Elizabeth Sapey

Human innate immunosenescence: causes and consequences for immunity in old age

The past decade has seen an explosion in research focusing on innate immunity. Through a wide range of mechanisms including phagocytosis, intracellular killing and activation of proinflammatory or antiviral cytokine production, the cells of the innate immune system initiate and support adaptive immunity. The effects of aging on innate immune responses remain incompletely understood, particularly in humans. Here we review advances in the study of human immunosenescence in the diverse cells of the innate immune system, including neutrophils, monocytes, macrophages, natural killer and natural killer T (NKT) cells and dendritic cells-with a focus on consequences for the response to infection or vaccination in old age.

COPD exacerbations · 2: Aetiology

Exacerbations of COPD are thought to be caused by complex interactions between the host, bacteria, viruses, and environmental pollution. These factors increase the inflammatory burden in the lower airways, overwhelming the protective anti-inflammatory defences leading to tissue damage. Frequent exacerbations are associated with increased morbidity and mortality, a faster decline in lung function, and poorer health status, so prevention or optimal treatment of exacerbations is a global priority. In order to evolve new treatment strategies there has been great interest in the aetiology and pathophysiology of exacerbations, but progress has been hindered by the heterogeneous nature of these episodes, vague definitions of an exacerbation, and poor stratification of known confounding factors when interpreting results. We review how an exacerbation should be defined, its inflammatory basis, and the importance of exacerbations on disease progression. Important aetiologies, with their potential underlying mechanisms, are discussed and the significance of each aetiology is considered.

Senescence in innate immune responses: reduced neutrophil phagocytic capacity and CD16 expression in elderly humans

Elderly humans are more susceptible to bacterial infections because of declining immune status. We have investigated the effect of aging on neutrophil bactericidal responses, comparing neutrophil function in healthy, young (23–35 years) and elderly (>65 years) volunteers. Superoxide generation in response to fMLP was slightly increased in neutrophils from elderly donors, and serum from the elderly was able to opsonize E. coli efficiently. In contrast, phagocytic index was significantly lower in neutrophils from the elderly, compared with young donors (P<0.005). CD11a and CD11b expression was not affected by age, but CD16 was significantly reduced in neutrophils from elderly donors (P<0.0001). CD16 expression and phagocytic index were measured in the same neutrophils using FITC‐labeled E. coli, PE‐conjugated anti‐CD16 antibody, and CD16 expression correlated with phagocytic index (r=0.83;P<0.05). In elderly patients with bacterial infection, CD16 expression remained low. We propose that reduced neutrophil CD16 expression and phagocytosis contribute to human immunesenescence.

Impaired neutrophil extracellular trap formation: a novel defect in the innate immune system of aged individuals

Neutrophil extracellular traps (NETs) are a recently discovered addition to the defensive armamentarium of neutrophils, assisting in the immune response against rapidly dividing bacteria. Although older adults are more susceptible to such infections, no study has examined whether aging in humans influences NET formation. We report that TNF‐α‐primed neutrophils generate significantly more NETs than unprimed neutrophils and that lipopolysaccharide (LPS)‐ and interleukin‐8 (IL‐8)‐induced NET formation exhibits a significant age‐related decline. NET formation requires generation of reactive oxygen species (ROS), and this was also reduced in neutrophils from older donors identifying a mechanism for reduced NET formation. Expression of IL‐8 receptors (CXCR1 and CXCR2) and the LPS receptor TLR4 was similar on neutrophils from young and old subjects, and neutrophils challenged with phorbol‐12‐myristate‐13‐acetate (PMA) showed no age‐associated differences in ROS or NET production. Taken together, these data suggest a defect in proximal signalling underlies the age‐related decline in NET and ROS generation. TNF‐α priming involves signalling through p38 MAP kinase, but activation kinetics were comparable in neutrophils from young and old donors. In a clinical setting, we assessed the capacity of neutrophils from young and older patients with chronic periodontitis to generate NETs in response to PMA and hypochlorous acid (HOCL). Neutrophil extracellular trap generation to HOCL, but not PMA, was lower in older periodontitis patients but not in comparison with age‐matched controls. Impaired NET formation is thus a novel defect of innate immunity in older adults but does not appear to contribute to the increased incidence of periodontitis in older adults.

Behavioral and structural differences in migrating peripheral neutrophils from patients with chronic obstructive pulmonary disease.

