Neutrophil responses to Mycobacterium tuberculosis infection in genetically susceptible and resistant mice

Abstract

The role of neutrophils in tuberculosis (TB) resistance and pathology is poorly understood. Neutrophil reactions are meant to target the offending pathogen but may lead to destruction of the host lung tissue, making the defending cells an enemy. Here, we show that mice of the I/St strain which are genetically susceptible to TB show an unusually high and prolonged neutrophil accumulation in their lungs after intratracheal infection. Compared to neutrophils from more resistant A/Sn mice, I/St neutrophils display an increased mobility and tissue influx, prolonged lifespan, low expression of the CD95 (Fas) apoptotic receptor, relative resistance to apoptosis, and an increased phagocytic capacity for mycobacteria. Segregation genetic analysis in (I/St x A/Sn)F2 hybrids indicates that the alleles of I/St origin at the chromosome 3 and 17 quantitative trait loci which are involved in the control of TB severity also determine a high level of neutrophil influx. These features, along with the poor ability of neutrophils to restrict mycobacterial growth compared to that of lung macrophages, indicate that the prevalence of neutrophils in TB inflammation contributes to the development of pathology, rather than protection of the host, and that neutrophils may play the role of a "Trojan horse" for mycobacteria.

FIG. 1.

Distinct patterns of neutrophil inflammation following TB challenge and neutrophil migration in I/St and A/Sn mice. Accumulation of Lyt-6G-positive cells following infection, as enumerated by fluorescence-activated cell sorter (a and b), and the presence of neutrophils containing mycobacteria in I/St lungs (c) are shown. Neutrophil influx to the lungs and peritoneal cavities shortly after a high-dose (10⁸ CFU/mouse) mycobacterial challenge was significantly higher in I/St mice than in A/Sn mice (d). In a transmigration assay of peptone-attracted peritoneal cavity cells (e), neutrophils selectively migrated through a microporous filter in response to mycobacterial sonicate (f and g). There was a sixfold interstrain difference in the migration capacity of neutrophils (g and h), which did not depend upon migration stimulus (h). A P of <0.01 between I/St and A/Sn mice in panels a, b, d, g, and h is given for three to six independent experiments (total n = 12 to 24), and error bars represent the standard deviations from a Student's t test.

FIG. 2.

I/St and A/Sn neutrophils acquired the identical activation phenotype during extravasation at week 3 postinfection. Down-regulation of CD62L (a) and up-regulation of CD11b (b) in the Lyt-6G⁺ cells after their transition from blood to the site of inflammation is shown. The expression of CD18 remained stable (c). The results of one out of two similar experiments performed with a mixture of cells obtained from three individual mice and gated for Lyt-6G are displayed.

FIG. 3.

I/St neutrophils are more resistant to apoptosis and have longer lifespan than A/Sn neutrophils. (A) I/St blood and lung neutrophils remained CD95 negative at week 3 following TB challenge, whereas a substantial proportion of A/Sn neutrophils acquired the CD95⁺ phenotype. (B) The proportion of live Annexin V⁻ propidium iodide⁻ cells was significantly (P < 0.05) higher, whereas the proportion of late-apoptotic-stage Annexin V⁺ propidium iodide⁺ cells was lower (P < 0.01) in I/St neutrophil cultures than in A/Sn neutrophil cultures after 8-h incubation. FITC, fluorescein isothiocyanate. (C) Microscopic appearance of live I/St and dead A/Sn neutrophils in 20-h cultures is shown. The results of one out of three similar experiments are displayed.

FIG. 4.

Phagocytic capacity of I/St neutrophils is significantly higher that that of A/Sn neutrophils. Following injection of peptone with mycobacteria (a, c, and d) or peptone with zymosan (b), neutrophil yield, percent phagocytosis, and phagocytic numbers were significantly higher (P < 0.01, mean ± standard error of the mean from four independent experiments, three mice each, Mann-Whitney U test) in peritoneal cavities of I/St mice than in those in A/Sn mice. Mycobacterial phagocytosis is opsonin dependent, and the serum from A/Sn mice has unimpaired opsonizing properties (e and f). hi, heat inactivated; NMS, normal mouse serum.

FIG. 5.

Inhibition of mycobacterial growth by I/St and A/Sn neutrophils is not elevated in the presence of exogenous IFN-γ (a) in contrast to the inhibitory activity of lung macrophages (b). Lung cells from infected I/St mice produce more IL-6 (c) and less active TGF-β₁ (d) in response to mycobacterial sonicate in vitro than their A/Sn counterparts. The results represent means ± standard deviations from two independent experiments, three mice per point each, total n = 6, assessed in enzyme-linked immunosorbent assay format individually. *, P < 0.05 by the Mann-Whitney U test.

FIG. 6.

Neutrophil influx to the lungs following TB challenge (3 weeks postinfection) is controlled by the QTLs involved in TB severity control. Variation in the neutrophil influx between F₂ mice indicates polygenic control (A). i alleles were of I/St origin; a alleles were of A/Sn origin. The recessive i allele of the D3Mit215 QTL (B) and the dominant i allele of the D17Mit175 QTL (C) determine a high level of neutrophil inflammation. In mice bearing their combination, accumulation of neutrophils in the lungs was significantly (P < 0.05, Mann-Whitney U test) higher than that in mice with other genotypes (D).