Adenosine deaminase 2 regulates the activation of the toll-like receptor 9 in response to nucleic acids
Liang Dong2,3, Wenwen Luo2, Skaldin Maksym2,4, Simon C. Robson5, Andrey V. Zavialov1,2()
. International Center for Aging and Cancer (ICAC), Hainan Medical University, Haikou 571199, China . Guangzhou Institute of Pediatrics, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou 510623, China . Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China . Joint Biotechnology Laboratory, University of Turku, Turku 20520, Finland . Center for Inflammation Research, Departments of Anesthesia and Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston 02215, USA
Human cells contain two types of adenosine deaminases (ADA) each with unique properties: ADA1, which is present in all cells where it modulates intracellular functions and extracellular signaling, and ADA2, which is secreted by immune cells. The exact intracellular functions of ADA2 remain undetermined and less defined than those of ADA1. ADA2 has distinct characteristics, such as low adenosine affinity, heparin-binding ability, and putative lysosomal entry. Here, we confirm that ADA2 is a lysosomal protein that binds toll-like receptor 9 (TLR9) agonists, specifically CpG oligodeoxynucleotides (CpG ODNs). We show that interferon-alpha (IFN-α) is secreted in response to TLR9 activation by CpG ODNs and natural DNA and markedly increases when ADA2 expression is downregulated in plasmacytoid dendritic cells (pDCs). Additionally, the pretreatment of pDCs with RNA further stimulates IFN-α secretion by pDCs after activation with CpG ODNs. Our findings indicate that ADA2 regulates TLR9 responses to DNA in activated pDCs. In conclusion, decreasing ADA2 expression or blocking it with specific oligonucleotides can enhance IFN-α secretion from pDCs, improving immune responses against intracellular infections and cancer.
Just Accepted Date: 28 June 2024Online First Date: 31 July 2024Issue Date: 29 October 2024
Cite this article:
Liang Dong,Wenwen Luo,Skaldin Maksym, et al. Adenosine deaminase 2 regulates the activation of the toll-like receptor 9 in response to nucleic acids[J]. Front. Med.,
2024, 18(5): 814-830.
Fig.1 ADA2 binds to dsDNA. (A) Gel-shift analysis of ADA2 (μg/mL) binding to plasmid DNA (μg/mL) in the presence of increasing salt concentration (mM). (B) Gel-shift analysis of ADA2 (μg/mL) binding to plasmid DNA (μg/mL) in the presence of different concentrations of DNase I (IU/mL). (C) Gel-shift analysis of ADA2 H88G and ADA2 (μg/mL) binding to plasmid DNA (μg/mL) in the presence or absence of different concentrations of ODN 2006 PTO (µM). (D) ADA2 binding to E. coli DNA was analyzed by a DNA ELISA assay. Each dot on the graph represents the mean value and standard deviation of three independent measurements.
Fig.2 Inhibition of the binding of ADA2 to ODN 2006 PTO biotin. (A) Inhibition of the binding of ADA2 to ODN 2006 PTO biotin with class B ODN 2006 PTO, ODN 2006 GC PTO, class A ODN 2216 PTO, and ODN 2336 PTO. (B) The inhibition of binding of ADA2 to ODN 2006 PTO biotin with class B ODN BW006 PTO, ODN D-SL01 GC PTO, class C ODN 2395 PTO, ODN M362 PTO, and ODN D-SL03 PTO. (C) ADA2 binding to ODNs PTO (ODN 2006 PTO and ODN 2216 PTO) and ODNs PD (ODN 2006 PD and ODN 2006-2006 PD). (D) ADA2 binding to ODN 2006 PD in a buffer with different pH values. Each dot on the graph represents the mean value and standard deviation of three independent measurements.
Fig.3 Inhibition of binding between ADA2 and ODN 2006 PD biotin. (A) Inhibition of binding between ADA2 and ODN 2006 PD biotin with class B (ODN 2006 PD), class A (ODN 2216 PD, ODN 2336 PD), or C (ODN 2395 PD) ODNs. (B) Inhibition of binding between ADA2 and ODN 2006 PD biotin with ODN 2006 PD, ODN GC2006 PD, ODN 2006 C-Met PD, poly T [24], or ODN 2006 Reverse PD, ODN 2006 (T changed to C) PD. (C) Inhibition of ADA2 binding to ODN 2006 PD biotin with modified class A ODN 2336 PD. (D) Inhibition of binding between ADA2 and ODN 2006 PD biotin with poly T of varying length. (E) Inhibition of binding between ADA2 and ODN 2006 PD biotin with RNA PTO and RNA PD. (F) The sequences of oligonucleotides. Each dot on the graph represents the mean value and standard deviation of three independent measurements.