RATIONALE There are increased neutrophils in the lungs of patients with chronic obstructive pulmonary disease (COPD), but it is unclear if this is due to increased inflammatory signal or related to the inherent behavior of the neutrophils. This is critical, because inaccurate or excessive neutrophil chemotaxis could drive pathological accumulation and tissue damage. OBJECTIVES To assess migratory dynamics of neutrophils isolated from patients with COPD compared with healthy smoking and nonsmoking control subjects and patients with α(1)-antitryspin deficiency. METHODS Migratory dynamics and structure were assessed in circulating neutrophils, using phase and differential interference contrast microscopy and time-lapse photography. The effect of COPD severity was studied. Surface expression of receptors was measured using flow cytometry. The in vitro effects of a phosphoinositide 3-kinase inhibitor (LY294002) were studied. MEASUREMENTS AND MAIN RESULTS COPD neutrophils moved with greater speed than cells from either control group but with reduced migratory accuracy, in the presence of IL-8, growth-related oncogene α, formyl-methionyl-leucyl-phenylalanine, and sputum. This was present across all stages of COPD. Structurally, COPD neutrophils formed fewer pseudopods during migration. There were no differences in surface expression of the receptors CXCR1, CXCR2, or FPR1. LY294002 reduced COPD neutrophil migratory speed while increasing chemotactic accuracy, returning values to normal. The inhibitor did not have these effects in healthy control subjects or patients with a similar degree of lung disease. CONCLUSIONS COPD neutrophils are intrinsically different than cells from other studied populations in their chemotactic behavior and migratory structure. Differences are not due to surface expression of chemoattractant receptors but instead appear to be due to differences in cell signaling.

Inter-relationships between inflammatory markers in patients with stable COPD with bronchitis: intra-patient and inter-patient variability

Background: Measurements of pulmonary biomarkers can be used to monitor airway inflammation in chronic obstructive pulmonary disease (COPD), but the variability of sampled biomarkers and their inter-relationships are poorly understood. A study was undertaken to examine the intra- and inter-patient variability in spontaneous sputum samples from patients in the stable state and to describe the relationship between biomarkers, cell counts and markers of disease. Methods: Sputum interleukin-1β, tumour necrosis factor α, interleukin 8, myeloperoxidase, leucotriene B4, growth-related oncogene α and differential cell counts were measured in patients with moderate to severe stable COPD (n = 14) on 11 occasions over a 1-month period. Results: There was significant variability in all inflammatory indices (median intra-patient coefficient of variation (CV) 35% (IQR 22–69), median inter-patient CV 102% (IQR 61–145)). Variability could be reduced by using a rolling mean of individual patient data points. Sample size calculations were undertaken to determine the number of patients required to detect a 50% reduction in neutrophil count. Using a crossover design of a putative effective treatment, the number needed using one data point per patient was 72, reducing to 23 when a mean of three data points was used. Significant correlations were demonstrated both between the inflammatory biomarkers themselves and between inflammatory biomarkers and markers of disease. Some relationships were not apparent when results from a single sample were used. The reliability of inter-relationships improved as more data points were used for each patient. Conclusions: Clear relationships exist between inflammatory biomarkers in patients with stable COPD. Sequential sampling reduced the variability of individual mediators and the potential number of patients needed to power proof of concept interventional studies.

Tumor Necrosis Factor–α rs361525 Polymorphism Is Associated with Increased Local Production and Downstream Inflammation in Chronic Obstructive Pulmonary Disease

RATIONALE Chronic obstructive pulmonary disease (COPD) has a genetic component, explaining susceptibility. Tumor necrosis factor (TNF)-alpha polymorphisms have been associated with COPD, but it is unclear if genotype influences clinical phenotype, protein expression, and bioactivity. OBJECTIVES To determine if a functional polymorphism was important by assessing TNF-alpha expression and activity and its association with clinical severity over time. METHODS Patients with COPD with rs361525 polymorphism were matched to patients with COPD without rs361525 polymorphism. TNF-alpha, its antagonists, and downstream mediators were measured in plasma and sputum. To determine TNF-alpha bioactivity, IL-8 secretion from primary bronchial epithelial cells (PBECs) was measured, and neutrophil migration was assessed using sputum from both subject groups in the presence and absence of TNF-alpha antibody. Subjects were followed annually and compared. MEASUREMENTS AND MAIN RESULTS Patients with polymorphism had more chronic bronchitis, a lower body mass index, and a greater annual decline in FEV(1) than patients with COPD without rs361525 polymorphism. TNF-alpha concentrations were 100-fold higher in airway secretions from the patients with the rs361525 polymorphism, with no difference in TNF-alpha antagonists. Their lung secretions contained more IL-8 and myeloperoxidase, consistent with downstream inflammation. Sputum from patients with rs361525 polymorphism induced greater secretion of IL-8 from PBECs and increased neutrophil migration. These effects could be abrogated by TNF-alpha antibody, demonstrating the bioactivity of TNF-alpha in lung secretions from this group. CONCLUSIONS This TNF-alpha polymorphism is associated with clinical features of disease including progression. There is clear evidence of TNF-alpha overexpression and bioactivity with neutrophilic inflammation. The polymorphism is likely to be a factor that influences a COPD disease phenotype and its progression.

