Altered regulation of CXCR4 expression during aging contributes to increased CXCL12-dependent chemotactic migration of CD4(+) T cells

Abstract

Chemokine-dependent migration of T lymphocytes assures recirculation of naïve T cells to secondary lymphoid organs and tissue-specific trafficking of memory-effector T cells. Previous studies carried out in rodents have demonstrated age-associated modulation of the expression of chemokine receptors such as CXCR4 and CCR5; however, little is known about the molecular mechanisms that regulate receptor expression and turnover in T cells, during advancing age in humans. Our recent results demonstrating increased chemotactic migration in response to CXCL12 in CD4(+) T cells obtained from the elderly, as compared to those from young donors, led us to hypothesize that increase in surface expression, because of altered endocytic regulation of CXCR4 on T cells during aging, might be directly responsible for increased migration toward CXCL12. Studies presented here demonstrate a significant increase in the surface expression of CXCR4 in CD4(+) T cells from elderly human donors, relative to those from the young. Additionally, CXCL12-mediated endocytosis of CXCR4 was differentially regulated during aging, which could be attributed to alterations in the ubiquitination of CXCR4. Thus, altered ubiquitination of CXCR4 may contribute to the increased surface expression and enhanced T-cell migration to chemotactic stimuli in the elderly.

Fig. 1. Increased surface expression of CXCR4 in CD4⁺ T cells during aging

(A). Left panel: Surface expression of CXCR4 in CD4⁺ T cells from young and elderly donors. CD4⁺ T cells obtained from young and elderly donors were labeled using antibody to CXCR4, followed by Alexa Fluor 488-conjugated secondary antibody staining and then fixed with 2% PFA. Right panel: Intracellular expression of CXCR4 in CD4⁺ T cells obtained from young and elderly donors. CD4⁺ T cells obtained from young and elderly donors were fixed using 2% PFA, and then permeabilized with 1xPBS buffer containing 0.1% Saponin. Cells were then stained using an antibody to CXCR4, followed by Alexa Fluor 488-conjugated secondary antibody. Cell surface and intracellular expression of CXCR4 were detected by flow cytometry. Cumulative data of percent cells expressing CXCR4 obtained from 10 independent donor pairs are provided. ** denotes statistical significance at p<0.001. (B). Flow cytometry of CD4⁺ T cells from young and elderly donors stained for intracellular and extracellular CXCR4 expression, using antibody to CXCR4, followed by secondary antibody coupled to fluorochrome. Representative data from one young (Left panel)) and elderly (Right panel) donor pair are provided. Intracellular CXCR4 staining was carried out using fixed, permeabilized CD4⁺ T cells, while extracellular staining was carried out on unfixed cells. Isotype stained cells served as negative controls. MFI indicates mean fluorescence intensity. (C). Surface and intracellular expression of CXCR4 in CD4⁺CD45RO⁺ and RA⁺ T cell subsets from young and elderly donors. Cumulative data of percent cells expressing CXCR4 obtained from 10 independent donor pairs are provided. (D). Increased levels of CXCR4 in CD4⁺ T cells from the elderly. A representative western blot of CXCR4 in CD4⁺ T cell lysates obtained from young and elderly donors are provided. β-actin was used as a protein loading control. Representative data from one donor pair out of 10 pairs tested are provided. (E). Quantitation of the specific band corresponding to CXCR4 protein was carried out by densitometry. Values represent mean integrated density ± SE obtained from 10 independent donor pairs. ** denotes statistical significance at p<0.001. (F). qRT-PCR analysis of CXCR4 mRNA from total RNA isolated from CD4⁺ T cells obtained from young and elderly donors. Data obtained from 10 donor pairs are provided. β-actin and GAPDH were used as reference genes and for normalization. Data are presented as fold induction.

