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. 2022 Jul 23;15(10):9286–9297. doi: 10.1007/s12274-022-4683-x

Hydroxyapatite nanoparticles drive the potency of Toll-like receptor 9 agonist for amplified innate and adaptive immune response

Qin Zeng 1,2,3,✉, Ruiqi Wang 4, Yuchen Hua 1,3, Hongfeng Wu 1,3, Xuening Chen 1,3, You-cai Xiao 4, Qiang Ao 2,3, Xiangdong Zhu 1,3,✉, Xingdong Zhang 1,2,3
PMCID: PMC9308403  PMID: 35911480

Abstract

The potency of Toll-like receptor 9 (TLR9) agonist to drive innate immune response was limited due to immune suppression or tolerance during TLR9 signaling activation in immune cells. Herein we addressed this problem by introducing hydroxyapatite nanoparticles (HANPs) to CpG ODN (CpG), a TLR9 agonist. The study revealed that HANPs concentration and duration-dependently reprogramed the immune response by enhancing the secretion of immunostimulatory cytokines (tumor necrosis factor α (TNFα) or IL-6) while reducing the production of immunosuppressive cytokine (IL-10) in macrophages in response to CpG. Next, the enhanced immune response benefited from increased intracellular Ca2+ in macrophage by the addition of HANPs. Further, we found exposure to HANPs impacted the mitochondrial function of macrophages in support of the synthesis of adenosine triphosphate (ATP), the production of nicotinamide adenine dinucleotide (NAD), and reactive oxygen species (ROS) in the presence or absence of CpG. In vaccinated mice model, only one vaccination with a mixture of CpG, HANPs, and OVA, a model antigen, allowed the development of a long-lasting balanced humoral immunity in mice without any histopathological change in the local injection site. Therefore, this study revealed that HANPs could modulate the intracellular calcium level, mitochondrial function, and immune response in immune cells, and suggested a potential combination adjuvant of HANPs and TLR9 agonist for vaccine development. graphic file with name 12274_2022_4683_Fig1_HTML.jpg

Electronic Supplementary Material

Supplementary material (TEM image, LDH activity, the Ca2+ release in PBS, qRT-PCR analysis, H&E staining, and IL-6 level in the injection site and serum) is available in the online version of this article at 10.1007/s12274-022-4683-x.

Keywords: hydroxyapatite nanoparticles, Toll-like receptor 9, intracellular calcium, mitochondrial function, adaptive immune response

Electronic Supplementary Material

12274_2022_4683_MOESM1_ESM.pdf (998.9KB, pdf)

Hydroxyapatite nanoparticles drive the potency of Toll-like receptor 9 agonist for amplified innate and adaptive immune response

Acknowledgements

We thank Tao Fu of Public lab platform in West China School of Basic Medical Sciences & Forensic Medicine of Sichuan University for help with TEM operation, and Xi Wu of the Analytical & Testing Center of Sichuan University for help with ICP measurement. We are grateful for the grants supported by Sichuan Science and Technology Program (Nos. 2020YFS0039 and 2020YFH0008), the National Natural Science Foundation of China (Nos. 81901685 and 32171333), and the Fundamental Research Funds for the Central Universities (No. YJ201915).

Contributor Information

Qin Zeng, Email: qzeng8156@scu.edu.cn.

Xiangdong Zhu, Email: zhu_xd1973@scu.edu.cn.

