Allergic Asthma Responses Are Dependent on Macrophage Ontogeny.

The ontogenetic composition of tissue-resident macrophages following injury, environmental exposure, or experimental depletion can be altered upon re-establishment of homeostasis. However, the impact of altered resident macrophage ontogenetic milieu on subsequent immune responses is poorly understood. Hence, we assessed the effect of macrophage ontogeny alteration following return to homeostasis on subsequent allergic airway responses to house dust mites (HDM). Using lineage tracing, we confirmed alveolar and interstitial macrophage ontogeny and their replacement by bone marrow-derived macrophages following LPS exposure. This alteration in macrophage ontogenetic milieu reduced allergic airway responses to HDM challenge. In addition, we defined a distinct population of resident-derived interstitial macrophages expressing allergic airway disease genes, located adjacent to terminal bronchi, and reduced by prior LPS exposure. These findings support that the ontogenetic milieu of pulmonary macrophages is a central factor in allergic airway responses and has implications for how prior environmental exposures impact subsequent immune responses and the development of allergy.

Nonclassical Monocytes Sense Hypoxia, Regulate Pulmonary Vascular Remodeling, and Promote Pulmonary Hypertension.

An increasing body of evidence suggests that bone marrow-derived myeloid cells play a critical role in the pathophysiology of pulmonary hypertension (PH). However, the true requirement for myeloid cells in PH development has not been demonstrated, and a specific disease-promoting myeloid cell population has not been identified. Using bone marrow chimeras, lineage labeling, and proliferation studies, we determined that, in murine hypoxia-induced PH, Ly6Clo nonclassical monocytes are recruited to small pulmonary arteries and differentiate into pulmonary interstitial macrophages. Accumulation of these nonclassical monocyte-derived pulmonary interstitial macrophages around pulmonary vasculature is associated with increased muscularization of small pulmonary arteries and disease severity. To determine if the sensing of hypoxia by nonclassical monocytes contributes to the development of PH, mice lacking expression of hypoxia-inducible factor-1α in the Ly6Clo monocyte lineage were exposed to hypoxia. In these mice, vascular remodeling and PH severity were significantly reduced. Transcriptome analyses suggest that the Ly6Clo monocyte lineage regulates PH through complement, phagocytosis, Ag presentation, and chemokine/cytokine pathways. Consistent with these murine findings, relative to controls, lungs from pulmonary arterial hypertension patients displayed a significant increase in the frequency of nonclassical monocytes. Taken together, these findings show that, in response to hypoxia, nonclassical monocytes in the lung sense hypoxia, infiltrate small pulmonary arteries, and promote vascular remodeling and development of PH. Our results demonstrate that myeloid cells, specifically cells of the nonclassical monocyte lineage, play a direct role in the pathogenesis of PH.

CHIP E3 ligase mediates proteasomal degradation of the proliferation regulatory protein ALDH1L1 during the transition of NIH3T3 fibroblasts from G0/G1 to S-phase.

ALDH1L1 is a folate-metabolizing enzyme abundant in liver and several other tissues. In human cancers and cell lines derived from malignant tumors, the ALDH1L1 gene is commonly silenced through the promoter methylation. It was suggested that ALDH1L1 limits proliferation capacity of the cell and thus functions as putative tumor suppressor. In contrast to cancer cells, mouse cell lines NIH3T3 and AML12 do express the ALDH1L1 protein. In the present study, we show that the levels of ALDH1L1 in these cell lines fluctuate throughout the cell cycle. During S-phase, ALDH1L1 is markedly down regulated at the protein level. As the cell cultures become confluent and cells experience increased contact inhibition, ALDH1L1 accumulates in the cells. In agreement with this finding, NIH3T3 cells arrested in G1/S-phase by a thymidine block completely lose the ALDH1L1 protein. Treatment with the proteasome inhibitor MG-132 prevents such loss in proliferating NIH3T3 cells, suggesting the proteasomal degradation of the ALDH1L1 protein. The co-localization of ALDH1L1 with proteasomes, demonstrated by confocal microscopy, supports this mechanism. We further show that ALDH1L1 interacts with the chaperone-dependent E3 ligase CHIP, which plays a key role in the ALDH1L1 ubiquitination and degradation. In NIH3T3 cells, silencing of CHIP by siRNA halts, while transient expression of CHIP promotes, the ALDH1L1 loss. The downregulation of ALDH1L1 is associated with the accumulation of the ALDH1L1 substrate 10-formyltetrahydrofolate, which is required for de novo purine biosynthesis, a key pathway activated in S-phase. Overall, our data indicate that CHIP-mediated proteasomal degradation of ALDH1L1 facilitates cellular proliferation.

