Autophagy efficacy and vitamin D status: Population effects
Introduction
Toll-like receptor (TLR) molecules count among the first line of defence against pathogens, recognising pathogen-associated molecular patterns (PAMPs). Lipoproteins (LPs) are pro-inflammatory cell wall PAMPs found in Gram-positive and Gram-negative bacteria. LPs play a role in diseases caused by medically relevant pathogens, such as Mycobacterium tuberculosis (M.tb), Streptococcus pneumoniae, Borrelia burgdorferi, and Neisseria meningitidis [1]. LPs with three fatty acids, such as those found in M.tb, are sensed by TLR2 in alliance with TLR1. TLR2/1 heterodimerization stimulates phagocytosis, leading to the recruitment of the class III PI(3)K autophagy complex, including Beclin1, VPS34, Rubicon and UVRAG [2]. The cytosolic microtubule-associated protein 1 light chain 3 beta (LC3-B, encoded by MAP1LC3B) is recruited through a series of downstream signalling events, mediated by autophagy-related (ATG) proteins. After binding phosphatidylethanolamine to form the lipidated LC3 (LC3-IIB), it associates with the autophagosome membrane, marking autophagosome presence. LC3-IIB, Beclin1 and lysosomal-associated membrane protein (LAMP) accelerate phagosome maturation [3]; essential to kill internalized pathogens [4]. LC3-IIB-positive phagosomes rapidly undergo lysosomal fusion and cargo degradation, coupled with secretion of anti-inflammatory cytokines such as IL10 and transforming growth factor beta 1 (TGFB1). In contrast, LC3-IIB-negative phagosomes, show impaired lysosomal fusion and decreased cargo degradation coupled with secretion of pro-inflammatory cytokines such as IL6 and IL1B [5], [6]. 1,25(OH)2D3 is a key regulator of LC3-IIB, relieving constitutive unliganded VDR repression of the MAP1LC3B gene, thereby inducing autophagy [7]. In addition, 1,25(OH)2D3 induces autophagy by activating BECN1 expression through cathelicidin antimicrobial peptide (hCAP18), often referred to by its antibacterial C-terminal domain, LL-37 [8].
Vitamin D is a steroid hormone with diverse functions including the maintenance of calcium and phosphate homeostasis, regulation of cell proliferation and differentiation, promotion of innate immune responses and reduction of inflammation [9]. Vitamin D3 synthesis in the skin, upon UVB exposure, is influenced by melanin pigmentation, making dark-skinned individuals more susceptible to 25(OH)D3 deficiency. This is reflected in the multi-ethnic South-African population, where serum 25(OH)D3 deficiency (<50 nM) appears to be more prevalent among Indian and African groups than in White individuals [9], [10]. Decreased solar UVB radiation in winter exacerbates deficiency in certain populations, reflected in the seasonal variation in 25(OH)D3 status in South Africans [10], [11], [12], with consequences for HIV and tuberculosis (TB) susceptibility [13]. The biological effects of vitamin D are mediated by binding of the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), to the vitamin-D receptor (VDR), which in turn binds to vitamin D response elements (VDREs) in target genes, to influence transcription. Given the pleiotropic role of vitamin D and the fact that more than 23,000 cell-specific genomic locations for VDREs have been observed [14], it is not surprising that vitamin D deficiency (serum 25(OH)D3 < 50 nM) [9], [15] has been associated with various pathologies, including cancer [16], TB [9], sepsis [17] and HIV infection [18].
Considering TB, mycobacterial antigen perception by TLR2/1 induces VDR and CYP27B1 expression to produce both receptor and active ligand. The VDR-1,25(OH)2D3 complex, activates a signalling cascade that induces CAMP to produce hCAP18 [19], which directly inhibits M.tb growth in vitro [19]. Besides direct bactericidal effects of hCAP18 that protects from M.tb, the regulatory effect of liganded VDR [7] on autophagy emphasizes the importance of maintaining adequate serum vitamin D levels (Deficient 25(OH)D3 < 50 nM; Normal 25(OH)D3 ≥ 50 nM; Excessive 25(OH)D3 greater than 125 nM, Institute of Medicine) [15].
