Neutrophil-mediated innate immune resistance to mycobacteria

Abstract

Neutrophils contain antimicrobial peptides with antituberculous activity, but their contribution to immune resistance to tuberculosis (TB) infection has not been previously investigated to our knowledge. We determined differential white cell counts in peripheral blood of 189 adults who had come into contact with patients diagnosed with active TB in London, United Kingdom, and evaluated them for evidence of TB infection and capacity to restrict mycobacterial growth in whole-blood assays. Risk of TB infection was inversely and independently associated with peripheral blood neutrophil count in contacts of patients diagnosed with pulmonary TB. The ability of whole blood to restrict growth of Mycobacterium bovis bacille Calmette Guérin and Mycobacterium tuberculosis was impaired 7.3- and 3.1-fold, respectively, by neutrophil depletion. In microbiological media, human neutrophil peptides (HNPs) 1-3 killed M. tuberculosis. The neutrophil peptides cathelicidin LL-37 and lipocalin 2 restricted growth of the organism, the latter in an iron-dependent manner. Black African participants had lower neutrophil counts and lower circulating concentrations of HNP1-3 and lipocalin 2 than south Asian and white participants. Neutrophils contribute substantially to innate resistance to TB infection, an activity associated with their antimicrobial peptides. Elucidation of the regulation of neutrophil antimicrobial peptides could facilitate prevention and treatment of TB.

Figure 1. Peripheral blood neutrophil counts at baseline and 6-week follow-up in PTB contacts and controls.

(A) Correlation between baseline and follow-up neutrophil count in PTB contacts. Peripheral blood neutrophil count was determined at baseline and 6 weeks later in 66 PTB contacts. Six-week counts correlated strongly and positively with baseline counts (Pearson’s r = 0.66; 95% confidence interval, 0.50 to 0.78; P < 0.001). (B) Correlation between baseline and follow-up neutrophil counts in control subjects. Peripheral blood neutrophil count was determined at baseline and 6 weeks later in 29 contacts of extrapulmonary TB. Six-week counts correlated strongly and positively with baseline counts (Pearson’s r = 0.67; 95% confidence interval, 0.40 to 0.83; P < 0.001). (C) Change in mean neutrophil counts between baseline and follow-up in PTB contacts versus control subjects. At baseline, PTB contacts had a higher mean neutrophil count than control subjects (4.2 × 10⁹/l versus 3.7 × 10⁹/l, respectively; P = 0.03). Mean neutrophil counts decreased in PTB contacts over 6 weeks (mean neutrophil count at baseline versus follow-up, 4.4 × 10⁹/l versus 4.1 × 10⁹/l, respectively; P = 0.02). Error bars represent SEM.

Figure 2. Effect of neutrophil depletion on containment of mycobaterial bioluminescence and secretion of antimicrobial peptides by whole blood.

(A) BCG-lux assay luminescence ratios in the presence and absence of neutrophils. Black bars, undepleted blood; white bars, depleted blood. Error bars represent SEM. Differences in luminescence ratios were compared using a paired Student’s t test. Experiments were conducted in blood of 6 healthy laboratory donors. (B) BCG-stimulated antimicrobial peptide concentration in the presence and absence of neutrophils. Error bars represent SEM. Differences in antimicrobial peptide concentrations in 96-hour supernatants were compared using Wilcoxon matched-pairs tests. Experiments were conducted in whole blood of 6 healthy laboratory donors. LL-37, human cathelicidin LL-37. (C) MTB-lux assay luminescence ratios in the presence and absence of neutrophils. Error bars represent SEM. Differences in luminescence ratios were compared using a paired Student’s t test. Experiments were conducted in blood of 6 healthy laboratory donors.

Figure 3. Effect of antimicrobial peptides on growth of Mycobacterium tuberculosis in microbiological broth media.

(A) Effect of lipocalin 2 on growth of MTB in iron-replete and iron-depleted broth. Effect of lipocalin 2 (5 μg/ml) on growth of an inoculum of 3 × 10⁴ CFU/ml MTB was determined in iron-replete (150 μM Fe) and iron-depleted (10 nM Fe) 7H9 broth medium. The mean of 6 replicates is shown. Error bars represent SEM. (B) Effect of cathelicidin LL-37 on the growth of MTB in broth. Effect of cathelicidin LL-37 (0.1–100 μg/ml) on growth of an inoculum of 6 × 10⁵ CFU/ml MTB was determined in iron-depleted (10 nM Fe) 7H9 broth medium. The mean of 4 replicates is shown. Error bars represent SEM. (C) Effect of HNP1–3 on the growth of MTB in broth. Effect of HNP1–3 (0.1–100 μg/ml) on growth of an inoculum of 6 × 10⁵ CFU/ml MTB was determined in iron-depleted (10 nM Fe) 7H9 broth medium. The mean of 4 replicates is shown. Error bars represent SEM.