Flow Cytometric Analysis of Myeloid Cells in Human Blood, Bronchoalveolar Lavage, and Lung Tissues.

Clear identification of specific cell populations by flow cytometry is important to understand functional roles. A well-defined flow cytometry panel for myeloid cells in human bronchoalveolar lavage (BAL) and lung tissue is currently lacking. The objective of this study was to develop a flow cytometry-based panel for human BAL and lung tissue. We obtained and performed flow cytometry/sorting on human BAL cells and lung tissue. Confocal images were obtained from lung tissue using antibodies for cluster of differentiation (CD)206, CD169, and E cadherin. We defined a multicolor flow panel for human BAL and lung tissue that identifies major leukocyte populations. These include macrophage (CD206(+)) subsets and other CD206(-) leukocytes. The CD206(-) cells include: (1) three monocyte (CD14(+)) subsets, (2) CD11c(+) dendritic cells (CD14(-), CD11c(+), HLA-DR(+)), (3) plasmacytoid dendritic cells (CD14(-), CD11c(-), HLA-DR(+), CD123(+)), and (4) other granulocytes (neutrophils, mast cells, eosinophils, and basophils). Using this panel on human lung tissue, we defined two populations of pulmonary macrophages: CD169(+) and CD169(-) macrophages. In lung tissue, CD169(-) macrophages were a prominent cell type. Using confocal microscopy, CD169(+) macrophages were located in the alveolar space/airway, defining them as alveolar macrophages. In contrast, CD169(-) macrophages were associated with airway/alveolar epithelium, consistent with interstitial-associated macrophages. We defined a flow cytometry panel in human BAL and lung tissue that allows identification of multiple immune cell types and delineates alveolar from interstitial-associated macrophages. This study has important implications for defining myeloid cells in human lung samples.

The mechanism of discrimination between oxidized and reduced coenzyme in the aldehyde dehydrogenase domain of Aldh1l1.

Aldh1l1, also known as 10-formyltetrahydrofolate dehydrogenase (FDH), contains the carboxy-terminal domain (Ct-FDH), which is a structural and functional homolog of aldehyde dehydrogenases (ALDHs). This domain is capable of catalyzing the NADP(+)-dependent oxidation of short chain aldehydes to their corresponding acids, and similar to most ALDHs it has two conserved catalytic residues, Cys707 and Glu673. Previously, we demonstrated that in the Ct-FDH mechanism these residues define the conformation of the bound coenzyme and the affinity of its interaction with the protein. Specifically, the replacement of Cys707 with an alanine resulted in the enzyme lacking the ability to differentiate between the oxidized and reduced coenzyme. We suggested that this was due to the loss of a covalent bond between the cysteine and the C4N atom of nicotinamide ring of NADP(+) formed during Ct-FDH catalysis. To obtain further insight into the functional significance of the covalent bond between Cys707 and the coenzyme, and the overall role of the two catalytic residues in the coenzyme binding and positioning, we have now solved crystal structures of Ct-FDH in the complex with thio-NADP(+) and the complexes of the C707S mutant with NADP(+) and NADPH. This study has allowed us to trap the coenzyme in the contracted conformation, which provided a snapshot of the conformational processing of the coenzyme during the transition from oxidized to reduced form. Overall, the results of this study further support the previously proposed mechanism by which Cys707 helps to differentiate between the oxidized and reduced coenzyme during ALDH catalysis.