Synergetic and antagonistic effects of combined calcitriol and interferon-β treatment on cytokine production by stimulated PBMCs

Highlights

• We assess calcitriol & interferon-β treatment effects on PBMC cytokine production.

• We find calcitriol significantly reduces IL-2, IFN-γ, TNF-α, IL-17 & IL-4 production.

• We find interferon-β reduces IL-6, TNF-α, & IL-17, and increases IL-2 & IL-10 production.

• We find a novel synergetic reduction in IL-6 and IL-17 by combined calcitriol & interferon-β.

• We find interferon-β attenuates the calcitriol reduction of IL-2 & IFN-γ.

Abstract

Background

We evaluated the effects of calcitriol and interferon-β on in vitro PBMC cytokine production from a cohort of 22 healthy adults not on medication.

Methods

PBMCs were incubated with calcitriol and/or 100 or 400 IU interferon-β or nothing, followed by stimulation with concanavalin A.

Results

When combined, calcitriol and interferon-β appeared to potentiate the effects of one another on reducing IL-6. Calcitriol significantly reduced the production of IL-2, IL-4, IL-6, and IFN-γ, while interferon-β significantly reduced production of IL-6 and TNF-α, and increased IL-10.

Discussion

This is the first study to evaluate the effects of combined calcitriol and interferon-β on cytokine production in PBMCs in vitro, demonstrating novel synergetic effects.

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.