Hannah Greenwood

Aging of the innate immune system

The innate immune system is composed of a network of cells including neutrophils, NK and NKT cells, monocytes/macrophages, and dendritic cells that mediate the earliest interactions with pathogens. Age-associated defects are observed in the activation of all of these cell types, linked to compromised signal transduction pathways including the Toll-like Receptors. However, aging is also characterized by a constitutive pro-inflammatory environment (inflamm-aging) with persistent low-grade innate immune activation that may augment tissue damage caused by infections in elderly individuals. Thus, immunosenescence in the innate immune system appears to reflect dysregulation, rather than exclusively impaired function.

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.

Habitual physical activity is associated with the maintenance of neutrophil migratory dynamics in healthy older adults

Highlights • Physical activity associates with neutrophil migration in the elderly.• Effects occur independently of inflammation or surface receptor expression.• Adiponectin levels may positively influence neutrophil migration.

Vitamin D deficiency in human and murine sepsis

Objectives: Vitamin D deficiency has been implicated as a pathogenic factor in sepsis and ICU mortality but causality of these associations has not been demonstrated. To determine whether sepsis and severe sepsis are associated with vitamin D deficiency and to determine whether vitamin D deficiency influences the severity of sepsis. Design, Setting, and Patients: Sixty-one patients with sepsis and severe sepsis from two large U.K. hospitals and 20 healthy controls were recruited. Murine models of cecal ligation and puncture and intratracheal lipopolysaccharide were undertaken in normal and vitamin D deficient mice to address the issue of causality. Measurements and Main Results: Patients with severe sepsis had significantly lower concentrations of 25-hydroxyvitamin D3 than patients with either mild sepsis or age-matched healthy controls (15.7 vs 49.5 vs 66.5 nmol/L; p = 0.0001). 25-hydroxyvitamin D3 concentrations were significantly lower in patients who had positive microbiologic culture than those who were culture negative (p = 0.0023) as well as those who died within 30 days of hospital admission (p = 0.025). Vitamin D deficiency in murine sepsis was associated with increased peritoneal (p = 0.037), systemic (p = 0.019), and bronchoalveolar lavage (p = 0.011) quantitative bacterial culture. This was associated with reduced local expression of the cathelicidin-related antimicrobial peptide as well as evidence of defective macrophage phagocytosis (p = 0.029). In the intratracheal lipopolysaccharide model, 1,500 IU of intraperitoneal cholecalciferol treatment 6 hours postinjury reduced alveolar inflammation, cellular damage, and hypoxia. Conclusions: Vitamin D deficiency is common in severe sepsis. This appears to contribute to the development of the condition in clinically relevant murine models and approaches to correct vitamin D deficiency in patients with sepsis should be developed.

Pulmonary Infections in the Elderly Lead to Impaired Neutrophil Targeting, Which Is Improved by Simvastatin

Rationale: Dysregulated neutrophil functions with age and sepsis are described. Statins are associated with improved infection survival in some observational studies, but trials in critically ill patients have not shown benefit. Statins also alter neutrophil responses in vitro. Objectives: To assess neutrophil migratory accuracy with age during respiratory infections and determine if and how a statin intervention could alter these blunted responses. Methods: The migratory accuracy of blood neutrophils from young (aged <35 yr) and old (aged >60 yr) patients in health and during a lower respiratory tract infection, community‐acquired pneumonia, and pneumonia associated with sepsis was assessed with and without simvastatin. In vitro results were confirmed in a double‐blind randomized clinical trial in healthy elders. Cell adhesion markers were assessed. Measurements and Main Results: In vitro neutrophil migratory accuracy in the elderly deteriorated as the severity of the infectious pulmonary insult increased, without recovery at 6 weeks. Simvastatin rescued neutrophil migration with age and during mild to moderate infection, at high dose in older adults, but not during more severe sepsis. Confirming in vitro results, high‐dose (80‐mg) simvastatin improved neutrophil migratory accuracy without impeding other neutrophil functions in a double‐blind randomized clinical trial in healthy elders. Simvastatin modified surface adhesion molecule expression and activity, facilitating accurate migration in the elderly. Conclusions: Infections in older adults are associated with prolonged, impaired neutrophil migration, potentially contributing to poor outcomes. Statins improve neutrophil migration in vivo in health and in vitro in milder infective events, but not in severe sepsis, supporting their potential utility as an early intervention during pulmonary infections. Clinical trial registered with www.clinicaltrialsregister.eu (2011‐002082‐38).

