IRF4 plays an important role in the development of Th2 and Th17 cells (14
). In Irf4
-/- mice, development of Th2 cells is decreased, suggesting that it plays a role in the differentiation process (14
). However, a role in differentiation does not preclude involvement in the regulation of specific cytokines in differentiated Th2 cells. In this report we demonstrate that IRF4 contributes to the heterogeneity of Th2 populations by increasing production of IL-4 and IL-10 and decreasing expression of Il9
, while having no effects on IL-5 or IL-13. IRF4 expression segregates in Th2 cells between IL-10-high and IL-10-low cells, directly binds to and transactivates the Il10
gene, and ectopic expression of IRF4 can increase IL-10 production from IL-10-low cells. Thus, IRF4 is an instructive factor in establishing Th2 heterogeneity.
IL-10 is a regulatory cytokine produced by a number of cells including Th2 cells and Il10
regulation in each cell type may be distinct. IL-10 plays a critical role in controlling inflammation in vivo by selectively suppressing the expression of pro-inflammatory cytokines. In this report we show that IL-10-high and -low cells have differing effects on co-cultured cells in vitro, and it is likely that functions differ in vivo as well. In various cell types, the molecular mechanism regulating the expression of IL-10 involves binding of IRF1 and STAT3 to the promoter region of the Il10
gene locus (26
), and the regulation of the level of IL-10 mRNA by Sp1 and Sp3 (35
). In Th2 cells, GATA-3 remodels the Il10
), and Jun family proteins bind the CNS3 region to induce Il10
). Moreover, IL-10 production requires repeated stimulation to be completely imprinted within the Th2 population (36
). In this report, we also show that IRF4 contributes to Il10
expression in Th2 and Th1 cells. The IL-12-dependent production of IL-10 in Th1 cells has been documented in human and mouse cells (26
). Clearly, additional factors contribute to the difference in IL-10 production by Th1 and Th2 cells, including that GATA-3 is not expressed in Th1 cells, c-jun and JunB bind to the Il10
CNS3 in Th2 but not Th1 cells (28
), and that Ets-1 is a negative regulator of IL-10 production in Th1 cells (39
). We demonstrate that in Th2 cells IRF4 binds directly to the Il10
locus and is able to transactivate Il10
regulatory element reporter plasmids. Our results parallel the recent description of a role for IRF4 in Treg cells where Il10
was one of the prominently regulated genes (15
). The fact that IRF4 binding sites exist in the Il10
promoter and CNS3 regions, and that it binds to the same regulatory element as Jun containing complexes suggests that these factors may cooperate. We did not observe strong interactions of IRF4 with GATA-3, suggesting that while these factors both contribute to Il10
expression, direct interactions are not required.
There are interactions of IRF4 with PU.1, which we previously demonstrated decreased IL-10 production (8
). In the absence of PU.1, IRF4 had greater potential to bind Il10
, transactivate the Il10
promoter and increase IL-10 production, suggesting that at least in part, PU.1 is able to modulate Il10
through the ability to interfere with IRF4 activity. It was surprising that we did not see increased IL-10 in Sfpi1lck-/-
control-transduced cells (), and this may be a result of the culture conditions required for retroviral transduction. The effects of PU.1-deficiency resulting in increased Th2 cytokine production are most dramatic when TCR stimulation is limiting (HCC, SLN and MHK, submitted). However, transduction of cells with limiting TCR stimulation was not efficient, and as a result of using optimal stimulation conditions for the retroviral transduction we did not observe altered IL-10 production in the absence of IRF4 transduction. The role of PU.1 must also be placed in the context of interactions with multiple transcription factors () and with the heterogeneity of the Th2 population where PU.1 is differentially expressed in subpopulations of cells. As previously noted, PU.1 is expressed highly in IL-4-low cells, and in this report is also expressed in IL-10-high cells. This might suggest that PU.1 is most highly expressed in an IL-4-low/IL-10-high population. However, it has thus far been difficult to sort cells stained for two cytokines preventing a more thorough analysis of this issue. As PU.1 expression is only present in a subpopulation of Th2, the effects of deficiency on IRF4 function might be obscured in a bulk Th2 population, unless IRF4 is overexpressed (). Moreover, as ectopic PU.1 expression decreased IL-5 and IL-13 production, as well as IL-4 and IL-10, it is clear that interactions with IRF4 only account for a portion of the observed function of PU.1 in Th2 cells.
The reciprocal regulation of Il10
in Th2 cells is striking and distinct from the IL-9 and IL-10-producing cells present in cultures primed with TGFβ and IL-4 (40
). While transduction of IRF4 in Th2 cells decreases Il9
expression, we observed only minimal IRF4 binding to the Il9
promoter in a ChIP assay. This suggested that the effects of IRF4 could be indirect, through the induction of IL-10. Indeed, neutralizing IL-10 in IL-10-high cells, that express low levels of Il9
, modestly increased Il9
mRNA (data not shown). However, it is not clear which IL-10 activated pathways might be responsible for this regulation. It is also not clear why a cell would be specialized to express only one of these cytokines. IL-9 is a pleiotropic cytokine involved in the pathologic and physiologic evolution of asthma by recruiting eosinophils and lymphocytes to the lung, inducing mucus hypersecretion, mast cells hyperplasia in concert with IL-4, IL-5 and IL-13 (42
), while IL-10 is a suppressive cytokine that may modulate many of these processes. It is possible that secretion of IL-9 by Th2 cells would only be effective if target cells did not receive a conflicting signal generated by IL-10. In this manner, Th2 heterogeneity may reflect functional specialization of cell types within the inflammatory microenvironment.
Increasing evidence suggests that the establishment of Th2 heterogeneity is not stochastic, but rather instructive, based on the expression of specific factors. As such, a growing list of transcription factors has specific effects on individual Th2 cytokines. IRF4 is induced following T cell activation, and expression is further increased following Th2 differentiation. Importantly, the level of IRF4 in IL-10-low cells is increased compared to that in recently activated T cells. As we have shown, IRF4 activates IL-10 and IL-4 production, while decreasing IL-9 and having little effect on IL-5 or IL-13. C-maf regulates IL-4 production but is not required for production of other Th2 cytokines (43
). Similarly, Pias1 increases IL-13 production without affecting IL-4 or IL-5 expression (44
). We have shown that PU.1 decreases expression of many Th2 cytokines, but increases expression of CCL22, a chemokine associated with Th2 inflammation (8
). BOB.1/OBF.1 regulates PU.1 expression in Th2 cells and also affects the potential for Th2 cytokine production (45
). Moreover, the expression levels of each of these factors, and other factors that contribute to Th2 cytokine production exist in gradients that correlate with cytokine producing phenotypes (). The similar level of expression of IRF4 in both IL-4- and IL-10-single positive cells supports the idea that other positive- or negative-acting factors overlay on the IRF4 gradient to generate the specific patterns of cytokine secretion. The mosaic of transcription factor gradients ultimately results in the heterogeneity observed in cytokine production from individual cells.
In this report, we have identified IRF4 as a regulator of Th2 heterogeneity by enhancing or decreasing the production of specific cytokines. IRF4 function, like GATA-3 as described in our previous report (8
), is limited by the expression of PU.1 in Th2 cells, which binds IRF4 and decreases binding to target genes including Il10
. Future work will examine how these factors interact to generate the population phenotype and what signals determine the expression of each factor within individual cells.