Project description:The importance of unanchored Ub in innate immunity has been shown only for a limited number of unanchored Ub-interactors. We investigated what additional cellular factors interact with unanchored Ub and whether unanchored Ub plays a broader role in innate immunity. To identify unanchored Ub-interacting factors from murine lungs, we used His-tagged recombinant poly-Ub chains as bait. These chains were mixed with lung tissue lysates and protein complexes were isolated with Ni-NTA beads. Sample elutions were subjected to mass spectrometry (LC-MSMS) analysis.
Project description:PPARγ is known for its anti-inflammatory actions in macrophages. However, which macrophage populations express PPARγ in vivo and how it regulates tissue homeostasis in the steady state and during inflammation is not completely understood. We show that lung and spleen macrophages constitutively expressed PPARγ, while other macrophage populations did not. Recruitment of monocytes to sites of inflammation was associated with induction of PPARγ as they differentiated to macrophages. Its absence in these macrophages led to failed resolution of inflammation, characterized by persistent, low-level recruitment of leukocytes. Conversely, PPARγ agonists supported an earlier cessation in leukocyte recruitment during resolution of acute inflammation and likewise suppressed monocyte recruitment to chronically inflamed atherosclerotic vessels. In the steady state, PPARγ deficiency in macrophages had no obvious impact in the spleen but profoundly altered cellular lipid homeostasis in lung macrophages. Reminiscent of pulmonary alveolar proteinosis, LysM-Cre x PPARγflox/flox mice displayed mild leukocytic inflammation in the steady-state lung and succumbed faster to mortality upon infection with S. pneumoniae. Surprisingly, this mortality was not due to overly exuberant inflammation, but instead to impaired bacterial clearance. Thus, in addition to its anti-inflammatory role in promoting resolution of inflammation, PPARγ sustains functionality in lung macrophages and thereby has a pivotal role in supporting pulmonary host defense. The two major subsets of monocytes (Ly-6C+ and Ly-6Clo) from 12-week old C57Bl/6 mice were sorted and the RNA extracted and hybridized to Affymetrix GeneChip® 430 2.0 arrays. We pooled leukocytes from 5 mice for each sort and sorted 3 to 4 separate times for 3 to 4 biological replicates.
Project description:This is an investigation of whole genome gene expression level in tissues of mice stimulated by LPS, FK565 or LPS + FK565 in vivo and ex vivo. We show that parenteral administration of a pure synthetic Nod1 ligand, FK565, induces site-specific vascular inflammation in mice, which is prominent in aortic root including aortic valves, slight in aorta and absent in other arteries. The degree of respective vascular inflammation is associated with persistent high expression of proinflammatory chemokine/cytokine genes in each tissue in vivo by microarray analysis, and not with Nod1 expression levels. The ex vivo production of proinflammatory chemokine/cytokine by Nod1 ligand is higher in aortic root than in other arteries from normal murine vascular tissues, and also higher in human coronary artery endothelial cells (HCAEC) than in human pulmonary artery endothelial cells (HPAEC), suggesting that site-specific vascular inflammation is at least in part ascribed to an intrinsic nature of the vascular tissue/cell itself.
Project description:The mononuclear phagocyte (MP) system consists of macrophages, monocytes, and dendritic cells. MP subtypes play distinct functional roles in steady-state and inflammatory conditions. Though murine MPs are well characterized, their human homologues remain poorly understood, particularly in the lung and draining lymph nodes (LNs). Understanding these homologies, and their similarities and differences with murine MPs, is critical for identifying human genetic targets and thus developing human immunotherapies. To address this gap, we performed transcriptome-based alignment across fifteen distinct human and nine distinct murine lung and LN MPs. As controls to validate the analytical quality of cross-species pulmonary MP analysis, human blood and murine spleen MPs were used. Constrained canonical correlation, t-SNE and catplot visualization was used to align human and mouse MPs and identify MP subtypes with maximal correspondence across species. Among the top marker genes expressed in corresponding human-mouse MP pairs, only 30-10% of the genes overlapped, indicating a need for caution when identifying human gene variants and functions from mice. For instance, SPP1 is highly expressed in human interstitial macrophages but not alveolar macrophages (AMs), whereas in mice SSP1 is only expressed in AMs. Moreover, human LN dendritic cells (DCs) align best with murine LN DCs but not murine splenic DCs indicating cross-species similarity in tissue. Lastly, in both species, CD88 was the most useful cell surface marker for distinguishing monocyte/macrophages from DCs. Overall, these data provide a reference for analyzing cross-species transcriptome data and evaluating whether specific murine genes are suitable guides to identify the functionality of human MPs, or conversely, whether pursuing specific human genes in mice, is likely to be a valid and fruitful approach.