Proinflammatory cytokine production by adherent donor blood cells stimulated by soluble LPS and phagocyted bacteria

Specific stimulation of receptors of the innate immune system by their purified ligands is commonly used in basic studies of inflammation and in the development of anti-inflammatory drugs. Based on location, receptors of the innate immunity can be classified into two groups: i) cell plasma membrane and on membranes of endosomes (Toll-like receptors (TLRs) and C-type lectin receptors), and recognizing the presence of pathogens in the extracellular space; ii) cytoplasmic sensors playing a special role in the recognition of intracellular pathogens (NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), bacterial DNA sensor cGAS, and Aim2 (absent in melanoma 2). Many experimental models of inflammation use bacterial lipopolysaccharides (LPSs) or other purified microbial molecules to simulate the innate immune response to microbes. In the present study, the response of human blood leukocytes to stimulation with soluble, highly purified LPS from gram-negative bacteria was compared with that induced by formalin-fixed, corpuscular E. coli. The data obtained demonstrate that LPS and bacteria induce similar levels of TNF and IL-6 by plastic-adherent leukocytes, whereas neither LPS nor whole bacteria induce a measurable IFNγ production. The LPS- and bacteria-induced cytokine production, however, drastically differed in the sensitivity to a broad-spectrum TLR inhibitor, peptide 5R667. The LPS-stimulated human leukocyte cytokine production, as expected, was highly sensitive to inhibition by the peptide, whereas production stimulated by corpuscular bacteria was not. The TLR-blocking peptide did not affect the ability of blood leukocytes to phagocytose E. coli as shown by flow cytometry data obtained using FITC-stained fixed bacteria. Because peptide 5R667 blocks several TLRs, including TLR4, TLR5, and TLR9, the differential sensitivity of LPS- and bacteria-induced cytokine production to 5R667 suggests that the intracellular pathogen sensors, most likely NOD1 and/or NOD2, essentially contribute to the bacteria-induced cytokine induction. These results show that LPS and phagocyted bacteria induce cytokine production via different mechanisms and also suggest that the models with corpuscular bacteria for simulating bacterially induced inflammation complement the models that using soluble TLR ligands; therefore, both models should be applied to properly reflect anti-bacterial immune response.