Intrapulmonary administration of leukotriene B(4) augments neutrophil accumulation and responses in the lung to Klebsiella infection in CXCL1 knockout mice

Abstract

In prior studies, we demonstrated that 1) CXCL1/KC is essential for NF-κB and MAPK activation and expression of CXCL2/MIP-2 and CXCL5/LPS-induced CXC chemokine in Klebsiella-infected lungs, and 2) CXCL1 derived from hematopoietic and resident cells contributes to host immunity against Klebsiella. However, the role of CXCL1 in mediating neutrophil leukotriene B(4) (LTB(4)), reactive oxygen species (ROS), and reactive nitrogen species (RNS) production is unclear, as is the contribution of these factors to host immunity. In this study, we investigated 1) the role of CXCL1 in LTB(4), NADPH oxidase, and inducible NO synthase (iNOS) expression in lungs and neutrophils, and 2) whether LTB(4) postinfection reverses innate immune defects in CXCL1(-/-) mice via regulation of NADPH oxidase and iNOS. Our results demonstrate reduced neutrophil influx, attenuated LTB(4) levels, and decreased ROS and iNOS production in the lungs of CXCL1(-/-) mice after Klebsiella pneumoniae infection. Using neutrophil depletion and repletion, we found that neutrophils are the predominant source of pulmonary LTB(4) after infection. To treat immune defects in CXCL1(-/-) mice, we intrapulmonarily administered LTB(4). Postinfection, LTB(4) treatment reversed immune defects in CXCL1(-/-) mice and improved survival, neutrophil recruitment, cytokine/chemokine expression, NF-κB/MAPK activation, and ROS/RNS production. LTB(4) also enhanced myeloperoxidase, H(2)O(2,) RNS production, and bacterial killing in K. pneumoniae-infected CXCL1(-/-) neutrophils. These novel results uncover important roles for CXCL1 in generating ROS and RNS in neutrophils and in regulating host immunity against K. pneumoniae infection. Our findings suggest that LTB(4) could be used to correct defects in neutrophil recruitment and function in individuals lacking or expressing malfunctional CXCL1.

Figure 1. MPO activity and LTB₄ production in the lungs is dependent on CXCL1 during K. pneumoniae infection

MPO activity (A) and LTB₄ levels (B) in homogenized (unlavaged) whole lungs of WT (C57Bl/6) and CXCL1^-/- mice infected with K. pneumoniae (10³ CFU/mouse) for 24 and 48 h. Data are presented as means ± SEM. n=6-8 mice/group. (* indicates p<0.05 compared with CXCL1^-/- mice). The levels of p67^phox, p47^phox, and iNOS (C) in K. pneumoniae-infected homogenized whole lungs of WT and CXCL1^-/- mice at 24 and 48 h post-infection. The blot is representative of three individual blots with identical results. Densitometric analysis of p67^phox, p47^phox and iNOS expression (D) in homogenized lungs. Densitometry was performed from three separate blots. Data are expressed as means ± SEM. n=6-8 mice/group. (* indicates p<0.05 compared to CXCL1^-/- mice). Levels of p67^phox and p47^phox in cytosolic and membrane fractions of the lung (E) upon K. pneumoniae infection. This is a representative blot of three independent experiments. GAPDH is a cytosolic marker, pan-cadherin is a plasma membrane marker. Densitometric analysis of p67^phox and p47^phox levels (F) in cytosol and membrane fractions of the lung following K. pneumoniae infection. Protein expression was quantitated from three separate blots. Data are shown as means ± SEM. For experiments E-F, a total of 6-8 mice were used in each group. (* indicates p<0.05 as compared to CXCL1^-/- mice.)

