Neutrophils Contribute to Severity of Tuberculosis Pathology and Recovery From Lung Damage Pre- and Posttreatment

Abstract

Background: Despite microbiological cure, about 50% of tuberculosis (TB) patients have poor lung recovery. Neutrophils are associated with lung pathology; however, CD16/CD62L-defined subsets have not been studied in TB. Using flow cytometry, we monitored frequencies, phenotype, and function of neutrophils following stimulation with Mycobacterium tuberculosis (Mtb) whole cell lysate (WCL) and ESAT-6/CFP-10 fusion protein (EC) in relation to lung pathology.

Methods: Fresh blood from 42 adult, human immunodeficiency virus (HIV)-negative TB patients were analyzed pre- and post-therapy, with disease severity determined using chest radiography and bacterial load. Flow cytometry was used to monitor frequencies, phenotype, and function (generation of reactive oxygen species [ROS], together with CD11b, tumor necrosis factor, and interleukin 10 [IL-10] expression) of neutrophils following 2-hour stimulation with Mtb-specific antigens.

Results: Total neutrophils decreased by post-treatment compared to baseline (P = .0059); however, CD16brCD62Lbr (segmented) neutrophils increased (P = .0031) and CD16dimCD62Lbr (banded) neutrophils decreased (P = .038). Banded neutrophils were lower in patients with severe lung damage at baseline (P = .035). Following WCL stimulation, ROS from segmented neutrophils was higher in patients with low Mtb loads even after adjusting for sex (P = .038), whereas IL-10-expressing CD16dimCD62Llo cells were higher in patients with mild damage (P = .0397) at baseline.

Conclusions: High ROS generation, low levels of banded neutrophils, and high levels of IL-10-expressing CD16dimCD62Llo neutrophils are associated with reduced lung pathology at diagnosis. Hence, neutrophils are potential early indicators of TB severity and promising targets for TB host-directed therapy.

Keywords: immunosuppression; inflammation; lung damage; neutrophils; tuberculosis.

Figure 1.

Longitudinal measurement of percentage blood neutrophil during antituberculosis treatment. A, Boxplots show median (horizontal line in box) and interquartile range of frequencies of granulocytes before and at treatment completion. The neutrophil counts in active TB patients were measured at baseline and at treatment completion (month 6). Neutrophil frequencies decreased upon treatment completion compared to baseline (n = 42). B, Similarly, granulocyte frequencies determined by flow cytometry also decreased after treatment. Immunofluorescence staining following 2-hour stimulation (or unstimulated [Nil]) with ESAT-6/CFP-10 fusion protein (EC), whole cell lysate (WCL), or phorbol 12-myristate 13-acetate (PMA) allowed for subset identification based on CD16 and CD62L expression levels. C and D, Segmented neutrophils decreased (C) and banded neutrophils increased (D) as homeostatic conditions were restored in the mild (not severe) group. E and F, CD16^dimCD62L^lo (E) and hypersegmented neutrophil frequencies (F) were unchanged between baseline and treatment completion. Each dot represents 1 individual patient. P values were obtained using the Wilcoxon signed-rank test. G, Boxplots show frequencies of granulocytes at baseline in good (n = 7) and poor (n = 4) recovery groups. The Wilcoxon signed-rank test was used to analyze differences between lung recovery groups at baseline. Abbreviation: TB, tuberculosis.

Figure 2.

Percentage of neutrophils in severity groups before and after treatment. A, CD16/CD62L-defined neutrophil frequencies in active TB patients at baseline were analyzed by flow cytometry within Ralph score–defined mild (n = 21, O) and severe (n = 21, ∆) lung disease groups. Banded (CD16^dimCD62L^br) and segmented (CD16^brCD62L^br) neutrophils were lower and higher, respectively, in the severe group compared to the mild group. Values 2 standard deviations above/below the mean cell count were considered outliers and excluded from analysis. Data are presented as boxplots and analyzed using Wilcoxon rank-sum test. B, Granulocyte frequencies decreased with treatment completion. This decrease was more significant in the severe damage and high Mycobacterium tuberculosis (Mtb) load groups compared to the mild damage and low Mtb load groups, respectively. C, Compared to baseline, banded neutrophils decreased significantly after treatment in the mild damage (n = 12) and low Mtb load (n = 11) groups, but not in the severe damage (n = 11) and high Mtb load groups (n = 11). D, In contrast, segmented neutrophils increased after treatment compared to baseline in the mild damage and low Mtb load groups but not in the severe damage and high Mtb load groups. Wilcoxon signed-rank test was used to analyze differences between treatment time within groups. Each dot represents a single patient. *P < .05; **P < .01. Abbreviations: 6M, month 6; BL, baseline; Ct, cycle threshold; ns, not significant; RS, Ralph score; TB, tuberculosis.

