Inhibition of proline tyrosine kinase 2 (Pyk2) phosphorylation during adherent-invasiveEscherichia coliinfection inhibits intra-macrophage replication and inflammatory cytokine release

Abstract

Adherent-invasive Escherichia coli (AIEC) have been implicated in the aetiology of Crohns Disease (CD). They are characterized by an ability to adhere to and invade intestinal epithelial cells, and to replicate intracellularly in macrophages resulting in inflammation. Proline-rich tyrosine kinase 2 (PYK2) has previously been identified as a risk locus for inflammatory bowel disease through genome wide association studies. It is overexpressed in patients with colorectal cancer, a major long-term complication of CD. Here we show that Pyk2 levels are significantly increased during AIEC infection of murine macrophages while an inhibitor of Pyk2, PF-431396 hydrate, significantly decreased intramacrophage AIEC numbers. Imaging flow cytometry indicated that Pyk2 inhibition blocked intramacrophage replication of AIEC with no change in the overall number of infected cells, but a significant reduction in bacterial burden per cell. This reduction in intracellular bacteria resulted in a 20-fold decrease in tumour necrosis factor alpha secretion by cells post-AIEC infection. These data demonstrate a key role for Pyk2 in modulating AIEC intracellular replication and associated inflammation and may provide a new avenue for future therapeutic intervention in CD.

Stratifying macrophages based on their infectious burden identifies novel host targets for intervention during Crohn’s disease associated adherent-invasiveEscherichia coliinfection

“…Six hours post-cell seeding, RAW 264.7 cells were treated with 100 ng/ml of lipopolysaccharide (LPS) and incubated overnight. RAW 264.7 cells in RPMI-1640 with 3% FBS without antibiotics, were then infected with LF82 carrying the p rpsM GFP plasmid (LF82 rpsM GFP) at a multiplicity of infection (MOI) of 100 for 1 hour (Li et al ., 2022). Post-infection, extracellular bacteria were removed by washing with fresh RPMI media (3% FBS) containing 50 μg/ml of gentamicin and the media was replaced with fresh RPMI media (3% FBS, 50 μg/ml gentamicin).…”

Stratifying macrophages based on their infectious burden identifies novel host targets for intervention during Crohn’s disease associated adherent-invasive Escherichia coli infection

“…Six hours post-cell seeding, RAW 264.7 cells were treated with 100 ng ml −1 of lipopolysaccharide (LPS) and incubated overnight. RAW 264.7 cells in RPMI-1640 with 3 % FBS without antibiotics, were then infected with LF82 carrying the p rpsM GFP plasmid (LF82 rpsM GFP) at a multiplicity of infection (MOI) of 100 for 1 h [ 16 ]. Post-infection, extracellular bacteria were removed by washing with fresh RPMI media (3 % FBS) containing 50 µg ml −1 of gentamicin.…”

“…Laser wavelength from relevant channels were Ch02 (488 nm, GFP fluorescence), Ch04 (bright field), and Ch05 (642 nm, nuclei). Quantification of intracellular bacteria was as previously described [ 16 ].…”