References

  • [1].Pulendran B, Arunachalam P S, O’Hagan D T. Emerging concepts in the science of vaccine adjuvants. Nat. Rev. Drug Discov. 2021;20:454–475. doi: 10.1038/s41573-021-00163-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [2].Vidya M K, Kumar V G, Sejian V, Bagath M, Krishnan G, Bhatta R. Toll-like receptors: Significance, ligands, signaling pathways, and functions in mammals. Int. Rev. Immunol. 2018;37:20–36. doi: 10.1080/08830185.2017.1380200. [DOI] [PubMed] [Google Scholar]
  • [3].Kayesh M E H, Kohara M, Tsukiyama-Kohara K. An overview of recent insights into the response of TLR to SARS-CoV-2 infection and the potential of TLR agonists as SARS-CoV-2 vaccine adjuvants. Viruses. 2021;13:2302. doi: 10.3390/v13112302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [4].Rodell C B, Arlauckas S P, Cuccarese M F, Garris C S, Li R, Ahmed M S, Kohler R H, Pittet M J, Weissleder R. TLR7/8-agonist-loaded nanoparticles promote the polarization of tumour-associated macrophages to enhance cancer immunotherapy. Nat. Biomed. Eng. 2018;2:578–588. doi: 10.1038/s41551-018-0236-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [5].Wei J J, Wu D, Zhao S S, Shao Y, Xia Y F, Ni D W, Qiu X Y, Zhang J P, Chen J, Meng F H, et al. Immunotherapy of malignant glioma by noninvasive administration of TLR9 agonist CpG nano-immunoadjuvant. Adv. Sci. 2022;9:2103689. doi: 10.1002/advs.202103689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [6].Zeng Q, Gammon J M, Tostanoski L H, Chiu Y C, Jewell C M. In vivo expansion of melanoma-specific T cells using microneedle arrays coated with immune-polyelectrolyte multilayers. ACS Biomater. Sci. Eng. 2017;3:195–205. doi: 10.1021/acsbiomaterials.6b00414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [7].Arunachalam P S, Walls A C, Golden N, Atyeo C, Fischinger S, Li C F, Aye P, Navarro M J, Lai L L, Edara V V, et al. Adjuvanting a subunit COVID-19 vaccine to induce protective immunity. Nature. 2021;594:253–258. doi: 10.1038/s41586-021-03530-2. [DOI] [PubMed] [Google Scholar]
  • [8].Butcher S K, O’Carroll C E, Wells C A, Carmody R J. Tolllike receptors drive specific patterns of tolerance and training on restimulation of macrophages. Front. Immunol. 2018;9:933. doi: 10.3389/fimmu.2018.00933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [9].Biswas S K, Lopez-Collazo E. Endotoxin tolerance: New mechanisms, molecules and clinical significance. Trends Immunol. 2009;30:475–487. doi: 10.1016/j.it.2009.07.009. [DOI] [PubMed] [Google Scholar]
  • [10].Iskander K N, Stepien D, Becker J M, Remick D G. Endotoxin tolerance impairs antimicrobial capacity in bacterial pneumonia and decreases murine lung inflammation. J. Am. Coll. Surg. 2011;213:S57. doi: 10.1016/j.jamcollsurg.2011.06.126. [DOI] [Google Scholar]
  • [11].Volpi C, Fallarino F, Pallotta M T, Bianchi R, Vacca C, Belladonna M L, Orabona C, De Luca A, Boon L, Romani L, et al. High doses of CpG oligodeoxynucleotides stimulate a tolerogenic TLR9-TRIF pathway. Nat. Commun. 2013;4:1852. doi: 10.1038/ncomms2874. [DOI] [PubMed] [Google Scholar]
  • [12].Trinchieri G, Sher A. Cooperation of Toll-like receptor signals in innate immune defence. Nat. Rev. Immunol. 2007;7:179–190. doi: 10.1038/nri2038. [DOI] [PubMed] [Google Scholar]
  • [13].Zhou Z M, Lin L T, An Y C, Zhan M X, Chen Y, Cai M Y, Zhu X J, Lu L G, Zhu K S. The combination immunotherapy of TLR9 agonist and OX40 agonist via intratumoural injection for hepatocellular carcinoma. J. Hepatocell. Carcinoma. 2021;8:529–543. doi: 10.2147/JHC.S301375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [14].Mackensen A, Galanos C, Engelhardt R. Modulating activity of interferon-γ on endotoxin-induced cytokine production in cancer patients. Blood. 1991;78:3254–3258. doi: 10.1182/blood.V78.12.3254.3254. [DOI] [PubMed] [Google Scholar]
