Comparison of Diabetic and Non-diabetic Human Leukocytic Responses to Different Capsule Types of Klebsiella pneumoniae Responsible for Causing Pyogenic Liver Abscess

Abstract

The major risk factor for Klebsiella liver abscess (KLA) is type 2 diabetes mellitus (DM), but the immunological mechanisms involved in the increased susceptibility are poorly defined. We investigated the responses of neutrophils and peripheral blood mononuclear cells (PBMCs) to hypervirulent Klebsiella pneumoniae (hvKP), the causative agent of KLA. DNA and myeloperoxidase levels were elevated in the plasma of KLA patients compared to uninfected individuals indicating neutrophil activation, but diabetic status had no effect on these neutrophil extracellular trap (NET) biomarkers in both subject groups. Clinical hvKP isolates universally stimulated KLA patient neutrophils to produce NETs ex vivo, regardless of host diabetic status. Ability of representative capsule types (K1, K2, and non-K1/K2 strains) to survive intra- and extra-cellular killing by type 2 DM and healthy neutrophils was subsequently examined. Key findings were: (1) type 2 DM and healthy neutrophils exhibited comparable total, phagocytic, and NETs killing against hvKP, (2) phagocytic and NETs killing were equally effective against hvKP, and (3) hypermucoviscous K1 and K2 strains were more resistant to total, phagocytic, and NETs killing compared to the non-mucoviscous, non-K1/K2 strain. The cytokine response and intracellular killing ability of type 2 DM as well as healthy PBMCs upon encounter with the different capsule types was also examined. Notably, the IL-12-IFNγ axis and its downstream chemokines MIG, IP-10, and RANTES were produced at slightly lower levels by type 2 DM PBMCs than healthy PBMCs in response to representative K1 and non-K1/K2 strains. Furthermore, type 2 DM PBMCs have a mild defect in its ability to control hvKP replication relative to healthy PBMCs. In summary, our work demonstrates that type 2 DM does not overtly impact neutrophil intra- and extra-cellular killing of hvKP, but may influence cytokine/chemokine production and intracellular killing by PBMCs.

Keywords: Klebsiella pneumoniae; cytokines; hypervirulent; liver abscess; neutrophil extracellular trap; neutrophils; peripheral blood mononuclear cells; type 2 diabetes.

Figure 1

Ex vivo NET formation by neutrophils isolated from a KLA patient after stimulation with hvKP. Neutrophils were exposed to a capsule type K1 strain at an MOI of 1:1 for 2 h, and representative immunofluorescence images were taken at a magnification of 100×. (A) Bacteria were stained red with mouse anti-Klebsiella spp. (primary) and Alexa Fluor 633-conjugated goat anti-mouse (secondary). (B) DNA was stained green with Sytox Green. (C) Human neutrophil elastase (NE) was stained blue with rabbit anti-NE (primary) and Pacific Blue-conjugated goat anti-rabbit (secondary). (D) To visualize co-localization of NET components and hvKP, images from each channel were merged. White arrows point to physical entrapment of hvKP within NETs.

Figure 2

NET formation in the plasma of healthy and type 2 DM individuals as well as non-diabetic and type 2 DM KLA patients. Concentrations of DNA and MPO, which serve as biomarkers for NET formation, were determined in plasma samples. Each dot represents data from one study subject. Horizontal bars indicate the mean. SEM values are presented in text. (A) Healthy: mean age = 51.8 ± 11.9 years; normal fasting blood glucose and HbA1c levels. Diabetic: mean age = 56.0 ± 3.8 years; mean HbA1c = 10.1 ± 2.1%. KLA non-diabetic: mean age = 59.1 ± 16.5 years; normal fasting blood glucose and HbA1c levels. KLA diabetic: mean age = 59.2 ± 10.7 years; mean HbA1c = 10.5 ± 3.3%. (B) Healthy: mean age = 54.4 ± 11.3 years; normal fasting blood glucose and HbA1c levels. Diabetic: mean age = 54.6 ± 7.2 years; mean HbA1c = 9.8 ± 1.4%. KLA non-diabetic: mean age = 58.7 ± 14.3 years; normal fasting blood glucose and HbA1c levels. KLA diabetic: mean age = 59.8 ± 9.6 years; mean HbA1c = 9.8 ± 3.3%. ^***p < 0.001.

