Whole blood assay MDP
Pattern recognition receptors (PRRs) are important players in early microbe recognition at mucosal surfaces. Nucleotide-binding oligomerization domain (NOD) 2 is a member of an intracellular family of PPRs, i.e. NOD-like receptors, involved in bacterial handling and autophagy, among other processes. Bacterial sensing in the gut epithelium is critical for the maintenance of homeostatic immune responses as genetic variants in the NOD2 locus, also known as IBD1, were found to be associated with local inflammation, including CD. Although environmental triggers and defects in NOD2 signaling is important in disease onset, the potential role played by this receptor in disease pathogenesis and chronic inflammation remains elusive.
We devised an assay to study the effect of chemical probes on NOD2-mediated inflammation in comparison with prednisolone, a steroidal anti-inflammatory drug routinely used to treat acute symptoms of inflammation in IBD. A pro-inflammatory cytokine response was elicited by stimulating whole blood from IBD patients (CD and UC) and healthy controls with a natural NOD2-agonist, the bacterial peptidoglycan component muramyl dipeptide (MDP). The anti-inflammatory effect of tested chemical probes was evaluated by measuring the levels of interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor (TNF) released in blood plasma over a 5-hour incubation.
Our results confirm the role of known players in NOD2-mediated pro-inflammatory cytokine responses, such as RIPK2 and p38, and point at MEK and SYK as additional targets of potential relevance for IBD treatment. Experiments to strengthen and validate our findings using different patient-derived cell assay, including primary colon tissue explants, are currently being performed.
Inhibitors of the NOD2 adaptor protein receptor-interacting serine/threonine-protein Kinase (RIPK) 2, and of the mitogen-activated protein kinases (MAPK) p38 and MEK1/2 were among the most effective probes in reducing MDP-induced pro-inflammatory cytokine release in whole blood from both IBD patients and healthy controls. There was no single most effective kinase inhibitor in the reduction of all four measured cytokines as compared with prednisolone. In IBD patients, the RIPK2-inhibitor HY-19764 significantly reduced all cytokines but it was less potent in inhibiting IL-8 compared to the other cytokines. Conversely, treatment with selected inhibitors of p38, i.e. VX-702 and Pamapimod, and MEK1/2, i.e. Trametinib and PD0325901, effectively reduced IL-8, whereas the magnitude of IL-1β, IL-6 and TNF reduction was similar or lower than that achieved with HY-19764 treatment. Additional probes, such as the spleen tyrosine kinase (SYK)-inhibitor, showed mixed inhibitory effect across IBD patients likely owing to the heterogeneity of our cohort in their clinical and treatment profiles. However, the latter finding may offer a functional insight into inter-patient variability and complement ongoing deep molecular characterization results on matched blood and colon biopsies for patient stratification.
![](/sites/www.ultra-dd.org/files/cell-assays/Whole_Blood_MPD_results01.png)
![Assay validation](/sites/www.ultra-dd.org/files/cell-assays/Whole_Blood_MPD_results02.png)
Fig 1 – Assay validation: modulation of MDP-induced pro-inflammatory cytokine response in whole blood.
Incubation with muramyl dipeptide (MDP) at 0,4 mg/mL induced pro-inflammatoy cytokine secretion after 4h in whole blood cultures from patients with ulcerative colitis (UC), Crohn’s disease (CD) and healthy controls (HC), compared with vehicle control (DMSO). Treatment with the steroideal anti-inflammatory drug prednisolone at 1 μM reduced MDP-induced production of all 4 measured cytokines in all groups. The bars show concentration values (mean + SD) from the number of blood donors indicated below the x axis.
![Effect of chemical probes](/sites/www.ultra-dd.org/files/cell-assays/Whole_Blood_MPD_results03.png)
Fig 2 – Effect of chemical probes on MDP-induced pro-inflammatory cytokine response in whole blood of IBD patients.
Whole blood cultures from patients with ulcerative colitis (n=6) and Crohn’s disease (n=9) were pre-treated with the indicated concentration of each chemical probe, or prednisolone as positive control, for 1 h. Muramyl dipeptide (MDP) at 0,4 mg/mL was added and blood incubated for 4 h.
The heat-map shows mean values (n=13-15) of the effect of each probe on the secretion of IL-1β, IL-6, IL-8 and TNF, measured as cytokine concentration in blood plasma normalized to that of vehicle control (DMSO) for each blood donor. Highlighted in red are probes tested at different concentrations: 0,2 and 1 µM for n=11-12 and 3 donors respectively.
![Effect of chemical probes](/sites/www.ultra-dd.org/files/cell-assays/Whole_Blood_MPD_results04.png)
Fig 3 – Effect of chemical probes on MDP-induced pro-inflammatory cytokine response in whole blood of healthy controls.
Whole blood cultures from healthy controls (n=6) were pre-treated with indicated concentration of each chemical probe, or prednisolone as positive control, for 1 h. Muramyl dipeptide (MDP) at 0,4 mg/mL was added and blood incubated for 4 h.
The heat-map shows mean values (n=5-6) of the effect of each probe on the secretion of IL-1β, IL-6, IL-8 and TNF, measured as cytokine concentration in blood plasma normalized to that of vehicle control (DMSO) for each blood donor.
![Effect of chemical probes](/sites/www.ultra-dd.org/files/cell-assays/Whole_Blood_MPD_results05.png)
Fig 4 – Effect of chemical probes on MDP-induced pro-inflammatory cytokine response in whole blood of IBD patients.
Whole blood cultures from patients with ulcerative colitis (n=4) and Crohn’s disease (n=4) were pre-treated with the indicated concentration of each chemical probe, or prednisolone as positive control, for 1 h. Muramyl dipeptide (MDP) at 0,4 mg/mL was added and blood incubated for 4 h.
The heat-map shows mean values (n=4-8) of the effect of each probe on the secretion of IL-1β, IL-6, IL-8 and TNF, measured as cytokine concentration in blood plasma normalized to that of vehicle control (DMSO) for each blood donor. Highlighted in red are probes tested at different concentrations: 0,1 (1 for PAK1) and 0,2 µM for n=3 and 5 donors respectively.