TREM-1 promotes survival during Klebsiella pneumoniae liver abscess in mice

Abstract

Klebsiella pneumoniae liver abscess (KPLA) is prevalent in East Asia. Liver abscess can develop after translocation of K. pneumoniae from a patient's bowel into the liver via the portal circulation. TREM-1 (triggering receptor expressed on myeloid cells 1) amplifies inflammatory signaling during infection, but its role in KPLA is poorly understood. We used an animal study to characterize the role of TREM-1 in KPLA. We compared survival rates, bacterial burdens in tissues, inflammatory cytokine levels, and histology findings between wild-type and Trem-1 knockout (KO) mice after oral inoculation of capsular type K1 K. pneumoniae. Translocation of K. pneumoniae to mesenteric lymph nodes and liver was examined, and intestinal permeability, antimicrobial peptide expression, and the clearance of K. pneumoniae in the small intestine were determined. In the absence of TREM-1, KPLA model mice showed increased K. pneumoniae dissemination, enhanced liver and systemic inflammation, and reduced survival. Impaired bacterial clearance in the small intestine causes enhanced K. pneumoniae translocation, which renders Trem-1 KO mice more susceptible to K. pneumoniae oral infection. In conclusion, TREM-1-mediated bacterial clearance in the small intestine is an important immune response against K. pneumoniae. TREM-1 deficiency enhances K. pneumoniae translocation in the small intestine and increases mortality rates in mice with KPLA.

FIG 1

TREM-1 gene expression in WT mice with KPLA. Upregulation of Trem-1 mRNA levels in liver at 24 and 48 h (n = 6, independent experiment) (A) and in distal small intestine at 48 h (n = 9, independent experiment) (B) after K. pneumoniae oral infection compared with that in the sham control by qRT-PCR. Data are expressed as means ± standard deviations (SD) for each group. Statistical significance was defined as P < 0.05 versus the sham control.

FIG 2

TREM-1 is important for host survival after oral administration of K. pneumoniae. (A) Kaplan-Meier survival plot of Trem-1 KO and WT mice (n = 21 per group, 4 independent experiments) following oral administration with K. pneumoniae (P = 0.026). (B) Trem-1 KO mice have increased histologic evidence of liver abscess and necrosis, as shown by pathological scores following K. pneumoniae challenge (n = 15 per group, 4 independent experiments; P = 0.013). Liver abscess and necrosis were examined by hematoxylin and eosin staining and observed under a microscope (magnification, ×200) in Trem-1 KO (C) and WT (D) mice 48 h after oral K. pneumoniae administration.

FIG 3

Enhanced translocation of K. pneumoniae in Trem-1 KO mice after oral administration of K. pneumoniae. (A to C) CFU of K. pneumoniae in MLN (P = 0.03) (A), liver (P = 0.001) (B), and blood (P = 0.036) (C) at 48 h after infection in WT and Trem-1 KO mice (n = 20, 4 independent experiments).

FIG 4

Trem-1 KO mice have increased local and systemic proinflammatory cytokine production. (A to C) Real-time PCR analysis of IL-6 (A), IL-1β (B), and TNF-α (C) expression in liver samples taken 48 h after infection (n = 7 each, 2 independent experiments). (D to F) Serum cytokine analysis of IL-6 (D), IL-1β (E), and TNF-α (F) expression in liver samples taken 48 h after infection (n = 7 each, 2 independent experiments).

FIG 5

TREM-1 is essential to prevent K. pneumoniae translocation from the intestine. Translocation of GFP-expressing K. pneumoniae from intestinal loops to MLNs (A), liver (B), and blood (C) is significantly increased in Trem-1 mice compared with WT KO mice (n = 9 each, 2 independent experiments).

FIG 6

Diminished clearance of K. pneumoniae in the small intestine of Trem-1 KO mice. K. pneumoniae cells (1,000 CFU/mouse) were injected into intestinal loops of WT and Trem-1 KO mice. Sections of the intestine were harvested 2 h after injection, and CFU were determined (n = 5 each, 3 independent experiments).

FIG 7

TREM-1 depletion does not significantly affect small intestine expression of antimicrobial peptides and tight junction components in K. pneumoniae-infected mice. (A) Real-time PCR analysis of various antimicrobial peptide expression in small intestine 4 h after injection of K. pneumoniae in the small intestine loop (n = 3 each, two independent experiments). (B) Real-time PCR analyses of mRNA expression of occludin, claudin-1, claudin-4, and ZO-1 in small intestine 4 h after injection of K. pneumoniae in the small intestine loop (n = 3 each, 2 independent experiments).

FIG 8

Neutrophil extracellular trap (NET) production in WT and Trem-1 KO neutrophils and TREM1-mediated NET DNA bactericidal effect. (A) Quantification of NET formation of PMA-stimulated, anti-TREM-1 antibody-pretreated, and control (Ctrl) neutrophils in WT and Trem-1 KO neutrophils (n = 3 each, 3 independent experiments). (B) Percentage of K. pneumoniae survival after incubation with PMA-stimulated, anti-TREM-1 antibody-pretreated, and control neutrophils in WT neutrophils (n = 3 each, 3 independent experiments). The percentages of K. pneumoniae survival were 72.4% in the PMA-stimulated group and 85.7% in the anti-TREM-1 antibody-pretreated group (*, P = 0.036).