Phosphoinositide 3-kinase inhibition restores neutrophil accuracy in the elderly: toward targeted treatments for immunosenescence - PubMed (original) (raw)

Phosphoinositide 3-kinase inhibition restores neutrophil accuracy in the elderly: toward targeted treatments for immunosenescence

Elizabeth Sapey et al. Blood. 2014.

Abstract

Immunosenescence is the functional deterioration of the immune system during natural aging. Despite increased susceptibility to bacterial infections in older adults, age-associated changes to neutrophil responses are only partially understood, and neutrophil migration has not been characterized in detail. Here we describe reduced chemotaxis but preserved chemokinesis toward a range of inflammatory stimuli in migrating neutrophils isolated from healthy older subjects. Cross-sectional data indicate that migratory behavior changes in the sixth decade of life. Crucially, aberrant migration may increase "bystander" tissue damage and heighten inflammation as a result of excess proteinase release during inaccurate chemotaxis, as well as reducing pathogen clearance. We show evidence of increased neutrophil proteinase activity in older adults, namely, raised levels of neutrophil proteinase substrate-derived peptides and evidence of primary granule release, associated with increased systemic inflammation. Inaccurate migration was causally associated with increased constitutive phosphoinositide 3-kinase (PI3K) signaling; untreated neutrophils from old donors demonstrated significant PI3K activation compared with cells from young donors. PI3K-blocking strategies, specifically inhibition of PI3Kγ or PI3Kδ, restored neutrophil migratory accuracy, whereas SHIP1 inhibition worsened migratory flaws. Targeting PI3K signaling may therefore offer a new strategy in improving neutrophil functions during infections and reduce inappropriate inflammation in older patients.

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Figures

Figure 1

Figure 1

Tracks of neutrophil migration from old and young healthy subjects. The images show the final neutrophil positions within the Insall chemotaxis chamber following 20 minutes of time-lapse recording of neutrophils from an old (A) or a young (B) donor. The chemoattractant source (CXCL8) was added at the top of the viewing field, creating a gradient diffusing down to the bottom of the viewing field. The tracks (shown in black) indicate the path of migration for each cell analyzed: each track begins where the cell was positioned at the start of recording and ends where the cell was positioned at the end of recording. Each image is representative of all recordings taken of neutrophils from each group for all chemoattractants tested. Bars represent 10 μm (A-B). Images were taken using a Zeiss Axiovert 100 microscope, magnification ×20 (A-B). Migration pathway arrows were generated using ImageJ software on manual tracking.

Figure 2

Figure 2

Chemokinesis, chemotaxis, and chemotactic index (accuracy) in neutrophils from younger and older subjects toward CXCL8 (A,C,E) and sputum (B,D,F). Neutrophils from healthy subjects (aged <35 or >65 years of age) migrated toward CXCL8 (100 nM) or pooled sputum collected from patients admitted to the hospital with an acute lower respiratory tract infection attributable to S. pneumoniae. Measurements were taken from 10 randomly selected cells from each individual, with 10 subjects in each group. The average results for each subject were calculated, and an overall average was used for comparisons between groups. Bars represent the mean migratory parameter with SD shown as the error line (A-F). Chemokinesis and chemotaxis were measured in micrometers per minute. Accuracy is a measure of the cell’s directional orientation and is expressed in a cs ranging from −1 to 1. * indicates significant difference in migratory parameter from negative control data (P < .05); §, significant difference in migratory parameter between young and old samples.

Figure 3

Figure 3

Association between reduced chemotaxis and chemotactic accuracy with increasing age with preserved chemokinesis. Migratory chemotaxis, chemotactic index, and chemokinesis were measured in healthy subjects aged 21 to 89 years of age (n = 70). Linear regression identified an age-associated reduction in (A) neutrophil chemotaxis (_R_2 = –0.4750, F = 56.99, P < .0001) and (B) chemotactic index (_R_2 = –0.5399, F = 73.94, P < .0001) with increasing age. Age did not affect (C) neutrophil chemokinesis (_R_2 = 0.004596, F = 0.2909, P = .5915).

Figure 4

Figure 4

PI3K activation in neutrophils from young and old healthy donors. PI3K activity was assessed by western blotting using an antibody to the phosphorylated p85 regulatory subunit of PI3K. β-actin was assessed as the loading control. Cells were unstimulated or incubated with CXCL8 (100 nM) for the times shown. Blots were run in duplicate. (A-B) Representative western blots for 1 young and 1 older donor. (C) The densitometric ratio of phospho-p85:β-actin for young and old adults (n = 5 each group). There was a significant difference in PI3K:β-actin densitometric ratio between young and old neutrophils across all time points. * indicates significant difference between young and old ratios (P < .05).

Figure 5

Figure 5

Chemokinesis, chemotaxis, and chemotactic index (accuracy) in neutrophils from young and old subjects toward CXCL8 in the presence of PI3K isoform selective inhibitors. Neutrophils from healthy subjects (aged <35 or >65 years of age) (n = 10 each group) migrated toward CXCL8 (100 nM) following incubation with carrier control or the denoted PI3K isoform selective inhibitor for 45 minutes. Measurements were taken from 10 randomly selected cells from each individual: from young donors (A,C,E) and from old donors (B,D,F). The average results for each subject were calculated, and an overall average was used for comparisons between groups. Bars represent the mean migratory parameter with SD shown as the error line (A-F). * indicates significant difference in migratory parameter from carrier control data (P < .05).

Figure 6

Figure 6

Chemokinesis and chemotaxis toward CXCL8 in the presence of a SHIP1 selective inhibitor. Neutrophils from healthy subjects (aged <35 or >65 years of age) (n = 6 each group) migrated toward CXCL8 (100 nM) following incubation with carrier control or the SHIP1 selective inhibitor for 45 minutes. Measurements were taken from 10 randomly selected cells from each individual. The average results for each subject were calculated, and an overall average was used for comparisons between groups. Bars represent the mean migratory parameter with SD shown as the error line. * indicates significant decrease in migratory parameter from carrier control data (P < .05).

Figure 7

Figure 7

The mean systemic concentrations of Aα-VAL 360 in young and older healthy adults. Aα-VAL360 is an NE-specific fibrinogen cleavage product. The cleavage product was measured by enzyme-linked immunosorbent assay in plasma in triplicate from all individuals, and healthy subjects aged over 65 and under 35 were compared (n = 20 each group). The mean concentration is shown for each group with the standard error given. * indicates significant difference from young adults (P < .05).

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