Figure 1.
Effect of CD4+CD25+Foxp3+cells on M. tb growth.
(A) Freshly isolated CD4+ cells and CD14+ monocytes from 6 individuals with LTBI were cultured with γ-irradiated M. tb H37Rv. After 4 days, CD4+CD25+ (85–90% Foxp3+) and CD4+CD25- (<5% Foxp3+) cells were isolated, as outlined in the methods. MDMs from the same donors were infected with H37Rv at a MOI of 1:2.5 (2.5 M. tb to one MDM). After 2 hr MDMs were washed to remove extracellular bacteria and M. tb-expanded CD4+CD25+ (85–90% Foxp3+) or CD4+CD25- (<5% Foxp3+) cells were cultured in Transwells, or directly in the same wells, at a T-cell:macrophage ratio of 1:9. After 2 hr and 7 days, CFU were measured and mean values, p values and SEs are shown. In 6 individuals with LTBI the number of CFU after 2 hr was 1.2 ± 0.3 × 106. (B) H37Rv infected MDMs and M. tb-expanded CD4+CD25+ (85–90% Foxp3+) cells in Transwells were cultured as in panel A. To some wells, anti-IFN-γ (10 μg/ml), anti-IL-22 (10 μg/ml), alone or together (5 + 5 μg/ml), or isotype control antibodies (10 μg/ml) were added on days 0 and 3. After 7 days, CFU were measured and data for 5 individual LTBI+ donors was shown. Mean values, p values and SEs are shown.
Figure 2.
A subset of Foxp3+ cells express D4GDI.
(A) CD4+ cells from PBMC of 6 donors with LTBI were cultured with autologous monocytes and γ-irradiated M. tb (10 μg/ml). After 4 days, CD4+CD25+ (85–90% Foxp3+) and CD4+CD25‑ (<5% Foxp3+) cells were isolated, and cultured overnight in serum-free medium. Culture supernatants were subjected to Western blotting with anti-D4GDI. Data from 2 individuals with LTBI are shown. (B) CD4+ cells and autologous monocytes from 6 donors were cultured as in panel A. After 4 days, the percentages of D4GDI+ cells in CD4+Foxp3+ and CD4+Foxp3‑ cells were determined by flow cytometry. Mean values, p values and SEs are shown. (C) A representative flow cytometry plot is shown. (D) PBMC from 5 individuals with LTBI were cultured with γ-irradiated M. tb H37Rv. After 4 days, the percentages ofCD4+Foxp3+D4GDI+IFN-γ+, CD4+Foxp3+D4GDI+IL-10+ and CD4+ Foxp3+D4GDI+TGF-β+ cells were determined by flow cytometry. A representative flow cytometry plot is shown.
Figure 3.
D4GDI inhibits growth of M. tb in macrophages.
(A) Recombinant D4GDI inhibits growth of M. tb. MDMs from 6 LTBI+ individuals were infected with H37Rv at a MOI of 1:2.5 (2.5 M. tb to one MDM), with or without recombinant D4GDI. After 7 days, CFU were measured. Mean values, p values and SEs are shown for CFU per well. (B) D4GDI siRNA reverses Treg-dependent M. tb growth inhibition in macrophages. CD4+ cells from PBMC of 5 donors with LTBI+ were transfected with siRNA for D4GDI or scrambled siRNA, then cultured with autologous monocytes and γ-irradiated M. tb (10 μg/ml). After 4 days, CD4+CD25+ (85–90% Foxp3+) and CD4+CD25- (<5% Foxp3+) cells were isolated, and cultured in Transwells with MDMs infected with H37Rv at a MOI of 1:2.5. After 7 days, CFU were measured. Mean values, p values and SEs are shown.
Figure 4.
D4GDI inhibits growth of M. tb in mice.
Ten wild type C57BL/6 mice were infected with 50–100 CFU of H37Rv by aerosol. Five mice were sacrificed after 24 hr and the bacterial burden in lungs was confirmed to be 50–100 CFU. The remaining five mice were sacrificed after 30 days. Total number of (A) CD4+ cells (B) CD4+CD25+Foxp3+ cells (C) CD4+CD25+Foxp3+D4GDI+ cells in the lungs of uninfected and M. tb-infected mice were measured by flow cytometry. (D) Twenty C57BL/6 wild type mice were infected with 50–100 CFU of M. tb H37Rv by aerosol. Ten infected mice were given recombinant D4GDI (10ng/ml) through tail vein injection on day 0, 4, 8, 12, 16, 20, 24 and 28 after infection. Thirty days later, bacterial burden in the lungs was measured by plating lung homogenates and counting CFU. Mean values, p values and SEs are shown.
Figure 5.
Apoptotic Foxp3+ cells are the source for D4GDI.
(A) Expression of D4GDI by apoptotic Foxp3+ cells. CD4+ cells and autologous monocytes from 6 persons with LTBI were cultured with γ-irradiated H37Rv. After 4 days, CD4+CD25+Foxp3+, CD4+CD25+Foxp3- and CD4+CD25-Foxp3- cells that expressed annexinV and/or D4GDI were identified by flow cytometry. Mean values, p values and SEs are shown. (B) A representative flow cytometry plot is shown.
Figure 6.
IL-1β and TNF-α contribute to D4GDI-dependent inhibition of M. tb growth.
