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. 2015 Feb 23;21(1):26-37.
doi: 10.2119/molmed.2014.00219.

A Coding Variant of ANO10, Affecting Volume Regulation of Macrophages, Is Associated With Borrelia Seropositivity

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A Coding Variant of ANO10, Affecting Volume Regulation of Macrophages, Is Associated With Borrelia Seropositivity

Christian Hammer et al. Mol Med. .
Free PMC article

Abstract

In a first genome-wide association study (GWAS) approach to anti-Borrelia seropositivity, we identified two significant single nucleotide polymorphisms (SNPs) (rs17850869, P = 4.17E-09; rs41289586, P = 7.18E-08). Both markers, located on chromosomes 16 and 3, respectively, are within or close to genes previously connected to spinocerebellar ataxia. The risk SNP rs41289586 represents a missense variant (R263H) of anoctamin 10 (ANO10), a member of a protein family encoding Cl(-) channels and phospholipid scramblases. ANO10 augments volume-regulated Cl(-) currents (IHypo) in Xenopus oocytes, HEK293 cells, lymphocytes and macrophages and controls volume regulation by enhancing regulatory volume decrease (RVD). ANO10 supports migration of macrophages and phagocytosis of spirochetes. The R263H variant is inhibitory on IHypo, RVD and intracellular Ca(2+) signals, which may delay spirochete clearance, thereby sensitizing adaptive immunity. Our data demonstrate for the first time that ANO10 has a central role in innate immune defense against Borrelia infection.