Pre-emptive or early adjuvant simvastatin therapy in elderly patients with infection and sepsis

Abstract Background The incidence, severity, morbidity, and mortality associated with sepsis increases with age, and statin treatment improves outcomes during infection. We characterised the effect of age and acute infection on key neutrophil functions, assessed whether physiologically relevant doses of simvastatin altered neutrophil functions, and, if benefits were seen, when statins could be used during septic infection. Methods Neutrophils extracted from the whole blood of healthy volunteers and patients with a lower respiratory tract infection (LRTI), pneumonia, or sepsis were assessed for migratory accuracy, phagocytosis, and production of neutrophil extracellular traps (NET) before and after in-vitro treatment with simvastatin. In addition, neutrophil function was assessed in healthy elderly volunteers, who were receiving simvastatin (80 mg/day for 2 weeks) or placebo as part of a crossover, double-blind, randomised controlled trial. Here we present data for neutrophil migration. Findings Neutrophils from healthy volunteers (n=70, aged 21–94 years) showed preserved chemokinesis (random movement) but reduced chemotaxis (directed migration) ( r 2 =−0·48, p Interpretation Neutrophil function in elderly people is compromised in health, and deteriorates further during infective episodes in accordance with the severity of the disease. Migratory accuracy can be improved with short-term in-vitro simvastatin therapy in health and mild infection but not in sepsis. Our data suggest that statin therapy might be a preventive or an early adjuvant intervention rather than a treatment in established sepsis. We are testing in a clinical trial whether simvastatin 80 mg for 7 days modifies neutrophil responses in elderly patients with pneumonia and sepsis. Funding British Journal of Anaesthesia, Royal College of Anaesthetists.

S96 Simvastatin as an adjuvant therapy for infection and sepsis–in-vitro and in-vivo studies suggest pre-emptive / early therapy in the elderly

Ageing is associated with increased episodes of sepsis and poorer outcomes. Statins are associated with improved outcomes during infection. We aimed to characterise the impact of age and acute severe infection on key neutrophil functions, assess whether physiologically relevant doses of simvastatin altered neutrophil functions and if benefits were seen, when during a septic episode statins could be utilised. Methods Neutrophils from healthy volunteers and patients with lower respiratory tract infections (LRTI), pneumonia and sepsis were assessed for migratory accuracy, phagocytosis and neutrophil extracellular trap production before and after in-vitro treatment with simvastatin. Healthy elderly volunteers received 80mg simvastatin or placebo in a cross over double-blind randomised controlled trial and neutrophil functions were assessed. Data presented is for migration. Results Neutrophils from healthy subjects (n = 70, aged 21–94) demonstrated preserved neutrophil movement) (R2 = -0.48, p < 0.0001) towards chemoattractants (data shown for IL-8). Neutrophil chemotaxis decreased after 60yrs (comparing <35 to >65yrs: mean difference (MD)1.25μm/min, p = 0.02). There was a progressive decrease in neutrophil chemotaxis in old patients with a LRTI, pneumonia and severe sepsis (MD compared to healthy control; LRTI (n = 10), 0.7μm/min, p = 0.04; pneumonia (n = 5), MD1.1μm/min, p = 0.02; sepsis (n = 22) MD1.6μm/min, p = 0.01) with “septic neutrophils” unable to mount targeted chemotaxis. Improvements to baseline were seen following recovery. In-vitro treatment of neutrophils from healthy older people with simvastatin (1µM) restored “old” neutrophil chemotaxis to that of “young” cells. Simvastatin also restored neutrophil migration from old patients with LRTI and pneumonia to baseline but not in patients with sepsis. Two weeks of oral simvastatin 80mg once daily therapy in healthy old volunteers (Age>65,n = 20) increased the accuracy of neutrophil migration (MD1.68μm/min, p = 0.02) replicating benchwork. Conclusions “Elderly” neutrophil function is compromised in health, and deteriorates during infective episodes, in accordance with the severity of the insult. Migratory accuracy can be improved with simvastatin therapy however neutrophil function in sepsis patients cannot be modulated during short term in-vitro therapy. Our data suggest statin therapy might be a preventative or an early adjuvant intervention rather than a treatment in established sepsis. We are testing whether simvastatin 80mg for seven days modifies neutrophil responses in elderly patients with pneumonia and sepsis (SNOOPI Trial). Abstract S96 Figure 1. Simavastatin 80mg once daily for 14 days improves directional migration (chemostaxis) of neutrophils from healthy elderly volunteers towards IL-8. *Student’s t-Test