Fig.4 ADA2 binds to ODN 2006 PTO in macrophage lysosomes. (A, B) ADA2 levels in cell lysates (A) and culture medium (B) of macrophages incubated with 0.5 μmol/L ODN PTO for 24 h. Monocytes were isolated from PBMCs and differentiated into macrophages with 40 ng/mL GM-CSF for 6 days. Macrophages were washed twice with PBS before lysis to remove the secreted ADA2. ADA2 levels were measured by ELISA. Error bars represent the standard deviation from four independent measurements. (C–F) Confocal microscopy of macrophages differentiated with 40 ng/mL GM-CSF for 6 days and treated with 0.5 μmol/L ODNs for 24 h. Cells were fixed and stained for ADA2 (blue), lysosomal marker LAMP2 (red), nuclear marker DAPI (white), and endoplasmic reticulum marker ER-tracker (red). ODNs were ODN 2006 PTO (D), ODN 2006 G5 PD (E), and ODN 2006 PTO FITC (F). Untreated cells are shown in (C).
Fig.5 Activation of pDCs by three classes of ODNs with PTO: (A) 1 μmol/L ODN with PTO was added to pDCs (0.0125 × 106 cells/mL) on day 1, and the amount of IFN-α (A) and IL-8 (B) released from the cells was analyzed by ELISA in 48 h. Class A ODNs are shown in blue, class B in red, and class C in green. (C, D) Class A ODN 2216 with PTO and class B ODN 2006 with PTO (1 μM) were added to pDCs (0.0125 × 106 cells/mL) on day 1 or day 2, and the amount of IFN-α (C) or IL-8 (D) released from the cells was analyzed by ELISA on the following day. (E, F) ODN with PTO (1 μM) was added to pDCs (0.05 × 106 cells/mL) on day 1 (E) or day 2 (F), and the amount of IFN-α released from the cells was analyzed by ELISA on the following day. Class A ODNs are shown in blue, class B in red, and class C in green. The error bars represent the standard deviation, and the results were obtained from four independent replicates.
Fig.6 (A) Human pDCs were isolated from PBMCs and cultured with 10 ng/mL IL-3 in an eight-well chamber. After 24 h, pDCs that were attached to the plastic were washed, fixed, and stained with anti-ADA2 antibodies (blue), ER-tracker (red) for endoplasmic reticulum staining, and DAPI (white) for nucleus staining. (B) Human pDCs were cultured with 10 ng/mL IL-3 in the presence of 0.5 μM ODN 2006 PTO FITC in an 8-well chamber. After 24 h, pDCs that were attached to the plastic were washed, fixed, and stained with anti-LAMP2 antibodies (red), anti-ADA2 antibodies (blue), and DAPI (white). (C) Colocalization of PTO-modified ODN 2006 with FITC, ADA2, and the lysosomal marker LAMP2.
Fig.7 Effects of knocking down or blocking of ADA2 during TLR9 activation in pDCs. In all experiments, human pDCs were isolated from PBMCs and cultured in the presence of 10 ng/mL IL-3. (A–D) pDCs (0.0125 × 106 cells/mL) were transfected with siRNAs using Lipofectamine RNAiMax transfection reagent on day 1. On day 2, PTO-modified ODNs (1 μmol/L) were added to the cells. The amount of IFN-α (A, C) and IL-8 (B, D) in the cell culture medium was analyzed on day 3 using ELISA. (E, F) The knockdown of ADA2 expression with siRNA in pDC promoted TLR9 activation by dsDNA. The cells (0.0125 × 106 cells/mL) were transfected with control (siRNA control) and specific (siRNA ADA2) siRNA. After 24 h, the cells were transfected with (E) E. coli genomic DNA and (F) genomic DNA from human THP1 cells using Lipofectamine 3000. (G) Analysis of ADA2 in the culture medium 2 days after the siRNA transfection of PMA-activated THP1 cells with ELISA. (H) Blocking ADA2 with poly U promoted TLR9 activation by PTO-modified ODN 2006. Human pDCs (0.02 × 106 cells/mL) were transfected with 0.2 µg Poly U or Poly U21 using Lipofectamine RNAiMax transfection reagent on day 1. On day 2, PTO-modified ODN 2006 (0.5 μmol/L) was added to the cells. The amount of IFN-α in the cell culture medium was analyzed on day 3 using ELISA [11]. The error bars represent the standard deviation, and the results were obtained from four independent replicates. In all experiments, the difference between the control siRNA and siRNA ADA2 was statistically significant (P < 0.008).
Fig.8 Scheme illustrates a proposed regulation mechanism of TLR9 activation by ADA2 in human pDCs.
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