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. 2022 Oct 1;58(4):295-303.
doi: 10.1097/SHK.0000000000001977. Epub 2022 Aug 23.

INTRAPULMONARY TREATMENT WITH A NOVEL TLR4 AGONIST CONFERS PROTECTION AGAINST KLEBSIELLA PNEUMONIA

Antonio Hernandez  1 , Jing Zhou  1 , Julia K Bohannon, Margaret A McBride  2 , Katherine N Gibson-Corley  2 , Naeem K Patil  1 , Allison M Owen  1 , Katherine R Burelbach  1 , Edward R Sherwood
Affiliations

INTRAPULMONARY TREATMENT WITH A NOVEL TLR4 AGONIST CONFERS PROTECTION AGAINST KLEBSIELLA PNEUMONIA

Antonio Hernandez et al. Shock. .

Abstract

Objectives: Nosocomial pneumonia is a common complication in critically ill patients. The goal of this study was to examine the efficacy of the Toll-like receptor 4 agonist 3-deacyl phosphorylated hexacyl disaccharide (3D PHAD), in a clinically relevant murine model of pneumonia, and assess the cellular mechanisms that mediate the protective response. Design: Mice received intrapulmonary 3D PHAD (20 μg) or vehicle for 2 consecutive days before challenge with intrapulmonary Klebsiella pneumoniae (2.3 × 10 3 colony-forming units). Mice were followed for 14-day survival, pulmonary K. pneumoniae burden, lung leukocyte profile, leukocyte phagocytic capacity, and cytokine production. Pneumonia severity and leukocyte recruitment were further assessed by histological evaluation. Setting: Research laboratory. Subjects: Wild-type, male C57BL/6 J mice. Interventions: Intrapulmonary treatment with 20 μg 3D PHAD for 2 consecutive days. Measurements and main results: Intrapulmonary treatment with 3D PHAD decreased lung K. pneumoniae colony-forming units and pneumonia severity with an associated improvement in survival compared with mice treated with vehicle. The numbers of neutrophils, monocytes, and macrophages in the lungs of 3D PHAD-treated mice were higher than those in vehicle-treated mice before infection but were not significantly different from vehicle-treated mice at 48 h after K. pneumoniae challenge. Lung innate leukocytes from 3D PHAD-treated mice had increased phagocytic capacity. Treatment with 3D PHAD alone increased cytokines in the lungs but decreased cytokines in plasma during K. pneumoniae pneumonia as compared with control. Conclusions: Intrapulmonary treatment with 3D PHAD augments innate immunity in the lung and facilitates resistance to K. pneumoniae pneumonia.

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Conflict of interest statement

The authors report no conflicts of interest.

Figures

Fig. 1.
Fig. 1.. Intrapulmonary treatment with 3D PHAD decreases bacterial burden and improves survival during K. pneumoniae pneumonia.
A, Mice were treated with intrapulmonary 20 μg 3D PHAD or vehicle at 48 and 24 h before K. pneumoniae (2.3 × 103 CFU) challenge, and separate cohorts of mice were killed at 6 and 8 h after infection to collect BAL for bacterial burden, and the third cohort was observed for 14-day survival. B, After treatment, mice were infected with intrapulmonary K. pneumoniae, and BAL was performed at 6 and 48 h after infection to assess K. pneumoniae burden. In the 6-h infection model, n = 10 per group, and the experiment was performed twice. In the 48-h infection model, n = 15 per group, and the experiment was performed thrice. C, After treatment, mice were infected with intrapulmonary K. pneumonia and followed for 14 days to assess survival; n = 15 per group, and this experiment was performed thrice.
Fig. 2.
Fig. 2.. Intrapulmonary 3D PHAD treatment induces innate leukocyte recruitment.
A, Mice were treated with intrapulmonary 20 μg 3D PHAD or vehicle at 48 and 24 h before intrapulmonary K. pneumoniae (2.3 × 103 CFU). Bronchoalveolar lavage was performed before infection and at 6 and 48 h after infection. B, Neutrophils, monocytes, and alveolar macrophages in BAL before and at 6 and 48 h after intrapulmonary K. pneumoniae challenge; n = 9 per group in the no-infection model, and the experiment was repeated twice; n = 10 in the 6-h infection model, and the experiment was repeated twice; n = 15 per group in the 48-h infection model, and the experiment was repeated thrice.
Fig. 3.
Fig. 3.. Intrapulmonary 3D PHAD treatment increases innate leukocyte numbers in the lung interstitium.
A, Mice were treated with intrapulmonary 20 μg 3D PHAD or vehicle at 48 and 24 h before bilateral lung harvest and homogenization for examination of lung leukocytes. B, Neutrophil, monocytes, interstitial macrophages, natural killer cell, and T cell numbers in lung homogenates were measured by flow cytometry; n = 10 per group, and the experiment was repeated twice.
Fig. 4.
Fig. 4.. Treatment with intrapulmonary 3D PHAD augments innate leukocyte phagocytic capacity.
Mice were treated with intrapulmonary 20 μg 3D PHAD or vehicle at 48 and 24 h before BAL and lung excision. Both lungs were homogenized. Bronchoalveolar lavage and lung homogenate were processed to isolate leukocytes at a concentration of 2 × 106/mL and incubated with 100 μg of pHrodo-labeled E. coli bioparticles for 60 min. pHrodo particle numbers were measured by flow cytometry; n = 9 per group, and the experiment was repeated twice.
Fig. 5.
Fig. 5.. Intrapulmonary 3D PHAD treatment alone induces cytokine production in the lungs but leads to decreased cytokine concentrations in plasma after K. pneumoniae challenge.
Mice were treated with intrapulmonary 20 μg 3D PHAD or vehicle at 48 and 24 h before K. pneumoniae (2.3 × 103 CFU) challenge for 48 h. Bronchoalveolar lavage was performed before and at 48 h after K. pneumoniae challenge. Cytokine concentrations in BAL were measured using a Magpix assay; n = 9 in the no-infection model, and it was repeated twice; n = 15 in the 48-h infection model, and it was repeated thrice.
Fig. 6.
Fig. 6.. Intrapulmonary 3D PHAD treatment alone induces plasma cytokine production, and it is not augmented upon K. pneumoniae challenge.
Plasma was harvested before and 48 h after K. pneumoniae challenge. Cytokine concentrations in BAL were measured using a Magpix assay; n = 9 in the no-infection model, and it was repeated twice; n = 15 in the 48-h infection model, and it was repeated thrice.
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