Chronic pulmonary LPS tolerance induces selective immunosuppression while maintaining the neutrophilic response

Abstract

LPS challenge causes potent activation of innate immunity. Because LPS is ubiquitously present in ambient air, repeated inhalation may lead to activation of the pulmonary immune response. If this activation is unregulated, chronic LPS inhalation would lead to persistent inflammation and organ damage. We hypothesized that the lung uses the mechanism of LPS tolerance to maintain the balance between hypoinflammatory and hyperinflammatory states. We developed a model of chronic pulmonary LPS tolerance induced by pulmonary exposure to 1 microg LPS for 4 consecutive days. Mice were challenged with 10 microg of LPS 24 h later. TNF-alpha protein was significantly decreased in the bronchoalveolar lavage fluid of tolerant versus nontolerant mice, whereas IL-6 levels were significantly increased in the tolerant group. Tolerant mice were also protected from airway hyperresponsiveness. M2 and M3 muscarinic receptor mRNA was significantly decreased in the lungs of tolerant mice, suggesting a mechanism for the decreased airway hyperresponsiveness. CXCL2 was significantly reduced in tolerant mice, but CXCL1 was equivalent between groups. No difference was seen in neutrophil recruitment to the alveolar space. Interestingly, LPS tolerance does not confer cross-tolerance to the Toll-like receptor (TLR) 2 stimulus Pam3Cys. TNF-alpha and IL-6 concentrations were significantly increased in LPS-tolerant mice challenged with Pam3Cys; however, chemokine concentrations were unaffected. Our data show that repeated LPS inhalation results in differential regulation of cytokines but does not inhibit neutrophil recruitment. This unrestricted neutrophil recruitment may represent a mechanism by which individuals may be protected from pulmonary bacterial infection and pneumonia.

Fig. 1. Physiologic parameters in response to LPS tolerance

A, Mouse weights were measured the day before, and each day, mice received intratracheal challenges. Complete differential was performed on blood collected from the tail vein and total white blood cell counts (B), lymphocyte counts (C), and hemoglobin concentrations (D) are represented. Data are expressed as mean ± SEM (n = 6–12 mice per group). There were no differences between the tolerant and nontolerant animals in any of the measured parameters.

Fig. 2. Acute-phase cytokine expression after induction LPS tolerance

A, Bronchoalveolar lavage TNF-α concentrations at the indicated time points post–final challenge and lung tissue TNF-α mRNA expression 1 h post–final challenge (B). C, Bronchoalveolar lavage IL-6 concentrations at the indicated time points post–final challenge and lung tissue IL-6 mRNA expression 1 h post–final challenge (D). Zero-hour samples were harvested immediately before the final LPS challenge. mRNA data were calculated as fold increase over 0-h nontolerant group and are expressed as mean ± SEM (n = 6–10 mice per group). **P < 0.01 compared with nontolerant group.

Fig. 3. TNF-soluble receptor expression in response to induction of LPS tolerance

TNF-SRI (A) and TNF-SRII (B) concentrations in BAL fluid at the indicated time points post–final challenge. Zero-hour samples were harvested immediately before the final LPS challenge. Data are expressed as mean ± SEM (n = 6–10 mice per group). **P < 0.01 and ***P < 0.0001 compared with the nontolerant group.

Fig. 4. Airway hyperresponsiveness and mediators of AHR in response to LPS tolerance

A, Airway hyperresponsiveness in response to increasing doses of aerosolized methacholine at 4 h post–final challenge. B, Bronchoalveolar lavage cysteinyl leukotriene concentrations at 2 h post–final challenge. C, Bronchoalveolar lavage tryptase concentrations at 2 h post–final challenge. Data are expressed as mean ± SEM (n = 6–10 mice per group). *P < 0.05 and **P < 0.01 compared with the nontolerant group.

Fig. 5. mRNA expression of M2 and M3 muscarinic acetylcholine receptors in nontolerant and tolerant mice at 1 h post–final challenge

Data are calculated as fold increase over 0-h nontolerant group and are expressed as mean ± SEM (n = 6 mice per group). *P < 0.05 and **P < 0.01 compared with the nontolerant group.

Fig. 6. Cytospin preparations of cells recovered from BAL fluid of naïve (unchallenged) (A), nontolerant (B), and LPS-tolerant (C) mice at 24 h post–final LPS challenge

Each is represented at 1,000× magnification. The cells from naïve mice are virtually all macrophages, whereas numerous neutrophils are observed in the other groups.

Fig. 7. Neutrophil influx and MPO activity in response to LPS tolerance

A, Bronchoalveolar lavage neutrophil numbers at various time points post–final challenge. Cytospins were prepared and quantified for collected cells, and data are represented as absolute cell number per mouse. B, After BAL, the right lung was homogenized and sonicated for MPO assay. Data are expressed as mean ± SEM (n = 6–10 mice per group). **P < 0.01 compared with the nontolerant group.

Fig. 8. Neutrophil chemotactic protein expression after induction LPS tolerance

A, Bronchoalveolar lavage KC (CXCL1) concentrations at the indicated time points post–final challenge and (B) lung tissue KC (CXCL1) mRNA expression 1 h post–final challenge. C, Bronchoalveolar lavage MIP-2 (CXCL2) concentrations at the indicated time points post–final challenge and lung tissue MIP-2 (CXCL2) mRNA expression (D) 1 h post–final challenge. Zero-hour samples were harvested immediately before the final LPS challenge. mRNA data are expressed as fold increase above naïve mice and are represented as mean ± SEM (n = 6–10 mice per group). *P < 0.05 and **P < 0.01 compared with the nontolerant group.

Fig. 9. Cross-tolerance to TLR-2 ligand PAM

LPS tolerance was induced by direct pulmonary exposure to LPS for 4 consecutive days (tolerant PAM). Mice were challenged with 25 μg PAM 24 h later. Control mice received PBS for 4 consecutive days and 25 μg PAM 24 h later (nontolerant PAM). TNF (A), IL-6 (B), KC (CXCL1) (C), MIP-2 (CXCL2) (D) expression in BAL fluid 2 h post-PAM challenge. Data are expressed as mean ± SEM (n = 6 mice per group). ***P < 0.0001 compared with the nontolerant group.