internal control for Bcl-xL and cyclin D2, respectively. Data are presented as means ± s.e.m. of two independent experiments. NS, not significant. *P < 0.05, **P < 0.01 (two-tailed unpaired Student’s t-test).
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f5: LRRK1 mediates BCR-dependent NF-κB activation.(a) BCR endocytosis of wild-type and Lrrk1−/− mice, monitored by flow cytometry at the indicated times after staining with biotinylated anti-IgM. Cells were incubated for 30 min on ice in the presence of biotinylated anti-IgM, and then incubated at 37 °C for 0, 5, 15, 30, or 60 min. The level of BCR remaining on the cell surface is presented as mean fluorescence intensity relative to that of the 0 min sample (defined as 100%). Results are representative of three similar experiments. (b) Phosphorylation status of Syk and PLCγ2 of wild-type and Lrrk1−/− B cells assessed by immunoblotting of whole-cell lysates obtained from B cells stimulated with anti-IgM. Data are representative of at least two independent experiments. (c) Ca2+ mobilization in response to stimulation with 10 μg/ml anti-IgM in wild-type and Lrrk1−/− splenocytes stained with anti-B220 (total B cell), anti-CD21, and anti-CD23 (FO, B220+ CD21loCD23hi; MZ, B220+ CD21hiCD23lo). Ca2+ flux was monitored by Indo-1AM imaging in the presence of 2 mM extracellular Ca2+, and all values are plotted as the FL5/FL4 fluorescence ratio (FL4 = 500–520 nm; FL5 = 400–420 nm). Data are representative of three independent experiments. (d,f) Activation status of NF-κB, NFAT, and MAPK signaling or induction of Bcl-xL, cyclin D2, and NFATc1/αA in wild-type and Lrrk1−/− B cells stimulated with anti-IgM, as determined by immunoblot analysis. Data are representative of three independent experiments. (e) Nuclear translocation of p65 after stimulation with anti-IgM in wild-type and Lrrk1−/− B cells assessed by immunoblot analysis. Lamin B and GAPDH serve as nuclear and cytosolic markers, respectively, and as loading controls. Data are representative of three independent experiments. (g) Quantitative RT-PCR analysis of Bcl-xL and cyclin D2 expression in B cells 6 h after stimulation with anti-IgM. GAPDH and β-actin were used as internal control for Bcl-xL and cyclin D2, respectively. Data are presented as means ± s.e.m. of two independent experiments. NS, not significant. *P < 0.05, **P < 0.01 (two-tailed unpaired Student’s t-test).

Mentions: Next, we sought to characterize potential signaling defects in Lrrk1−/− B cells. Because LRRK1 has been reported to regulate endosomal trafficking of EGFR by binding to Grb217, and BCR endocytosis also requires Grb225, we first tested the effect of LRRK1 on BCR internalization. In both wild-type and Lrrk1−/− B cells, BCR cross-linking led to a rapid decrease in the level of BCR remaining on the surface within 30 min after BCR stimulation similarly, indicating that LRRK1 is not critical for BCR internalization (Fig. 5a). We next examined the activation of various intracellular BCR signaling cascades in wild-type and Lrrk1−/− B cells. Tyrosine phosphorylation status of Syk and PLCγ2 did not differ significantly between wild-type and Lrrk1−/− B cells (Fig. 5b). In addition, the absence of LRRK1 did not affect the BCR-induced increase in intracellular Ca2+ in total B cell, which is mediated by PLCγ2 (Fig. 5c). This Ca2+ increase is larger in MZ B cells than in FO B cells26. Given that Lrrk1−/− mice have a higher percentage of MZ B cells than wild-type mice, we tested Ca2+ mobilization separately in MZ and FO B cells in order to accurately evaluate the Ca2+ response. LRRK1 did not contribute to Ca2+ flux in either subset (Fig. 5c). Taken together, these observations suggest that BCR-mediated proximal signaling is not significantly impaired in Lrrk1−/− B cells.


LRRK1 is critical in the regulation of B-cell responses and CARMA1-dependent NF-κB activation.

