Sonja T H M Kolanowski1, Suzanne N Lissenberg-Thunnissen1, Diba Emal1, S Marieke van Ham1,2, Anja Ten Brinke3. 1. Division Research and Landsteiner Laboratory, Sanquin Blood Supply, Department of Immunopathology, Academic Medical Center, University of Amsterdam, P.O. Box 9190, 1006 AD, Amsterdam, The Netherlands. 2. Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands. 3. Division Research and Landsteiner Laboratory, Sanquin Blood Supply, Department of Immunopathology, Academic Medical Center, University of Amsterdam, P.O. Box 9190, 1006 AD, Amsterdam, The Netherlands. a.tenbrinke@sanquin.nl.
Abstract
OBJECTIVE: To elucidate if TLR4-mediated MyD88 and TRIF signalling by the clinically applicable Lipopolysaccharide (LPS)-derivative monophosphoryl lipid A (MPLA) in primary human dendritic cells requires LPS cofactors LPS-binding protein (LBP) and CD14. METHODS: Cytokine production by monocyte-derived DCs stimulated with MPLA or LPS was determined using ELISA. To investigate involvement of CD14 for action of LPS or MPLA, CD14 was inhibited using blocking antibodies or down-modulated using specific siRNA. To assess involvement of LBP monocyte-derived DCs were stimulated in serum-free culture medium in absence or presence of purified LBP. RESULTS: LBP and CD14 are not required for and do not enhance the capacity of MPLA to induce MyD88- and TRIF-dependent pro-inflammatory IL-6 and TNF-α. Interestingly, although CD14 is required for TRIF-dependent downstream events in mice, we show that in human CD14 is redundant for MPLA-induced TRIF-dependent chemokine production. CONCLUSIONS: These findings provide novel insight in the modes of action of MPLA in human and show that, compared to LPS, MyD88 and TRIF signalling in dendritic cells by MPLA is not mediated nor amplified by TLR4 cofactors. This gives insight why MPLA induces immune activation without provoking toxicity in human and clarifies why MPLA can be used as activating compound for clinically applicable immuno-activatory cellular products grown in serum-free regimens.
OBJECTIVE: To elucidate if TLR4-mediated MyD88 and TRIF signalling by the clinically applicable Lipopolysaccharide (LPS)-derivative monophosphoryllipid A (MPLA) in primary human dendritic cells requires LPS cofactors LPS-binding protein (LBP) and CD14. METHODS: Cytokine production by monocyte-derived DCs stimulated with MPLA or LPS was determined using ELISA. To investigate involvement of CD14 for action of LPS or MPLA, CD14 was inhibited using blocking antibodies or down-modulated using specific siRNA. To assess involvement of LBP monocyte-derived DCs were stimulated in serum-free culture medium in absence or presence of purified LBP. RESULTS:LBP and CD14 are not required for and do not enhance the capacity of MPLA to induce MyD88- and TRIF-dependent pro-inflammatory IL-6 and TNF-α. Interestingly, although CD14 is required for TRIF-dependent downstream events in mice, we show that in humanCD14 is redundant for MPLA-induced TRIF-dependent chemokine production. CONCLUSIONS: These findings provide novel insight in the modes of action of MPLA in human and show that, compared to LPS, MyD88 and TRIF signalling in dendritic cells by MPLA is not mediated nor amplified by TLR4 cofactors. This gives insight why MPLA induces immune activation without provoking toxicity in human and clarifies why MPLA can be used as activating compound for clinically applicable immuno-activatory cellular products grown in serum-free regimens.
Authors: Antonio Hernandez; Naeem K Patil; Cody L Stothers; Liming Luan; Margaret A McBride; Allison M Owen; Katherine R Burelbach; David L Williams; Edward R Sherwood; Julia K Bohannon Journal: Pharmacol Res Date: 2019-11-02 Impact factor: 7.658
Authors: Sebastian Wirsching; Marina Machtakova; Frauke Borgans; Leah Pretsch; Michael Fichter; Maximiliano L Cacicedo; Héloïse Thérien-Aubin; Katharina Landfester; Stephan Gehring Journal: Front Pediatr Date: 2022-09-13 Impact factor: 3.569