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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 1  |  Issue : 1  |  Page : 45-50

Vitamin D attenuates viral-induced inflammation in adipocytes of obese individuals


1 Translational Research in Respiratory Diseases, Meakins-Christie Laboratories, Research Institute of the McGill University Health Center, Montréal, QC, Canada
2 Translational Research in Respiratory Diseases, Meakins-Christie Laboratories, Research Institute of the McGill University Health Center, Montréal, QC, Canada; Mohammed Bin Rashid University of Medicine and Health Sciences, College of Medicine, Dubai, United Arab Emirates

Date of Submission25-Nov-2021
Date of Decision16-Dec-2021
Date of Acceptance20-Dec-2021
Date of Web Publication19-Jan-2022

Correspondence Address:
Saba Al Heialy
Mohammed Bin Rashid University of Medicine and Health Sciences, College of Medicine, Dubai.
United Arab Emirates
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/abhs.abhs_19_21

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  Abstract 

Background: The clinical association between obesity and increased risk of infection is well established; however, the role of adipocytes remains unknown. Adipocytes are important players in the meta-inflammation observed in obese individuals. Moreover, adipocytes are now emerging as potential viral reservoirs for viruses such as SARS-CoV-2, the virus behind the COVID-19 pandemic, due to increased expression of virus receptors [angiotensin converting enzyme-2 (ACE2) and TMPRSS2]. Moreover, obesity has been linked to vitamin D deficiency. We hypothesized that vitamin D supplementation can attenuate the viral-induced inflammation in adipocytes of obese subjects and potentially regulate the expression of viral receptors. Materials and Methods: Adipocytes were differentiated in vitro from subcutaneous human pre-adipocytes obtained from nonobese and obese individuals. Poly(I:C) (10 μg/mL), which binds to toll-like receptor-3 (TLR3), was used to mimic viral infection, in the absence and presence of 100 nM of 1α,25-dihydroxyvitamin D3 for 24 hours. Adipocytes were collected for RNA extraction. qRT-PCR was performed to assess the expression of TLR3, IL-8, IL-6, TNF-α, IFN-β, ACE2, TMPRSS2. Results: Pre-stimulation with Poly(I:C), adipocytes from obese individuals showed higher expression of TLR3, TNF-α, IFN-β, ACE2, and TMPRSS2 highlighting the inflammatory status of obese adipocytes. Following stimulation with Poly(I:C), expression of TLR3, IL-8, TNF-α, and IFN-β were significantly increased in obese adipocytes compared to nonobese. Vitamin D supplementation was able to decrease significantly TLR3, IL-8, and IFN-β expression. Expression of IL-6, ACE2, and TMPRSS2 were increased in both nonobese and obese adipocytes in response to Poly (I:C) with significant effect of vitamin D supplementation on IL-6 and TMPRSS2 expression in obese adipocytes. Conclusion: Vitamin D supplementation provides a potential therapeutic advantage in the viral-induced inflammation seen in adipocytes especially in relation to obesity. Our results also suggest that vitamin D can be used to regulate the expression of receptors and proteases involved in SARS-CoV-2 viral entry.

Keywords: Adipocytes, inflammation, obesity, viral infection, vitamin D


How to cite this article:
Gaudet M, Mogas A, Heialy SA. Vitamin D attenuates viral-induced inflammation in adipocytes of obese individuals. Adv Biomed Health Sci 2022;1:45-50

How to cite this URL:
Gaudet M, Mogas A, Heialy SA. Vitamin D attenuates viral-induced inflammation in adipocytes of obese individuals. Adv Biomed Health Sci [serial online] 2022 [cited 2022 Aug 18];1:45-50. Available from: http://www.abhsjournal.net/text.asp?2022/1/1/45/335721




  Background Top


Obesity, a state of excessive accumulation of white adipose tissue, is one of the major public health burdens of the 21st century. Adipose tissue is emerging as not only an endocrine organ for lipid storage but also a site of chronic immune activation and inflammation through its release of pro-inflammatory cytokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, and adipokines such as leptin [1]. Through its expression of toll-like receptors (TLRs) and responsiveness to lipopolysaccharides, the adipose tissue participates in innate immune responses [2]. In obesity, dysfunction of adipose tissue is characterized by hypertrophied adipocytes, fibrosis, and elevated inflammation [3]. Mostly composed of mature adipocytes, adipose tissue also contains pre-adipocytes, fibroblasts, and immune cells. Interestingly, adipose tissue and their residing cells have been shown to be the target of viruses ranging from human immunodeficiency virus (HIV) [4] to coronaviruses such as SARS-CoV [5]. Studies have also suggested that adipocytes specifically from obese and diabetic patients show an increased expression of the viral receptor, angiotensin converting enzyme-2 (ACE-2). Adipocytes can then be the target of the virus or serve as a viral reservoir [6]. These data add to the body of literature establishing an association between obesity and infection [7,8].