Figure 2. Neutrophil depletion impairs LTB₄, iNOS, and NADPH oxidase component expression in lung tissues during K. pneumoniae infection

LTB₄ levels (A) in the lungs of neutrophil-depleted WT and CXCL1^-/- mice at 24 and 48 h following K. pneumoniae infection (10³ CFUs/mouse). WT and CXCL1^-/- mice were i.p. injected with anti-Gr-1/Ly6G or control Ab at 12 and 2 h before i.t. infection with K. pneumoniae. Data are presented as means ± SEM. (* indicates p < 0.05 compared with control Ab-administered mice.) Protein levels of p67^phox, p47^phox, and iNOS in whole lung homogenates (B) of neutrophil-depleted WT and CXCL1^-/- mice after K. pneumoniae infection (10³ CFUs/mouse). This blot is representative of three separate blots. Densitometric analysis (C) of p67^phox, p47^phox, and iNOS levels in the lungs of neutrophil-depleted WT and CXCL1^-/- mice following K. pneumoniae (10³ CFUs/mouse) infection. Western blots from three independent blots experiments were used to quantify protein levels compared to GAPDH. Data are expressed as mean ± SE. For experiments A-C, n=6-9 mice/group. (* indicates p<0.05 as compared to control Ab-administered mice.) Role of hematopoietic and resident cells in LTB₄ production. Levels of LTB₄ (D) in the lungs of i.t. neutrophil repleted (10⁷ cells/mouse) neutropenic WT and CXCL1^-/- mice at 48 h following K. pneumoniae infection. Data are presented as means ± SEM. (* indicates p<0.05 compared to depleted (non-repleted) mice.) (E) Bone marrow chimeras were generated and LTB₄ levels in lungs were measured at 48 h post K. pneumoniae challenge. A total of 5-7 mice/group were used. (* indicates p < 0.05 compared with CXCL1->CXCL1 mice.)

Figure 3. CXCL1 is essential for Klebsiella-induced expression of p67^phox, p47^phox, and ROS/RNS production by neutrophils

Levels of p67^phox and p47^phox (A) in bone marrow-derived neutrophils from WT and CXCL1^-/- mice after infection with K. pneumoniae (MOI of 1). This is a representative blot from three separate experiments. Densitometric analysis from three separate blots (B) shows the expression of p67^phox and p47^phox in K. pneumoniae stimulated-neutrophils that were normalized against GAPDH. (* indicates p<0.05 as compared to CXCL1^-/- neutrophils). MPO activity and nitrite and H₂O₂ release (C) in WT and CXCL1^-/- neutrophils stimulated with K. pneumoniae. The levels of MPO, nitrite, and H₂O₂ were measured in infected neutrophils at 30, 60, and 180 min post-infection. Experiments were performed in triplicates. Bacterial killing capacity (D) of K. pneumoniae-infected neutrophils from WT and CXCL1^-/- deficient mice was determined by assessing extracellular and intracellular CFUs at 30, 60, and 180 mins post-infection with K. pneumoniae (MOI of 1). Relative phagocytosis (E) of K. pneumoniae-infected WT and CXCL1^-/- neutrophils at 30 min post-treatment (MOI of 1). For experiments A-E, a total of 5-8 mice/group were used. (* indicates p<0.05 compared with CXCL1^-/- neutrophils).

Figure 4. Impaired survival, bacterial clearance, neutrophil influx, and cytokine/chemokine production in the lungs of CXCL1^-/- mice are restored by exogenous LTB₄

