Epstein-Barr virus-induced gene 3 (EBI3) associates with p28 to form IL-27 and with IL-12p35 to form IL-35. IL-27Ra -/-mice studies indicate that IL-27 negatively regulates Th17 cell differentiation. However, no EBI3, p28 or p35-deficiency studies that directly address the role of EBI3, p28 or p35 on Th17 cells have been done. Here, we demonstrate that spleen cells derived from EBI3 -/-mice produce significantly higher levels of IL-17 as well as IL-22 upon stimulation with OVA. In vitro derived EBI3 -/-Th17 cells also produced significantly higher levels of IL-17 and IL-22 than WT cells. The frequency of IL-17-producing cells was also elevated when EBI3 -/-cells were cultured under Th17 conditions. In addition, spleen cells from EBI3 -/-mice immunized with Listeria monocytogenes produced significantly elevated levels of IL-17 and IL-22. Furthermore, the Th17 transcription factor RORct was significantly enhanced in EBI3 -/-cells. Finally, EBI3 -/-mice exhibited a reduced bacterial load following an acute challenge with L. monocytogenes or a re-challenge of previously immunized mice, suggesting that EBI3 negatively regulates both innate and adaptive immunity. Taken together, these data provide direct evidence that EBI3 negatively regulates the expression of IL-17, IL-22 and RORct as well as protective immunity against L. monocytogenes.

Protein kinase C theta (PKCtheta) is essential for T cell activation, as it is required for the activation of NF-kappaB and expression of IL-2. PKCtheta has also been shown to affect NFAT activation and Th2 differentiation. To better understand the role of PKCtheta in the regulation of T helper cells, we used PKCtheta-deficient DO11.10 transgenic T cells to study its role in vitro. DO11.10 Th1 cells deficient in PKCtheta produced significantly less TNF-alpha and IL-2. The expression of Th2 cytokines, including IL-4, IL-5, IL-10, IL-13 and IL-24 was significantly reduced in PKCtheta-deficient T cells. Moreover, the expression of the Th2 transcription factor, GATA3, was significantly reduced in PKCtheta-deficient T cells. Overexpression of GATA3 by retroviral infection in PKCtheta-deficient T cells resulted in increased expansion of IL-4-producing T cells and higher IL-4 production than that of wild type Th2 cells. IL-5, IL-10, IL-13 and IL-24 expressions were also rescued by GATA3 overexpression. Our observations suggest that PKCtheta regulates Th2 cytokine expression via GATA3.

SummaryPIM (proviral integration site) kinases are a distinct class of serine/threonine-specific kinases consisting of PIM1, PIM2 and PIM3. PIM2 is known to function in apoptosis pathways. Expression of PIM2 is highly induced by pro-inflammatory stimuli but the role of PIM2 in the expression of pro-inflammatory cytokines is unclear. In this study, we showed that over-expression of PIM2 in HeLa cells as well as in human umbilical vein endothelial cells enhanced interleukin-1b (IL-1b) -induced and tumour necrosis factor-a-induced IL-6 expression, whereas over-expression of a kinase-dead PIM2 mutant had the opposite effect. Studies with small interfering RNA specific to PIM2 further confirmed that IL-6 expression in HeLa cells requires PIM2. To investigate the function of PIM2 further, we generated PIM2-deficient mice. It was found that IL-6 production was significantly decreased from PIM2-deficient spleen cells after stimulation with lipopolysaccharide. Taken together, we demonstrated an important function of PIM2 in controlling the expression of the pro-inflammatory cytokine IL-6. PIM2 inhibitors may be beneficial for IL-6-mediated diseases such as rheumatoid arthritis.

The product of the Epstein-Barr virus-induced gene 3 (EBI3) associates with p28, an IL-12p35-related protein, to form IL-27. EBI3 also associates with IL-12p35 to form a distinct heterodimer. The function of EBI3 in innate as well as adaptive immunity to intracellular bacteria has not been established. Here we demonstrate that EBI3 deficient mice exhibit a reduced bacterial load following an acute challenge with Listeria monocytogenes or a re-challenge of animals previously immunized with L. monocytogenes, suggesting EBI3 plays a negative role in both innate and adaptive immunity. The enhanced protective immunity in EBI3-deficient mice is not due to modulation of the Th1 immune response since the production of IFNγ and TNFα was not significantly altered. Importantly, spleen cells from EBI3-deficient mice immunized with L. monocytogenes produced significantly higher levels of IL-17 than wild type cells. Furthermore splenocytes as well as Th17 cells from EBI3-deficient mice secreted increased levels of IL-17. Elevated numbers of IL-17-producing cells were observed when EBI3 deficient CD4+ T cells were cultured under conditions to induce Th17 cell differentiation. Taken together, these data provide direct evidence that EBI3 is an important negative regulator of protective immunity against L. monocytogenes as well as IL-17 expression.