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Monophosphoryl lipid A-induced pro-inflammatory cytokine expression does not require CD14 in primary human dendritic cells

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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.

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Fig. 1
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Abbreviations

LPS:

Lipopolysaccharide

MPLA:

Monophosphoryl lipid A

LBP:

LPS-binding protein

MPLAs:

MPLA derived from S. minnesota re595

LPSs:

LPS derived from S. typhimurium

MPLAe:

MPLA derived from E. coli r515

LPSe:

LPS derived from E. coli O111B4

RANTES:

Chemokine CCL5

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Acknowledgments

We thank Gijs van Schijndel and Miranda Dieker for technical assistance. We thank Prof. dr. van der Schoot, Sanquin Blood Supply for provision of mouse anti-human CD14.22 antibodies.

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Authors and Affiliations

  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

    Sonja T. H. M. Kolanowski, Suzanne N. Lissenberg-Thunnissen, Diba Emal, S. Marieke van Ham & Anja ten Brinke

  2. Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands

    S. Marieke van Ham

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  1. Sonja T. H. M. Kolanowski
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Corresponding author

Correspondence to Anja ten Brinke.

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This work was supported by a grant from the Joghem van Loghem foundation.

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The authors declare that there is no conflict of interest.

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Responsible Editor: John Di Battista.

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Fig. S1

MPLA does not require LBP to induce cytokine production. (A,B) IL-6 production by DCs stimulated with a titration of LBP in serum-free IMDM in presence of (A) 10 ng/ml LPSe, one representative experiment out of 6 is shown, or (B) 10 μg/ml MPLAe, a representative experiment (n = 7). (C,D) TNF-α (C) and IL-6 (D) production by DCs stimulated with indicated concentrations of MPLAs in presence or absence of LBP. Cytokine production is shown relative to stimulation with comparable amounts of MPLAs in absence of LBP, n = 8. For statistical analysis a paired t test was performed (EPS 92 kb)

Fig. S2

RANTES production is solely TRIF dependent in human moDCs. To verify that production of chemokine RANTES was TRIF dependent, MyD88 or TRIF was down-regulated using siRNA prior to stimulation of iDCs with MPLAe. To down-regulate MyD88 of TRIF iDCs were electroporated with control siRNA (siC) or specific siRNA targeting MyD88 (siM) or TRIF (siT). Two days after electroporation iDCs were stimulated with MPLAe. Production of TNF-α and IL-6 was lower in MyD88- and TRIF down-regulated DCs compared to control siRNA-treated DCs (A, B) while production of RANTES was only decreased in TRIF down-regulated DCs, but not MyD88 down-regulated DCs (C). Concentration of cytokines TNF-α (A), IL-6 (B) or chemokine RANTES (C) was determined in supernatant harvested 24 h after stimulation with MPLA, siMyD88, n = 17, siTRIF n = 8, a paired t test was performed for statistical analysis, values were compared to siC condition *: p < 0.05 (EPS 117 kb)

Fig. S3

CD14 is not required for MPLAe-mediated cytokine production. (A-C) Cytokine production by DCs stimulated with LPSe in the presence of control antibodies (CTRL, grey lines) or CD14-blocking antibodies (a-CD14, black lines). LPSe-stimulated production of TNF-α (A), IL-6 (B), a representative experiment (n = 7). (C) LPSe responsiveness by a-CD14 treatment is quantified as described in materials & methods, n = 4. (D,E) MPLAe-stimulated production of TNF-α (D) or IL-6 (E), one representative experiment out of 9 is shown. (F,G) MPLAe-stimulated production of TNF-α (F), IL-6 (G), as a  % of CTRL condition n = 6. (C, F, G) A one sample t test was performed for statistical analysis. *: p < 0.05, **: p < 0.01 (EPS 145 kb)

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Kolanowski, S.T.H.M., Lissenberg-Thunnissen, S.N., Emal, D. et al. Monophosphoryl lipid A-induced pro-inflammatory cytokine expression does not require CD14 in primary human dendritic cells. Inflamm. Res. 65, 449–458 (2016). https://doi.org/10.1007/s00011-016-0927-0

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