TLR2 and TLR4 co-activation utilizes distinct signaling pathways for the production of Th1/Th2/Th17 cytokines in neonatal immune cells.

Co-activation of TLR2 and TLR4 by gram negative and gram positive bacterial ligands induces a robust pro-inflammatory response in inflammatory cells. In order to understand the signaling mechanism, we aimed to delineate the signaling molecules involved in TLR2 and TLR4 co-activation in neonatal immune cells for the production of Th1/Th2/Th17 inflammatory cytokines. For this, we pretreated cord blood and peripheral blood mononuclear and human mast cells with specific signaling molecule inhibitors such as BAY117082, PD98059 and LY294002 and then stimulated with LPS and PGN and assayed for cytokines IL-6, IL-12/IL-23p40 (Th1), IL-13 (Th2), IL-23 (Th17) and RANTES secretion. We found that upon co-stimulation the phosphorylation of NFκBp65, ERK1/2 and Akt was found to be higher than when stimulated with individual ligands in CBMCs. Also, when compared to adult cells, neonatal cells were more potent in the activation of ERK and Akt through TLR2 and TLR4 co-activation. In addition, neonatal cells possess similar capacity to activate NFκB as that of adult cells for IL-6 secretion. Furthermore, all three signaling molecules were found to be involved in the production of Th17 cytokines which is detrimental during inflammation induced by infection in neonates whereas NFκB is mainly involved in the induction of pro-inflammatory response and Th2 cytokines production. In conclusion, different signaling molecules were utilized for the production of different cytokines in immune cells.

TLR-mediated inflammatory response to neonatal pathogens and co-infection in neonatal immune cells.

Neonates heavily depend on the innate immune system for defence against invading pathogens. Toll-like receptors (TLRs) represent a primary line of host defence and play an important role in orchestrating the inflammatory response to invading pathogens. The most commonly infecting pathogens in neonates are E. coli, Klebsiella pneumoniae and Staphylococcus aureus. Also, co-infection with more than one organism is common in neonatal sepsis. Therefore, we aimed to study the TLR2 and TLR4 mediated neonatal inflammatory response to these pathogens. For this, we stimulated mononuclear cells from cord blood with LPS, PGN, E. coli, K.pneumoniae andS.aureus and analyzed the surface expression of TLR2 and TLR4 on CD14(+)CD16(+) and CD14(dim)CD16(+) and its inflammatory response in comparison with peripheral blood. We found that the TLR2 and TLR4 were differentially expressed on both monocyte subpopulations. Cytokines such as IL-6, IL-1ß, IL-23, IL-10, IL-13, MCP-1 and IL-8 were measured using ELISA and we observed that although, neonatal cells were able to produce similar levels of the classical pro-inflammatory (IL-6, IL-1ß) and anti-inflammatory (IL-10, IL-13) cytokines as that of adult cells, the amounts of IL-23 and MCP-1 were lower in CBMCs while the chemokine IL-8 was higher in CBMCs when compared with PBMCs. In addition, using Human Inflammation Antibody array technique we found that multiple cytokine production was impaired in cord blood when cells were co-infected with LPS and PGN. In conclusion, the TLR-mediated inflammatory response to neonatal pathogens is differentially regulated by different pathogens.

Inflammatory mediators of systemic inflammation in neonatal sepsis.

OBJECTIVE AND DESIGN: Sepsis refers to severe systemic inflammation in response to invading pathogens. To understand the molecular events that initiate the systemic inflammatory response, various inflammatory mediators were analyzed in neonatal sepsis samples and compared with normal samples. MATERIALS AND METHODS: We initially measured the levels of the various classical inflammatory mediators such as acute phase proteins [C-reactive protein (CRP) and procalcitonin (PCT)], granule-associated mediators (NE, MPO and NO), proinflammatory cytokines [tumour necrosis factor-α (TNFα), IL-1ß and IL-6), antiinflammatory cytokines (IL-10 and IL-13) and chemokines [IL-8 and monocyte chemotactic protein (MCP-1)] and novel cytokines (IL-12/IL-23p40, IL-21 and IL-23) using ELISA. We also used the human inflammation antibody array membrane to profile the inflammatory proteins that are involved in neonatal sepsis. RESULTS: There were significantly higher levels of CRP (5.4 ± 0.70 mg/L), PCT (1.500 ± 0.2400 µg/L); NE (499.2 ± 22.01 µg/L), NO (54.22 ± 3.131 µM/L); TNFα (396.6 ± 37.40 pg/mL), IL-1ß (445.3 ± 34.25 pg/mL), IL-6 (320.9 ± 43.38 pg/mL); IL-8 (429.5 ± 64.08 pg/mL) MCP-1 (626.25 ± 88.91 pg/mL), IL-10 (81.80 ± 9.45 pg/mL), IL-12/IL-23p40 (30.25 ± 0.6 pg/mL), IL-21 (8,263.3 ± 526.8 pg/mL) and IL-23 (6,083 ± 781.3 pg/mL) in neonates with sepsis compared to normal. The levels of MPO (21.20 ± 3.099 ng/mL) were downregulated, whereas there was no change in IL-13 (188.7 ± 10.63 pg/mL) levels in septic neonates when compared with normal. Using the human inflammation antibody array membrane, we detected the presence of 17 inflammatory proteins such as IL-3, IL6R, IL12p40, IL-16, TNFα, TNFß, TNF R1, chemokines I-309, IP-10 (IFN-γ inducible protein 10), MCP-1, MCP-2, MIP 1ß (macrophage inflammatory protein), MIP-1δ, eotaxin-2, growth factors TGFß1 (transforming growth factor beta), PDGF (platelet derived growth factor), and cell adhesion molecule ICAM-1 (intracellular adhesion molecule) that were upregulated whereas RANTES which was downregulated in neonatal sepsis. CONCLUSION: The simultaneous secretion and release of multiple mediators such as proinflammatory cytokines and chemokines, cell adhesion molecules, and growth factors were found to be involved in the initiation of systemic inflammation in neonatal sepsis. Therefore, measuring the concentration of multiple mediators may help in the early detection of neonatal sepsis and help to avoid unnecessary antibiotic treatment.