Hip Fracture Leads to Transitory Immune Imprint in Older Patients

Figures

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  Figure 1. Longitudinal analysis of innate phenotype. (A) Number of neutrophils (103 cells/mm3); (B) Gating strategy depicting neutrophils staining based on size/structure and CD62L/CD16 expression; (C) Percentage of inflammatory neutrophils expressing CD16+CD62Lhigh; (D) Percentage of suppressive neutrophils expressing CD16+CD62Llow; (E) Number of monocytes (103cells/mm3); (F) Gating strategy depicting monocytes staining based on size/structure criteria, HLADR, CD14, and CD16 expression; (G) Percentage of conventional CD14+CD16− monocytes; (H) Percentage of intermediate CD14+ CD16+ monocytes; (I) Percentage of non-conventional CD14−CD16+ monocytes; (J) Representative histogram of CCR2 expression within conventional monocytes (black; control isotype in gray overlay); (K) Representative histogram of CX3CR1 expression within non-conventional monocytes (black; control isotype in gray overlay); (L) CCR2 expression within conventional monocytes (expressed in mean fluorescence intensity); (M) CX3CR1 expression within non-conventional monocytes (expressed in mean fluorescence intensity). Data are plotted for age-matched control individuals (CTR) or for hip fracture patients at different times of…

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  Figure 2. Ability of monocytes from HFP to produce pro-inflammatory cytokines upon stimulation. (A) Representative flow cytometry profile of cytokines secretion (TNF-α and IL-1β upper panel; IL-8 and IL-6 bottom panel) in unstimulated monocytes (US, left panel) or in LPS conditions (right panel). Monocytes were identified according to size/structure, HLADR, CD14, and CD16 expression; (B) Percentages of monocytes secreting (B) TNF-α, (C) IL-1 β, (D) IL-8, and (E) IL-6. Data are plotted for age-matched control individuals (CTR) or for hip fracture patients at different times of follow-up (pre-surgery: PRE; post-surgery: POST; during hospitalization: HOSP; at hospital discharge: DISCHARGE and between 6 and 12 months post-fracture: LONG TERM). Each dot represents an individual. The lanes indicate the medians. Statistical significance is determined by the nonparametric Mann–Whitney test: p < 0.05 was considered significant.

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  Figure 3. Longitudinal analysis of adaptive phenotype. Number (103 cells/mm3) of (A) total lymphocytes, (B) T-cells, (C) CD4+ T-cells, (D) CD8+ T-cells, (E) CD19+ B cells, and of (F) CD56+ NK cells. Data are plotted for age-matched control individuals (CTR) or for hip fracture patients at different times of follow-up (pre-surgery: PRE; post-surgery: POST; during hospitalization: HOSP; at hospital discharge: DISCHARGE and between 6 and 12 months post-fracture: LONG TERM). Each dot represents an individual. The lanes indicate the medians. Statistical significance is determined by the nonparametric Mann–Whitney test: p < 0.05 was considered significant.

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  Figure 4. Functionality of CD4+ and CD8+ T cells in HF patients (longitudinal analysis) compared to control. Proliferation was evaluated by the % of cells expressed Ki67+ for (A) CCR7+ CD45RA+ naïve CD4+, (B) memory CD4+, (C) CCR7+ CD45RA+ naïve CD8+, and (D) memory CD8+. Activation was evaluated by the % of cells expressed CD38+ within (E) memory CD4+ and (F) CD8+. Priming capacity was evaluated by the polyfunctionality index within (G) CD4+ and (H) CD8+. Data are plotted for age-matched control individuals (CTR) or for hip fracture patients at different times of follow-up (pre-surgery: PRE; post-surgery: POST; during hospitalization: HOSP; at hospital discharge: DISCHARGE and between 6 and 12 months post-fracture: LONG TERM). Each dot represents an individual. The lanes indicate the medians. Statistical significance is determined by the nonparametric Mann–Whitney test: p < 0.05 was considered significant.

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  Figure 5. Longitudinal analysis of immune checkpoint expression within T cells. Surface membrane expression within memory CD4+ (Left) and memory CD8+ T cells (Right) for PD-1 (A,B upper panels) LAG-3 (C,D middle panels) and 2-B4 expression (E,F bottom panels). All results are expressed in mean fluorescence intensity. Data are plotted for age-matched control individuals (CTR) or for hip fracture patients at different times of follow-up (pre-surgery: PRE; post-surgery: POST; during hospitalization: HOSP; at hospital discharge: DISCHARGE and between 6 and 12 months post-fracture: LONG TERM). Each dot represents an individual. The lanes indicate the medians. Statistical significance is determined by the nonparametric Mann–Whitney test: p < 0.05 was considered significant.