The Intracellular Growth of M. tuberculosis Is More Associated with High Glucose Levels Than with Impaired Responses of Monocytes from T2D Patients

Abstract

Diabetes mellitus, a metabolic disease characterized by hyperglycemia and poor glucose control, is a risk factor for Mycobacterium tuberculosis (M. tuberculosis) infection and the development of active tuberculosis. To evaluate whether M. tuberculosis infection susceptibility is associated with an intrinsic factor in monocytes from type 2 diabetes (T2D) patients or it is associated with hyperglycemia per se, we analyzed TLR-2 and TLR-4 expression by flow cytometry and the cytokines IL-1β, IL-6, IL-8, IL-10, and TNF-α by cytometric bead array assays, either stimulated with TLR-2 and TLR-4 ligands or infected with M. tuberculosis in the whole blood from T2D patients (n = 43) and healthy subjects (n = 26) or in CD14+ monocytes from healthy subjects cultured in high glucose (HG) (30 mM). The intracellular growth of M. tuberculosis was evaluated by CFU counts at 0, 1, and 3 days in both monocytes from T2D patients and monocytes from healthy subjects cultured in HG. We did not find significant differences in TLR expression, cytokine production, or growth of M. tuberculosis in monocytes from T2D patients compared with those in monocytes from healthy subjects. Despite these results, in vitro assays of monocytes cultured with 30 mM glucose led to significantly increased TLR-2 and TLR-4 basal expression compared to those of monocytes cultured with 11 mM glucose (P < 0.05). Conversely, the production of IL-6 by TLR-2 ligand stimulation, of IL-1β, IL-6, and IL-8 by TLR-4 ligand stimulation, and of IL-8 by M. tuberculosis infection significantly decreased in monocytes cultured in HG (P < 0.05). Additionally, the intracellular survival of M. tuberculosis increased in monocytes in HG after day 3 of culture (P < 0.05). In conclusion, HG decreased IL-8 production and the intracellular growth control of M. tuberculosis by monocytes, supporting the hypothesis that hyperglycemia plays an important role in the impaired immune responses to M. tuberculosis in patients with T2D.

Figure 1

TLR cell surface expression in monocytes in T2D patients and healthy subjects. TLR surface expression was analyzed in whole blood samples by surface staining with AbM anti-human TLR-2PE or TLR-4PE and analyzed by flow cytometry. The monocyte region was analyzed, and TLR expression was reported as MFI. A representative experiment examining (a) TLR-2 and (b) TLR-4 in T2D and healthy subjects by cytometry is shown. Additionally, the surface expression of (c) TLR-2 and (d) TLR-4 in healthy subjects (open circle, n = 20) and T2D patients with HbA1c > 9 (closed circle, n = 16) is shown. The median is represented by a horizontal line.

Figure 2

Cytokine production after TLR-specific stimulation or M. tuberculosis infection and intracellular growth control in monocytes from T2D patients and healthy subjects. Cytokine production elicited by activation through TLRs or M. tuberculosis infection was measured in the culture supernatants. Interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-α) protein levels were measured by a CBA-based assay and reported as pg/ml. Monocytes from healthy subjects (open circle, n = 17) and T2D patients with HbA1c > 9 (closed circle, n = 12) were stimulated with (a) Pam3Cys or (b) LPS or infected with (c) M. tuberculosis. Mann-Whitney U test, healthy subjects vs. T2D patients with HbA1c > 9. Monocytes from T2D patients and healthy subjects were infected with M. tuberculosis (MOI of 10) for 1 h; after the indicated time, cells were lysed with 1% SDS, and the mycobacteria were serially diluted and quantified in 7H10 agar media after 21 incubation days, which was reported as CFU. (d) M. tuberculosis growth in healthy subjects (open circle, n = 20) and T2D patients with HbA1c > 9 (closed circle, n = 19). The median is represented by a horizontal line.

Figure 3

Effect of high glucose on TLR-2 and TLR-4 surface expression in human monocytes from healthy subjects. Monocytes were cultured in high glucose (30 mM) or RPMI medium for 24 h and then stained with specific monoclonal antibodies against human CD14, and the TLR and monocyte regions were analyzed. A representative analysis of the effect of HG on (a) TLR-2 and (b) TLR-4 is shown. Expression of (c) TLR-2 and (d) TLR-4 in monocytes cultured in RPMI medium (closed circles) or under HG (30 mM) (gray circles) (n = 8). Mann-Whitney U test, ^∗P < 0.05, medium vs. HG. The median is represented by a horizontal line.

Figure 4

Effect of high glucose on cytokine production after TLR-specific stimulation or M. tuberculosis infection and intracellular growth control of the mycobacteria in human monocytes. Monocytes were infected with M. tuberculosis (MOI of 10) for 1 h. Subsequently, cells were transferred to RPMI medium or HG (30 mM) and cultured for 0, 1, and 3 days. After the indicated time, the supernatants were analyzed for IL-1β, IL-6, IL-8, IL-10, and TNF-α cytokines, as determined by the CBA-based assay. The cells were lysed with 1% SDS, and the mycobacteria were serially diluted and plated on 7H10 agar media. After 21 days of incubation, CFU were determined. Cytokine production after (a) P3Cys TLR-2 ligand and (b) LPS TLR-4 ligand stimulation or (c) M. tuberculosis infection in culture supernatants in RPMI medium (open circles) and HG (gray circles) (n = 6). The growth control of (d) M. tuberculosis in RPMI medium (open circles) and HG (closed circles) is shown (n = 6). Mann-Whitney U test, ^∗P < 0.05, RPMI medium vs. HG. The median is represented by a horizontal line.