Synergetic and antagonistic effects of combined calcitriol and interferon-β treatment on cytokine production by stimulated PBMCs
Graphical abstract
Introduction
Interferon-β is a first-line treatment for persons with relapsing-remitting multiple sclerosis (MS), acting clinically to reduce the frequency of relapse and the risk of conversion to clinically definite MS (Hawkins and Wolinsky, 2000). The mode of action of interferon-β, while uncertain, is thought to act by anti-inflammatory mechanisms, including increased IL-10 expression (Rudick et al., 2004), T-cell migration inhibition (Leppert et al., 1996), reduced antigen presentation (Markowitz, 2007) and restoration of blood brain barrier integrity (Veldhuis et al., 2003), or acting against potentially aetiologic human herpesviruses associated with MS (Alvarez-Lafuente et al., 2004, Hong et al., 2002). Another compound of increasing import in MS is vitamin D, the active form of which has also been found to have potent immunomodulatory effects, and the diagnostic form of which has been found in observational studies to be associated with reduced relapse risk (Mowry et al., 2010, Simpson et al., 2010), reduced disability (Weinstock-Guttman et al., 2011), and reduced conversion to clinically definite MS (Martinelli et al., 2013).
Recently, we have demonstrated a novel interplay between interferon-β and vitamin D sufficiency, with interferon-β therapy being associated with higher levels of serum 25(OH)D, this apparently acting by an enhancement of 25(OH)D production from UV exposure, and interferon-β and 25(OH)D positively interacting to reduce relapse risk (Simpson et al., 2012). This latter interaction is of particular interest, as it is unclear how interferon-β and vitamin D are acting on MS, whether their effects are simply cumulative or if each is directly affecting the activity of the other.
While trials are being undertaken to evaluate the clinical effects of combined interferon-β and vitamin D supplementation (Dorr et al., 2012, Golan et al., 2013, Smolders et al., 2011, Soilu-Hanninen et al., 2012), a useful start in the evaluation of the biological mechanisms underlying this potential interaction is to evaluate immunological activity in a controlled in vitro design. The production of cytokines from peripheral blood mononuclear cells (PBMCs) in response to a standardised antigenic challenge is a useful measure of immune responsiveness, as this can gauge how the immune system will respond to antigenic challenge in vivo, and it has been demonstrated that in-vitro treatment with both interferon-β (Coclet-Ninin et al., 1997, Comabella et al., 2002, Liu et al., 2001, Noronha et al., 1993, Rosztoczy et al., 1986, Wang et al., 2000) and calcitriol (Ardizzone et al., 2009, Khoo et al., 2011a, Khoo et al., 2011b), as well as in-vivo treatment of subjects with interferon-β (Bustamante et al., 2013b, Dressel et al., 2006, Graber et al., 2007, Khademi et al., 2000, Liu et al., 2001, Ossege et al., 1998, Rothuizen et al., 1999, Rudick et al., 1998) and cholecalciferol (Kimball et al., 2011), act upon the production of cytokines and chemokines from stimulated peripheral blood mononuclear cells. In pursuit of a greater understanding of the mechanism of this interferon-β and vitamin D interaction on immune activity, we have sought here to evaluate the effect of single and combined pre-treatment with physiologically relevant levels of calcitriol and interferon-β on cytokine production from PBMCs from a cohort of 22 healthy adults.
Section snippets
Materials & methods
A cohort of 22 healthy subjects not on medication was recruited for the study. Forty mls of blood was taken by standard venepuncture. All participants gave informed consent. This study was reviewed and approved by the Southern Tasmania HREC.
Serum 25(OH)D was measured by radioimmunoassay (Diasorin, Stillwater, MN, USA).
Collected blood was layered over histopaque (Sigma), spun at 400g for 30 min, and the interface containing the PBMC was removed and washed twice in incomplete RPMI-1640 (JRH
Results
Twenty-two subjects were recruited for the study. The majority (59.1%) of subjects were female, and the cohort had median serum 25(OH)D of 69.5 nmol/L. Other cohort characteristics and the distributions of PBMC cytokine concentrations are shown in Table 1.
Discussion
We have demonstrated the effect of combined treatment of calcitriol and interferon-β on the production of cytokines by stimulated PMBCs, demonstrating evidence of a novel two-way synergetic effect on the production of IL-6, an amelioration of the modulatory effects of calcitriol on IL-4 and IFN-γ by interferon-β, and some evidence for an enhancement of the suppressive effect of calcitriol on IL-2 by interferon-β. Individually, calcitriol treatment reduced production of IL-2, IL-4, IL-6, and
Author contributions
The study was conceived by NS, SSJ and BT. Funding was obtained by NS, SSJ and BT. Participant recruitment and sample processing and analysis were done by NS. Statistical analysis was done by SSJ, with advice by LB. Initial drafting of the manuscript was done by SSJ. All authors participated in critical revision of manuscript and approve it for submission. SSJ had access to all the data in the study and takes full responsibility for the data presented and the decision to submit the article for
Funding
This study was funded by a grant from the Royal Hobart Hospital Research Foundation. SSJ is supported by a Multiple Sclerosis Research Australia Postdoctoral Research Fellowship. IvM is supported by a Future Fellowship from the Australian Research Council.
Acknowledgements
We would like to thank the study participants for their kind participation in the study. We would also like to thank Professor Andrew Kemp of the University of Sydney for his immunological advice in the interpretation of our findings.
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