Figure 3

Ex vivo interactions between healthy as well as poorly controlled type 2 DM neutrophils and different hvKP capsule types. Neutrophils isolated from healthy donors (n = 8; mean age = 40.0 ± 11.7 years; normal fasting blood glucose and HbA1c levels) or type 2 DM donors with poor glycemic control (n = 8; mean age = 48.5 ± 13.9 years; mean HbA1c = 10.6 ± 1.7%) were pretreated with DNase I, cytochalasin D, DNase I + cytochalasin D, or left untreated. This was followed by infection with the K1 strain SGH04, K2 strain NUH14 and K28 strain NUH29. As a control, strains were also inoculated into infection media (RPMI + 10% pooled healthy human serum or RPMI + 10% pooled diabetic human serum) without neutrophils. (A,C) Bacterial survival was expressed as percentage change from inoculum, calculated with the equation: (CFU_+neutrophils at 2-h/CFU_{−neutrophils} at 0-h) × 100. (B,D) Percentage neutrophil killing was calculated with the equation: 100—[(CFU_+neutrophils at 2-h/CFU_{−neutrophils} at 2-h) × 100]. ^*Statistical analyses in the graphs were by ANOVA. #Denotes bars with negative values that were not visually represented in the graph. Error bars represent SEM. ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

Figure 4

Cytokine production by healthy and poorly controlled type 2 DM PBMCs induced by various hvKP capsule types. PBMCs isolated from healthy donors (n = 12; mean age = 45.0 ± 12.5 years; normal fasting blood glucose and HbA1c levels) or type 2 DM donors with poor glycemic control (n = 16; mean age = 54.8 ± 13.7 years; mean HbA1c = 10.3 ± 1.6%) were infected with the K1 strain SGH04 and the K28 strain NUH29, or left uninfected. Kanamycin was added at 2-h post-infection to kill off extracellular bacteria and culture supernatant was collected at 24-h post-infection for CBA analysis. (A) The K1 isolate induced higher production of IL-6, IL-8, IL-10, IL-12/IL-23p40, G-CSF, MCP-1, and TNFRII than the non-K1/K2 isolate. (B) Type 2 DM PBMCs generated a slightly lower production of IL-12p70, IFNγ, MIG, IP-10, and RANTES than healthy PBMCs. Error bars represent SEM. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

Figure 5

Intracellular survival of different hvKP capsule types in healthy and poorly controlled type 2 DM PBMCs. PBMCs isolated from healthy donors (n = 8; mean age = 58.1 ± 8.6 years; normal fasting blood glucose and HbA1c levels) or type 2 DM donors with poor glycemic control (n = 10; mean age = 60.3 ± 9.5 years; mean HbA1c = 9.8 ± 3.2%) were infected with the K1 strain SGH04 and the K28 strain NUH29. Kanamycin was added at 2-h post-infection to kill off extracellular bacteria, and the number of intracellular bacteria was quantified at 4-, 8-, and 24-h post-infection. (A) Percentage killing between 4 and 8 h post-infection. (B) Percentage killing between 4 and 24 h post-infection. Error bars represent SEM. ^*p < 0.05, ^**p < 0.01.

Figure 6

TNF receptor protein levels in the plasma of healthy individuals and KLA patients. Each dot represents data from one study subject. Horizontal bars indicate the mean. Healthy: mean age = 52.4 ± 10.7 years. A-KLASS (Day 0/1): mean age = 60.1 ± 13.0 years. A-KLASS (Day 28): mean age = 60.5 ± 13.6 years. (A) TNFRI levels in the plasma of healthy individuals as well as KLA patients at the time of enrollment into the clinical trial and 1 month after the A-KLASS treatment. (B) TNFRII levels in the plasma of healthy individuals as well as KLA patients at the time of enrollment into the clinical trial and 1 month after the A-KLASS treatment. ^**p < 0.01, ^****p < 0.0001.