We previously showed that the signalling efficacy of the TLR2/1-VDR pathway is population-specific, influenced by environment (ultraviolet index, UVI), 25(OH)D3 status, DNA methylation of VDR and SNPs in VDR, TLR1 and TIRAP (toll-interleukin-1 receptor domain-containing adapter protein) [10]. No population-specific data exists regarding vitamin D regulation of autophagy and whether this regulation is influenced by the reported variance in TLR2/1 signalling. We hypothesized that TLR2/1-stimulated autophagy is population-specific, influenced by serum 25(OH)D3 levels and can be boosted with 1,25(OH)2D3 supplementation. We evaluated the effect of in vitro 1,25(OH)2D3 supplementation on autophagy, by stimulating monocytes-macrophages from populations of diverse skin types with a TLR2/1 agonist, imitating mycobacterial lipoprotein. TLR2, VDR, hCAP18, Beclin1, LC3-IIB and immune-related Th1, Th2 and Th17 cytokine protein levels, as well as CYP24A1 mRNA level were quantified to determine the efficacy of signal transduction from TLR2/1 stimulation, through vitamin D mediation, to autophagy and the associated inflammatory response. Our findings suggest a population-specific autophagic response that is influenced by 25(OH)D3 sufficiency.
Section snippets
Study participants
Ethics was cleared by the Human Research Ethics Committee (HREC) of the South African National Blood Service (SANBS, clearance certificate number 2010/01 and 2013/08) and the Ethics Committee, Faculty of Science, University of Johannesburg (2010/06/03), according to ethical principles of the Declaration of Helsinki. The study population consisted of individuals of African and European descent living in South Africa. In line with vitamin D production, inversely correlating with skin
Population-specific efficacy of TLR2/1-VDR signalling in the context of autophagy is influenced by 1,25(OH)2D3
Autophagy is a dynamic process, with LC3-IIB continuously being formed and degraded upon autophagosome-lysosome fusion within the cell. The continuous formation and degradation of LC3-IIB-marked autophagosomes by lysosomes is called autophagic flux. Measuring the number of LC3-IIB-marked autophagosomes at any point in time reflects steady state autophagy (LC3-IIB/SS). Inhibiting autophagosome-lysosome fusion and consequent LC3-IIB degradation, reflects cumulative state autophagy (LC3-IIB/CS).
Discussion
TLR2/1 signalling is linked to autophagy through transcriptional actions of the 1,25(OH)2D3-VDR complex. Population-specific efficacy has been reported for TLR2/1-VDR signalling [10], but has not been studied for autophagy. Here we report that the autophagic response (LC3-IIB) is population-specific, enhanced by in vitro 1,25(OH)2D3 and promoted by higher serum 25(OH)D3, TLR2, hCAP18, and lower VDR and pro-inflammatory cytokines (IL6, TNFA) levels.
In response to TLR2/1 stimulation, a positive
Conclusion
Taken together, the results point to differential overall efficacy between the two populations regarding signalling of autophagy involving TLR2, VDR, CYP24A1 mRNA, hCAP18 and Beclin1 (Fig. 6). Differential levels of the components of TLR2/1-VDR signalling and vitamin D sufficiency primarily guide this. Thus, the markers of autophagy to TLR2/1 stimulation and in vitro 1,25(OH)2D3 supplementation is differentially expressed and show differential relations with partner components in the signal
Credit authorship contribution statement
Abhimanyu: Conceptualization, Methodology, Formal analysis, Investigation, Validation, Data curation, Writing - original draft, Writing - review & editing, Visualization, Supervision, Project administration. Vanessa Meyer: Investigation, Methodology, Formal analysis, Writing - original draft, Writing - review & editing. Brandon R. Jones: Investigation, Data curation, Writing - review & editing. Liza Bornman: Conceptualization, Methodology, Resources, Writing - review & editing, Visualization,
Acknowledgements
The authors would like to thank Dr H-A Byth-Illing, Department of Biochemistry, University of Johannesburg for assistance with flow cytometry and Dr Gerald McInerney, Dr Bastian Thaa, Karolinska Institute for discussions on LC3-IIB quantification.
Funding
Abhimanyu was supported by a postdoctoral fellowship from the Claude Leon Foundation. This work was supported by grants to LB; the National Research Foundation of South Africa [NRF, Grant No. 81774] and Cancer Association of South Africa (CANSA). The funders played no role in study design or in the collection and analysis of data.
Conflict of Interest
The authors declare that they have no conflict of interest.
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- 1
Present address: Wellcome Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Anzio Rd, Observatory, 7925, Western Cape, South Africa.
- 2
Present address: School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, WITS, Johannesburg 2050, Gauteng, South Africa.