Excessive Reactive Oxygen Species Inhibit IL-17A+ γδ T Cells and Innate Cellular Responses to Bacterial Lung Infection.

Aims: Excessive reactive oxygen species (ROS) are detrimental to immune cellular functions that control pathogenic microbes; however, the mechanisms are poorly understood. Our aim was to determine the immunological consequences of increased ROS levels during acute bacterial infection. Results: We used a model of Streptococcus pneumoniae (Spn) lung infection and superoxide dismutase 3-deficient (SOD3-/-) mice, as SOD3 is a major antioxidant enzyme that catalyses the dismutation of superoxide radicals. First, we observed that in vitro, macrophages from SOD3-/- mice generated excessive phagosomal ROS during acute bacterial infection. In vivo, there was a significant reduction in infiltrating neutrophils in the bronchoalveolar lavage fluid and reduced peribronchial and alveoli inflammation in SOD3-/- mice 2 days after Spn infection. Annexin V/propidium iodide staining revealed enhanced apoptosis in neutrophils from Spn-infected SOD3-/- mice. In addition, SOD3-/- mice showed an altered macrophage phenotypic profile, with markedly diminished recruitment of monocytes (CD11clo, CD11bhi) in the airways. Further investigation revealed significantly lower levels of the monocyte chemokine CCL-2, and cytokines IL-23, IL-1ß, and IL-17A in Spn-infected SOD3-/- mice. There were also significantly fewer IL-17A-expressing gamma-delta T cells (γδ T cells) in the lungs of Spn-infected SOD3-/- mice. Innovation: Our data demonstrate that SOD3 deficiency leads to an accumulation of phagosomal ROS levels that initiate early neutrophil apoptosis during pneumococcal infection. Consequent to these events, there was a failure to initiate innate γδ T cell responses. Conclusion: These studies offer new cellular and mechanistic insights into how excessive ROS can regulate innate immune responses to bacterial infection.

Aspirin-triggered resolvin D1 reduces pneumococcal lung infection and inflammation in a viral and bacterial coinfection pneumonia model.

Formyl peptide receptor 2/lipoxin A4 (LXA4) receptor (Fpr2/ALX) co-ordinates the transition from inflammation to resolution during acute infection by binding to distinct ligands including serum amyloid A (SAA) and Resolvin D1 (RvD1). Here, we evaluated the proresolving actions of aspirin-triggered RvD1 (AT-RvD1) in an acute coinfection pneumonia model. Coinfection with Streptococcus pneumoniae and influenza A virus (IAV) markedly increased pneumococcal lung load and neutrophilic inflammation during the resolution phase. Fpr2/ALX transcript levels were increased in the lungs of coinfected mice, and immunohistochemistry identified prominent Fpr2/ALX immunoreactivity in bronchial epithelial cells and macrophages. Levels of circulating and lung SAA were also highly increased in coinfected mice. Therapeutic treatment with exogenous AT-RvD1 during the acute phase of infection (day 4-6 post-pneumococcal inoculation) significantly reduced the pneumococcal load. AT-RvD1 also significantly reduced neutrophil elastase (NE) activity and restored total antimicrobial activity in bronchoalveolar lavage (BAL) fluid (BALF) of coinfected mice. Pneumonia severity, as measured by quantitating parenchymal inflammation or alveolitis was significantly reduced with AT-RvD1 treatment, which also reduced the number of infiltrating lung neutrophils and monocytes/macrophages as assessed by flow cytometry. The reduction in distal lung inflammation in AT-RvD1-treated mice was not associated with a significant reduction in inflammatory and chemokine mediators. In summary, we demonstrate that in the coinfection setting, SAA levels were persistently increased and exogenous AT-RvD1 facilitated more rapid clearance of pneumococci in the lungs, while concurrently reducing the severity of pneumonia by limiting excessive leukocyte chemotaxis from the infected bronchioles to distal areas of the lungs.

IL-17A and serum amyloid A are elevated in a cigarette smoke cessation model associated with the persistence of pigmented macrophages, neutrophils and activated NK cells.

