Simultaneous analysis of host and pathogen interactions during an in vivo infection reveals local induction of host acute phase response proteins, a novel bacterial stress response, and evidence of a host-imposed metal ion limited environment.

A fundamental goal in the study of infections is to understand the dynamic interplay between host and pathogen; however, direct in vivo interrogation of this disease process via transcriptional profiling has been lacking. Here we describe the development and application of novel bacterial RNA amplification technology to simultaneously identify key elements of both host and pathogen responses in a murine infection model. On the bacterial side, we found induction of an unusual pattern of stress response genes, a response to host-induced metal ion limitation, and a failure to achieve stationary phase in vivo. On the mammalian side, we observed the surprising induction of several genes encoding acute phase response proteins including hepcidin, haptoglobin, complement C3 and metallothionein 1 at the site of infection, as well as other mediators of innate immunity. Thus, our results reveal host-pathogen cross-talk not predicted by previous in vitro analyses and provide the framework to eavesdrop on a broad array of host-pathogen interactions in vivo. As described here, the comprehensive examination of host-pathogen interactions during an infection is critical to the discovery of novel approaches for intervention not predicted by current models.

Cbl-mediated ubiquitinylation is required for lysosomal sorting of epidermal growth factor receptor but is dispensable for endocytosis.

Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identified Cbl-mediated ubiquitinylation of EGFR as a mechanism of ligand-induced EGFR down-regulation. However, the role of endogenous Cbl in EGFR down-regulation and the precise step in the endocytic pathway regulated by Cbl remain unclear. Using Cbl-/- mouse embryonic fibroblast cell lines, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and efficient degradation of EGFR. Further analyses using Chinese hamster ovary cells with a temperature-sensitive defect in ubiquitinylation confirm a crucial role of the ubiquitin machinery in Cbl-mediated EGFR degradation. However, internalization into early endosomes did not require Cbl function or an intact ubiquitin pathway. Confocal immunolocalization studies indicated that Cbl-dependent ubiquitinylation plays a critical role at the early endosome to late endosome/lysosome sorting step of EGFR down-regulation. These findings establish Cbl as the major endogenous ubiquitin ligase responsible for EGFR degradation, and show that the critical role of Cbl-mediated ubiquitinylation is at the level of endosomal sorting, rather than at the level of internalization.

IL-6 production by pulmonary dendritic cells impedes Th1 immune responses.

Mucosal tissues, such as the lung, are continually exposed to both foreign and environmental Ags. To counter the potential inflammatory tissue injury of chronic Th1-mediated responses against these Ags, mucosal sites may skew toward Th2 immune responses. However, the mechanism by which this occurs is unknown. Dendritic cells (DC), as orchestrators of the immune response, skew Th1/Th2 differentiation by cytokine secretion and expression of specific cell surface markers. We compared DC from mucosal and systemic locations. In this study, we show that the lung lacks a CD8alpha(+) DC subpopulation and contains DC that appear less mature than splenic DC. Furthermore, we demonstrate that pulmonary DC produce significant levels of IL-6 and fail to produce the Th1-polarizing cytokine IL-12. Importantly, we demonstrate that IL-6 negatively regulates IL-12 production, as pulmonary DC from IL-6(-/-) mice produce significant levels of IL-12 and induce Th1 polarization of naive CD4(+) T cells. Furthermore, we demonstrate that IL-6 is sufficient to explain the differential polarizing abilities of pulmonary and splenic DC, as splenic DC cocultures supplemented with IL-6 polarize naive T cells toward Th2, and pulmonary DC cultures in which IL-6 was removed with neutralizing Ab resulted in more Th1 polarization, pointing to IL-6 as the mechanism of Th2 polarization in the lung. We propose that the Th2 response seen in the lung is due to DC-mediated inhibition of Th1 responses via IL-6 production, rather than enhanced Th2 responses, and that this regulation decreases the likelihood of chronic inflammatory pathology in the lung.

ErbB2 degradation mediated by the co-chaperone protein CHIP.