  • [15].Chen J, Ivashkiv L B. IFN-γ abrogates endotoxin tolerance by facilitating Toll-like receptor-induced chromatin remodeling. Proc. Natl. Acad. Sci. USA. 2010;107:19438–19443. doi: 10.1073/pnas.1007816107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [16].Vaeth M, Zee I, Concepcion A R, Maus M, Shaw P, Portal-Celhay C, Zahra A, Kozhaya L, Weidinger C, Philips J, et al. Ca2+ signaling but not store-operated Ca2+ entry is required for the function of macrophages and dendritic cells. J. Immunol. 2015;195:1202–1217. doi: 10.4049/jimmunol.1403013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [17].Trebak M, Kinet J P. Calcium signalling in T cells. Nat. Rev. Immunol. 2019;19:154–169. doi: 10.1038/s41577-018-0110-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Schappe M S, Szteyn K, Stremska M E, Mendu S K, Downs T K, Seegren P V, Mahoney M A, Dixit S, Krupa J K, Stipes E J, et al. Chanzyme TRPM7 mediates the Ca2+ influx essential for lipopolysaccharide-induced Toll-like receptor 4 endocytosis and macrophage activation. Immunity. 2018;48:59–74.e5. doi: 10.1016/j.immuni.2017.11.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].De Dios R, Nguyen L, Ghosh S, Mckenna S, Wright C J. CpG-ODN-mediated TLR9 innate immune signalling and calcium dyshomeostasis converge on the NFκB inhibitory protein IκBβ to drive IL1α and IL1β expression. Immunology. 2020;160:64–77. doi: 10.1111/imm.13182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [20].Chen X N, Wang M L, Chen F Y, Wang J, Li X F, Liang J, Fan Y J, Xiao Y M, Zhang X D. Correlations between macrophage polarization and osteoinduction of porous calcium phosphate ceramics. Acta Biomater. 2020;103:318–332. doi: 10.1016/j.actbio.2019.12.019. [DOI] [PubMed] [Google Scholar]
  • [21].Zhang K, Zhou Y, Xiao C, Zhao W L, Wu H F, Tang J Q, Li Z T, Yu S, Li X F, Min L, et al. Application of hydroxyapatite nanoparticles in tumor-associated bone segmental defect. Sci. Adv. 2019;5:eaax6946. doi: 10.1126/sciadv.aax6946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Hua Y C, Wu J J, Wu H F, Su C, Li X F, Ao Q, Zeng Q, Zhu X D, Zhang X D. Exposure to hydroxyapatite nanoparticles enhances Toll-like receptor 4 signal transduction and overcomes endotoxin tolerance in vitro and in vivo. Acta Biomater. 2021;135:650–662. doi: 10.1016/j.actbio.2021.09.006. [DOI] [PubMed] [Google Scholar]
  • [23].Wang X P, Ihara S, Li X, Ito A, Sogo Y, Watanabe Y, Yamazaki A, Tsuji N M, Ohno T. Rod-scale design strategies for immune-targeted delivery system toward cancer immunotherapy. ACS Nano. 2019;13:7705–7715. doi: 10.1021/acsnano.9b01271. [DOI] [PubMed] [Google Scholar]
  • [24].Wu, H. F.; Hua, Y. C.; Wu, J. J.; Zeng, Q.; Yang, X.; Zhu, X. D.; Zhang, X. D. The morphology of hydroxyapatite nanoparticles regulates clathrin-mediated endocytosis in melanoma cells and resultant anti-tumor efficiency. Nano Res., in press, 10.1007/s12274-022-4220-y.
  • [25].Zhao H, Wu C H, Gao D, Chen S P, Zhu Y D, Sun J, Luo H R, Yu K, Fan H S, Zhang X D. Antitumor effect by hydroxyapatite nanospheres: Activation of mitochondria-dependent apoptosis and negative regulation of phosphatidylinositol-3-kinase/protein kinase B pathway. ACS Nano. 2018;12:7838–7854. doi: 10.1021/acsnano.8b01996. [DOI] [PubMed] [Google Scholar]
  • [26].Valdinocci D, Simões R F, Kovarova J, Cunha-Oliveira T, Neuzil J, Pountney D L. Intracellular and intercellular mitochondrial dynamics in parkinson’s disease. Front. Neurosci. 2019;13:930. doi: 10.3389/fnins.2019.00930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Khurana A, Allawadhi P, Khurana I, Allwadhi S, Weiskirchen R, Banothu A K, Chhabra D, Joshi K, Bharani K K. Role of nanotechnology behind the success of mRNA vaccines for COVID-19. Nano Today. 2021;38:101142. doi: 10.1016/j.nantod.2021.101142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].Pujari-Palmer S, Chen S, Rubino S, Weng H, Xia W, Engqvist H, Tang L P, Ott M K. In vivo and in vitro evaluation of hydroxyapatite nanoparticle morphology on the acute inflammatory response. Biomaterials. 2016;90:1–11. doi: 10.1016/j.biomaterials.2016.02.039. [DOI] [PubMed] [Google Scholar]
