# Chronic Cholesterol Exposure Disrupts Macrophage Polarization and Cytokine Secretion in a 3D Microenvironment

## TL;DR

Excess cholesterol disrupts how macrophages develop and function, leading to mixed immune responses that could worsen inflammation in cholesterol-rich environments.

## Contribution

This study reveals that chronic cholesterol exposure primes monocytes and disrupts macrophage polarization, leading to noncanonical hypersecretory states.

## Key findings

- Cholesterol causes cytotoxicity in monocytes and alters their metabolism, leading to a primed state.
- Cholesterol-loaded macrophages show reduced surface markers but increased secretion of both pro-inflammatory and regulatory cytokines.
- Cholesterol exposure uncouples macrophage surface phenotype from cytokine output, promoting mixed immune responses.

## Abstract

Cholesterol is essential
for membrane organization and signaling,
but excess cholesterol is increasingly linked to immune dysregulation.
How chronic cholesterol loading shapes macrophage differentiation
and polarization remains unclear. Here, we examined the effects of
sustained cholesterol exposure on THP-1 monocytes and their polarization
within 3D collagen hydrogels. At the monocyte stage, cholesterol caused
cytotoxicity above 3 mg/mL, with an early decline in reactive oxygen
species and metabolic remodeling marked by cholesterol accumulation,
tricarboxylic acid cycle suppression, and redox imbalance. Subcytotoxic
doses preserved cell count but altered metabolic profiles, indicating
a primed state. Differentiation into uncommitted M0 macrophages produced
only minimal phenotypic changes, though modest increases in IL-10,
IFN-γ, and IP-10 suggested early functional effects. Under M1
polarization, cholesterol-loading macrophages showed reduced expression
of CD80, CD86, and HLA-DR, yet secreted higher levels of both pro-inflammatory
(IL-12p70, IFN-γ, IL-17A, MCP-1, IL-2) and regulatory (IL-10,
IL-4) cytokines. Under M2 polarization, canonical markers CD206, CD105,
and CD163 were diminished, while secretion of TGF-β1, IL-10,
TNF-α, and IL-12p70 was increased. Across both conditions, cholesterol
consistently uncoupled surface phenotype from cytokine output, producing
a noncanonical hypersecretory state. These findings suggest that cholesterol
primes monocytes and disrupts macrophage polarization, redirecting
them toward mixed, hypersecretory phenotypes independent of stimulus.
This work links cholesterol-induced metabolic stress to altered macrophage
plasticity, with implications for maladaptive immune responses in
cholesterol-rich environments.