Mortality (A) in WT or CXCL1^-/- mice infected with 10³ CFUs of K. pneumoniae and administered with LTB₄ (100 ng/mouse) or vehicle (BSA) 1 h later, and survival was assessed up to 15 days. Data are presented as % survival (n=20 mice/group) and analyzed using Wilcoxon signed-rank test. * indicates the difference between LTB₄ or vehicle (BSA) control treated CXCL1^-/- mice (p<0.05) and # indicates the difference between WT+LTB₄ and CXCL1^-/-+LTB₄ mice.) Bacterial clearance (B, C) in the lungs and dissemination were examined in lung homogenates of LTB₄ or BSA administered WT and CXCL1^-/- mice at 48 and 96 h post-K. pneumoniae challenge (10³ CFUs/mouse). Data are presented as Mean ± SE (n=5-6 mice/group). (* indicates p<0.05 compared with BSA (vehicle) administered mice.) Cellular infiltration (D,E) in airspaces at 48 h after i.t. treatment with LTB₄ (100 ng/mouse) or vehicle (BSA) control. Concentrations of CXCL2/MIP-2, IL-6, TNF-α, and CXCL5/LIX (F) in BALF from CXCL1^-/- or WT mice infected with 10³ CFUs of K. pneumoniae and administered with LTB₄ or vehicle (BSA). For experiments D-F, n=6-8 mice/group. (* indicates p<0.05 as compared to BSA administered mice).

Figure 5. Defective K. pneumoniae-induced activation of NF-κB, MAPKs, and expression of ICAM-1 in the lungs of CXCL1^-/- mice is corrected by LTB₄

Activation of NF-κB and MAPKs and expression of ICAM-1 and VCAM-1 (A) in K. pneumoniae-infected and LTB₄ administered homogenized whole lungs at 48 h post-infection. WT and CXCL1^-/- mice were infected with 10³ CFU of K. pneumoniae cells via the i.t. route and treated with LTB₄ (100 ng/mouse) or vehicle administration 1 h later. Data are presented as a representative of three independent blots/experiments. Densitometric analysis (B) of activation of NF-κB and MAPKs and expression of ICAM-1 and VCAM-1 in the lung homogenates after LTB₄ or vehicle treatment. Data represent the means ± SEM of arbitrary densitometric units for each band from three independent blots/experiments. (* indicates p<0.05 as compared to BSA treated C57Bl6 or CXCL1^-/- mice). Expression levels of p67^phox, p47^phox, and iNOS (C) in homogenized lungs of K. pneumoniae-infected and i.t.-treated with vehicle or LTB₄ (100 ng) 1 h post-infection. This blot is representative of three independent blots. Densitometric analysis of iNOS, p67^phox, and p47^phox levels in lung (D) homogenates from three independent experiments (p<0.05). (* indicates p< 0.05 compared with C57Bl6 or CXCL1^-/- vehicle (BSA) administered mice (n=5-6/group). For experiments A-D, a total of 6-9 mice/group was used.

Figure 6. Impaired bacterial killing, ROS and RNS generation, and phagocytosis in K. pneumoniae-stimulated CXCL1^-/- neutrophils is restored by LTB₄

(A) Bacterial killing capacity of K. pneumoniae-infected, LTB₄-treated neutrophils from WT and CXCL1^-/- deficient mice was determined at 30, 60, and 180 mins by assessing intracellular and extracellular CFUs. Relative phagocytosis (B) of K. pneumoniae-infected WT and CXCL1^-/- neutrophils at 30 min post-treatment (1 MOI). Relative phagocytosis index was calculated as described in Materials and Methods. MPO release, intracellular MPO, NO, and H₂O₂ production (C) in K. pneumoniae-infected WT and CXCL1^-/- neutrophils at 30 and 180 mins post-LTB₄ treatment. For experiments A-C, a total of 7-9 mice/group were used. (* indicates p<0.05 as compared to BSA treated neutrophils).

Figure 7. Scheme depicting CXCL1-dependent bacterial clearance in the lung following K. pneumoniae infection

K. pneumoniae induces CXCL1 production by hematopoietic and resident cells, causing neutrophil accumulation in the lungs. Neutrophil influx into the lungs is important for LTB₄ production by bone marrow-derived cells. LTB₄ subsequently activates NF-κB and MAPKs, resulting in the production of cytokines/chemokines and the generation of ROS/RNS. Cytokines/chemokines and ROS/RNS regulate each other through signaling cascades. Cytokines/chemokines and ROS/RNS can regulate the production of LTB4 via a positive feedback loop. ROS/RNS ultimately lead to bacterial clearance from the lungs.