  • [29].Lebre F, Sridharan R, Sawkins M J, Kelly D J, O’Brien F J, Lavelle E C. The shape and size of hydroxyapatite particles dictate inflammatory responses following implantation. Sci. Rep. 2017;7:2922. doi: 10.1038/s41598-017-03086-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [30].Grandjean-Laquerriere A, Tabary O, Jacquot J, Richard D, Frayssinet P, Guenounou M, Laurent-Maquin D, Laquerriere P, Gangloff S. Involvement of Toll-like receptor 4 in the inflammatory reaction induced by hydroxyapatite particles. Biomaterials. 2007;28:400–404. doi: 10.1016/j.biomaterials.2006.09.015. [DOI] [PubMed] [Google Scholar]
  • [31].Masson J D, Thibaudon M, Bélec L, Crépeaux G. Calcium phosphate: A substitute for aluminum adjuvants? Expert Rev. Vaccines. 2017;16:289–299. doi: 10.1080/14760584.2017.1244484. [DOI] [PubMed] [Google Scholar]
  • [32].Zeng Q, Jewell C M. Directing Toll-like receptor signaling in macrophages to enhance tumor immunotherapy. Curr. Opin. Biotechnol. 2019;60:138–145. doi: 10.1016/j.copbio.2019.01.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [33].Khalifehzadeh R, Arami H. The CpG molecular structure controls the mineralization of calcium phosphate nanoparticles and their immunostimulation efficacy as vaccine adjuvants. Nanoscale. 2020;72:9603–9615. doi: 10.1039/C9NR09782A. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [34].Cejudo-Guillen M, Ramiro-Gutiérrez M L, Labrador-Garrido A, Díaz-Cuenca A, Pozo D. Nanoporous silica microparticle interaction with Toll-like receptor agonists in macrophages. Acta Biomater. 2012;8:4295–4303. doi: 10.1016/j.actbio.2012.07.026. [DOI] [PubMed] [Google Scholar]
  • [35].Qing F Z, Wang Z, Hong Y L, Liu M, Guo B, Luo H R, Zhang X D. Selective effects of hydroxyapatite nanoparticles on osteosarcoma cells and osteoblasts. J. Mater. Sci. Mater. Med. 2012;23:2245–2251. doi: 10.1007/s10856-012-4703-6. [DOI] [PubMed] [Google Scholar]
  • [36].Zhao R, Xie P F, Zhang K, Tang Z R, Chen X N, Zhu X D, Fan Y J, Yang X, Zhang X D. Selective effect of hydroxyapatite nanoparticles on osteoporotic and healthy bone formation correlates with intracellular calcium homeostasis regulation. Acta Biomater. 2017;59:338–350. doi: 10.1016/j.actbio.2017.07.009. [DOI] [PubMed] [Google Scholar]
  • [37].Zhao X X, Ng S, Heng B C, Guo J, Ma L, Tan T T Y, Ng K W, Loo S C J. Cytotoxicity of hydroxyapatite nanoparticles is shape and cell dependent. Arch. Toxicol. 2013;87:1037–1052. doi: 10.1007/s00204-012-0827-1. [DOI] [PubMed] [Google Scholar]
  • [38].Petersen O H, Gerasimenko J V, Gerasimenko O V, Gryshchenko O, Peng S. The roles of calcium and ATP in the physiology and pathology of the exocrine pancreas. Physiol. Rev. 2021;101:1691–1744. doi: 10.1152/physrev.00003.2021. [DOI] [PubMed] [Google Scholar]
  • [39].Lombardi M, Gabrielli M, Adinolfi E, Verderio C. Role of ATP in extracellular vesicle biogenesis and dynamics. Front. Pharmacol. 2021;72:654023. doi: 10.3389/fphar.2021.654023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [40].Navarro M N, De Las Heras M M G, Mittelbrunn M. Nicotinamide adenine dinucleotide metabolism in the immune response, autoimmunity and inflammageing. Br. J. Pharmacol. 2022;179:1839–1856. doi: 10.1111/bph.15477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [41].Weeratna R D, Millan C L B, Mccluskie M J, Davis H L. CpG ODN can re-direct the Th bias of established Th2 immune responses in adult and young mice. FEMS Immunol. Med. Microbiol. 2001;32:65–71. doi: 10.1111/j.1574-695X.2001.tb00535.x. [DOI] [PubMed] [Google Scholar]
  • [42].Jiang H, Wang Q, Li L, Zeng Q, Li H M, Gong T, Zhang Z R, Sun X. Turning the old adjuvant from gel to nanoparticles to amplify CD8+ T cell responses. Adv. Sci. 2018;5:1700426. doi: 10.1002/advs.201700426. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

12274_2022_4683_MOESM1_ESM.pdf (998.9KB, pdf)

Hydroxyapatite nanoparticles drive the potency of Toll-like receptor 9 agonist for amplified innate and adaptive immune response

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