A two‐step human culture system replicates intestinal monocyte maturation cascade: Conversion of tissue‐like inflammatory monocytes into macrophages

Abstract

Monocyte maturation program into macrophages (MΦ) is well defined in murine gut under homeostatic or inflammatory conditions. Obviously, in vivo tracking of monocytes in inflamed tissues remains difficult in humans. Furthermore, in vitro models fall short in generating the surrogates of transient extravasated tissue inflammatory monocytes. Here, we aimed to unravel environmental cues that replicated the human monocyte “waterfall” process in vitro by first, generating tissue‐like inflammatory monocytes, which were then shifted toward MΦ. Purified CD14+CD16− monocytes, cultured with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), IFN‐γ and IL23, differentiated into CD14+CD163− cells that displayed a monocyte‐like morphology. In vitro generated inflammatory CD14+CD163− (inflammatory monocyte‐like cells) cells promoted IL‐1β‐dependent memory Th17 and Th17/Th1 responses, like the CD14+CD163− mo‐like cells that accumulate in inflamed colon of Crohn's disease patients. Next, in vitro generated inflammatory monocyte‐like cells converted to functional CD163+ MΦ following exposure to TGF‐β and IL10. Gene set enrichment analysis further revealed a shared molecular signature between converted CD163+ MΦ and MΦ detected in various inflamed nonlymphoid and lymphoid diseased tissues. Our findings propose a two‐step in vitro culture that recapitulates human monocyte maturation cascade in inflamed tissue. Manipulation of this process might open therapeutic avenues for chronic inflammatory disorders.

Behind the monocyte’s mystique: uncovering their developmental trajectories and fates

“…Furthermore, basic homeostatic cues such as neuronal, circadian rhythms, sleep, diet, and the microbiome have also been recently found to have a significant impact on monocyte fate [ 83 , 84 ]. In particular, the gut is constantly exposed to a low-grade inflammation caused by commensal microbiota and their products, resulting in monocytes undergoing a loss of Ly6C and concomitant gain in MHCII expression, which has been described as the ‘monocyte waterfall’ [ 68 , 85 ] that have also been described in other inflammatory settings [ 86 ]. In particular, major transcriptomic changes were already found between Ly6C hi monocytes and TRMs early after engraftment [ 87 ].…”

The Conversion of Human Tissue‐Like Inflammatory Monocytes Into Macrophages

“…Purified monocytes will be cultured in the presence of a cocktail of cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFNγ), and interleukin 23 (IL-23), mimicking inflammatory conditions. The combination of three cytokines is required for optimal generation of Infl mo-like cells displaying morphology, phenotype, and function of their tissue counterpart (Bsat et al, 2020). After 6 days, a few cells are collected and stained for phenotypic analysis.…”

“…Monocytes' maturation cascade from pro-inflammatory tissue-like monocytes to antiinflammatory Mφ occurs in intestinal mucosa at homeostasis in mice and humans. The following protocol uses an anti-inflammatory cocktail, i.e., TGFβ plus IL-10 that shifts or converts CD14 + HLA-DR + CD64 + CD163 − Infl mo-like cells into cells displaying phenotype, molecular profile, and phagocytic function of Mφ (Bsat et al, 2020). Cell culture, from Basic Protocol 2, enriched in CD14 + HLA-DR + CD64 + CD163 − Infl mo-like cells will be supplemented with transforming growth factor beta 1 (TGFβ1) plus IL-10.…”

“…Furthermore, in vitro culture systems of human monocyte differentiation into Mφ fall short in first generating the surrogates of transiently extravasated tissue inflammatory monocytes that can be subsequently shifted to mature Mφ. Here, we present the method to purify circulating human monocytes and culture conditions to first generate the surrogates of tissue-like inflammatory monocytes, and next, their conversion into Mφ that display phenotypic, functional, and molecular features with ex vivo tissue Mφ (Bsat, Chapuy, Rubio, & Sarfati, 2020). This in vitro model will allow further investigations of the functional properties of human surrogates at each step of their differentiation.…”

The relationship between extreme inter-individual variation in macrophage gene expression and genetic susceptibility to inflammatory bowel disease

“…The progressive adaptation of monocytes to the intestinal environment has been described as a waterfall (Hegarty et al 2023 ; Joeris et al 2017 ; Mowat et al 2017 ). Bsat et al ( 2020 ) described a system to replicate this cascade based upon sequential cultivation in CSF2 (GM-CSF), interferon-gamma and IL23. However, this system reflects an inflammatory milieu.…”