Role of Liver Kinase 1B in Platelet Activation and Host Defense During Klebsiella pneumoniae-Induced Pneumosepsis

Abstract

Pneumonia is the most common cause of sepsis, with Klebsiella pneumoniae frequently implicated as a causative pathogen. Platelets play a crucial role in host defense during sepsis, and their activation is essential for effective immune responses, which is at least in part induced through activation of the collagen receptor glycoprotein (GP)VI. Platelets require energy for their activation, and Liver kinase B1 (LKB1) is a key regulator of energy metabolism. We sought to determine the role of LKB1 in platelet function and host response during K. pneumoniae-induced pneumosepsis. Platelet-specific-Lkb1-deficient mice were generated and compared to control littermates. Platelet counts were unaffected by Lkb1 deficiency in naïve mice. However, Lkb1-deficient platelets exhibited significant hyperreactivity to GPVI stimulation, an effect not observed after stimulation of the thrombin receptor protease-activated receptor 4. During K. pneumoniae infection, platelets of both Lkb1-deficient and control mice became equally hyporesponsive to GPVI stimulation, without differences between genotypes. Platelet-specific Lkb1 deficiency did not alter bacterial outgrowth or dissemination, inflammatory responses, or lung pathology. These findings suggest that while Lkb1 plays a role in regulating platelet activation in response to GPVI stimulation, it does not significantly impact platelet activation or the host response during pneumonia-induced sepsis.

Keywords: microbes–host interaction; microbial infection; molecular and cell biology.

Figure 1

Generation of platelet-specific-Lkb1-deficient mice and the effect of platelet-specific Lkb1 deficiency on platelet counts and activation in naïve mice and mice with Klebsiella-induced pneumosepsis. (A) Representative Western blot of platelet lysates for Lkb1, showing effective knockdown of platelet Lkb1 in Stk11^fl/fl × Pf4-Cre mice. (B) Boxplots displaying the platelet counts in blood obtained in naïve (uninfected) mice and mice 40 h after infection with Klebsiella pneumoniae via the airways; n = 8 per genotype for each timepoint. (C) Boxplots comparing the median fluorescence intensity (MFI) for P-selectin, CD63, or GPIIbIIIa in active conformation on the platelet surface of Stk11^fl/fl × Pf4-Cre and Stk11^fl/fl littermate control mice (naïve and 40 h after infection). Each column represents a treatment with the platelet agonist indicated or vehicle (PBS-BSA); n = 8 per group for naïve mice and n = 16 per group for mice 40 h after infection. The p-values are derived from BH-adjusted Wilcoxon tests; p-values between genotypes at specific timepoints are denoted by ‘*’; * p < 0.05, ** p < 0.01. The p-values between timepoints within the same genotype are denoted by ‘#’; ## p < 0.01, ### p ≤ 0.001, #### p ≤ 0.0001.

Figure 2

Platelet-specific Lkb1 deficiency does not impact bacterial growth or dissemination during Klebsiella-induced pneumosepsis. Bacterial counts are quantified as colony forming units (CFU) per ml at the primary site of infection and distant organs. (A) Lung and BALF and (B) Blood, Liver, and Spleen at 24 and 40 h after K. pneumoniae infection via the airways. (For (B), bacterial dissemination to blood, liver, and spleen is quantified as CFU per ml at 24 h and 40 h post K. pneumonia infection.) n = 8 per group at t = 24 h; n = 16 per group at t = 40 h.

Figure 3

Pathology scores. (A) Lung pathology scores from H/E-stained slides, evaluated by a pathologist blinded to group identity, and visualized in three panels: non-infected naïve mice, and mice 24 and 40 h after K. pneumoniae infection. (B,C) Representative microphotographs (magnification ×20) of H/E-stained slides from the lungs of naïve mice, Stk11^fl/fl mice, and Stk11^fl/fl × Pf4-Cre mice, respectively. Scale bars represent 200 μm. (D,E) Representative microphotographs (magnification ×20) of H/E-stained slides of lungs of mice 40 h post K. pneumoniae infection via the airways; Stk11^fl/fl and Stk11^fl/fl × Pf4-Cre mice, respectively. Scale bars represent 200 μm. Asterisks represent perivascular and peribrochial edemas and collections of neutrophils in the peribronchovascular interstitium. Arrowheads point to interstitial hemorrhages. Black arrows indicate endothelitis. White arrows indicate bronchitis.