Figure 3.

CD11b expression by neutrophils before and after treatment. A, Patients with severe lung damage (top panel, n = 11) or high Mycobacterium tuberculosis (Mtb) load (n = 11, middle panel) showed higher frequencies of CD11b⁺CD16^dimCD62L^lo neutrophils upon 2-hour stimulation with Mtb-specific antigens (ESAT-6/CFP-10 fusion protein and whole cell lysate [WCL]) at baseline compared to treatment completion; meanwhile, patients with mild lung damage (n = 12) and low Mtb load (n = 11) showed no significant difference in the frequency of this phenotype with treatment time. B, Patients with high Mtb load (n = 11) showed significant difference in the frequency of CD11b-expressing banded neutrophils (CD11b⁺CD16^dimCD62L^br) with WCL stimulation following treatment compared to baseline; the low Mtb load group (n = 11) showed none. Wilcoxon signed-rank test was used to analyze differences between treatment time within groups. *P < .05; **P < .01. Abbreviations: 6M, month 6; BL, baseline; EC, ESAT-6/CFP-10 fusion protein; ns, not significant; WCL, whole cell lysate.

Figure 4.

Neutrophil oxidative indices (NOIs) of neutrophil subsets. A, Pretreatment: low CD62L-expressing subsets CD16^dimCD62L^lo (n = 42) and CD16^brCD62L^lo (hypersegmented neutrophils, n = 27) produced more reactive oxygen species (ROS) than CD16^dimCD62L^br (banded, n = 42) or CD16^brCD62L^br (segmented, n = 42) neutrophils at diagnosis following 2 hours of stimulation with whole cell lysate (WCL). B, After treatment, the WCL-stimulated neutrophil subsets had relatively similar ROS generation capacities. P values were obtained using the Kruskal-Wallis test with Dunn posttest comparison. *P < .05; **P < .01. C, Spearman rank correlation of NOI of total granulocytes with bacterial burden at baseline. There was a weak correlation of the NOI of WCL-stimulated granulocytes with Mycobacterium tuberculosis loads in active TB patients, which was not observed with ESAT-6/CFP-10 fusion protein or with phorbol 12-myristate 13-acetate. D and E, CD16^dimCD62L^br (banded, D) and CD16^dimCD62L^br (segmented, E) neutrophils showed a similar correlation (moderately and weakly, respectively) as granulocytes. Abbreviations: Ct, cycle threshold; EC, ESAT-6/CFP-10 fusion protein; NOI, neutrophil oxidative index; PMA, phorbol 12-myristate 13-acetate; TB, tuberculosis; WCL, whole cell lysate.

Figure 5.

Neutrophil oxidative indices (NOIs) of neutrophil subsets in cycle threshold value–defined severity groups at diagnosis. A, At baseline, granulocytes, CD16^dimCD62L^br neutrophils (banded), and CD16^brCD62L^br neutrophils (segmented) had higher NOIs following 2-hour whole cell lysate stimulation in patients with low Mycobacterium tuberculosis (Mtb) load (n = 20) compared to those with high Mtb load (n = 18). B, There were no significant differences in NOI between mild (n = 20) and severe (n = 21) lung damage groups at baseline. Wilcoxon rank-sum test was used to analyze differences between groups at baseline. *P < .05; **P < .01; ***P < .005. Abbreviations: Ct, cycle threshold; NOI, neutrophil oxidative index; ns, not significant; RS, Ralph score.

Figure 6.

Frequency of interleukin 10 (IL-10)⁺ neutrophils pretreatment following whole cell lysate (WCL) stimulation. A, Following 2-hour stimulation with WCL, the baseline (BL) frequencies of IL-10–expressing CD16^dimCD62L^lo neutrophils were higher in patients with mild damage (n = 19) than in patients with severe damage (n = 20). There were no significant differences in tumor necrosis factor (TNF)–expressing neutrophil frequencies between ATB severity groups at diagnosis. Wilcoxon rank-sum test was used to analyze differences between groups at BL. B, Frequency of IL-10–expressing banded (IL-10⁺CD16^dimCD62L^br) neutrophils is higher at month 6 (6M) compared to BL in patients with severe lung damage. C, IL-10–expressing segmented (IL-10⁺CD16^brCD62L^br) neutrophil frequencies are higher at BL compared to 6M in the mild lung damage group. Wilcoxon signed-rank test was used to analyze differences between treatment time within groups. *P < .05. Abbreviation: ns, not significant.