S16 Simvastatin improves neutrophil migration in elderly patients with septic pneumonia and reduces 6-month mortality and re-admissions: results of the snoopi trial

Introduction and objectives Community acquired pneumonia is a leading infectious cause of death in the elderly and the commonest source of sepsis. Neutrophil functions decline with age, and deteriorate further in sepsis.1 Restoring neutrophil function may improve sepsis outcomes. Recent in-vitro and in-vivo studies suggest simvastatin improves aspects of neutrophil function.2 Adjuvant statin therapy in severely critically ill patients has failed to improve outcomes and may be associated with increased morbidity,4,5 however our ASEPSIS study suggested that early intervention with statins may reduce the progression of sepsis in a ward-based cohort of milder sepsis patients.6 In light of this, we investigated whether oral treatment with simvastatin improved neutrophil function and clinical outcomes in elderly patients with septic pneumonia. Methods ‘SNOOPI’ was a phase-4, randomised controlled trial comparing 7-days of 80mg simvastatin with placebo in patients aged 55 years or over admitted to hospital with septic pneumonia.3 The primary outcome was changes in neutrophil extracellular trap (NETs) formation by day3/4 compared with baseline. Secondary outcomes included neutrophil migration, safety and tolerability, length of stay, readmissions and mortality. Results 61 patients were recruited acute admissions unit at the Queen Elizabeth Hospital Birmingham between 2013 and 2015, with 31 patients randomised to simvastatin and 30 to placebo. Groups were well matched for baseline characteristics, pneumonia and sepsis severity, co-morbidities and biochemical and haematological parameters. There was no significant difference in the primary end-point of change in NETS at day3/4. Directional neutrophil migration (chemotaxis) was significantly improved in patients who received simvastatin at day 3/4 (0.35 ± 0.16 μm/min vs. −0.15 ± 0.17 μm/min; p = 0.033). Simvastatin was well tolerated with no SUSARS, even with the co-prescription of macrolides. At 6-months, patients in the simvastatin group were less likely to have been admitted to hospital or died compared to those in the placebo group (OR: 0.44; 95% CI: 0.21–0.91; p = 0.02) (Figure 1). Conclusions The current study suggests that early intervention with statins in septic pneumonia patients may improve patient outcomes. We propose that one of the mechanistic drivers may be the restoration of sepsis-associated dysregulated neutrophil function. Further larger studies are warranted to confirm whether early intervention with statins in patients with sepsis confer an overall survival benefit. References Alves-Filho JC, Spiller F, Cunha FQ. Neutrophil paralysis in sepsis. Shock 2010;34(Suppl 1):15–21. Sapey E, Greenwood H, Walton G, et al. Phosphoinositide 3-kinase inhibition restores neutrophil accuracy in the elderly: toward targeted treatments for immunosenescence. Blood 2014;123(2):239–248. Greenwood H, Patel J, Mahida R, et al. Simvastatin to modify neutrophil function in older patients with septic pneumonia (SNOOPI): study protocol for a randomised placebo-controlled trial. Trials 2014;15:332. McAuley DF, Laffey JG, O’Kane CM, et al. Simvastatin in the acute respiratory distress syndrome. N Engl J Med 2014;371:1695–1703. The National Heart L, Blood Institute ACTN. Rosuvastatin for sepsis-associated acute respiratory distress syndrome. N Engl J Med 2014;370:2191–2200. Patel JM, Snaith C, Thickett DR, et al. Randomised double-blind placebo-controlled trial of 40 mg/day of atorvastatin in reducing the severity of sepsis in ward patients (ASEPSIS Trial). Critical Care 2012;16(6):R231. Abstract S16 Figure 1 Kaplan-Meier Curve showing the time (in-days)to either death or re-admission to hospital in patients’ allocated to simvastatin or placebo

Meeting report: British Society for Research on Ageing (BSRA) annual scientific meeting 2012, Aston University, Birmingham, 3rd to 4th July 2012.

The focus of the British Society for Research on Ageing (BSRA) annual scientific meeting 2012 was aging mechanisms and mitigants. The themes covered included epigenetics, stem cells and regeneration, aging pathways and molecules, the aging bladder and bowel, as well as updates from the New Dynamics of Ageing (NDA) programme. The topics incorporated new directions for staple aging research in caloric restriction (CR), inflammation, immunesenescence, neurodegeneration, homeostasis and stress resistance, as well as newer research areas such as bioengineering of tissues, including the internal anal sphincter and thymus.