Research ArticleVIROLOGY

Cofilin hyperactivation in HIV infection and targeting the cofilin pathway using an anti-α4β7 integrin antibody

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Science Advances  09 Jan 2019:
Vol. 5, no. 1, eaat7911
DOI: 10.1126/sciadv.aat7911
  • Fig. 1 HIV gp120-CCR5 signaling activates cofilin in memory CD4 T cells.

    (A) Resting CD4 T cells were treated with CXCR4-utilizing HIV gp120 (IIIB). Cofilin phosphorylation was measured with intracellular staining and flow cytometry using an anti-human p-cofilin antibody. (B) Resting memory CD4 T cells were infected with CCR5-tropic HIV-1(AD8) in the presence or absence of PTX. Cofilin phosphorylation was measured with intracellular staining and flow cytometry using an anti-human p-cofilin antibody. (C) Resting memory CD4 T cells were treated with CCR5-utilizing gp120(BAL) in the presence or absence of maraviroc. Cofilin phosphorylation was measured with intracellular staining and flow cytometry using an anti-human p-cofilin antibody. FSC, forward scatter.

  • Fig. 2 Cofilin hyperactivation in HIV infection.

    (A) Flowchart of the clinical study. (B) Development of the reverse-phase cofilin microarray for profiling cofilin phosphorylation. Synthetic peptides or cell lysates were serially diluted (1:1) and printed onto the microarray slides, which were then stained with antibodies against either total cofilin (right) or phospho-cofilin (left). P-cofilin-S3, a synthetic cofilin peptide with serine 3 phosphorylated; cofilin-S3, a similar peptide with no serine 3 phosphorylation. A431 or HeLa cells were not treated or treated with human epithelial growth factor (EGF) or pervanadate (Perv). (C) Relative levels of p-cofilin in blood resting CD4 T cells from HIV-infected patients with ART (HIV + ART), without ART (HIV), or healthy control (HC) donors were profiled. Box plots show interquartile range, median, and range. There were no statistically significant differences in the total protein levels of the resting CD4 T cells from HC, HIV, and HIV + ART (see Materials and Methods). (D and E) The correlation between levels of p-cofilin and plasma viral load (D) and CD4 T cell count (E) in untreated patients was plotted using Spearman rank correlation tests (Ln, natural logarithm). (F) In ART-treated patients, IRs had significantly higher levels of cofilin phosphorylation than did INRs. (G) A subgroup of ART-naïve patients was subsequently treated with ART following p-cofilin profiling. IRs had significantly higher levels of cofilin phosphorylation than INRs.

  • Fig. 3 Quantification of effects of cofilin hyperactivation on T cell migration.

    (A) A3R5.7 T cells were treated with different dosages of R10015 for 1 hour. Phospho-cofilin and total cofilin were quantified by Western blot. (B) The relative ratio of p-cofilin/cofilin in response to R10015 treatment was plotted (n = 4 independent experiments). (C) R10015 inhibits cofilin phosphorylation and T cell chemotaxis in response to CXCL12. A3R5.7 cells were treated with different dosages of R10015 for 1 hour and then added to the upper chamber of a 24-well Transwell plate. The lower chamber was filled with CXCL12 (40 ng/ml), and cell migration to the lower chamber was quantified (n = 3 independent experiments). (D) The linear correlation between T cell migration and levels of cofilin phosphorylation. The x axis is the relative ratio of p-cofilin/cofilin derived from (B); the y axis is the number of migrating cells derived from (C).

  • Fig. 4 Act-1 modulates the cofilin pathway through PTX-sensitive Gαi signaling.

    (A) A3R5.7 T cells were treated with different dosages of R10015 for 1 hour. Cofilin phosphorylation was quantified with intracellular staining and flow cytometry. Shown are the histogram in (A) and the density plot in (K). (B) Resting CD4 T cells were stimulated with CXCL12 (50 ng/ml) for various times. Cofilin phosphorylation was quantified with intracellular staining and flow cytometry. Shown are the histogram in (B) and the density plot in (L). (C and D) Resting memory CD4 T cells were not treated (C) or treated with PTX (D) for 1 hour and then stimulated with Act-1 (1 μg/ml) for various times. Shown are the histograms in (C) and (D) and the density plots in (M and N). (E) Naïve CD4 T cells (cultured in IL-7) were also similarly stimulated with Act-1. Cofilin phosphorylation was quantified with intracellular staining and flow cytometry. Shown are the histograms in (E) and the density plots in (O). The mean fluorescence intensities (MFIs) of p-cofilin staining are also shown on the histograms. Statistical analyses of the MFI of p-cofilin staining in (A) to (E) are presented in (F) to (J). Results in (B) to (E) were representative of three independent experiments using blood CD4 T cells from three individual donors. SSC, side scatter.

