In this study, we have addressed the role of IL-10 derived from endogenous, unmanipulated B cells in spontaneous chronic autoimmune disease. By deleting Il10 specifically in B cells in lupus prone mice we demonstrate that B cell-secreted IL-10 has no protective effect in lupus. This was reflected in equally severe organ disease, similar degrees of immune system activation, and indistinguishable survival rates in MRL.Faslpr mice that lack IL-10 specifically in B cells. Consistent with those results, using reporter mice, we found that B cells were only a minor source of IL-10 in vivo. By deleting Il10 in B cells from birth in this model we maximized the opportunity for IL-10+ B cells to exert regulatory effects without making prior assumptions at what stage of disease those might ensue. The extent of deletion, though not 100%, as with every Cre-loxP system, was 10–20 fold, which we believe should have been more than enough to reveal a phenotype if B cell-secreted IL-10 were truly regulating spontaneous lupus in a biologically significant fashion. Thus, our work indicates that B cell-derived IL-10 is not a principal regulator of disease in murine lupus.
Based on the existing paradigm (12
) and prior results in MRL.Faslpr
), our findings are unexpected. Endogenous IL-10+
B cells do regulate the immune response in infections with bacteria, viruses and parasites (35
). Directly pertinent to autoimmunity, IL-10-producing B cells contained disease in EAE (4
) and AIA (11
). An important difference between these diseases and lupus in MRL.Faslpr
mice could be the nature and kinetics of the initiating stimulus. In infectious disease models, as well as induced “autoimmunity” models such as EAE and AIA, the immune response is incited by inoculation with a pathogen or an antigen in combination with an adjuvant, resulting in sudden onset of immune response and pathology. In contrast, spontaneous chronic autoimmune diseases like lupus have a gradual onset. B cells might require abrupt and strong stimulation for robust IL-10 production. Thus B cell-derived IL-10 could be critical in pathogen responses and induced models of autoimmunity whereas syndromes of chronic autoimmunity in humans—such as SLE, rheumatoid arthritis, type 1 diabetes and multiple sclerosis—and mice may not be regulated by B cell-secreted IL-10, even if IL-10+
B cells might be therapeutic when infused.
It has been described that CD24hi
B cells from healthy individuals suppress T helper 1 cell differentiation in vitro in a partially IL-10 dependent manner (38
). In contrast, CD24hi
B cells from SLE patients lacked an equivalent suppressive capacity and expressed less IL-10 after stimulation. These results, although in vitro, are consistent with our findings that in lupus B cells do not bring their regulatory potential to bear.
To define the cells that produce IL-10 in the context of ongoing autoimmunity we used 10BiT reporter mice on the MRL.Faslpr
background. B cells represented only a minor fraction of Il10
transcribing cells and had low expression levels. Importantly, the vast majority of B cells with a CD1dhi
phenotype did not spontaneously synthesize IL-10 mRNA in vivo. From these data, we cannot confirm that there are bona fide, discrete, IL-10 producing, Breg subsets at least during active murine lupus. However, there was a considerable plasmablast population that transcribed Il10
. This argues that the stimuli that lead to the acquisition of IL-10 competence frequently induce plasmablast differentiation at the same time. Similar findings were reported for Vert-X C57BL/6 mice, another IL-10 reporter mouse, after challenge with different immunogens (35
). Whether the B cells that gave rise to IL-10 competent plasmablasts had a specific phenotype is unclear. Because plasmablasts are short-lived, our results imply that in lupus IL-10 producing B cell progeny represent a transient activation state and not a stable cell lineage with homeostatic regulation.
Both resting (39
) and activated (3
) B cells can suppress immune responses. Signals that have repeatedly been found to induce IL-10 production in B cells are Toll-like receptor activation, particularly in combination with B cell receptor ligation, and CD40 stimulation (41
). In MRL.Faslpr
mice self-reactive B cells are spontaneously activated via TLR7/9 and B cell receptor cross-linking by immune complexes (42
). Further CD40L deficient MRl.Faslpr
mice do not develop nephritis or make rheumatoid factor and anti-dsDNA autoantibodies (43
) arguing that CD40-CD40L interactions occur in this strain. Hence it is reasonable to assume that B cells receive signals in vivo that are known to induce IL-10. Chronic exposure to those signals, however, might have a different outcome than acute stimulation or other factors might impede a regulatory B cell phenotype in MRL.Faslpr
Recently, it was reported that deletion of all mature B cells, including B10 cells, in young preautoimmune NZB/W F1
mice accelerates disease onset and decreases survival time (12
mice had exacerbated nephritis paralleled by a reduction of B10 cells (13
). Both studies were interpreted to support a protective effect of B10 cells in lupus. However, in these studies the total mature B cell population was either depleted or genetically impaired, but it was not directly tested whether the observed effects were actually caused by the lack of B10 cells or any other IL-10 producing B cell population. Many other mechanisms could explain the observed effects. Altered activation state of macrophages after uptake of Ab coated B cells during the depletion process, indirect effects on the T cell compartment owing to lack of global B cell interactions, structural changes in lymphoid architecture or skewing of residual or regenerating B cell compartments could all potentially account for these earlier findings.
Hypothetically it is possible that B cells can employ IL-10 independent mechanisms to suppress an immune response that are potent enough to compensate for Il10
deficiency. However, in vivo evidence for such mechanisms has yet to be presented. Rather, in essentially all papers on Bregs in which a mechanism of regulation has been demonstrated, IL-10 is implicated (4
). Of greatest relevance to the present data, in MRL.Faslpr
mice regulatory effects of infused B cells are clearly IL-10 dependent (15
). Even if Bregs were to possess inhibitory means apart from IL-10, published studies indicate that Il10
transcription would at least identify most regulatory B cells. Yet, the fraction of Il10
transcribing B cells in MRL.Faslpr
mice was very small. Our data therefore does not favor the interpretation that other factors than IL-10 account for suppressive effects of endogenous Bregs in lupus. In any case, it is important to emphasize that the previously-implicated mechanism of B cell regulation in this instance was not validated when directly tested.
Our results, along with studies of B cell-targeted therapies in humans (1
) and mice (45
), suggest that B cells have a net pathogenic role in lupus that is not substantially counterbalanced by their IL-10 dependent regulatory functions. Using lupus prone mice bearing an IL-10 reporter transgenic locus we did not identify distinct B cell subsets that are enriched for Il10
transcription (other than plasmablasts), calling into question the existence of specific Breg populations, at least in the context of ongoing lupus. Further, our findings should precipitate a rethinking of whether endogenous B cells exist that regulate spontaneous chronic autoimmunity and emphasize the need to define the variables that govern B cell regulatory capacity in vivo.