Monocyte-derived macrophages and dermal fibroblasts-derived ECM making small talk

last updated: 2023-10-03
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TitleMonocyte-derived macrophages and dermal fibroblasts-derived ECM making small talk
Publication TypeConference Abstract -ISI Web of Science Indexed
Year of Publication2023
AuthorsChaves S., Macedo J., Reis D. P., da Silva L. P., and Marques A. P.

INTRODUCTION: 3D in vitro skin models allow studying molecular pathways that otherwise are only
possible to explore using animal models. The incorporation of immune cells, such as macrophages,
has been implemented to create immunocompetent analogues. Yet, the human biology
representation in those models is limited, among other aspects, by the minimal extracellular matrix
(ECM) mimicry[1]. In order to tackle this limitation, we have established a protocol to extract
fibroblasts-own ECM. In this work, we hypothesised that those ECM extracts were not able to
promote macrophage activation, which could compromise the physiologic phenotype of the model.
METHODS: Structural (strECM) and soluble (sECM) ECM components were extracted from fibroblasts
cultures using an in-house established approach. Blood monocytes were differentiated into
macrophages (MDMs) with M-CSF (50ng/mL) and exposed to a range of ECM concentrations (5, 25
and 50 μg/mL - sECM or 250, 500 and 1000 μg/mL - strECM). The response to the ECM was also
assessed in the presence of TNFα (50ng/mL and 100ng/mL), to understand its influence on a proinflammatory
signal. Pro-inflammatory control cells were attained with LPS/IFNγ (10ng/mL,20ng/mL).
MDMs response was monitored after 24h of exposure by RT-PCR (NF-kB, STAT1, PPARG and JMJD3),
flow cytometry (CD14, CD86, CD197, CD319, CD206,) and immunofluorescence (CD86, CD206)
analyses, and multiplexing of cells’ supernatants (IL-6, CXCL10, TNF-α, CCL18, CCL22).
RESULTS: Exposure to the ECM extracts led to downregulation of transcription factors associated
with pro-inflammatory pathways (NF-kB, STAT1) and unchanged mRNA expression of antiinflammatory
(PPARG and JMJD3) genes. Moreover, lower/unchanged levels of pro-inflammatory
surface markers (CD86, CD197, CD319) were detected. All cytokine’s levels were similar to the
unstimulated MDMs condition, with the exception of CXCL10 that was increased in the presence of
the lowest sECM concentration. When MDMs were simultaneously exposed to ECM and TNFα, only
the expression of the anti-inflammatory molecule CD206 was observed.
DISCUSSION & CONCLUSIONS: Fibroblast-derived ECM support MDMs basal or anti-inflammatory
phenotype, even if obtained from different donors. Moreover, the presence of ECM seems to
attenuate the response to a pro-inflammatory stimulus. Overall, our study shows that cells-own ECM
do not activate macrophages, warranting the representation of the physiologic skin phenotype
although might have a role in an inflammatory microenvironment.
ACKNOWLEDGEMENTS: ERC-2016-COG-726061 and PD/BD/14301/2018
REFERENCES: Chau, D.Y.S., et al., The development of a 3D immunocompetent model of human skin.
Biofabrication, 2013. 5(3): p. 035011.

Conference NameMeeting of the European Chapter of the Tissue Engineering and Regenerative Medicine International Society (TERMIS-EU)
Date Published2023-07-17
PublisherMary Ann Liebert, Inc., publishers
Conference LocationManchester, UK
KeywordsImmunity, Immunomodulation, macrophage, skin
Peer reviewedno

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