  • Fig. 5 Targeting the cofilin pathway using the anti–human α4β7 antibody Act-1.

    (A) Resting CD4 T cells were treated with different dosages of R10015 for 1 hour. Phospho-cofilin and total cofilin were quantified by Western blot. (B) Act-1 promotes T cell chemotaxis. Resting CD4 T cells were pretreated with R10015 or dimethyl sulfoxide (DMSO) for 1 hour and then stimulated with Act-1 or a control mouse immunoglobulin G for an additional 15 min. Cells were then added to the upper chamber of a 24-well Transwell plate. The lower chamber was filled with CXCL12 (40 ng/ml). Cell migration to the lower chamber was enumerated. (C) Act-1 selectively promotes the migration of the α4β7+ CD4 T cells. Migrating T cells in the lower chamber were stained with the anti-α4β7 antibody Act-1, followed by staining with Alexa Fluor 647–conjugated goat anti-mouse secondary antibodies. The percentage of the α4β7high CD4 T cells was quantified with flow cytometry. (D) The number of migrating α4β7high CD4 T cells in (C) was also enumerated. Results in (C) and (D) were representative of five independent experiments using blood CD4 T cells from five individual donors.

  • Fig. 6 Model of cofilin hyperactivation in HIV infection and therapeutic targeting of the cofilin pathway.

    Early HIV signaling through chemokine coreceptors (CCR5 and CXCR4) and late chronic immune activation may trigger cofilin hyperactivation, impairing CD4 T cell migration and homing to lymphoid tissues such as GALTs. ART alone is not sufficient to restore T cell motility. For early, low levels of cofilin hyperactivation, stimulation of the upstream regulators of cofilin through antibodies (e.g., stimulating chemokine or integrin receptors such as the α4β7 receptor), receptor agonists, or activators of G proteins and GTPases may repair cofilin-mediated T cell migratory defect. For late, high levels of cofilin hyperactivation, it may need to use LIMK (the cofilin kinase) activators or the cofilin phosphatase (slingshot) inhibitors to directly inhibit cofilin dephosphorylation to restore actin dynamics. Therapeutically, targeting cofilin to restore T cell motility may bring two major benefits: (i) For uninfected cells, the restoration of CD4 T cell circulation and homing to lymphoid tissues such as GALTs may help immune reconstitution; (ii) for latent HIV+ T cells, the restoration of T cell circulation and homing to lymphoid tissues may lead to their reactivation and eventual containment by the restored immune system, reducing latent viral reservoirs persistent in the peripheral blood and in tissues.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/1/eaat7911/DC1

    Fig. S1. Selective infection of memory CD4 T cells by CCR5-utilizing HIV-1.

    Fig. S2. R10015 does not alter the CXCR4 receptor surface density.

    Fig. S3. Surface expression of α4β7 on different subsets of human blood resting CD4 T cells, as measured by surface staining and flow cytometry.

    Fig. S4. R10015 does not alter the α4β7 receptor surface density.

    Fig. S5. Act-1 is not a chemoattractant.

    Fig. S6. Act-1 selectively promotes the migration of the α4β7high CD4 T cells.

    Fig. S7. Act-1 does not selectively promote the migration of the CCR7+ CD4 T cells.

    Fig. S8. Act-1 stimulation does not activate resting CD4 T cells.

    Fig. S9. Act-1 is not effective in rescuing T cell motility with high levels of cofilin hyperactivation.

    Fig. S10. Accumulation of the α4β7high CD4 T cells in the peripheral blood of HIV-infected patients.

    Table S1. Characteristics of clinical study participants.

    Table S2. Patient enrollment and grouping.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Selective infection of memory CD4 T cells by CCR5-utilizing HIV-1.
    • Fig. S2. R10015 does not alter the CXCR4 receptor surface density.
    • Fig. S3. Surface expression of α4β7 on different subsets of human blood resting CD4 T cells, as measured by surface staining and flow cytometry.
    • Fig. S4. R10015 does not alter the α4β7 receptor surface density.
    • Fig. S5. Act-1 is not a chemoattractant.
    • Fig. S6. Act-1 selectively promotes the migration of the α4β7high CD4 T cells.
    • Fig. S7. Act-1 does not selectively promote the migration of the CCR7+ CD4 T cells.
    • Fig. S8. Act-1 stimulation does not activate resting CD4 T cells.
    • Fig. S9. Act-1 is not effective in rescuing T cell motility with high levels of cofilin hyperactivation.
    • Fig. S10. Accumulation of the α4β7high CD4 T cells in the peripheral blood of HIV-infected patients.
    • Table S1. Characteristics of clinical study participants.
    • Table S2. Patient enrollment and grouping.

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