While global success in cessation advocacy has seen smoking rates fall in many developed countries, persistent lung inflammation in ex-smokers is an increasingly important clinical problem whose mechanistic basis remains poorly understood. In this study, candidate effector mechanisms were assessed in mice exposed to cigarette smoke (CS) for 4 months following cessation from long term CS exposure. BALF neutrophils, CD4+ and CD8+ T cells and lung innate NK cells remained significantly elevated following smoking cessation. Analysis of neutrophil mobilization markers showed a transition from acute mediators (MIP-2α, KC and G-CSF) to sustained drivers of neutrophil and macrophage recruitment and activation (IL-17A and Serum Amyoid A (SAA)). Follicle-like lymphoid aggregates formed with CS exposure and persisted with cessation, where they were in close anatomical proximity to pigmented macrophages, whose number actually increased 3-fold following CS cessation. This was associated with the elastolytic protease, MMP-12 (macrophage metallo-elastase) which remained significantly elevated post-cessation. Both GM-CSF and CSF-1 were significantly increased in the CS cessation group relative to the control group. In conclusion, we show that smoking cessation mediates a transition to accumulation of pigmented macrophages, which may contribute to the expanded macrophage population observed in COPD. These macrophages together with IL-17A, SAA and innate NK cells are identified here as candidate persistence determinants and, we suggest, may represent specific targets for therapies directed towards the amelioration of chronic airway inflammation.

SAA drives proinflammatory heterotypic macrophage differentiation in the lung via CSF-1R-dependent signaling.

Serum amyloid A (SAA) is expressed locally in chronic inflammatory conditions such as chronic obstructive pulmonary disease (COPD), where macrophages that do not accord with the classic M1/M2 paradigm also accumulate. In this study, the role of SAA in regulating macrophage differentiation was investigated in vitro using human blood monocytes from healthy subjects and patients with COPD and in vivo using an airway SAA challenge model in BALB/c mice. Differentiation of human monocytes with SAA stimulated the proinflammatory monokines IL-6 and IL-1ß concurrently with the M2 markers CD163 and IL-10. Furthermore, SAA-differentiated macrophages stimulated with lipopolysaccharide (LPS) expressed markedly higher levels of IL-6 and IL-1ß. The ALX/FPR2 antagonist WRW4 reduced IL-6 and IL-1ß expression but did not significantly inhibit phagocytic and efferocytic activity. In vivo, SAA administration induced the development of a CD11c(high)CD11b(high) macrophage population that generated higher levels of IL-6, IL-1ß, and G-CSF following ex vivo LPS challenge. Blocking CSF-1R signaling effectively reduced the number of CD11c(high)CD11b(high) macrophages by 71% and also markedly inhibited neutrophilic inflammation by 80%. In conclusion, our findings suggest that SAA can promote a distinct CD11c(high)CD11b(high) macrophage phenotype, and targeting this population may provide a novel approach to treating chronic inflammatory conditions associated with persistent SAA expression.

Glutathione peroxidase-1 reduces influenza A virus-induced lung inflammation.

Oxidative stress caused by excessive reactive oxygen species production is implicated in influenza A virus-induced lung disease. Glutathione peroxidase (GPx)-1 is an antioxidant enzyme that may protect lungs from such damage. The objective of this study was to determine if GPx-1 protects the lung against influenza A virus-induced lung inflammation in vivo. Male wild-type (WT) or GPx-1(-/-) mice were inoculated with HKx31 (H3N2, 1 × 10(4) plaque-forming units), and bronchoalveolar lavage fluid (BALF)/lung compartments were analyzed on Days 3 and 7 after infection for inflammatory marker expression, histology, and viral titer. WT mice infected with HKx31 had significantly more BALF total cells, macrophages, neutrophils, and lymphocytes at Days 3 and 7 compared with naive WT animals (n = 5-8; P < 0.05). However, infected GPx-1(-/-) mice had significantly more BALF inflammation, which included more total cells, macrophages, and neutrophils, compared with WT mice, and this was abolished by treatment with the GPx mimetic ebselen. BALF inflammation persisted in GPx-1(-/-) mice on Day 10 after infection, and GPx-1(-/-) mice had significantly more influenza-specific CD8(+) T cells in spleen compared with WT mice (n = 3-4; P < 0.05). Infected GPx-1(-/-) mice had greater peribronchial and parenchymal inflammation than WT mice, and viral titer was significantly reduced in GPx-1(-/-) mice at Day 3 (n = 5; P < 0.05). Gene expression analysis revealed that infected GPx-1(-/-) mice had higher whole lung TNF-α, macrophage inflammatory protein (MIP)-1α, MIP-2, KC, and matrix metalloproteinase (MMP)-12 mRNA compared with infected WT mice. GPx-1(-/-) mice had more MIP-2 protein in BALF at Day 3 and more active MMP-9 protease in BALF at Days 3 and 7 than WT mice. These data indicate that GPx-1 reduces influenza A virus-induced lung inflammation.