The moderate efficacy of recombinant IFNβ in patients with MS suggests the obvious conclusion that type I interferon is therapeutic rather than pathogenic in that disease [19
]. It should be noted, however, that the clinical development programs which led to the approval of IFNβ did not define its mechanism of action. Nor has it been clear whether IFNβ offers a benefit different from that seen after administration of IFNα. In fact, the differential effects of IFNα and IFNβ are difficult to demonstrate. In general, the gene expression programs that are induced by IFNα versus IFNβ are largely overlapping [34
]. While subtle differences in the binding properties of each of the interferons to IFNAR, their common receptor, have been predicted based on analysis of their amino acid sequence and mutation studies, and there are demonstrated differences in engaging downstream signaling components by the two type I interferon subtypes, their functional impact on gene expression is quite comparable [32
In light of the frequent administration of therapeutic IFNβ, it is perhaps surprising that gene expression analysis of patients with relapsing remitting multiple sclerosis (RRMS) (untreated with IFNβ) has demonstrated an interferon signature similar to the more classic signature seen in many patients with SLE [31
]. Van Baarsen and colleagues were among the first to discern the typical signature reflecting type I interferon activation in whole blood in their study of 29 patients with RRMS and 25 healthy controls [54
]. Along with a signature of immunoglobulin-related transcripts, one of the most prominent groups of transcripts was enriched in interferon-induced genes. The authors performed several analyses of the differentially expressed genes in their dataset in comparison with genes defined as either type I or type II (IFNγ)-inducible based on data in the literature, and concluded that type I interferon-inducible genes were increased in RRMS patients compared with control subjects whereas type II-induced genes were comparable between the two groups.
Van Baarsen and colleagues went further, however, and analyzed the gene program with a view towards predicting whether bacteria - which tend to activate the immune response through NF-κB-activating TLR2 or TLR4 path-ways - or viruses - which tend to activate the immune response through TLR3, TLR7 or TLR9 pathways and utilize MyD88 - are more likely to be responsible for the gene program observed in the patients. The NF-κB program was not different between patients and controls, but the interferon-induced gene program, similar to that induced by viruses, was differentially expressed. The study also compared the pattern of overexpressed genes in the RRMS patients with those induced in macaques by smallpox infection, and found that more than 50% of the patients clustered with the virus-infected macaques. The differentially expressed genes that characterized this subset of RRMS patients corresponded to those that describe a common response pathway characterizing innate immune responses to microbes [54
A role for type I interferon in RRMS is also supported by demonstration of IFNα, IFNβ, and MxA protein in brain lesions of patients with MS [56
]. In acute lesions, astrocytes stained positive for IFNβ, macro-phages expressed more IFNα, and endothelial cells some-times expressed both IFNα and IFNβ. Chronic lesions were more likely to be positive for IFNα [56
]. MXA, a type I interferon-inducible gene product, is present in astrocytes, in infiltrating T lymphocytes, and in endothelial cells - and the presence of nearby plasmacytoid dendritic cells suggests that the interferon is produced locally [57
]. MXA protein in peripheral blood of RRMS patients and elevated serum levels of type I interferon are also detected [55
]. Since the assays used to detect type I interferon activity in MS sera are distinct from those that have been used by others to quantify that activity in SLE patients, the relative levels cannot be compared. Based on the requirement for IFNγ priming to detect MxA protein in IFNAR-positive WISH epithelial cells cultured with MS sera, however, it seems probable that the levels are likely to be lower in most MS patients than in SLE patients with detectable interferon activity. One interpretation of the data demonstrating local type I interferon and its induced protein products in MS brain is that the interferon is providing an immunosuppressive effect [56
The paradigm of IFNα promoting systemic autoimmunity versus IFNβ reducing local inflammatory disease as an approach to understanding the role of type I interferons might apply to patients with MS treated with IFNβ. Consistent with the hypothesis that type I interferon inhibits TNF production are data from a study of RRMS patients treated for 18 to 24 months with IFNβ compared with patients not treated with IFNβ [31
]. IL-12, TNF, and IFNγ levels were elevated in the plasma or culture supernatants from MS patients compared with controls, but TNF and IFNγ levels were significantly lower in patients treated with IFNβ compared with those not treated. Of interest, TNF levels in whole blood cultures stimulated with lipopolysaccharide and IFNγ in supernatants of cultures stimulated with myelin basic protein were not different from levels in healthy controls in patients who had been treated with IFNβ, but did increase further in RRMS patients who had not been treated. At least in the case of the TNF data, the results would support an inhibitory effect of IFNβ downstream of TLR4 that reduces target gene expression.
A comprehensive analysis of IFNβ responders and nonresponders was recently published [59
]. The study analyzed 47 patients with RRMS (29 responders and 18 nonresponders, with responders defined based on no increase in the Expanded Disability Status Sale and no relapses during 2 years of treatment). Comparison of baseline gene expression profiles in PBMC identified differentially expressed genes in the two groups. Of great interest, type I interferon-inducible genes were generally overexpressed in the nonresponder patients and represented the pathway most significantly associated with nonresponse to IFNβ. When assessed after 3 months of therapy, most IFNβ clinical responders showed a robust cellular response with increased expression of interferon-inducible genes, while the nonresponder group showed modest or no increases in levels of expression of those genes. In fact, a prediction algorithm identifying the eight genes that best predicted IFNβ responders from nonresponders included five typical type I interferon-inducible genes (IFIT1, IFIT2, IFIT3, IFI44, and OASL).