MDMs from 5 to 6 healthy donors with LTBI were infected with H37Rv, and control and infected MDMs were cultured with or without recombinant D4GDI. After 6 hr, culture supernatants were collected and levels of 27 different cytokines and chemokines were measured by multiplex ELISA. (A) IL-1β production. (B) TNF-α production. (C) G-CSF production. (D) IL-6 production. (E) MCP-1 production. (F) IL-2production. (G) IL-10 production. (H) Effect of IL-1β, IL-27, G-CSF, TNF-α, SIVA and MMP12 siRNAs on D4GDI-dependent M. tb growth inhibition. MDMs from 5 donors were transfected with siRNA for IL-1β, IL-27, G-CSF, TNF-α, SIVA, MMP-12 or scrambled siRNA (control siRNA), and infected with H37Rv at a MOI of 1:2.5 (2.5 M. tb to one MDM). Some infected MDMs were cultured with recombinant D4GDI (10 ng/ml). After 7 days, CFU were measured. Mean values, p values and SEs are shown. *p<0.05, **p<0.005.
Figure 7.
Reactive oxygen species (ROS) contribute to D4GDI-induced IL-1β, TNF-α and G-CSF dependent growth inhibition.
MDMs from 5 donors were infected with H37Rv at a MOI of 1:2.5 (2.5 M. tb to one MDM). To some wells, recombinant D4GDI (10 ng/ml) or hydrogen peroxide (10 μM) were added. (A) After 24 hr, cells were labeled with 2′,7′-dichlorofluorescein, and ROS expression was measured by flow cytometry. (B) MDMs from 6 donors were cultured. N-acetylacysteine was added to some wells and CFU were measured after 7 days. (C) IL-1β or TNF-α regulate D4GDI-dependent ROS production. MDMs from 5 donors were transfected with siRNA for IL-1β, TNF-α, G-CSF, IL-27or scrambled siRNA (control siRNA), and infected with H37Rv at a MOI of 1:2.5. Some infected MDMs were cultured with recombinant D4GDI (10 ng/ml). After 24 hr, cells were labeled with 2′,7′-dichlorofluorescein, and ROS expression was measured by flow cytometry. Mean values and SEs are shown. *p<0.05, **p<0.005.
Figure 8.
D4GDI enhances apoptosis of M. tb-infected MDMs.
(A) Apoptosis of control and M. tb-infected MDMs in the presence of D4GDI. MDMs from 5 donors were infected with H37Rv at a MOI of 1:2.5 (2.5 M. tb to one MDM). To some control and M. tb-infected MDM wells, recombinant D4GDI (10 ng/ml) was added. After 3 days, a TUNEL assay was performed to identify apoptotic cells by flow cytometry. MDMs were transfected with siRNA for IL-1β, TNF-α, G-CSF, or scrambled siRNA, and infected with H37Rv at a MOI of 1:2.5 (2.5 M. tb to one MDM). Some infected MDMs were cultured with recombinant D4GDI (10 ng/ml). After 3 days, a TUNEL assay was performed to determine the apoptotic cells by flow cytometry. Mean values and SEs are shown. *p<0.05, ***p<0.0005.
Figure 9.
Effect of CD4+CD25+Foxp3+cells and D4GDI on M. tb expression of dosR.
(A) MDMs from 7 donors with LTBI were infected with M. tb and cultured with autologous M. tb-expanded CD4+CD25+ (85–90% Foxp3+) or CD4+CD25- (<5% Foxp3+) cells in Transwells. After 7 days, mycobacterial mRNA was quantified by real-time PCR. Mean values and SEs are shown. (B) Effect of D4GDI on DosR expression. MDMs from 5 healthy donors were infected with H37Rv, with or without D4GDI (10 ng/ml). After 7 days, DosR expression was quantified by real time PCR. ** P = 0.004. (C) Effect of CD4+CD25+FoxP3+ cells on the growth of a M. tb DosR deletion mutant. MDMs from four donors with LTBI were infected with the H37Rv strains shown, and M. tb-expanded CD4+CD25+ (85–90% Foxp3+) cells were added in Transwells, at a T-cell:macrophage ratio of 1:9. After 7 days, CFU were measured. Mean values and SEs are shown. Mean values, p values and SEs are shown.
Figure 10.
D4GDI expression by TB patients.
(A) D4GDI is highly expressed in pleural fluid of TB patients. Pleural fluid and serum from 10 patients were collected, and equal amounts of protein were subjected to Western blotting with a monoclonal antibody to D4GDI. Data from 8 patients are shown. (B) PBMC from TB patients express reduced amounts of D4GDI. PBMC from 5 donors with LTBI and 5 TB patients were cultured with γ-irradiated H37Rv (10 μg/ml). After 5 days, culture supernatants were subjected to Western blotting with monoclonal antibody to D4GDI. Data from 2 patients and 2 individuals with LTBI+ are shown. (C) Intracellular staining for FOXP3+D4GDI+ cells. CD4+ cells from PBMC of 5 donors with LTBI+ and 5TB patients were cultured with autologous monocytes in medium alone, or with γ-irradiated H37Rv (10 μg/ml). After 4 days, immunolabeling and flow cytometry were performed. (D) A representative flow cytometry plot is shown. Mean values, p values and SEs are shown.