Pharmacological validation of targets regulating CD14 during macrophage differentiation

Abstract

The signalling receptor for LPS, CD14, is a key marker of, and facilitator for, pro-inflammatory macrophage function. Pro-inflammatory macrophage differentiation remains a process facilitating a broad array of disease pathologies, and has recently emerged as a potential target against cytokine storm in COVID19. Here, we perform a whole-genome CRISPR screen to identify essential nodes regulating CD14 expression in myeloid cells, using the differentiation of THP-1 cells as a starting point. This strategy uncovers many known pathways required for CD14 expression and regulating macrophage differentiation while additionally providing a list of novel targets either promoting or limiting this process. To speed translation of these results, we have then taken the approach of independently validating hits from the screen using well-curated small molecules. In this manner, we identify pharmacologically tractable hits that can either increase CD14 expression on non-differentiated monocytes or prevent CD14 upregulation during macrophage differentiation. An inhibitor for one of these targets, MAP2K3, translates through to studies on primary human monocytes, where it prevents upregulation of CD14 following M-CSF induced differentiation, and pro-inflammatory cytokine production in response to LPS. Therefore, this screening cascade has rapidly identified pharmacologically tractable nodes regulating a critical disease-relevant process.

CD14 Is Involved in the Interferon Response of Human Macrophages to Rubella Virus Infection

“…The downmodulation of CD14 appears to be supportive for RuV infection of MΦs and could potentially be involved in RuV maintenance, as it was shown in association with M2 MΦs in granulomas of PID patients [ 40 ]. The identification of high serum soluble CD14 level in COVID19 patients highlights its potential as a therapeutic target [ 60 , 61 ]. A CRISPR/Cas9 screen identified genes involved in downmodulation or upregulation of CD14 and as such pharmacological compounds that target these processes [ 61 ].…”

“…The identification of high serum soluble CD14 level in COVID19 patients highlights its potential as a therapeutic target [ 60 , 61 ]. A CRISPR/Cas9 screen identified genes involved in downmodulation or upregulation of CD14 and as such pharmacological compounds that target these processes [ 61 ]. Furthermore, reactivation and persistence of RNA viruses such as RuV holds many open questions including the potential cellular reservoir.…”

Tumor immunology CRISPR screening: present, past, and future

“…These PRRs activate critical transcriptional programs that initiate the localized immune response through the upregulation of chemokines, cytokines, proinflammatory mediators, and antigen-processing machinery. Pooled knockout screens have identified genes that modulate the expression of PRRs [46] as well as their downstream mediators [47][48][49][50], by enriching for either: (i) genetic perturbations in negative regulators with heightened PRR expression, or (ii) positive regulator perturbations with attenuated PRR expression. TLR signaling transduced either by MyD88 or TRIF, ultimately converges on nuclear factor (NF)-κB and interferon-regulatory factor signaling [51].…”

New Strategies to Promote Macrophage Cholesterol Efflux

“…At the end of the resting period, cells were harvested to determine protein expression levels. A co-receptor for bacterial lipopolysaccharide, CD14 is a key marker for macrophage function, and the upregulation of CD14 expression has been used as a readout of the differentiation of THP-1 monocytes into macrophages ( 46 , 80 ). The CD14 expression level was proportional to the period of THP-1 monocyte culture, suggesting that the number of THP-1 monocytes sensitized by the high-density culture environment increases as a function of culture time ( Figure 2A ).…”