Moreover, obesity is associated with low levels of vitamin D. In addition to its role in calcium homeostasis and bone metabolism, vitamin D exerts immunomodulatory functions [9]. It has been suggested that the low levels of vitamin D in obesity are due to the low expression of vitamin D metabolizing enzymes such as 25-hydroxylase (CYP2J2) and 1α-hydroxylase (CYP27B1) in adipose tissue [10]. The consequence of this deficiency in vitamin D may have detrimental effects on immune responses. Low-levels of vitamin D have been linked to certain infections such as hepatitis, influenza, and COVID-19 [11-14]. Although the role of vitamin D supplementation in treatment of noncommunicable diseases has been controversial, a meta-analysis study in 2019, looking at 11,321 participants across 25 randomized control trials, showed that vitamin D supplementation protected against upper respiratory infections especially in patients with low levels of vitamin D (<25 nmol/L) [15]. The effects of vitamin D supplementation on immune cells has been extensively described. In macrophages, vitamin D promotes the induction of antimicrobial proteins and in monocytes it can downregulate the expression of TLR2 and TLR4 [16]. With regards to cells of the adaptive immune system, vitamin D inhibits the expression of Th1 and Th17 cytokines in vitro [17,18]. However, little is known on its effect on adipocytes as a remote source of inflammatory mediators and a viral reservoir.

In this study, we hypothesized that supplementation of adipocytes with vitamin D may decrease the pro-inflammatory status in obesity and moreover decrease it in response to viral infection. Results obtained from this study can shape our knowledge on the role of vitamin D as a potential therapeutic agent in viral infections.


  Materials and methods Top


Pre-adipocyte and adipocyte cell culture and treatment

Subcutaneous Human Pre-adipocytes from nonobese and obese subjects were purchased from ATCC (VA, USA) and ZenBio (NC, USA). [Table 1] shows the data on pre-adipocytes obtained from nonobese and obese subjects. Pre-adipocytes were cultured and differentiated in vitro as previously described [19].
Table 1: Data of adipocytes from nonobese and obese subjects

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Mature adipocytes were starved with DMEM/Ham’s F-12 (1:1, v/v) media supplemented with: 0.01M HEPES pH 7.4, 0.5% fetal bovine serum, and 100 U/mL penicillin/streptomycin overnight. Cells were then stimulated with 10 μg/mL of Poly(I:C) (Invivogen, CA, USA), and treated with 100 nM of 1α,25-dihydroxyvitamin D3 (VitD) (Sigma-Aldrich, Ontario, Canada) for 24 hours. Adipocytes were then processed for RNA extraction.

RNA extraction and quantitative reverse transcription polymerase chain reaction

Extraction of total RNA from adipocytes and cDNA synthesis was preformed as previously described [19]. [Table 2] shows the forward and reverse primers used. mRNA expression was measured using AdvanTech 2X qPCR MasterMix (Diamed, Ontario, Canada). The reaction was as follows: 5 µL of Mastermix, 1 µL of diluted cDNA (1/5), 0.5 µL of forward and reverse primers (10 µM), and 3.5 µL of nuclease free H2O. Each sample was tested in duplicates and the quantitative reverse transcription polymerase chain reaction (qPCR) amplification was performed using CFX96 thermal cycler (Bio-Rad, Hercules, 130 CA, USA) and cycler conditions were preformed according to the manufacturer’s protocol. The ∆∆CT method was used to measure gene expression for both detection methods: amount of target = 2−∆∆CT.
Table 2: Forward and reverse primers and house keeping gene

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Statistical analysis

Standard statistical two-tailed t-tests and one-way ANOVA using Tukey’s multiple comparison test were performed to test for statistical significance between data groups using GraphPad Prism 8 (GraphPad, San Diego, CA, USA).


