CXCL1 regulates pulmonary host defense to Klebsiella Infection via CXCL2, CXCL5, NF-kappaB, and MAPKs

Abstract

Pulmonary bacterial infections are a leading cause of death. Since the introduction of antibiotics, multidrug-resistant Klebsiella pneumoniae became an escalating threat. Therefore, development of methods to augment antibacterial defense is warranted. Neutrophil recruitment is critical to clear bacteria, and neutrophil migration in the lung requires the production of ELR(+) CXC chemokines. Although lung-specific CXCL1/keratinocyte cell-derived chemokine (KC) transgene expression causes neutrophil-mediated clearance of K. pneumoniae, the mechanisms underlying KC-mediated host defense against K. pneumoniae have not been explored. In this study, we delineated the host defense functions of KC during pulmonary K. pneumoniae infection using KC(-/-) mice. Our findings demonstrate that KC is important for expression of CXCL2/MIP-2 and CXCL5/LPS-induced CXC chemokine, and activation of NF-κB and MAPKs in the lung. Furthermore, KC derived from both hematopoietic and resident cells contributes to host defense against K. pneumoniae. Neutrophil depletion in mice before K. pneumoniae infection reveals no differences in the production of MIP-2 and LPS-induced CXC chemokine or activation of NF-κB and MAPKs in the lung. Using murine bone marrow-derived and alveolar macrophages, we confirmed KC-mediated upregulation of MIP-2 and activation of NF-κB and MAPKs on K. pneumoniae infection. Moreover, neutralizing KC in bone marrow-derived macrophages before K. pneumoniae challenge decreases bacteria-induced production of KC and MIP-2, and activation of NF-κB and MAPKs. These findings reveal the importance of KC produced by hematopoietic and resident cells in regulating pulmonary host defense against a bacterial pathogen via the activation of transcription factors and MAPKs, as well as the expression of cell adhesion molecules and other neutrophil chemoattractants.

Figure 1. Mortality in KC^-/- mice infected with Kp

(A) Enhanced mortality in CXCL1/KC^-/- mice following i.t. Kp challenge. Mice were i.t. infected with 10³ CFU Kp per mouse and survival was assessed up to 15 days. Data are from two separate experiments (n=10 mice/group). Significance between groups was examined by Wilcoxon rank sign test and asterisk indicates the difference between KC^-/- and KC^+/+ mice (p < 0.05). (B-C) Impaired bacterial clearance in the lungs and spleens in KC^-/- mice challenged i.t. with Kp. The CFUs were examined in homogenates obtained from the lungs and spleens at 24 and 48 h post Kp challenge (n=5/group from 2 independent experiments; p< 0.05; significant differences between KC^–/– and KC^+/+ mice. (D-E) KC^-/- mice have reduced cellular/neutrophil accumulation in the airspaces (BALF) after Kp (10³ CFU/mouse) inoculation. (F) Attenuated neutrophil influx in the lung parenchyma, as measured by MPO activity, in KC^-/- mice following Kp infection. (n=6/group from 2 separate experiments for D-F; p < 0.05; significant differences between KC^–/– and KC^+/+ mice.

Figure 2. Kp-induced cytokine/chemokine production in KC^-/- mice

(A-C) KC-dependent signaling regulates the expression of MIP-2 and LIX in the lungs post Kp inoculation. Mice were infected with Kp, BALF was collected and MIP-2, LIX and TNF-α concentrations were measured at 24 and 48 h post infection. A total of 4-6 animals were used at each time point in each group. Significant differences between KC^-/- and control (KC^+/+) mice are indicated by asterisks; *, p < 0.05.

Figure 3. Activation of Kp-mediated NF-κB and MAPKs and expression of ICAM-1 and VCAM-1 in lung tissues of KC^-/- mice

(A) KC mediated regulation of IKK (an upstream kinase of NF-κB) and NF-κB/p65 and IκBα in the lungs of KC^-/- mice post Kp infection. The blots are a representative of 3 independent experiments with similar results. (B) Reduced activation of the NF-κB/p65 subunit of NF-κB as detected by p65 DNA binding in the nuclear extracts of murine lungs at 24 and 48 h following Kp infection (n = 3–4 mice per group at a time point.). (C) Attenuated upregulation of MAPKs and cell adhesion molecules (VCAM-1 and ICAM-1) in the lungs of KC^-/- mice following Kp inoculation. Results are representative of three separate experiments with identical results. Values that are significantly different between KC^–/– and KC^+/+ are indicated by asterisks (p < 0.05). (D) Densitometric analysis of Western blots from three independent experiments to quantify the protein levels of IKK, NF-κB, and phospho-IKK, -NF-κB, -IκBα and -MAPKs as well as ICAM-1, VCAM-1, total p38 following Kp infection. The results obtained were normalized against GAPDH and expressed as mean ± SE. The difference between the KC^-/- and KC^+/+ animals was determined and the difference was expressed as p < 0.05.

Figure 4. Role of bone marrow and non-bone marrow cells in host defense against Kp

KC produced from bone marrow and non-bone marrow/resident cells following Kp infection is essential for host defense. Bone marrow chimeras were generated by lethal irradiation of KC^-/- and KC^+/+ mice and reconstituted with bone marrow cells via tail injection. (A) Bacterial CFU in the lungs at 48 h post-Kp infection. (B-C) Total WBC and neutrophil counts in BALF at 48 h post Kp infection. (D) KC levels in BALF at 48 h following Kp challenge. A total of 5-7 mice/group were used in 2 independent experiments and statistical significance was expressed as p < 0.05.