Morimoto K, Baba Y, Shinohara H, Kang S, Nojima S, Kimura T, Ito D, Yoshida Y, Maeda Y, Sarashina-Kida H, Nishide M, Hosokawa T, Kato Y, Hayama Y, Kinehara Y, Okuno T, Takamatsu H, Hirano T, Shima Y, Narazaki M, Kurosaki T, Toyofuku T, Kumanogoh A - Sci Rep (2016)

LRRK1 mediates BCR-dependent NF-κB activation.(a) BCR endocytosis of wild-type and Lrrk1−/− mice, monitored by flow cytometry at the indicated times after staining with biotinylated anti-IgM. Cells were incubated for 30 min on ice in the presence of biotinylated anti-IgM, and then incubated at 37 °C for 0, 5, 15, 30, or 60 min. The level of BCR remaining on the cell surface is presented as mean fluorescence intensity relative to that of the 0 min sample (defined as 100%). Results are representative of three similar experiments. (b) Phosphorylation status of Syk and PLCγ2 of wild-type and Lrrk1−/− B cells assessed by immunoblotting of whole-cell lysates obtained from B cells stimulated with anti-IgM. Data are representative of at least two independent experiments. (c) Ca2+ mobilization in response to stimulation with 10 μg/ml anti-IgM in wild-type and Lrrk1−/− splenocytes stained with anti-B220 (total B cell), anti-CD21, and anti-CD23 (FO, B220+ CD21loCD23hi; MZ, B220+ CD21hiCD23lo). Ca2+ flux was monitored by Indo-1AM imaging in the presence of 2 mM extracellular Ca2+, and all values are plotted as the FL5/FL4 fluorescence ratio (FL4 = 500–520 nm; FL5 = 400–420 nm). Data are representative of three independent experiments. (d,f) Activation status of NF-κB, NFAT, and MAPK signaling or induction of Bcl-xL, cyclin D2, and NFATc1/αA in wild-type and Lrrk1−/− B cells stimulated with anti-IgM, as determined by immunoblot analysis. Data are representative of three independent experiments. (e) Nuclear translocation of p65 after stimulation with anti-IgM in wild-type and Lrrk1−/− B cells assessed by immunoblot analysis. Lamin B and GAPDH serve as nuclear and cytosolic markers, respectively, and as loading controls. Data are representative of three independent experiments. (g) Quantitative RT-PCR analysis of Bcl-xL and cyclin D2 expression in B cells 6 h after stimulation with anti-IgM. GAPDH and β-actin were used as internal control for Bcl-xL and cyclin D2, respectively. Data are presented as means ± s.e.m. of two independent experiments. NS, not significant. *P < 0.05, **P < 0.01 (two-tailed unpaired Student’s t-test).
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f5: LRRK1 mediates BCR-dependent NF-κB activation.(a) BCR endocytosis of wild-type and Lrrk1−/− mice, monitored by flow cytometry at the indicated times after staining with biotinylated anti-IgM. Cells were incubated for 30 min on ice in the presence of biotinylated anti-IgM, and then incubated at 37 °C for 0, 5, 15, 30, or 60 min. The level of BCR remaining on the cell surface is presented as mean fluorescence intensity relative to that of the 0 min sample (defined as 100%). Results are representative of three similar experiments. (b) Phosphorylation status of Syk and PLCγ2 of wild-type and Lrrk1−/− B cells assessed by immunoblotting of whole-cell lysates obtained from B cells stimulated with anti-IgM. Data are representative of at least two independent experiments. (c) Ca2+ mobilization in response to stimulation with 10 μg/ml anti-IgM in wild-type and Lrrk1−/− splenocytes stained with anti-B220 (total B cell), anti-CD21, and anti-CD23 (FO, B220+ CD21loCD23hi; MZ, B220+ CD21hiCD23lo). Ca2+ flux was monitored by Indo-1AM imaging in the presence of 2 mM extracellular Ca2+, and all values are plotted as the FL5/FL4 fluorescence ratio (FL4 = 500–520 nm; FL5 = 400–420 nm). Data are representative of three independent experiments. (d,f) Activation status of NF-κB, NFAT, and MAPK signaling or induction of Bcl-xL, cyclin D2, and NFATc1/αA in wild-type and Lrrk1−/− B cells stimulated with anti-IgM, as determined by immunoblot analysis. Data are representative of three independent experiments. (e) Nuclear translocation of p65 after stimulation with anti-IgM in wild-type and Lrrk1−/− B cells assessed by immunoblot analysis. Lamin B and GAPDH serve as nuclear and cytosolic markers, respectively, and as loading controls. Data are representative of three independent experiments. (g) Quantitative RT-PCR analysis of Bcl-xL and cyclin D2 expression in B cells 6 h after stimulation with anti-IgM. GAPDH and β-actin were used as internal control for Bcl-xL and cyclin D2, respectively. Data are presented as means ± s.e.m. of two independent experiments. NS, not significant. *P < 0.05, **P < 0.01 (two-tailed unpaired Student’s t-test).
Mentions: Next, we sought to characterize potential signaling defects in Lrrk1−/− B cells. Because LRRK1 has been reported to regulate endosomal trafficking of EGFR by binding to Grb217, and BCR endocytosis also requires Grb225, we first tested the effect of LRRK1 on BCR internalization. In both wild-type and Lrrk1−/− B cells, BCR cross-linking led to a rapid decrease in the level of BCR remaining on the surface within 30 min after BCR stimulation similarly, indicating that LRRK1 is not critical for BCR internalization (Fig. 5a). We next examined the activation of various intracellular BCR signaling cascades in wild-type and Lrrk1−/− B cells. Tyrosine phosphorylation status of Syk and PLCγ2 did not differ significantly between wild-type and Lrrk1−/− B cells (Fig. 5b). In addition, the absence of LRRK1 did not affect the BCR-induced increase in intracellular Ca2+ in total B cell, which is mediated by PLCγ2 (Fig. 5c). This Ca2+ increase is larger in MZ B cells than in FO B cells26. Given that Lrrk1−/− mice have a higher percentage of MZ B cells than wild-type mice, we tested Ca2+ mobilization separately in MZ and FO B cells in order to accurately evaluate the Ca2+ response. LRRK1 did not contribute to Ca2+ flux in either subset (Fig. 5c). Taken together, these observations suggest that BCR-mediated proximal signaling is not significantly impaired in Lrrk1−/− B cells.

