Macrophages are key orchestrators of the inflammatory and repair responses in the lung, and the diversity of their function is indicated by their polarized states and distinct subpopulations and localization in the lung. Here, we characterized the pulmonary macrophage populations in the interstitial and alveolar compartments during the induction and resolution of acute lung injury induced by Pseudomonas aeruginosa infection. We identified macrophage subpopulations and polarity according to FACS analysis of cell surface protein markers, combined with cell sorting for gene expression using real-time PCR. With these techniques, we validated a novel, alternatively activated (M2) marker (transferrin receptor), and we described three interstitial and alveolar macrophage subpopulations in the lung whose distribution and functional state evolved from the induction to resolution phases of lung injury. Together, these findings indicate the presence and evolution of distinct macrophage subsets in the lung that serve specific niches in regulating the inflammatory response and its resolution. Alterations in the balance and function of these subpopulations could lead to nonresolving acute lung injury.
MHC tetramers are an essential tool for characterizing antigen-specific CD4+ T cells. However, their ex vivo analysis is limited by the large sample requirements. Here we demonstrate a combinatorial staining approach that allows simultaneous characterization of multiple specificities to address this challenge. As proof of principle, we analyse CD4+ T-cell responses to the seasonal influenza vaccine, establishing a frequency hierarchy and examining differences in memory and activation status, lineage commitment and cytokine expression. We also observe cross-reactivity between an established epitope and recent variant and provide a means for probing T-cell receptor cross-reactivity. Using cord blood samples, we correlate the adult frequency hierarchy with the naive precursor frequencies. Last, we use our combinatorial staining approach to demonstrate that rheumatoid arthritis patients on therapy can mount effective responses to influenza vaccination. Together, these results demonstrate the utility of combinatorial tetramer staining and suggest that this approach may have broad applicability in human health and disease.
Objective. Recognition of citrullinated antigens such as vimentin, fibrinogen, and α-enolase is associated with rheumatoid arthritis (RA). Emerging data suggest that the matrix protein aggrecan is also recognized as a citrullinated antigen. This study was undertaken to directly visualize Cit-aggrecan-specific T cells and characterize them in patients with RA.Methods. Citrullinated aggrecan peptides with likely DRB1 * 04:01 binding motifs were predicted using a previously published scanning algorithm. Peptides with detectable binding were assessed for immunogenicity by HLA tetramer staining, followed by single cell cloning. Selectivity for citrullinated peptide was assessed by tetramer staining and proliferation assays. Ex vivo tetramer staining was then performed to assess frequencies of aggrecan-specific T cells in peripheral blood. Finally, disease association was assessed by comparing T cell frequencies in RA patients and controls and correlating aggrecan-specific T cells with levels of aggrecan-specific antibodies.Results. We identified 6 immunogenic peptides, 2 of which were the predominant T cell targets in peripheral blood. These 2 epitopes were citrullinated at HLA binding residues and shared homologous sequences. RA patients had significantly higher frequencies of Cit-aggrecan-specific T cells than healthy subjects. Furthermore, T cell frequencies were significantly correlated with antibodies against citrullinated aggrecan.Conclusion. Our findings indicate that T cells that recognize citrullinated aggrecan are present in patients with RA and correlate with antibodies that target this same antigen. Consequently, aggrecan-specific T cells and antibodies are potentially relevant markers that could be used to monitor patients with RA or at-risk subjects.
Tissue inhibitor of metalloproteinases-3 (TIMP-3) has emerged as a key mediator of inflammation. Recently, we reported that the resolution of inflammation is impaired in Timp3 2/2 mice after bleomycininduced lung injury. Here, we demonstrate that after LPS instillation (another model of acute lung injury), Timp32/2 mice demonstrate enhanced and persistent neutrophilia, increased numbers of infiltrated macrophages, and delayed weight gain, compared with wild-type (WT) mice. Because macrophages possess broad immune functions and can differentiate into cells that either stimulate inflammation (M1 macrophages) or are immunosuppressive (M2 macrophages), we examined whether TIMP-3 influences macrophage polarization. Comparisons of the global gene expression of unstimulated or LPS-stimulated bone marrow-derived macrophages (BMDMs) from WT and Timp3 2/2 mice revealed that Timp3 2/2 BMDMs exhibited an increased expression of genes associated with proinflammatory (M1) macrophages, including Il6, Il12, Nos2, and Ccl2. Microarray analyses also revealed a baseline difference in gene expression between WT and Timp3 2/2 BMDMs, suggesting altered macrophage differentiation. Furthermore, the treatment of Timp3 2/2 BMDMs with recombinant TIMP-3 rescued this altered gene expression. We also examined macrophage function, and found that Timp3 2/2 M1 cells exhibit significantly more neutrophil chemotactic activity and significantly less soluble Fas ligand-induced caspase-3/7 activity, a marker of apoptosis, compared with WT M1 cells. Macrophage differentiation into immunosuppressive M2 cells is mediated by exposure to IL-4/IL-13, and we found that Timp3 2/2 M2 macrophages demonstrated a lower expression of genes associated with an anti-inflammatory phenotype, compared with WT M2 cells. Collectively, these findings indicate that TIMP-3 functions to moderate the differentiation of macrophages into proinflammatory (M1) cells.Keywords: resolution of inflammation; macrophage; metalloproteinase; lung injury Several immunosuppressive mechanisms have evolved to ensure that acute, generally beneficial inflammation does not progress to chronic, destructive inflammation. Beyond reversal of the initiating event (e.g., bacterial clearance and wound closure), active host processes, such as the release of the anti-inflammatory cytokines IL-4, IL-10, and IL-13, among other processes (1), actively repress the proinflammatory properties of various cell types. Macrophages, through their ability to clear neutrophils and release anti-inflammatory cytokines, are thought to be important in the resolution of inflammation (2).Generally speaking, macrophages are key effectors promoting the shift from a proinflammatory to an anti-inflammatory environment, and can be broadly classified into two groups: classically activated (M1) and alternatively activated (M2) macrophages, which can be further subdivided into specialized M2 groups (3). M1 macrophages are induced by proinflammatory Th1 cytokines, such as IFN-g, and are characterized by the production of proinfl...
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