Inescapable need for neutrophils as mediators of cellular innate immunity to acute Pseudomonas aeruginosa pneumonia

Abstract

Pseudomonas aeruginosa is a leading cause of pneumonia, and many components of the innate immune system have been proposed to exert important effects in preventing lung infection. However, a vigorous experimental system to identify an overriding, key effector mediating innate immunity to lung infection has not been utilized. As many of the important components of innate immunity are involved in recruitment and activation of polymorphonuclear neutrophils (PMNs) to infected tissues, we hypothesized that the cells and factors needed for their proper recruitment to the lung comprised the major mediators of innate immunity. In neutropenic mice, intranasal (i.n.) doses of P. aeruginosa as low as 10 to 100 CFU/mouse produced a fatal lung infection, compared with 10(7) to >10(8) CFU for nonneutropenic mice. There was only a very modest increased mortality in mice lacking mature lymphocytes and no increased mortality in mice depleted of alveolar macrophages when administered i.n. P. aeruginosa. Recombinant mouse granulocyte colony-stimulating factor increased survival of neutropenic mice after i.n. P. aeruginosa inoculation. MyD88(-/-) mice, which cannot recruit PMNs to the lungs, were highly susceptible to fatal P. aeruginosa lung infection, with bacterial doses of <120 CFU being lethal. Activation of a MyD88-independent pathway for PMN recruitment to the lungs in MyD88(-/-) mice resulted in enhanced protection against P. aeruginosa lung infection. Overall, in the absence of PMNs, mice cannot resist P. aeruginosa lung infection from extremely small bacterial doses. There is an inescapable requirement for local PMN recruitment and activation to mediate innate immunity to P. aeruginosa lung infection.

FIG. 1.

(A) Survival curves of female 6- to 8-week-old C3H/HeN mice treated with Cy (150 mg/kg i.p. once daily every other day for 3 days) or MAb RB6-8C5 (200 μg i.p.) and subsequently challenged i.n. with P. aeruginosa strain PAO1, IT4, or 17003. Median survival of mice given Cy or MAb RB6-8C5 and P. aeruginosa was significantly lower than that of mice treated only with Cy or MAb RB6-8C5 (P < 0.01, log rank test). Each group contained eight mice. Values in parentheses indicate the average P. aeruginosa CFU inoculum. (B) Effect of cyclophosphamide (150 mg/kg i.p. once daily every other day for 3 days) on murine white blood cell, absolute neutrophil, absolute lymphocyte, and absolute monocyte blood counts. Data are means ± standard errors of the means (in cells/mm³) for four mice per group.

FIG. 2.

Survival curves of Rag^−/− mice and wild-type counterparts (C57BL/6) after i.n. administration of P. aeruginosa strain PAO1. Median survival of wild-type mice was higher than that of Rag^−/− mice (P = 0.0028, log rank test). Each group contained eight mice. Values in parentheses indicate the average P. aeruginosa CFU inoculum.

FIG. 3.

Histological analysis of murine lungs after MAb RB6-8C5 administration. Histological sections of lungs from C3H/HeN mice that were given PAO1 (100 CFU) only (A), MAb RB6-8C5 (0.200 mg i.p. once only) (B), or MAb RB6-8C5 followed by P. aeruginosa strain PAO1 (100 CFU i.n. 24 h after RB6-8C5 administration) (C). Mice were sacrificed 24 h after either MAb RB6-8C5 or PAO1 challenge. Magnification, ×40. Images in panels A and B show normal histologic appearance, whereas the section in panel C shows consolidation and histologic changes consistent with an acute pneumonia.

FIG. 4.

Effects of r-mGCSF on murine white blood cell, absolute neutrophil, absolute lymphocyte, and absolute monocyte blood counts. C3H/HeN mice were administered MAb RB6-8C5 (0.200 mg, i.p. once on day −1). Mice were then randomized to receive either 0.150 mg/kg r-mGCSF i.p. or 0.150 mg/kg albumin i.p. daily for five doses, starting on day zero. Data are means ± standard errors of the means of cells/mm³ for four mice per group. White bars, r-mGCSF; dark bars, albumin (control).

FIG. 5.

Survival curves of neutropenic C3H/HeN mice treated i.p. with either albumin (control) or r-mGCSF and challenged i.n. with the P. aeruginosa strain indicated in each survival curve graph and the dose (CFU/mouse) indicated in parentheses. P values by log-tank test: PAO1, P = 0.05; IT4, P = 0.008; 170003, P = 0.02; PA14, P = 0.06; N13, P = 0.12; 6077, P = 0.002. Each group contained eight mice.

FIG. 6.

Survival curves of WT C57BL/6 or MyD88^−/− mice after i.n. challenge with P. aeruginosa strain PAO1, PA14, or N8. Median survival of MyD88^−/− mice infected i.n. with P. aeruginosa was significantly lower (P ≤ 0.025, log-rank test) than that of WT C57BL/6 mice infected i.n. with P. aeruginosa (all showed 100% survival). Each group contained eight mice. Values in parentheses indicate the average P. aeruginosa CFU inoculum.

FIG. 7.

Murine BAL fluid analysis for the presence of neutrophils in lung sections from MyD88^−/− mice administered various bacterial preparations. BAL fluid from 6- to 8-week-old female MyD88^−/− mice was obtained 24 h after i.n. administration of the following: 50 CFU of P. aeruginosa strain PAO1 (A); 10 μg of E. coli strain 0111:B4 LPS (B); 10⁸ CFU of paraformaldehyde-killed E. coli strain HB101 (C); 10 μg of poly(I:C) (D); 10⁸ CFU of live E. coli strain HB101 (E). Differential cell counts were determined from Wright-Giemsa-stained cytospins (100 cells per slide were counted). Objective lens, 10×. Only panel E shows the presence of polymorphonuclear lymphocytes in the BAL fluid.

FIG. 8.

Survival curves of MyD88 ^−/− mice after i.n. administration of live or paraformaldehyde (PAF)-killed E. coli strain HB101 followed by challenge with 38 CFU of P. aeruginosa strain PAO1 or 256 CFU of strain PA14 i.n. The RB6, live HB101 group was given MAb RB6-8C5, 0.200 mg i.p., once on day zero, E. coli strain HB101 i.n. on day 1 (24 h); or 38 CFU of PAO1 i.n. on day 2 (48 h). Median survival of mice given live HB101 and subsequent i.n. P. aeruginosa was higher than that of mice treated with PAF-HB101 (P = 0.0012 for PAO1 group, log rank test). Each group contained eight mice. Values in parentheses indicate the average P. aeruginosa inoculum CFU.