An age-related numerical and functional deficit in CD19 + CD24 hi CD38 hi B cells is associated with an increase in systemic autoimmunity

Autoimmunity increases with aging indicative of reduced immune tolerance, but the mechanisms involved are poorly defined. In recent years, subsets of B cells with immunoregulatory properties have been identified in murine models of autoimmune disorders, and these cells downregulate immune responses via secretion of IL10. In humans, immature transitional B cells with a CD19+CD24hiCD38hi phenotype have been reported to regulate immune responses via IL10 production. We found the frequency and numbers of CD19+CD24hiCD38hi cells were reduced in the PBMC pool with age. IL10 expression and secretion following activation via either CD40, or Toll‐like receptors was also impaired in CD19+CD24hiCD38hi B cells from healthy older donors. When investigating the mechanisms involved, we found that CD19+CD24hiCD38hi B‐cell function was compromised by age‐related effects on both T cells and B cells: specifically, CD40 ligand expression was lower in CD4 T cells from older donors following CD3 stimulation, and signalling through CD40 was impaired in CD19+CD24hiCD38hi B cells from elders as evidenced by reduced phosphorylation (Y705) and activation of STAT3. However, there was no age‐associated change in expression of costimulatory molecules CD80 and CD86 on CD19+CD24hiCD38hi cells, suggesting IL10‐dependent immune suppression is impaired, but contact‐dependent suppressive capacity is intact with age. Finally, we found a negative correlation between CD19+CD24hiCD38hi B‐cell IL10 production and autoantibody (Rheumatoid factor) levels in older adults. We therefore propose that an age‐related decline in CD19+CD24hiCD38hi B cell number and function may contribute towards the increased autoimmunity and reduced immune tolerance seen with aging.

Vitamin D deficiency contributes directly to the acute respiratory distress syndrome (ARDS)

Rationale Vitamin D deficiency has been implicated as a pathogenic factor in sepsis and intensive therapy unit mortality but has not been assessed as a risk factor for acute respiratory distress syndrome (ARDS). Causality of these associations has never been demonstrated. Objectives To determine if ARDS is associated with vitamin D deficiency in a clinical setting and to determine if vitamin D deficiency in experimental models of ARDS influences its severity. Methods Human, murine and in vitro primary alveolar epithelial cell work were included in this study. Findings Vitamin D deficiency (plasma 25(OH)D levels <50 nmol/L) was ubiquitous in patients with ARDS and present in the vast majority of patients at risk of developing ARDS following oesophagectomy. In a murine model of intratracheal lipopolysaccharide challenge, dietary-induced vitamin D deficiency resulted in exaggerated alveolar inflammation, epithelial damage and hypoxia. In vitro, vitamin D has trophic effects on primary human alveolar epithelial cells affecting >600 genes. In a clinical setting, pharmacological repletion of vitamin D prior to oesophagectomy reduced the observed changes of in vivo measurements of alveolar capillary damage seen in deficient patients. Conclusions Vitamin D deficiency is common in people who develop ARDS. This deficiency of vitamin D appears to contribute to the development of the condition, and approaches to correct vitamin D deficiency in patients at risk of ARDS should be developed. Trial registration UKCRN ID 11994.

Aberrant neutrophil functions in stable chronic obstructive pulmonary disease: the neutrophil as an immunotherapeutic target.

Chronic obstructive pulmonary disease (COPD) is a common, progressive and debilitating chronic inflammatory condition affecting the lungs, with significant systemic manifestations and co-morbidities. Smoking cigarettes is the main risk factor, but only a fifth of smokers have clinically significant airflow obstruction and the inflammation persists after smoking cessation. This suggests that smoking (and exposure to other inhaled toxins) may be necessary but not sufficient to cause COPD. Neutrophils are believed central to COPD and their accumulation and degranulation are associated with tissue damage, increased inflammation and disordered tissue repair. It was assumed that neutrophil activity and function were appropriate in COPD, responding to the presence of high levels of inflammation in the lung. However more recent studies of neutrophil function (including migration, reactive oxygen species generation, degranulation, phagocytosis and extracellular trap (NET) production) suggest that there is a general impairment in COPD neutrophil responses that predispose towards increased inflammation and reduced bacterial clearance. This may be amenable to correction and manipulating neutrophil intracellular pathways (such as phosphoinositide-3-kinase signalling) appears to restore some key COPD neutrophil responses. Targeting neutrophil intra-cellular signalling may provide a means to normalise neutrophil behaviour in COPD. This could lead to improvements in disease outcomes by reducing extraneous inflammatory burden. However further studies are needed to determine if these findings are relevant in vivo and whether this would impact positively upon health and disease.