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It was hypothesized that the catalyst nanoceria can increase inflammation/oxidative stress from the basal and reduce it from the elevated state. Macrophages clear nanoceria. To test the hypothesis, M0 (non-polarized), M1- (classically activated, pro-inflammatory), and M2-like (alternatively activated, regulatory phenotype) RAW 264.7 macrophages were nanoceria exposed. Inflammatory responses were quantified by IL-1β level, arginase activity, and RT-qPCR and metabolic changes and oxidative stress by the mito and glycolysis stress tests (MST and GST). Morphology was determined by light microscopy, macrophage phenotype marker expression, and a novel three-dimensional immunohistochemical method. Nanoceria blocked IL-1β and arginase effects, increased M0 cell OCR and GST toward the M2 phenotype and altered multiple M1- and M2-like cell endpoints toward the M0 level. M1-like cells had greater volume and less circularity/roundness. M2-like cells had greater volume than M0 macrophages. The results are overall consistent with the hypothesis.

Regulatory properties of macrophages associated with alternative activation serve to limit the exaggerated inflammatory response during pneumonia caused by Pseudomonas aeruginosa infection. Arginase-1 is an important effector of these macrophages believed to play an essential role in decreasing injury and promoting repair. We investigated the role of arginase-1 in the control of inflammatory immune responses to P. aeruginosa pneumonia in mice that exhibit different immunologic phenotypes. C57BL/6 mice with conditional knockout of the arginase-1 (Arg1) gene from myeloid cells (Arg1(ΔM)) or BALB/c mice treated with small molecule inhibitors of arginase were infected intratracheally with P. aeruginosa. Weight loss, mortality, bacterial clearance, and lung injury were assessed and compared, as were the characterization of immune cell populations over time post-infection. Myeloid arginase-1 deletion resulted in greater morbidity along with more severe inflammatory responses compared to littermate control mice. Arg1(ΔM) mice had greater numbers of neutrophils, macrophages, and lymphocytes in their airways and lymph nodes compared to littermate controls. Additionally, Arg1(ΔM) mice recovered from inflammatory lung injury at a significantly slower rate. Conversely, treatment of BALB/c mice with the arginase inhibitor S-(2-boronoethyl)-L-cysteine hydrochloride (BEC) did not change morbidity as defined by weight loss, but mice at day 10 post-infection treated with BEC had gained significantly more weight back than controls. Neutrophil and macrophage infiltration were similar between groups in the lung parenchyma, and neutrophil migration into the airways was reduced by BEC treatment. Differences seem to lie in the impact on T cell subset disposition. Arg1(ΔM) mice had increased total CD4+ T cell expansion in the lymph nodes, and increased T cell activation, IFNγ production, and IL-17 production in the lymph nodes, lung interstitium, and airways, while treatment with BEC had no impact on T cell activation or IL-17 production, but reduced the number of T cells producing IFNγ in the lungs. Lung injury scores were increased in the Arg1(ΔM) mice, but no differences were observed in the mice treated with pharmacologic arginase inhibitors. Overall, myeloid arginase production was demonstrated to be essential for control of damaging inflammatory responses associated with P. aeruginosa pneumonia in C57BL/6 mice, in contrast to a protective effect in the Th2-dominant BALB/c mice when arginase activity is globally inhibited.

BackgroundAzithromycin treatment improves clinical parameters in patients with CF, and alters macrophage activation from a pro-inflammatory (M1) phenotype to a pro-fibrotic, alternatively activated (M2) phenotype. The transcriptional profile of cells from patients receiving azithromycin is unknown.MethodsGene expression in association with macrophage polarization, inflammation, and tissue remodeling was assessed from sputum samples collected from patients with CF. Transcriptional profiles and clinical characteristics, including azithromycin therapy, were compared.ResultsExpression of NOS2 and TNFα was decreased in subjects receiving azithromycin, whereas expression of M2-associated genes was unaffected. Principal component analysis revealed gene expression profiles consistent with M1- (MMP9, NOS2, and TLR4) or M2-polarization (CCL18, fibronectin, and MR1) in select subject groups. These expression signatures did not significantly correlate with clinical characteristics.ConclusionsPro-inflammatory gene expression was low in subjects receiving AZM. Genes were stratified into groupings characteristic of M1- or M2-polarization, suggesting that overall polarization status is distinct among patient groups.

Background Chronic airway inflammation characterizes patients with cystic fibrosis (CF). The role of alternative macrophage activation in this disease course is unknown. Objective We evaluated markers of alternative and classical macrophage activation in the lungs of patients with CF and evaluated these characteristics in the context of Pseudomonas aeruginosa ( PA ) infection, immunomodulatory drug therapy and pulmonary function. Methods Bronchoalveolar lavage or spontaneously expectorated sputum samples were collected from 48 CF patients. Clinical data were related to macrophage surface expression of mannose receptor (MR) (up-regulated in alternatively activated macrophages) and TLR4 (up-regulated in classically activated macrophages). Also, the activity of the alternatively activated macrophage effector molecule arginase was compared among patient groups, and pro- and anti-inflammatory cytokines produced by alternatively and classically activated macrophages were measured. Results There were significant differences between PA -infected and -uninfected patients in several clinical measurements. PA -infected patients exhibited increased use of azithromycin, up-regulation of MR on CD11b+ cells and increased arginase activity in their lung samples, and had a strong inverse relationship between MR and arginase activity to FEV 1 . Upon further analysis, PA -infected patients who were treated with azithromycin had the highest arginase activity and the highest number of macrophages that were MR+TLR4−, and both of these markers were inversely related to the FEV 1 . Conclusions Our findings suggest an increase in both MR and arginase expression as pulmonary function declines in PA -infected patients with CF. These markers of an alternatively activated macrophage phenotype give cause for future study to define the function of macrophage activation states in the CF lung.

Sulfamethoxazole-trimethoprim and zidovudine (AZT), drugs used often in combination in patients infected with HIV, were investigated for their effects on B cell development in a mouse model. BALB/c mice were randomized to receive oral doses of AZT, sulfamethoxazole-trimethoprim, or the combination via oral gavage for up to 28 days. Immune cell populations in the spleen, lung, and peripheral blood were examined, and toxicity to B lineage subtypes in the bone marrow was investigated by phenotypic analysis via flow cytometry. Pre-pro-B, pro-B, early pre-B, and late pre-B cells were assayed for apoptosis and analyzed for cell cycle profile. Total as well as B cell splenic and bone marrow cellularities were significantly decreased by using the drugs concomitantly, while B cell populations in the lungs and percentage in the peripheral blood were not affected. Combination therapy caused significant increases in apoptosis in B cells and granulocytes in the bone marrow, with the late pre-B cell population being the most depleted. The proliferative expansion and differentiation of early pre-B cells (B220+/CD43+/BP-1+/HSA+) to the late pre-B cell (B220+/CD43-/IgM-) stage was blocked, with early pre-B cells accumulating in the proliferative phases of the cell cycle. This apoptosis increase is likely due to elevated blood sulfamethoxazole concentrations that were observed in mice also receiving AZT. Concurrent sub-chronic administration of AZT and sulfamethoxazole-trimethoprim adversely affected B lymphocyte development in mouse bone marrow.