Fig. 2. CCR5 surface expression in resting and activated CD4⁺ T cells during aging

(A& B). Surface expression of CCR5 in CD4⁺ T cells from young and elderly donors. CD4⁺ T cells obtained from young and elderly donors were either left untreated (A) or activated with PMA/Ionomycin for 12h (B), stained using antibody to CCR5 followed by Alexa Fluor 555-conjugated secondary antibody. Surface expression of CCR5 was detected by flow cytometry. Cumulative data obtained from a minimum of 8 independent donor pairs are provided. ** denotes statistical significance at p<0.001; * denotes statistical significance at p<0.009. (C). Dose dependent migration of CD4⁺ T cells from young and elderly donors to CCL8 gradient. CD4⁺ T cells (3×10⁵) from young and elderly donors were placed in the upper chamber of a trans-well migration plate (5μM pore) and allowed to migrate for 4h at 37°C towards a gradient generated by either 1, 10, 50 or 100nM of CCL8. The number of migrating cells present in the lower chamber was assessed by Trypan Blue staining, and the percentage of migrating cells is provided. Values represent mean ± SE from 4 independent donor pairs. (D). Chemotactic migration of CD4⁺ T cells from young and elderly donors to CCL8 gradient. CD4⁺ T cells were either left untreated or pretreated with PMA/Ionomycin for 12h. Data are representative of 10 independent donor pairs and represent percentage of migrating CD4⁺ T cells from young and elderly donors in response to CCL8.

Fig. 3. CXCR4 is differentially ubiquitinated in CD4⁺ T cells during aging

(A). Lowered expression of low molecular weight CXCR4 isoforms in CD4⁺ T cells from the elderly. CD4⁺ T cell lysates obtained from a pool of 5 young and 5 elderly donors were immunoprecipitated with an antibody to CXCR4. The immunoprecipitate was resolved using SDS-PAGE and detected using an antibody to CXCR4. Representative blot from one immune-precipitation assay is provided. Arrows depict approximate molecular weight of the CXCR4 isoforms derived from molecular weight standards, and approximate number of ubiquitin tags that could account for the increase in molecular weight. (B). Decreased monoubiquitination and increased polyubiquitination (>8Ub-linkages) of CXCR4 in CD4⁺ T cells, accompanies aging. Cell lysates of CD4⁺ T cells obtained from 3 independent pools of 5, 3 and 3 young and elderly donors were immunoprecipitated with an antibody to CXCR4. Resolved immunoprecipitates were immunoblotted with antibody to ubiquitin. Long exposure indicates extended exposure of the film to demonstrate the appearance of intense high molecular weight CXCR4 isoforms denoting polyubiquitination. Arrows indicate ubiquitinated species attributable to 1, 3 and 8 ubiquitin linkages and are based on approximately 8.5kDa increments in molecular weight.

Fig. 4. Inhibition of USP14 results in increased CXCR4 surface expression in CD4⁺ T cells

Surface expression of CXCR4 in CD4⁺ T cells from young and elderly donors. CD4⁺ T cells obtained from young and elderly donors were either left untreated or pretreated with IU-1 (50μM; 4h), and then either activated with CXCL12 (100nM; 1h), or not. T cells were then stained using an antibody to CXCR4, followed by Alexa Fluor 488-conjugated secondary antibody. Surface expression of CXCR4 was detected by flow cytometry. Data obtained from 3 independent donor pairs are provided. ** denotes statistical significance at p<0.009; * denotes statistical significance at p<0.05.

Fig. 5. Decreased ligand-induced degradation of CXCR4 in CD4⁺ T cells accompanies aging

(A). Representative western blot of the predominant CXCR4 isoforms in CD4⁺ T cell lysates obtained from young and elderly donors that were either left untreated or treated with CXCL12 for 1h and 3h. β-actin was used as a control for equal protein loading. (B). Percent degradation of CXCR4 following CXCL12 treatment at 1 and 3h relative to control (untreated) obtained from young and elderly donors are presented. Values represent mean ±S.E. obtained from a minimum of 6 independent donor pairs. ** denotes statistical significance at p<0.003; * denotes statistical significance at p<0.05.