Hypervirulent Klebsiella pneumoniae Strains Modulate Human Dendritic Cell Functions and Affect TH1/TH17 Response

Abstract

Hypervirulent Klebsiella pneumoniae (Hv-Kp) strains have emerged as pathogens causing life-threatening, invasive disease even in immunocompetent hosts. Systemic dissemination usually occurs following perturbations of the gut microbiota and is facilitated by Hv-Kp resistance to phagocytosis and complement activity. Hv-Kp are usually associated with K1 or K2 capsular types, produce several iron uptake systems (e.g., aerobactin and salmochelin) and are often but not invariably, capsular material hyper-producers (hypermucoviscous phenotype: HMV). Whether Hv-Kp escape the immune response at mucosal site is unknown. In this work, we studied the effects of Hv-Kp on human dendritic cells (DCs), central players of the IL-23/IL-17 and IL-12/IFN-γ axis at mucosal sites, essential for pathogen clearance. Four Hv-Kp and HMV strains were selected and their activity on DC maturation and cytokine production was compared to that of non-virulent Kp strains with classic or HMV phenotypes. While the maturation process was equally induced by all Kp strains, significant differences between virulent and non-virulent strains were found in the expression of genes for cytokines involved in T-cell activation and differentiation. The non-virulent KP04C62 and the classic Kp, KPC157 induced high expression of T_H1 (IL-12p70 and TNFα) and T_H17 cytokines (IL-23, IL-1β and IL-6), while Hv-Kp poorly activated these cytokine genes. Moreover, conditioned media from DCs cultured with non-virulent Kp, either classical or hypercapsulated, induced the activation of IL-17 and IFN-γ genes in preactivated CD4⁺-cells suggesting their T_H17/T_H1 differentiation. Conditioned media from Hv-Kp poorly activated IL-17 and IFN-γ genes. In summary, our data indicate that Hv-Kp interfere with DC functions and T-cell differentiation and suggest that the escape from the IL-23/IL-17 and IL-12/IFN-γ axes may contribute to pathogen dissemination in immunocompetent hosts.

Keywords: TH differentiation; dendritic cells (DCs); hypermucoviscous K. pneumoniae; hypervirulent; immune response; inflammatory cytokines.

Figure 1

Maturation of DCs induced by K. pneumoniae strains. DCs were cultured with medium alone (unstimulated, US) or with live bacterial cells. Data were collected with a cytofluorimeter and are expressed as median fluorescence intensity (± IQR ×1.5) of three different experiments. The maturation level of each sample was compared (with the exception of US) and no significant statistical differences were observed.

Figure 2

Cytokine expression by DCs cultured in presence of live K. pneumoniae strains; the global p-value obtained by ANOVA is reported.

Figure 3

IFN-γ and IL-17 gene expression by pre-activated CD4⁺ T-cells. The bar-graph shows data (mean ± SE) of three different experiments. Data are expressed as fold-change with respect to unstimulated (US) cultures; the global p-value obtained by ANOVA is reported.

Figure 4

NF-kB (p65) activation and p38-MAPK phosphorylation by DCs. Dendritic cells were cultured in the absence (US) or presence of K. pneumoniae strains or LPS as a positive control. Cells were analyzed by Western blot analysis. Data from one representative experiment out of three performed are shown. Data are expressed as the fold increase of each experimental point over unstimulated control. A t-test was performed comparing KP04C62 (*) and KPC157 (#) (non-Hv-HMV strains) with the Hv-HMV Kp strains. We considered statistical significance as a p-value < 0.05 (*, #); p-value < 0.01, (**, ##).