Figure 5. Kp-induced cytokine/chemokine production in lungs of neutrophil depleted KC^+/+ and KC^-/- mice

(A-D) KC derived from neutrophils does not contribute to MIP-2 and LIX production. Mice were pretreated with anti-Gr1 or control mAb at 12 and 2 h prior to infection with 10³ CFU of Kp. Cytokine/chemokines levels were determined at 24 and 48 h post Kp infection (n=6-8 animals per group; * p < 0.05).

Figure 6. Activation of Kp-induced NF-κB and MAPKs in lung tissues of neutrophil depleted KC^+/+ and KC^-/- mice

(A) Wild-type and KC^-/- mice challenged with 10³ CFU i.t. were euthanized at 24 and 48 h post infection and lungs were used for western blotting. The membranes were probed with activated NF-κB and MAPK Abs. (B) Densitometric analysis of Western blots from three independently performed experiments to quantify the protein levels of IKK, NF-κB, ICAM-1, VCAM-1 and phospho-IKK, -NF-κB, and -MAPKs following Kp challenge. The data obtained from these experiments were normalized against GAPDH and expressed as mean ± SE from 2 independent experiments). The significance between the KC^-/- and KC^+/+ mice was indicated as p < 0.05.

Figure 7. Activation of NF-κB and MAPKs in BMMs of KC^-/- mice infected with Kp

(A) Decreased activation of NF-κB and MAPKs in BMMs obtained from KC^-/- mice post Kp infection. Activation of IKK, NF-κB and MAPKs was detected at 24 and 48 h following Kp stimulation. The blot is a representative of three independent experiments with identical results. (B) Densitometric analysis of IKK, NF-κB and MAPK activation in BMMs up to 60 min following stimulation with Kp (MOI of 1). Data expressed as mean ± SE of three blots from three mice in each group (p < 0.05). (C) Attenuated production of MIP-2 in BMMs obtained from KC^-/- mice post Kp infection. BMMs were stimulated with Kp at an MOI of 1 and supernatants collected at 18 h post infection were used to determine MIP-2, KC and TNF-α release. The data obtained from 3 independent experiments.

Figure 8. Effect of KC neutralization on the production of cytokines/chemokines in BMMs following Kp challenge

(A-C) BMMs from KC^+/+ mice produce KC, MIP-2 and TNF-α during differentiation in the absence of Kp infection on day 7. Supernatants were collected 1, 3, 5 and 7 days culture and cytokine/chemokine levels were assessed by sandwich ELISA. Data are representative of 2 independent experiments using 3 mice/group (p < 0.05). (D-E) Blocking KC using Ab attenuated KC and MIP-2 but not TNF-α production in BMMs. BMMs were pretreated with KC neutralizing Ab 2 h prior to Kp infection (MOI of 1). Supernatants were collected 2, 4 and 8 h after Kp challenge and cytokine/chemokines levels were measured by sandwich ELISA. Data are pooled from 2 independent experiments using 5 mice/group and significant difference was expressed as p < 0.05.

Figure 9. Effect of KC blocking on activation of NF-κB and MAPKs in BMMs challenged with Kp

(A) KC blocking decreased activation of NF-κB and MAPKs in BMMs obtained from KC^+/+ (C57Bl/6) mice following pulmonary Kp infection.Activation of NF-κB and MAPKs was detected at various time points after stimulation.The blot is a representative of four blots with identical results. (B) Densitometric analysis of IKK, NF-κB, IκBα and MAPK activation in BMMs at 2, 4 and 8 h post-stimulation with an MOI of 1. Data expressed as mean ± SE of 3 independent experiments in each group (p < 0.05).

Figure 10. Effect of KC disruption on cytokine/chemokine production and activation of NF-κB and MAPKs in AMs following Kp infection

(A-C) Decreased production of MIP-2 in alveolar macrophages obtained from KC^-/- mice to Kp challenge. Alveolar macrophages were stimulated with Kp at an MOI of 1 and supernatants collected at 18 h post infections were used to determine MIP-2, TNF-α, and KC levels. The data obtained from 2 independent experiments using 4 mice in total. (D) Reduced activation of NF-κB and MAPKs in alveolar macrophages obtained from KC^-/- mice following Kp infection. Activation of IKK, NF-κB and MAPKs was detected at various time points following Kp stimulation (MOI of 1). The blot is a representative of three experiments with identical results. (E) Densitometric analysis of IKK, NF-κB and MAPK activation in BMMs up to 8 h following Kp stimulation with an MOI of 1. Data expressed as mean ± SE of three blots from three mice in each group from 2 independent experiments (p < 0.05).

Figure 11. Schematic depicting the functions of KC leading to neutrophil accumulation following Kp infection

KC activates of NF-κB and MAPK causes upregulation of chemokines, such as KC, MIP-2 and LIX and adhesion molecules, including VCAM- and ICAM-1. In turn, these events ultimately cause neutrophil recruitment to clear the bacterial infection from the lungs.