The Cholinergic System Contributes to the Immunopathological Progression of Experimental Pulmonary Tuberculosis

Abstract

The cholinergic system is present in both bacteria and mammals and regulates inflammation during bacterial respiratory infections through neuronal and non-neuronal production of acetylcholine (ACh) and its receptors. However, the presence of this system during the immunopathogenesis of pulmonary tuberculosis (TB) in vivo and in its causative agent Mycobacterium tuberculosis (Mtb) has not been studied. Therefore, we used an experimental model of progressive pulmonary TB in BALB/c mice to quantify pulmonary ACh using high-performance liquid chromatography during the course of the disease. In addition, we performed immunohistochemistry in lung tissue to determine the cellular expression of cholinergic system components, and then administered nicotinic receptor (nAChR) antagonists to validate their effect on lung bacterial burden, inflammation, and pro-inflammatory cytokines. Finally, we subjected Mtb cultures to colorimetric analysis to reveal the production of ACh and the effect of ACh and nAChR antagonists on Mtb growth. Our results show high concentrations of ACh and expression of its synthesizing enzyme choline acetyltransferase (ChAT) during early infection in lung epithelial cells and macrophages. During late progressive TB, lung ACh upregulation was even higher and coincided with ChAT and α7 nAChR subunit expression in immune cells. Moreover, the administration of nAChR antagonists increased pro-inflammatory cytokines, reduced bacillary loads and synergized with antibiotic therapy in multidrug resistant TB. Finally, in vitro studies revealed that the bacteria is capable of producing nanomolar concentrations of ACh in liquid culture. In addition, the administration of ACh and nicotinic antagonists to Mtb cultures induced or inhibited bacterial proliferation, respectively. These results suggest that Mtb possesses a cholinergic system and upregulates the lung non-neuronal cholinergic system, particularly during late progressive TB. The upregulation of the cholinergic system during infection could aid both bacterial growth and immunomodulation within the lung to favor disease progression. Furthermore, the therapeutic efficacy of modulating this system suggests that it could be a target for treating the disease.

Keywords: acetylcholine; choline acetyltransferase; cholinergic; immune response; mycobacterium tuberculosis; nAChR antagonism; pulmonary inflammation; tuberculosis.

Figure 1

Kinetics of ACh, ChAT, and the α7 nAChR subunit during pulmonary TB. Kinetics of ACh quantification determined by HPLC in BALB/C mice lung pairs infected with the Mtb strain H37Rv (box-and-whisker plots of ACh values). (A) Horizontal black lines within the boxes represent the median, while the lower and upper boundaries represent the 25th and 75th percentile, respectively. The upper and lower whiskers correspondingly outspread from the box toward the maximum and minimum values (n = 4 mice per time point/group). One-way ANOVA was used for significance testing, with Bonferroni's post-test for comparisons using lungs of mice from day 1 as the control group (*P < 0.05, **p < 0.01). Representative immunohistochemistry micrographs detecting ChAT and the nAChR α7 subunit (B–G). Representative sections are shown by a 40x objective; bar, 50 um. At day 7 postinfection, there is mild ChAT immunostaining in the bronchial epithelium (arrow) (B). Two weeks after infection, organized nodules constituted by inflammatory cells that correspond to granulomas showed some cells that exhibit ChAT immunostaining (arrow). (C). After 60 days of infection, occasional macrophages with cytoplasmic vacuoles located in pneumonic areas show strong ChAT immunostaining (arrowheads) (D). After one week of infection, some macrophages exhibit intense immunolabeling (arrowhead) of the α7 nAChR subunit (E). Two weeks after infection, some cells in a granuloma show α7 nAChR immunostaining (arrow) (F). After 60 days of infection, numerous vacuolated macrophages and lymphocytes located in pneumonic patches exhibit α7 nAChR immunostaining (arrowheads) (G).

Figure 2

Administration of nAChR antagonists during late infection with Mtb H37Rv reduces lung bacillary burden but does not reduce pneumonia. (A) After 60 days of infection with Mtb strain H37Rv BALB/c mice received saline solution (control mice, black bars) or a nAChR antagonist (dihydro-beta-erythroidine [DHβE] gray bars or methyllycaconitine [MLA] white bars). After 30 and 60 days of treatment initiation, quantification of colony forming units (CFU) determined bacilli load in the lungs. (B) Percentage of pneumonic areas of the mice infected lungs determined by automated morphometry. Automatized reconstruction of infected lungs after 60 days of treatment with saline solution (C), DHβE (D), or MLA (E). Graphs represent pooled data from two experiments (N = 7 mice) and individual groups display median + interquartile range at each time-point. Significance testing was done using the Kruskal-Wallis test with Dunn's multiple comparison. NS refers to non-significant difference between groups (**P < 0.01).