  Results Top


VitD treatment reduces adipocytes responsiveness to viral-induced stimulation

Pre-adipocytes from nonobese and obese subjects were differentiated in vitro as previously described [19]. Mature adipocytes were stimulated with Poly(I:C) (10 μg/mL) with or without VitD treatment (100 nM) for 24 hours. Following stimulation and treatment of adipocytes, mRNA expression was evaluated by qRT-PCR. We observed significant differences in TLR3 mRNA expression at the baseline, prior to Poly(I:C) stimulation, where adipocytes from obese show a significant increase, compared to adipocytes from nonobese subjects [Figure 1]A. As expected, Poly(I:C) stimulation increased TLR3 expression in both groups, and this effect was observed to a higher extent in adipocytes from obese. However, VitD significantly decreased TLR3 mRNA expression following Poly(I:C) in adipocytes from nonobese subjects.
Figure 1: Expression of TLR3, IL-8, IL-6, and IFN-β in adipocytes is downregulated by VitD. Adipocytes from obese (n = 6) and nonobese (n = 6) patients were stimulated with 10 μg/mL of Poly(I:C) and treated with 100 nM of 1α,25-dihydroxyvitamin D3 for 24 hours. RNA was extracted for quantitative reverse transcription polymerase chain reaction (qPCR). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

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VitD treatment downregulates Poly(I:C)-induced inflammatory markers in adipocytes

The mRNA expression of IL-8, IL-6, TNF-α, and IFN-β were also measured by qRT-PCR. Poly(I:C)-stimulated adipocytes from both the groups had an increase in mRNA of inflammatory markers IL-6 and IL-8 compared to untreated cells, with a higher overall expression in adipocytes from obese subjects [Figure 1B]–E] for IL-8 and TNF-α. VitD treatment significantly reduced IL-8, IL-6, and IFN-β expression induced by Poly(I:C) in adipocytes from obese subjects [[Figure 1B], C, E] compared to adipocytes from nonobese, for the exception of IL-6. It should also be noted that significant higher basal expression of TNF-α and IFN-β was measured in adipocytes from obese group. There was no difference in IL-6 and IL-8 mRNA expression between obese and nonobese in the absence of Poly(I:C) stimulation. IL-8 and IFN-β mRNA was also significantly higher after Poly(I:C) stimulation in adipocytes from the obese group [[Figure 1B] and E].

ACE2 and TMPRSS2 gene expression is regulated by VitD supplementation

At the baseline, prior to Poly(I:C) stimulation, ACE2 expression was significantly higher in adipocytes from obese subjects (P = 0.05, [Figure 2]A) compared to the nonobese subjects. Similarly, prior to Poly(I:C) stimulation, TMPRSS2 was also significantly increased in adipocytes from obese ([Figure 2]B, P < 0.05) subjects. Furthermore, adipocytes stimulated with Poly(I:C) had an increase in mRNA expression of ACE2 andTMPRSS2 compared to untreated cells and this effect was more evident in adipocytes from obese [[Figure 2]A and B]. Overall, an important decrease in ACE2 and TMPRSS2 was seen when VitD was added to Poly(I:C)-stimulated adipocytes, with a significant decrease of TMPRSS2 expression from the obese group [[Figure 2]B and C]. There was no statistically significant decrease of ACE2 expression in response to VitD.
Figure 2: Expression of TMPRSS2 is decreased by VitD in obese adipocytes. Adipocytes from obese (n = 6) and nonobese (n = 6) patients were stimulated with 10 μg/mL of Poly(I:C) and treated with 100 nM of 1α,25-dihydroxyvitamin D3 for 24 hours. RNA was extracted for quantitative reverse transcription polymerase chain reaction (qPCR). *P < 0.05

