Triggering Receptor Expressed on Myeloid Cells-1 (TREM-1) Contributes to Bordetella pertussis Inflammatory Pathology

Abstract

Whooping cough (pertussis) is a severe pulmonary infectious disease caused by the bacteria Bordetella pertussis. Pertussis infects an estimated 24 million people annually, resulting in >150,000 deaths. The NIH placed pertussis on the list of emerging pathogens in 2015. Antibiotics are ineffective unless administered before the onset of the disease characteristic cough. Therefore, there is an urgent need for novel pertussis therapeutics. We have shown that sphingosine-1-phosphate receptor (S1PR) agonists reduce pertussis inflammation without increasing bacterial burden. Transcriptomic studies were performed to identify this mechanism and allow for the development of pertussis therapeutics that specifically target problematic inflammation without sacrificing bacterial control. These data suggested a role for triggering receptor expressed on myeloid cells-1 (TREM-1). TREM-1 cell surface receptor functions as an amplifier of inflammatory responses. Expression of TREM-1 is increased in response to bacterial infection of mucosal surfaces. In mice, B. pertussis infection results in Toll-like receptor 9 (TLR9)-dependent increased expression of TREM-1 and its associated cytokines. Interestingly, S1PR agonists dampen pulmonary inflammation and TREM-1 expression. Mice challenged intranasally with B. pertussis and treated with ligand-dependent (LP17) and ligand-independent (GF9) TREM-1 inhibitors showed no differences in bacterial burden and significantly reduced tumor necrosis factor-α (TNF-α) and C-C motif chemokine ligand 2 (CCL-2) expression compared to controls. Mice receiving TREM-1 inhibitors showed reduced pulmonary inflammation compared to controls, indicating that TREM-1 promotes inflammatory pathology, but not bacterial control, during pertussis infection. This implicates TREM-1 as a potential therapeutic target for the treatment of pertussis.

Keywords: S1P; bordetella; host-directed therapeutics; host-pathogen interactions; pertussis; pulmonary infection; trem-1.

FIG 1

Transcriptomic analysis of TREM-1-associated genes following B. pertussis infection. (A) Differential expression of TREM-1-associated genes identified by whole-lung transcriptomics. Gene expression was compared using RNA that had been isolated from whole-lung tissue of C57BL/6 mice 4 days postinfection with B. pertussis or PBS sham infection (black bars). White bars represent gene fold induction in infected animals treated with vehicle or S1PR agonist. (B) Canonical pathway analysis performed with the Ingenuity Pathway Analysis software identified TREM-1 signaling as being significantly associated with genes whose expression is increased by B. pertussis infection compared to PBS (black bars) and in infected, water control-treated versus S1PR agonist (AAL-R, 0.5 mg/kg)-treated mice (white bars). The expression of TREM-1-associated genes at 4 days postinfection was confirmed by qRT-PCR (C). Fold induction is calculated by normalization to PBS-challenged mice. Each data point represents the mean of triplicate technical replicates from one of four individual mice (n = 4).

FIG 2

B. pertussis-induced expression of TREM-1 is TLR9 dependent. qRT-PCR analysis of lung tissue harvested from C57BL/6, TLR9 KO, or TLR4 KO mice at 7 days postinfection with B. pertussis. Data are normalized to PBS-challenged animals of the same genetic background. Each datapoint represents the mean value obtained from triplicate samples from one of four individual mice (n = 4); ***, P < 0.005; NS, P > 0.05.

FIG 3

TREM-1 does not contribute to bacterial control in B. pertussis. We used ligand-dependent (LP17) and ligand-independent (GF9) mechanisms to inhibit TREM-1 activity. LP17 binds potential TREM-1 ligands, preventing receptor binding. GF9 prevents transmembrane interactions between TREM-1 and its binding partner DAP12 (A). Bacterial burden was assessed at 4 and 7 dpi by plating the lungs of B. pertussis-challenged C57BL/6 mice receiving TREM-1 inhibitors (gray bars) LP17 (5 mg/kg) (B) or GF9 (20 mg/kg) (C) or vehicle control (black bars) on Bordet-Gengou agar plates. Vehicle controls consisted of 5 mg/kg scrambled peptide (LP17 studies) or PBS (GF9 studies). Each data point represents the average of three technical replicates obtained from one of four individual mice (n = 4). Data are shown as mean with standard deviation. P values were determined by two-way ANOVA using Sidak’s multiple-comparison test; NS, P > 0.05.

FIG 4

TREM-1 promotes inflammatory responses to B. pertussis. Lungs were isolated from B. pertussis-challenged mice treated with TREM-1 inhibitors LP17 (5 mg/kg) or GF9 (20 mg/kg) at 4 dpi to assess expression of CCL2 (A), CXCL3 (B), and TNF-α (C) by qRT-PCR. qRT-PCR data are normalized to uninfected animals. Black bars represent B. pertussis-challenged, vehicle-treated animals. Gray bars represent B. pertussis-challenged animals treated with TREM-1 inhibitors LP17 (5 mg/kg) or GF9 (20 mg/kg). TNF-α protein levels were increased in response to B. pertussis infection (black bars) and decreased in response to treatment with GF9 (gray bars) (D). Data represent mean and standard deviation values obtained from triplicate technical replicates and four biological replicates (n = 4). P values were determined by two-way ANOVA using Sidak’s multiple-comparison test; *, P < 0.05; **, P < 0.01; ***, P < 0.005.

FIG 5

TREM-1 pathogenesis in B. pertussis infection. Lungs were isolated from B. pertussis-challenged mice treated with TREM-1 inhibitors LP17 (5 mg/kg, gray dots) or GF9 (20 mg/kg, gray dots) or vehicle (black dots; 5 mg/kg scrambled peptide for LP17 studies and PBS for GF9 studies) at 7 dpi. Lung tissues were stained with hematoxylin and eosin for assessment of histopathology. Three blinded investigators scored slides on a scale of 1 to 9 using our established semiquantitative system (A). Representative images are shown for infected C57BL/6 mice treated with vehicle (B), LP17 (C), or GF9 (D). Each data point represents one biological replicate (n = 4); *, P < 0.05.