| Upon immune challenge, DCs are able to produce a range of different cytokines that depend on the initial stimulus and help determine the nature of the resulting innate and adaptive responses. Identifying regulators of these initial DC responses is important for understanding the development of downstream responses and potentially influencing their outcome.;We have developed high throughput screening methods using a lentiviral shRNA library, together with microarray analysis, to identify novel regulators of cytokine production in primary BMDCs. shRNA constructs targeting 300 phosphatases were screened for their ability to modulate production of IL-12/23p40, as measured by ELISA. Nine genes were selected as positive regulators and nine as negative regulators.;A pilot screen identified the E3 ubiquitin ligase specificity factor, Fbxw11, as a gene required for IL-12/23p40 production. Induction of co-stimulatory molecules and additional cytokines were also decreased in DCs in which Fbxw11 was knocked down. Fbxw11 is known to be involved in ubiquitination of IkappaBalpha, along with its homologue betaTrCP1. While MEFs containing a gene trap inserted into the Fbxw11 gene showed reduced degradation of IkappaBalpha some degradation still occurred following TLR stimulation. In addition, levels of IkappaBalpha were reduced in Fbxw11 GT MEFs prior to stimulation, which would lead us to expect NFkappaB activity to be increased at baseline. However, both kappaB activity and NFkappaB binding were significantly decreased in unstimulated cells. Binding and activity could be induced by TLR stimulation, but activity did not reach WT levels.;The decrease in basal kappaB activity could be explained by a reduction in production of NFkappaB subunits or an increase in the fraction of NFkappaB bound by IkappaBs in the cytoplasm. Fbxw11 has also been implicated in the processing of p100, which would be consistent with reduced levels of NFkappaB components seen in Fbxw11 GT MEFs. While DNA-binding activity of p50 and p65 is induced to WT levels, this does not translate to similar induction of kappaB activity. This may be due to a requirement for RelB, which is not induced, or because of changes in the balance of NFkappaB dimers translocated to the nucleus. |