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  Discussion Top


Multiple epidemiological studies have established obesity as a significant risk factor for increased susceptibility to viral infections and increased severity of the infection. Mechanisms such as dysregulation of the immune responses have been suggested as potential explanations to this association. However, adipocytes are emerging as a significant source of inflammatory markers and more studies are needed to understand their contribution. In this study, we focused on the differences between adipocytes from nonobese and obese subjects in response to viral infection using Poly(I:C) stimulation. We were also interested in the role of vitamin D as a regulator of inflammatory responses. Results showed that at baseline, adipocytes from obese subjects show an increase in inflammatory markers such as TNF-α, IFN-β, IL-8. Our results showed that TLR3 expression was significantly increased in adipocytes from obese subjects in the absence and presence of Poly(I:C) stimulation. TLR3 recognizes double-stranded viral RNA as well as RNA from dying endogenous cells. Although TLR3 has beneficial roles in the induction of immune responses to SARS-CoV-2, it may lead to a surge of pro-inflammatory mediators via NFκB pathway [20. Therefore, reducing its expression by VitD may have beneficial effects. Studies have shown that in vitro adipocytes highly express TLR3 and that the receptor is functionally active [21]. In vivo studies have also shown that visceral adipose tissue (VAT) of a high-fat diet animal model express higher level of TLR3. Moreover, VAT from obese female subjects expressed higher levels of TLR3. Ablation of TLR3 resulted in a decrease in macrophage infiltration and prevented obesity in vivo [22]. Interestingly, our results showed that VitD significantly decreased TLR3 expression following Poly(I:C) stimulation in adipocytes obtained from nonobese subjects. To our knowledge, this is the first report on the effect of VitD on TLR3 expression in adipocytes. Other studies have shown an effect of VitD on TLR2, 4, and 9 [16,23].

Our results strengthen the knowledge that obesity leads to increased inflammation where we saw that adipocytes from obese subjects expressed higher levels of TNF-α, IL-8, and IFN-β. IFN-β is a part of the Type I interferon family along with IFN-α and are important immune mediators bridging innate and adaptive immune responses. TLR activation and signaling leads to type I interferon production. The role of type I interferons in obesity is controversial with studies showing detrimental and protective roles [24]. In vivo studies have shown that obesity leads to interferon I responses, which contributes to obesity-associated pathogenesis. Engagement of the type I IFN/IFN-α receptor (IFNAR) activated inflammatory mediators such as STAT3, IL-6, and TNF-α [25]. Our study shows that the type I IFN/IFNAR axis is activated in human adipocytes through Poly(I:C) with increases in IFN-β, TNF-α, IL-6 and more significantly in adipocytes obtained from obese subjects. VitD was able to significantly inhibit this axis as seen with decreases in TLR3, IFN-β, TNF-α, and IL-6.

We were also interested in the potential role of vitamin D supplementation in COVID-19 patients. Recently, a lot of interest has been seen with regards to vitamin D as a potential therapy for COVID-19 patients. SARS-CoV-2 uses ACE2 and TMPRSS2 for their entry into the cell [26]. Interestingly, ACE2 has been shown to be expressed in VAT at a higher rate than lung tissue [6]. In vivo studies have shown that ACE2 gene expression was increased in mouse adipocytes after 4 months in high-fat diet [27]. We have previously shown that ACE2 expression was increased in lung epithelial cells of obese subjects compared to nonobese subjects [19]. Results from this study indicate the same pattern in human adipocytes where adipocytes from obese subjects show a significant increase in ACE2 expression compared to nonobese. This adds to the literature suggesting adipocytes can potentially act as a reservoir for viruses such as SARS-CoV-2. VitD was able to decrease the expression of ACE2 and TMPRSS2. This is of great importance as VitD deficiency is 35% higher in obese subjects and 24% higher in overweight subjects compared to healthy subjects [28].

In summary, our study highlights the effect of obesity on increasing pro-inflammatory mediator production in response to viral infection in adipocytes, a cell that is established as a significant source of mediators and viral reservoir. VitD showed a significant effect on the expression of these identified mediators highlighting the immunomodulatory role of VitD. Future studies should address the effect of VitD at the protein level. This can be of importance in viral infections such as SARS-CoV-2 infection not only by decreasing the production of pro-inflammatory cytokines but also decreasing pathways for viral entry. This study sheds further light on the potential therapeutic properties of VitD.

Acknowledgements

The authors are grateful to Al Jalila Foundation, as this work was supported in part by the Al Jalila Foundation.

Authors’ contributions

M.G. and S.A.H. conceived the research concept; M.G. and S.A.H. developed research design; M.G. and A.M. performed field work and data analysis; M.G. and S.A.H. prepared the first draft and all reviewers reviewed and approved final draft of the manuscript. All authors are responsible for the contents and integrity of this manuscript.

Ethical statement

Ethical review and approval were obtained from McGill University Health Centre Research Ethics Board (2021–6961). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

Financial support and sponsorship

Funding was provided by the Mohammed Bin Rashid University of Medicine and Health Sciences Internal Research Grant (MBRU-CM-RG2020-06).

Conflicts of interest

There are no conflicts of interest.

Data availability statement

Not applicable.

Patients’ consent

Not applicable.

Study limitations

Not applicable.



 
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