The Tumor Suppressor ARF Regulates Innate Immune Responses in Mice

Abstract

The innate immune system is the first line of defense against invading organisms, and TLRs are the main sensors of microbial components, initiating signaling pathways that induce the production of proinflammatory cytokines and type I IFNs. An antiviral action for the tumor suppressor alternative reading frame (ARF) has been reported; however, the precise role of ARF in innate immunity is unknown. In this study, we show that ARF plays an important role in regulation of inflammatory responses. In peritoneal macrophages and bone marrow-derived macrophages from ARF-deficient animals, the induction of proinflammatory cytokines and chemokines by TLR ligands was severely impaired. The altered responses of ARF−/− cells to TLR ligands result from aberrant activation of intracellular signaling molecules including MAPKs, IκBα degradation, and NF-κB activation. Additionally, animals lacking ARF were resistant to LPS-induced endotoxic shock. This impaired activation of inflammation in ARF−/− mice was not restricted to TLRs, as it was also shown in response to non-TLR signaling pathways. Thus, ARF−/− mice were also unable to trigger a proper inflammatory response in experimental peritonitis or in 12-O-tetradecanoylphorbol-13-acetate–induced edema. Overexpression of ARF, but not its downstream target p53, rescued the ARF-deficient phenotype, increasing TLR4 levels and restoring inflammatory reaction. An increase in the E2F1 protein levels observed in ARF−/− macrophages at basal condition and after LPS stimulation may be involved in the impaired response in this system, as E2F1 has been described as an inflammatory suppressor. These results indicate that tumor suppressor ARF is a new regulator of inflammatory cell signaling.

NADPH Oxidase Modifies Patterns of MHC Class II–Restricted Epitopic Repertoires through Redox Control of Antigen Processing

“…It could be argued that was assessed using MOG in a reconstituted system with increasing concentrations of H 2 O 2 . Following incubation at 37˚C, relative quantities of remaining substrate (MOG ) and cleavage products (MOG [42][43][44][45][46][47][48][49][50][51][52][53][54][55] and MOG [35][36][37][38][39][40][41] ) were quantified by reverse-phase HPLC following peak identification by MALDI-TOF MS. Data are presented as the abundance of the cleavage product MOG [42][43][44][45][46][47][48][49][50][51][52][53][54][55] relative to MOG (corresponding destruction of the I-A b binding region) and was calculated using the following formula: RPA = (P x /S x )/(P 0 /S 0 ), where RPA indicates relative peptide abundance; P x indicates product (MOG [42][43][44][45][46][47][48][49][50][51]…”

“…To achieve this, T cell hybridomas were first stably transfected with an NFA-T-enhanced IL-2 promoter-driven eGFP construct, followed by multiple rounds of selection by FACS to enrich those hybridoma clones that expressed eGFP only following presentation of their cognate peptide epitope by APCs (25). When added to BMMfs that were previously pulsed with HEL protein, we found that WT and Cybb 2/2 BMMfs were equally able to induce eGFP expression in two of the hybridomas (Hb1.9 and H46.13), indicating that the processing/presentation efficiency of HEL [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] and HEL [48][49][50][51][52][53][54][55][56][57][58][59][60][61][62] peptides were unaffected by NOX2 (Fig. 2D).…”

“…The relative abundance of the peptides was quantified using the area under corresponding peaks. Abundance of the cleavage product MOG [42][43][44][45][46][47][48][49][50][51][52][53][54][55] relative to MOG (corresponding destruction of the I-A b binding region) was calculated using the following formula: RPA = (P x /S x )/(P 0 /S 0 ), where RPA indicates relative peptide abundance, P x indicates product (MOG [42][43][44][45][46][47][48][49][50][51][52][53][54][55] ) of sample, S x indicates substrate (MOG ) of sample, P 0 indicates product (MOG [42][43][44][45][46][47][48][49][50][51][52][53][54][55] ) without hydrogen peroxide, and S 0 indicates substrate (MOG ) without hydrogen peroxide.…”

“…To further interrogate the ability of NOX2 to alter processing patterns of a single Ag, we measured the efficiency of WT and Cybb 2/2 BMMfs to process four distinct HEL epitopes (HEL [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] , HEL [31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] , HEL [48][49][50][51][52][53][54][55][56][57][58][59][60][61][62] , and HEL 74-90 ) from whole HEL protein and present them to epitope-specific CD4 + T cell hybridomas (Hb1.9, H30.44, H46.13, and B04, respectively) (26). To achieve this, T cell hybridomas...…”

“…qPCR and qPCR array qPCR array technology was used to screen for transcriptional changes in genes of interest to EAE (51,52). The relative expression of 84 genes relating to myelination, T cell activation and signaling, adaptive immunity, cytokines, chemokines, inflammation, apoptosis, cell adhesion, cell stress, immune receptors, and immune/CNS-related transcription factors were examined in the lumbar spinal cord of WT and Cybb 2/2 mice using the mouse multiple sclerosis RT 2 Profiler PCR array (SABiosciences).…”