Bottom Line: Concomitantly, B cells lacking LRRK1 exhibited a profound defect in proliferation and survival upon BCR stimulation, which correlated with impaired BCR-mediated NF-κB activation and reduced expression of NF-κB target genes including Bcl-xL, cyclin D2, and NFATc1/αA.Furthermore, LRRK1 physically interacted and potently synergized with CARMA1 to enhance NF-κB activation.Our results reveal a critical role of LRRK1 in NF-κB signaling in B cells and the humoral immune response.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunopathology, World Premier International (WPI) Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan.

ABSTRACT
B-cell receptor (BCR) signaling plays a critical role in B-cell activation and humoral immunity. In this study, we discovered a critical function of leucine-rich repeat kinase 1 (LRRK1) in BCR-mediated immune responses. Lrrk1(-/-) mice exhibited altered B1a-cell development and basal immunoglobulin production. In addition, these mice failed to produce IgG3 antibody in response to T cell-independent type 2 antigen due to defects in IgG3 class-switch recombination. Concomitantly, B cells lacking LRRK1 exhibited a profound defect in proliferation and survival upon BCR stimulation, which correlated with impaired BCR-mediated NF-κB activation and reduced expression of NF-κB target genes including Bcl-xL, cyclin D2, and NFATc1/αA. Furthermore, LRRK1 physically interacted and potently synergized with CARMA1 to enhance NF-κB activation. Our results reveal a critical role of LRRK1 in NF-κB signaling in B cells and the humoral immune response.

No MeSH data available.


Related in: MedlinePlus