Figure 3

Administration of nAChR antagonists during late tuberculosis infection increases the mRNA transcripts of proinflammatory mediators in the lungs. After 60 days of infection with the Mtb strain H37Rv, groups of BALB/c mice were treated with saline solution (control mice, black bars) or dihydro-beta-erythroidine (DHβE, a nAChR antagonist, gray bars). After 90 and 120 days of infection, the indicated gene expression of the proinflammatory molecules in the lungs was quantified by RT-PCR using the 2^ΔΔCTmethod in relation to the expression of the β-actin housekeeping gene: TNG-α (A) IFN-γ (B) iNOS (C) and IL-17A (D). Graphs represent pooled data from two experiments (N = 7 mice) and individual groups display mean + interquartile range at each time-point. Significance testing was done using the Kruskal-Wallis test with Dunn’s multiple comparison. NS refers to a non-significant difference between groups (*P < 0.05, **P < 0.01, *** P < 0.001).

Figure 4

Administration of nAChR antagonists synergizes with antibiotic therapy reducing bacillary burden and pneumonia. (A) Lung colony forming unit (CFU) determination in mice receiving the indicated treatments for 7, 14 and 30 days, following 60 days of infection with an MDR strain. (B) Percentage of lung area affected by pneumonia determined by automated morphometry. Graphs represent pooled data from two experiments (N = 6 mice) and individual groups display median + interquartile range at each time-point. Significance testing was done using the Kruskal-Wallis test with Dunn's multiple comparison. NS refers to a non-significant difference between groups (*P < 0.05, **P < 0.01). Lower panel (C–E) shows low power micrographs of the lungs after 90 days of the indicated treatment.

Figure 5

Mtb growth is stimulated by ACh and is blocked by nAChR antagonists. Optical density served as a surrogate of the quantity of live bacteria after the addition of Owen's reagent and was confirmed by CFU counting. The optical density of 3 × 10⁵ CFUs of the Mtb strain H37Rv in liquid culture increases after incubation with nanomolar and micromolar concentrations of ACh (gray circles). Bacteria exposed to nM concentrations and 1 µM concentrations of ACh showed a significant increase in optical density compared with bacterial and solvent controls, indicating bacterial proliferation (A). Increase in mycobacterial CFU burden in bacteria incubated with the ACh concentration that presented the highest optical density (66 nM) compared with bacterial and solvent controls was confirmed through the determination and counting of CFUs (B). Conversely, the incubation of 6 × 10⁵ CFUs of the Mtb strain H37Rv after the addition of incremental concentrations of nAChR antagonists (gray circles) reduced their optical density (C) and CFU burden (D) compared with bacterial and solvent controls. Data expressed as the mean SEM of three wells and are representative of three independent experiments. ANOVA P < 0.001. Bonferroni's multiple comparison of values from the bacterial control (black circles), which was not exposed to any compound, with bacteria exposed to the solvent control (saline solution), isoniazid (INH), as well as different concentrations of ACh (A) or nicotinic antagonists (B) is shown (*P < 0.05, **P < 0.01 ***P < 0.001 ****P < 0.0001), NS refers to a non-significant difference between groups.

Figure 6

Mycobacterium tuberculosis produces different acetylcholine concentrations during its growth curve and induces acetylcholine secretion in MH-S cells as measured in supernatants and lysates of Mtb H37Rv recovered during three different growth phases. Acetylcholine was detected in Mtb supernatants exclusively during its lag phase and was present in bacterial lysates during all three growth phases but predominated during the logarithmic phase (day 10). (A) Acetylcholine concentrations in supernatants of MH-S cells. Acetylcholine concentrations were below the limit of detection (<LOD) in the supernatants of uninfected MH-S cells and in supernatants of MH-S cells infected with avirulent or heat-inactivated Mtb strains. In contrast, infection with live Mtb virulent strains generated detectable acetylcholine concentrations in MH-S cell supernatants (B). Data expressed as the mean + SEM of three wells and are representative of two independent experiments using 3 x 10⁵ CFUs of Mtb. ANOVA P < 0.001. The limit of detection of acetylcholine for the assay was 3 nmol.