NOTCH1 and DLL4 are involved in the human tuberculosis progression and immune response activation

Abstract

Tuberculosis (TB) is the leading cause of mortality among infectious diseases worldwide. The study of molecular targets for therapy and diagnosis suggested that Notch signaling is an important pathway for the maintenance of the immune response during Mycobacterium tuberculosis (Mtb) infection. We evaluated the participation of the Notch pathway in the modulation of immune response during Mtb infection, and observed that patients with active TB had increased DLL4 expression in intermediate and non-classic monocytes. Further, patients with moderate and advanced lung injury have higher Notch1 expression in CD4⁺ T cells when compared to patients with a minimal lung injury. When we considered the severity of disease in active TB patients, the expression of the DLL4 in intermediate monocytes and the expression of Notch1 in CD4⁺ T cells are positively correlated with the degree of lung injury. In vitro, PBMCs treated with the Notch pharmacological inhibitor reduced the production of IL-17A and IL-2, whereas anti-hDLL4 treatment promoted a significant increase in TNF-α and phagocytosis. We suggest that Notch1 and DLL4 are associated with immune response activation in human tuberculosis, and can be a novel target to be exploited in the future in the searching of biomarkers.

Keywords: Immune response; Mycobacterium tuberculosis; Notch signaling; Progression biomarker.

Figure 1.. Expression of Notch1 receptor in CD4⁺ T lymphocytes from patients with active TB.

The expression of Notchl and Notch2 receptors in PBMCs from control subjects and patients with active tuberculosis was evaluated by flow cytometry. A) Representative dotplot of distribution of CD4⁺ lymphocyte populations. B and C) frequency (%) and number of the CD4⁺ T cells. D) Representative histogram the frequency (%) of Notchl in CD4⁺ T cells from control subjects and patients with active tuberculosis. E and F) frequency (%) and expression (Mean Fluorescence Intensity - MFI) of Notchland Notch2 receptors in CD4⁺ T lymphocyte population. G) Representative histogram the frequency (%) of Notchl in CD4⁺ T cells from patients with active tuberculosis. H and I) frequency (%) and expression (Mean Fluorescence Intensity - MFI) of Notchl receptor in the CD4⁺ T cells from patients with minimum (MIN), moderate (MOD) and advanced (ADV) pulmonary lesions. The bars represent the mean ± standard deviation of each group. Control subjects (n=ll) and patients with active TB (n=l2) were used to evaluate of Notchland Notch2. Patients with minimum (n=4), moderate/advanced (n=8) pulmonary lesions were used to evaluate of Notchl. The results were analyzed with the t-test. ***P <0.0005, *P <0.05.

Figure 2.. Expression of DLL4 in subpopulations of monocytes from patients with active TB.

Mononuclear cells from control subjects and active TB patients were labeled with anti-CD16 and anti-CD14 antibodies to divide monocyte subpopulations into classical (CD14⁺ CD16⁻), intermediate (CD14⁺⁺ CD16⁺) and non-classical (CD14⁺ CD16⁺⁺) monocytes. A) Distribution of subpopulations of monocytes (number of cells). B) Representative histogram the frequency (%) of DLL4 ligands in classical monocyte subpopulations from active tuberculosis patients and control subjects. C) Frequency (%) of DLL1 and DLL4 ligands in classical monocyte subpopulations. D) Representative histogram the frequency (%) of DLL4 ligands in intermediate monocyte subpopulations from patients with active tuberculosis and control subjects. E) Frequency (%) of DLL1 and DLL4 ligands in subpopulations of intermediate monocytes. F) Representative histogram the frequency (%) of DLL4 ligands in non-classical monocyte subpopulations from patients with active tuberculosis and control subjects. G) Frequency (%) of DLL1 and DLL4 ligands in non-classical monocyte subpopulations. The bars represent the mean ± standard deviation of each group. Control subjects (n=11) and patients with active TB (n=12). The results were analyzed using the t-test; * P <0.05.

Figure 3.. Quantification of cytokine levels in plasma and global signature of the gene expression of Notch targets and cytokines from patients with active TB.

A-N) Cytokines levels were evaluated in plasma samples from control subjects and patients with active TB through the Magnetic Luminex Assay Kit. The bars represent the mean ± standard deviation of each group and each spot is representative of an individual. Control group (n=13) and patients with active TB (n=12). Gene expression of Notch targets and cytokines were plotted as radar charts, in which the frequencies of higher producers (%) were determined based on the overall median value of each target gene as a cutoff point for individuals with “low” or “high” gene expression. Radar plots assume that each axis shows the proportion of individuals with high levels of a given target gene. The relevant values (≥ 50%) are indicated (*). G) Representation of control individuals (black). H) Representation of patients with active TB (gray). The results were analyzed through the t-test. **** P <0.0005, ** P <0.005, * P <0.05.

Figure 4.. Correlation between expression of Notch1 and DLL4 with immunological mediators in patients with active TB.

The expression of Notch1 receptor on CD4⁺ T cells was correlated with plasma levels of (A and B) IFN-γ, (C and D) IL-17A and (E and F) IL-2. Correlation studies between expression (MFI) of DLL4 in CD14⁺⁺ CD16⁻ monocytes and plasma levels of IL-17A (G). Correlation studies between expression (MFI) of DLL4 in CD14⁺⁺ CD16⁺ cells and plasma levels of IL- 17A (H). The analyses were performed using Spearman’s statistical test. Each spot is representative of an individual and the values of r and p are specified in the graphs. Patients with active TB (n=12).

Figure 5.. Effect of Notch pathway modulation in cytokine release and monocytes phagocytosis in response to Mtb in vitro infection.

IL-17A, IL-2 and TNF-α were evaluated in culture supernatant of PBMCs, previously treated with GSI, anti-hDLL1, or anti-hDLL4 for 24 h and infected with Mtb H37Rv for 72 h, using the Magnetic Luminex Assay Kit. (A) Experimental design, (B – D) Cytokine quantitation of IL-17, Il-2 and TNF-α, respectively. (E) Experimental design of phagocytic activity analysis - PBMCs from control subjects were pretreated with anti-hDLL1 and anti-hDLL4 for 24 hours and then infected with the Mtb H37Rv strain. (F) Phagocytosis analysis of this experiment was performed using the resazurin metabolism assay by viable internalized bacteria. The bars represent the mean ± standard deviation (n=6). The bars represent the mean ± standard deviation (n=6). The results were analyzed through the t-test. ** P <0.005, * P <0.05.