Blockade of TLR9 agonist-induced type I interferons promotes inflammatory cytokine IFN-γ and IL-17 secretion by activated human PBMC☆
Introduction
Type I interferons (IFN) (IFN-α/β) are increasingly recognized as immune modulators that can inhibit or promote autoimmune diseases, including multiple sclerosis (MS), systemic lupus erythematosus (SLE), rheumatoid arthritis, and diabetes[61]. In SLE, a central role for type I IFN, in particular IFN-α, in disease pathogenesis is strongly indicated by studies of SLE patients and murine models of lupus (reviewed in [8]). In patients with active SLE, levels of IFN-α and expression of IFN-responsive genes are often elevated and correlated with the severity of disease [7], [11], [12], [34], [54]. Moreover, IFN-α therapy is known to induce clinical SLE [49]. Finally, in certain murine models of SLE, genetic type I IFN receptor deficiency suppresses autoimmune disease [37], [56].
It is now known that the specific source of IFN-α in SLE is the plasmacytoid dendritic cell (pDC) [55]. A current model of SLE envisions that engagement of pDC Toll-like receptors (TLRs) by circulating nucleic acid-containing autoimmune complexes (autoICs) stimulates pDC secretion of IFN-α. Subsequently, IFN-α acts to drive monocyte differentiation and immature myeloid DC maturation which, in turn, leads to the presentation of nuclear self-antigen to autoreactive T helper (Th) cells and breaking of tolerance[51]. Activated Th cells then help autoreactive B cells differentiate into autoantibody-producing plasma cells. In SLE, the effects of pathogenic autoantibodies and autoIC deposition results in inflammation and tissue damage, with release of cell debris that may provide a new source of self antigen to perpetuate the disease[41]. Currently, a major area of interest in designing new SLE therapies is on breaking the cycle of autoimmune disease by blocking the activity of circulating, pDC-derived type I IFN [51], [58].
The direct effects of inflammatory Th1-type cells also appear to contribute to autoimmune-mediated tissue injury in SLE [3], [9], [15], [61], [62], [63]. A Th1 predominance has been reported in some SLE patients, as indicated by increased systemic and/or local levels of the inflammatory cytokine IFN-γ, and the pro-inflammatory IFN-γ-inducers, IL-12 p70, and IL-18 [6], [15], [28], [52], [53], [64], [70], [71]. In addition, the activity of inflammatory Th1 cells is strongly implicated in mediating certain forms of lupus, including lupus glomerular nephritis, and cutaneous, and CNS lupus [2], [15], [17], [33], [66], [70].
The role of pDC-derived type I IFN in influencing autoimmune inflammatory Th cell responses in SLE is unknown. Type I IFN can have both positive and negative regulatory effects on inflammatory Th cell functions. Thus, although type I IFN are often considered to be promoters of anti-viral Th1-mediated immunity [42], [57], we and others have demonstrated that these IFN can act as potent inhibitors of Th1-mediated inflammation, and have shown that the basis for this effect is, at least in part, through their regulation of myeloid DC function [13], [22], [24], [44], [45], [47], [48], [67]. Indeed, it is this latter, Th1-inhibitory effect that appears to contribute to the beneficial effects of IFN-β therapy in the treatment of relapsing remitting MS, an inflammatory Th1-mediated autoimmune disease [14], [32].
The mechanisms involved in the opposing effects of type I IFN on human inflammatory Th cell-mediated immunity are not well understood, and they are the focus of our laboratory’s research. We observe that type I IFN regulate human myeloid DC expression of pro-inflammatory IFN-γ-inducing cytokines, and that depending on the timing of exposure, type I IFN can either promote or inhibit the differentiation of human naïve Th cells into IFN-γ-secreting Th1-type effector cells [67], [47]. Based on these and other findings, we hypothesized that in SLE, constant triggering of pDC TLR9 by autoICs results in high levels of circulating type I IFN that act to inhibit Th cell-mediated autoimmune inflammation. To explore this hypothesis, we developed a unique model of human PBMC stimulation to examine the effects of TLR9-induced type I IFN on PBMC cytokine secretion. In this model, normal PBMC are pretreated with the TLR9 agonist, CpG-A, and Th cells within the PBMC are subsequently activated with superantigen, while APC are activated with CD40L. Our results indicate that when pDC TLR9 triggering precedes APC cell activation and Th activation, the overall effect is to inhibit PBMC secretion of, respectively, the pro-inflammatory cytokines, IL-12 p70 and IL-23, and the inflammatory cytokines, IFN-γ and IL-17. Moreover, our data also indicate that this inhibitory effect is specifically mediated by pDC TLR9 agonist-induced type I IFN.
Section snippets
Cytokines and other cell culture reagents
The following recombinant human (rh) cytokines and other molecules were used during cell culture: Staphylococcal Enterotoxin A (SEA) (Toxin Technology; Sarasota, FL); rhIFN-β (Avonex; Biogen Idec, Cambridge, MA), rhIFN-α-a2 (Biosource) rhIFN-γ (BD/Pharmingen; San Diego, CA); rh interleukin (IL)-12 p70 (Hoffman-La Roche; Nutley, NJ), rhIL-23 (R&D Systems; Minneapolis, MN), and rhIL-10 (BD/Pharmingen); CpG-A, oligodeoxynucleotide (ODN) 2336 (Coley Pharmaceuticals, Wellesley, MA); B18R
A model of human PBMC stimulation
We developed a unique model of human PBMC stimulation in order to examine how, in SLE and other autoimmune diseases, constant triggering of pDC TLR9 by autoICs might regulate Th cell autoimmune inflammatory responses. In this model (see Fig. 6A), sustained pDC TLR9 stimulation by circulating SLE autoICs is mimicked by pretreating normal PBMC for 18 h with the pDC TLR9 ligand, CpG-A. Subsequently, stimulation of autoreactive Th cells with nuclear self antigen is mimicked by using the bacterial
Discussion
The studies presented herein demonstrate that pre-exposure to TLR9 agonist-induced type I IFN can inhibit activated PBMC secretion of the inflammatory cytokines, IFN-γ and IL-17, and pro-inflammatory cytokines, IL-12 p70 and IL-23. Our results indicate that type I IFN-mediated inhibition of PBMC IFN-γ secretion is largely due to a suppression of IFN-γ secretion by activated Th cells. Moreover, they suggest that inhibition of PBMC IFN-γ is mediated by a negative regulatory effect of type I IFN
Acknowledgements
The authors thank Dr. Ann Marshak Rothstein, and Dr. Robert Lafyatis for their helpful discussions and critical reviews of the manuscript.
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This research was funded by NIH/NIAID Grants R01AI44209 to G.A.v.S., and R21A1061433 to G.A.v.S. and J.M.v.S.