ErbB2 overexpression contributes to the evolution of a substantial group of human cancers and signifies a poor clinical prognosis. Thus, down-regulation of ErbB2 signaling has emerged as a new anti-cancer strategy. Ubiquitinylation, mediated by the Cbl family of ubiquitin ligases, has emerged as a physiological mechanism of ErbB receptor down-regulation, and this mechanism appears to contribute to ErbB2 down-regulation induced by therapeutic anti-ErbB2 antibodies. Hsp90 inhibitory ansamycin antibiotics such as geldanamycin (GA) induce rapid ubiquitinylation and down-regulation of ErbB2. However, the ubiquitin ligase(s) involved has not been identified. Here, we show that ErbB2 serves as an in vitro substrate for the Hsp70/Hsp90-associated U-box ubiquitin ligase CHIP. Overexpression of wild type CHIP, but not its U-box mutant H260Q, induced ubiquitinylation and reduction in both cell surface and total levels of ectopically expressed or endogenous ErbB2 in vivo, and this effect was additive with that of 17-allylamino-geldanamycin (17-AAG). The CHIP U-box mutant H260Q reduced 17-AAG-induced ErbB2 ubiquitinylation. Wild type ErbB2 and a mutant incapable of association with Cbl (ErbB2 Y1112F) were equally sensitive to CHIP and 17-AAG, implying that Cbl does not play a major role in geldanamycin-induced ErbB2 down-regulation. Both endogenous and ectopically expressed CHIP and ErbB2 coimmunoprecipitated with each other, and this association was enhanced by 17-AAG. Notably, CHIP H260Q induced a dramatic elevation of ErbB2 association with Hsp70 and prevented the 17-AAG-induced dissociation of Hsp90. Our results demonstrate that ErbB2 is a target of CHIP ubiquitin ligase activity and suggest a role for CHIP E3 activity in controlling both the association of Hsp70/Hsp90 chaperones with ErbB2 and the down-regulation of ErbB2 induced by inhibitors of Hsp90.

Conservation of CD1 intracellular trafficking patterns between mammalian species.

Dendritic cells (DC) are potent APCs that sample Ags from the surrounding environment and present them to naive T cells using cell surface Ag-presenting molecules. The DC in both lymphoid and nonlymphoid tissues express high levels of CD1, a cell surface glycoprotein capable of presenting lipids and glycolipids to T cells. Distinct group 1 CD1 isoforms (CD1a, -b, -c) in man are known to traffic to different parts of the endocytic system where microbial Ags may be sampled. Guinea pigs are the only known rodent species that express the group 1 CD1 proteins. Therefore, we examined the expression and trafficking of guinea pig CD1 (gpCD1) isoforms on isolated DC. Confocal microscopy using mAbs specific for individual gpCD1 isoforms revealed differential trafficking of two distinct CD1b isoforms within DC. Colocalization of MHC class II was observed with the gpCD1b1 isoform, consistent with localization in the late endosomes of DC. In contrast, the gpCD1b3 isoform lacks an endosomal sorting motif and remains on the cell surface. Following incubation with Mycobacterium tuberculosis lipoarabinomannan, colocalization of endocytosed lipoarabinomannan with the gpCD1b1 isoform was observed but not with the gpCD1b3 isoform, which remained primarily on the cell surface. These data demonstrate that guinea pig DC express CD1 isoforms with unique trafficking patterns that recapitulate the patterns seen for human CD1 isoforms. This suggests evolutionary pressure for a conserved mechanism in mammals that allows CD1 to sample lipid Ags from various subcompartments of the endocytic system.

Negative regulation of Lck by Cbl ubiquitin ligase.

The Cbl-family ubiquitin ligases function as negative regulators of activated receptor tyrosine kinases by facilitating their ubiquitination and subsequent targeting to lysosomes. Cbl associates with the lymphoid-restricted nonreceptor tyrosine kinase Lck, but the functional relevance of this interaction remains unknown. Here, we demonstrate that T cell receptor and CD4 coligation on human T cells results in enhanced association between Cbl and Lck, together with Lck ubiquitination and degradation. A Cbl(-/-) T cell line showed a marked deficiency in Lck ubiquitination and increased levels of kinase-active Lck. Coexpression in 293T cells demonstrated that Lck kinase activity and Cbl ubiquitin ligase activity were essential for Lck ubiquitination and negative regulation of Lck-dependent serum response element-luciferase reporter activity. The Lck SH3 domain was pivotal for Cbl-Lck association and Cbl-mediated Lck degradation, with a smaller role for interactions mediated by the Cbl tyrosine kinase-binding domain. Finally, analysis of a ZAP-70-deficient T cell line revealed that Cbl inhibited Lck-dependent mitogen-activated protein kinase activation, and an intact Cbl RING finger domain was required for this functional effect. Our results demonstrate a direct, ubiquitination-dependent, negative regulatory role of Cbl for Lck in T cells, independent of Cbl-mediated regulation of ZAP-70.