Ageing of the Innate Immune System

Neutrophils

Neutrophils are the most abundant leukocyte in circulation and provide immediate frontline protection against rapidly dividing bacteria, fungi and yeast. The first step in neutrophil anti-microbial defence is their extravasation from the bloodstream and migration to the site of infection. Whilst age appears to have no effect upon the speed at which neutrophils migrate towards chemotactic signals in vitro [15], the directional accuracy of neutrophil migration to inflammatory agonists (e.g. interleukin-8 (IL-8, CXCL8)) as well as bacterial peptides (e.g. formyl-methionine-leucine- phenylalanine (fMLP)) is significantly reduced [15]. Recently, we attributed this age-related aberration in neutrophil chemotaxis to constitutive activation of phosphoinositide 3-kinase (PI3-K), a lipid kinase implicated in cell polarisation and propulsion via its generation of phosphoinositol 3,4,5-triphosphate (PIP3) at the leading edge of the cell [15]. Selective inhibition of the PI3-K isoforms PI3-K 8 and Y in neutrophils from older adults significantly improved their migratory accuracy towards IL-8 [15]. Conversely, increasing PIP3 levels in neutrophils from young donors, resulted in reduced chemotaxis similar to that seen in older donors [15]. Given that in a longitudinal study of older adults, reduced neutrophil chemotaxis at baseline was associated with “non-survival” following infectious episodes [16], our work on PI3-K suggests that therapeutic targeting of this kinase could be a novel approach by which to improve patient outcome in times of infection [15]. Whilst age-related impairments in neutrophil migration have been observed in vitro [15, 17], conflicting data has emerged from in vivo studies, where neutrophil chemotaxis has been reported to be either unaffected with age [18] or significantly reduced [19, 20]. In the most recent of these studies, Nacionales and colleagues recovered significantly fewer neutrophils from the bronchoalveolar lavage fluid of aged mice in a model of pneumonia infection [20]. Reduced expression of adhesion molecules on the surface of activated endothelium could be one mechanism that explains the aberrant migration of neutrophils in aged mice [19].

Upon arrival at sites of infection, neutrophils attempt to contain invading pathogens via phagocytosis. To date, no age-related impairments have been reported in the ability of neutrophils to engulf non-opsonised particles [21]. However, neutrophils from older adults exhibit significantly reduced phagocytic activity towards complement and immunoglobulin (Ig)-opsonised pathogens [22,23]. We have attributed the age-related impairment in neutrophil uptake of Ig-coated microbes to reduced expression of CD16, an Fc receptor, whose surface density positively correlates with the uptake of Ig-opsonised pathogens [22] . In contrast, the surface expression of complement receptors are comparable between neutrophils from young and old donors [24], suggesting intrinsic signalling defects are likely to be responsible for reduced phagocytic activity of neutrophils from older adults towards complement- coated pathogens. Once phagocytosed, pathogens are exposed to a harsh microbicidal environment, the creation of which requires the generation of reactive oxygen species (ROS). Currently, the effect that physiological ageing has on neutrophil ROS generation is unclear, with study outcomes seemingly dependent upon the type of activating stimulus. For example, no age-associated differences have been described for ROS generation initiated by Candida albicans or Escherichia coli (E. coli) stimulation [23, 25], whereas neutrophils from older adults generate significantly fewer ROS when challenged with Staphylococcus aureus (S. aureus), fMLP or granulocyte macrophage-colony stimulating factor (GM-CSF) [17,23,25].

In a seminal paper published in 2004, Brinkmann and co-workers demonstrated that following microbial or pro-inflammatory cytokine stimulation, neutrophils release into the extracellular environment their nuclear DNA [26]. Termed neutrophil extracellular traps (NETs), these structures are decorated with an array of granule and cytosol-derived peptides and proteases and have been shown to capture and in some cases disarm gram positive and negative bacteria [26]. To our knowledge, only two studies have investigated whether NET formation is altered during

“healthy” ageing. In response to stimulation with the diacylglycerol mimetic phorbol 12-myristate 13-acetate (PMA) or, methicillin-resistant S. aureus (MRSA), Tseng et al. found in vitro NET production by neutrophils from aged mice was significantly lower than that of neutrophils from young mice, a defect they also observed in an in vivo model of MRSA infection [27]. In agreement with these findings, we showed that human ageing also results in impaired NET formation [28]. Compared to those isolated from their older counterparts, we found tumour necrosis factor-alpha (TNF-a) primed neutrophils from younger adults generated significantly more NETs following IL-8 or lipopolysaccharide stimulation [28]. Mechanistically, this aberration in NET formation was attributed to an age-related decline in ROS generation, a crucial prerequisite for NET production [28, 29].

Aside from the controversy that exists regarding the impact of age on ROS production [17, 23, 25], neutrophils from older adults clearly exhibit defects in several key defensive mechanisms, namely chemotaxis [15, 19, 20], phagocytosis of opsonised pathogens [22, 23] and NET formation [27, 28]. Given this near global impairment in neutrophil function, alterations to a generic signalling element rather than defects in molecules specific to each anti-microbial defence strategy is likely to explain the aberrations in neutrophil function that occur with age. In support of this idea, ageing in rodents is associated with a significant increase in neutrophil membrane fluidity, which coincides with a marked reduction in neutrophil function [30]. Moreover, human studies have shown the recruitment of receptors into lipid rafts, specialised microdomains enriched for phospholipids and cholesterol that assist in signal transduction, is markedly reduced with age [17, 31]. That proximal signalling defects contribute at least in part to the impaired functions of aged neutrophils is further demonstrated by the fact that no age-related differences in ROS production [17, 28, 30] or NET formation [28] have been reported for neutrophils treated with PMA, a stimulus that activates cells independently of surface membrane receptors .

 
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