The conclusions from the study of this initial cohort were validated in a second cohort including 15 responders and 15 nonresponders [59
]. Consistent with the increased level of interferon-inducible gene transcripts in the nonresponder group, baseline phosphorylated-STAT1 levels were higher in nonresponder monocytes than in responder monocytes. In addition, type I interferon bioactivity was higher in the nonresponders than in responders or healthy donors. The authors of this highly informative study performed in vitro
stimulation experiments to compare signaling downstream of IFNAR as well as in response to TLR ligands, and found roughly comparable responses in the two patient groups - with the exception of production of IFNα in response to lipopolysaccharide, which was significantly lower in responders than in nonresponders or healthy donors, as was expression of IFNAR1.
The interpretation of these results suggests a complex role for the type I interferon system in MS: consistent with the Van Baarsen and colleagues study, a subset of RRMS showed a type I interferon signature in blood in the absence of treatment, with Comabella and colleagues showing increased bioactive type I interferon in the nonresponder group - an observation confirmed in a recent report [59
]. The Comabella and colleagues study suggests that the high interferon group, those cases that do not respond to IFNβ, has an interferon pathway that is constitutively activated but is not further activated by administration of recombinant IFNβ. As the non-responder group obviously has poorer outcomes than the IFNβ responders, one is led to the speculation that increased production of type I interferon in MS patients contributes to disease and refractoriness to therapy. Similar to mechanisms suggested relevant to SLE, myeloid dendritic cells in the nonresponder RRMS patients studied by Comabella and colleagues showed increased expression of the costimulatory molecule CD86, suggesting that those cells might be capable of effective activation of self-reactive T cells.
One interpretation of the different profiles in the IFNβ responders and nonresponders is that when presented with an innate immune stimulus (such as lipopolysaccharide), the responder monocytes engage cellular mechanisms that reduce the capacity of the cells to produce type I interferon while the cells from non-responder patients do not ramp down that pathway. Impaired production of inhibitors of the Jak-STAT pathways activated by interferon binding to IFNAR was not demonstrated by the authors, as SOCS1, SOCS2 and PIAS1 expression was comparable between responders and nonresponders. Taken together, the data draw attention to the regulatory mechanisms that modulate innate immune responses downstream of TLRs, with TLR4 the relevant pathway in the RRMS patients.
Consideration of the demonstrated increased type I interferon bioactivity, increased expression of interferon-inducible genes, and stimulatory dendritic cell phenotype in IFNβ-treated patients who do not respond to that treatment raises the possibility that, similar to the situation in SLE, type I interferon might be a pathogenic mediator in that subset of RRMS patients and might be an appropriate therapeutic target. Additional studies that characterize this interesting nonresponder group more completely from the immunologic and serologic parameters will be of great interest. Although autoantibodies are not presumed to play as significant a pathogenic role in MS as T cells, it will be interesting to know whether the interferon high nonresponder group demonstrates higher levels of relevant autoantibodies than the interferon low responder group - as is the case in interferon high SLE patients [18
]. The induction of BAFF by IFNβ has been demonstrated in MS as in other diseases and could be a mechanism that contributes to increased humoral immunity. It will also be productive to compare T-cell responses to relevant self-antigens, such as myelin basic protein, in the IFN high group - the prediction being that self-reactive T cells will be expanded or more readily activated by antigen-presenting cells in those patients.
The somewhat counterintuitive data presented by Comabella and colleagues leave hanging the issue of how IFNβ results in a beneficial effect in those patients who do respond. One should note there is general agreement that recombinant IFNβ produces only modest responses in some patients. One prediction that could be tested using samples from the published study cohorts is that patients who go on to respond to IFNβ therapy are those with more robust TNF production. While the mechanisms that account for inhibition of TNF by type I interferon are not fully elucidated, the cytokine data do show reduction in TNF in patients who complete 18 to 24 months of IFNβ therapy, many of whom are presumably clinical responders [31
]. Augmentation of IL-10 by IFNβ through an IFNγ-dependent pathway might also contribute to amelioration of disease activity [60
There seem to be three categories of defect that are associated with the IFNβ nonresponder RRMS patients: production of interferon is high; in the setting of the in vivo
stimuli that characterize MS, IFNAR1 expression and signaling through TLR4 are not reduced in the nonresponders as they are in the responders; and capacity to further activate transcription of type I interferon-inducible genes is abrogated. The latter alteration might be due to a system in overdrive in which all available transcription factors are engaged; in effect, the patient's immune system is desensitized to further activation by IFNβ. It should be noted that extremely high-level expression of gene transcripts typically associated with inflammatory states, such as CXCL10 and PBEF1, achieves levels that are substantially higher in the responders after 3 months of IFNβ therapy [59
]. This concurrence of improved clinical activity and increased expression of proinflammatory mediators, at least at the transcript level, indicates that increased proinflammatory gene expression does not necessarily translate into increased inflammation. Perhaps the extremely high expression of IL1RN (IL-1 receptor antagonist) transcripts in the treated responders provides balance that counters the proinflammatory mediators.