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Published on: May 22, 2014
Monophosphoryl lipid A-induced pro-inflammatory cytokine expression does not require CD14 in primary human dendritic
Sonja T H M Kolanowski1, Suzanne N Lissenberg-Thunnissen1, Diba Emal1
1Division 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.
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.
Insights
Monophosphoryl lipid A (MPLA) activates immune cells via Toll-like receptor 4 (TLR4) without needing LPS-binding protein (LBP) or CD14 cofactors. This TLR4 signaling pathway clarifies MPLA
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Toll-like receptor 4 (TLR4) signaling is crucial for innate immunity, typically activated by lipopolysaccharide (LPS).
- Monophosphoryl lipid A (MPLA), a clinically relevant LPS derivative, also engages TLR4.
- LPS cofactors, LPS-binding protein (LBP) and CD14, are known to facilitate LPS-TLR4 interactions.
Purpose of the Study:
- To determine if MPLA-induced TLR4 signaling in human dendritic cells requires LPS cofactors LBP and CD14.
- To investigate the roles of MyD88 and TRIF pathways in MPLA-mediated signaling.
- To understand the implications for MPLA's clinical application and safety profile.
Main Methods:
- Primary human dendritic cells (DCs) were stimulated with MPLA or LPS.
- Cytokine production (IL-6, TNF-α) was measured using ELISA.
- CD14 involvement was assessed via antibody inhibition and siRNA knockdown; LBP involvement was studied in serum-free conditions.
Main Results:
- MPLA induced pro-inflammatory cytokines IL-6 and TNF-α via MyD88 and TRIF pathways independently of LBP and CD14.
- CD14 was not required for MPLA-induced TRIF-dependent chemokine production in human DCs, unlike in mice.
- LBP and CD14 did not enhance MPLA's capacity to induce these signaling pathways.
Conclusions:
- MPLA-mediated MyD88 and TRIF signaling in human dendritic cells does not require TLR4 cofactors LBP and CD14.
- This cofactor-independent mechanism explains MPLA's immune activation without toxicity in humans.
- Findings support MPLA's use in serum-free conditions for clinically applicable immuno-activatory cellular products.