Figures

Figure 1
Figure 1
Manhattan plot of genome-wide association analysis. The red horizontal line designates the threshold for genome-wide significance, corrected for number of tested SNPs.
Figure 2
Figure 2
ANO10 but not R263H-ANO10 generates volume-activated whole cell currents in Xenopus oocytes. (A) Current–voltage relationships of whole cell currents activated by cell swelling (IHypo, 50 % reduced extracellular osmolarity) in Xenopus oocytes. R263H-ANO10 does not produce IHypo. (B) Current overlay (voltage clamp [Vc] = ±100 mV) demonstrates typical time dependent inactivation of IHypo. (C) IHypo in oocytes expressing (left to right, respectively): AQP1 and ANO10; AQP1 and ANO10-R263H; or AQP1, ANO10, and ANO10-R263H. Coexpression of ANO10-R263H suppressed currents produced by wt ANO10. (D) Oocyte bursting after exposure to hypotonic bath solution. Fraction of burst oocytes was reduced by expression of ANO10. Oocytes survived in the absence of AQP1. (E) Summary of whole cell currents activated by Hypo and the PLA2-activator melittin (100 nmol/L). (F) Summary of time-dependent activation of whole cell currents in cells expressing ANO10, LRRC8A or coexpressing both. All oocytes expressed AQP1. Mean ± SEM (number of oocytes); *significant activation by Hypo (paired t test); #significant difference (by unpaired t test) when compared with ANO10 alone (E) or with ANO10 plus AQP1 (ANO10+AQP1) (F).
Figure 3
Figure 3
ANO10 affects volume-activated whole cell currents in HEK293 cells. (A) Whole cell currents (voltage clamp [Vc] = ± 100 mV) activated by cell swelling (IHypo, 33% reduced extracellular osmolarity) in ANO10-expressing cells. (B) Swelling induced currents (IHypo) in ANO10-expressing cells relative to mock transfected cells and inhibition by NPPB (50 μmol/L), NS3728 (5 μmol/L) and TinhAO1 (20 μmol/L). (C) Current–voltage (I–V) curves indicating loss of IHypo with complete elimination of Ca2+ and preincubation with BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; 50 μmol/L, 30 min). (D) Regulation of cell volume in the presence of Hypo (regulatory volume decrease, RVD) in mock-transfected cells or cells overexpressing ANO10 (flow cytometry). (E) RVD in mock-transfected cells or cells overexpressing ANO10 and inhibition by NPPB, NS3728 and TinhAO1. (F) Reshrinkage of cells exposed to hypotonic bath solution (RVD), measured in single cells loaded with calcein. (G) Comparison of RVD (measured by calcein fluorescence) obtained in mock-transfected and ANO10-overexpressing cells. Mean ± SEM (number of cells); *significant inhibition (paired t test); #significant difference to mock (unpaired t test).
Figure 4
Figure 4
R263H inhibits volume regulation, IHypo and intracellular Ca2+ signaling in HEK293 cells. (A) Whole cell currents (voltage clamp [Vc] = ± 100 mV) activated by cell swelling (IHypo, 33 % reduced extracellular osmolarity) in cells expressing ANO10 and R263H-ANO10 (R263H). (B) Current–voltage relationships for IHypo and inhibition of IHypo by removal of Cl from the extracellular bath solution (5Cl). (C) Regulation of cell volume in the presence of Hypo (regulatory volume decrease, RVD) in cells expressing ANO10 or R263H (flow cytometry). (D) Effect of cell swelling on intracellular [Ca2+] in cells expressing ANO10 or R263H or mock transfected cells, as measured by the Ca2+ sensor GCAMP2. (E) Summary of the effects of cell swelling on [Ca2+]i (485/405 fluorescence emission ratio) in ANO10 and R263H expressing cells. (F) Collected recordings of the effects of cell swelling on [Ca2+]i, measured by Fura2. (G) Collected recordings of the effects of ER-store emptying by cyclopiazonic acid (CPA; 10 μmol/L) on [Ca2+]i, measured by Fura2. (H) Confocal images of cells expressing ANO10 or R263H suggesting weak membrane expression. (I) Live staining of ANO10-GFP (green) and ER (ER-tracker; red) suggesting ER localization of ANO10. (J) Membrane biotinylation of cells expressing ANO10 or R263H, suggesting low membrane expression of ANO10, which is even reduced for R263H. Mean ± SEM (number of experiments); #significant difference when compared with mock (analysis of variance [ANOVA]); §significant difference when compared with ANO10 (ANOVA). Bar = 20 μm. Numbers are given in the graph in parenthesis. z, z-scan, side view; DIC, differential interference contrast.
Figure 5
Figure 5
Role of ANO10 for volume regulation in macrophages. (A) RT-PCR analysis of anoctamin expression in THP-1 macrophages. (B) Western blot indicating knockdown of ANO10-expression by siRNA. (C) ANO10 (green) and peripheral actin (rhodamin-phalloidin) of THP-1 cells suggesting dominant intracellular location of ANO10. (D) Summary trace for reshrinkage of cells exposed to hypotonic bath solution (RVD), measured in single cells loaded with calcein. RVD was abolished after siRNA-knockdown of ANO10. (E) Summary of RVD measured by absolute fluorescence change. (F) I/V curves indicating reduced IHypo in R263H-expressing cells. (G) Migration assay in Boyden chambers. MCP-1 induced migration was inhibited by siRNA knockdown of ANO10 and anoctamin inhibitors TinhAO1 (20 μmol/L), NPPB (50 μmol/L) or tannic acid (TA, 10 μmol/L). (H) THP-1 cells exposed to red-fluorescent cherry-labeled B. garinii. Accumulation of cytosolic fluorescence, indicating progressing phagocytosis of Borrelia by THP-1 cells. (I) Increase in fluorescence intensity as a measure of phagocytic activity. (J) Exposure of THP-1 cells to cherry-labeled B. garinii. (K) Release of TNFα upon exposure to B. garinii was not affected by siRNA-knockdown of ANO10. Mean ± SEM (number of cells or assays). #Significant difference when compared with scrambled, MCP-1 alone, mock or con (ANOVA); §significant increase in migration and phagocytosis, respectively (unpaired t test). M, marker; RT, reverse transcriptase; con, control; crbld: scrambled control RNA; siANO10: siRNA against ANO10. Bar = 20 μm.

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