Abstract
The obligate intracellular bacterium, Chlamydia pneumoniae, has been identified as a risk factor for several chronic inflammatory diseases in addition to respiratory tract infections. The dissemination of C. pneumoniae from respiratory tract to secondary sites of infection occurs via infected monocyte / macrophage line cells, in which C. pneumoniae can persist as an antibiotic-refractory phenotype. To allow more detailed studies on the epithelium-monocyte/macrophage transition of the infection, new in vitro bioassays are needed. To this end, a coculture system with human continuous cell lines was established. Respiratory epithelial HL cells were infected with C. pneumoniae and THP-1 monocytes were added into the cultures at 67 h post infection. After a 5 h coculture, THP-1 cells were collected with a biotinylated HLA antibody and streptavidin-coated magnetic beads and C. pneumoniae genome copy numbers in THP-1 determined by quantitative PCR. The assay was optimized for cell densities, incubation time, THP-1 separation technique and buffer composition, and its robustness was demonstrated by a Z' value of 0.6. The mitogen-activated protein kinase (MAPK) inhibitors: SP600125 (JNK inhibitor), SB203580 (p38 inhibitor) and FR180204 (ERK inhibitor) suppressed the transfer of C. pneumoniae from HL to THP-1 cells, making them suitable positive controls for the assay. Based on analysis of separate steps of the process, the MAPK inhibitors suppress the bacterial entry to THP-1 cells. The transfer of C. pneumoniae from epithelium to phagocytes represents a crucial step in the establishment of persistent infections by this pathogen, and the presented methods enables future studies to block this process by therapeutic means.
Highlights
Chlamydia pneumoniae is a gram-negative, obligate intracellular bacterium, whose primary site of infection is the respiratory tract (Kuo et al, 2015)
The epithelial human lung (HL) cells were infected with C. pneumoniae and at 67 h post infection, representing a late stage of the replication cycle of C. pneumoniae, uninfected THP-1 monocytes were added to the wells in a ratio of 1:1
In control samples collected from cultures with uninfected HL cells, minor nonspecific amplification yielded a in apparent genome copy numbers (GE) numbers of 13.4, more than 2200 times smaller than in infection samples (S/B = 2209)
Summary
Chlamydia pneumoniae is a gram-negative, obligate intracellular bacterium, whose primary site of infection is the respiratory tract (Kuo et al, 2015). It causes various acute illnesses such as pharyngitis, sinusitis and it is a cause of 10% of community acquired pneumonia (CAP) (Kuo et al, 1995). In addition to respiratory tract infections, C. pneumoniae has been identified as a risk factor for several chronic inflammatory diseases such as asthma (Smith-Norowitz et al, 1971; Webley and Hahn, 2017), atherosclerosis (Campbell and Kuo, 2004; Filardo et al, 2015) and Alzheimer's disease and neurovascular complications (Little et al., 2004; Balin PhD et al, 2017; Richard, 2018). C. pneumoniae DNA has been detected from post mortem Alzheimer's brain samples (Balin et al, 1998) and the infection has been found to induce amyloid plague
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