A PNPLA3-NACC1-RIPK3 pathway mediates macrophage necroptosis and inflammation in MASLD
Xinjia Wang1, Lu Bian1, Zhuoying Feng1
1Department of Microbial Science in Health, Cleveland Clinic, Cleveland, Ohio, USA.
Background:
The 148M variant of PNPLA3 is a major genetic risk factor for metabolic dysfunction-associated steatotic liver disease (MASLD), yet its macrophage-specific role remains unclear. We investigated how PNPLA3-148M alters macrophage behavior and multicellular liver pathology under lipotoxic stress.
Methods:
We used a human induced pluripotent stem cell (iPSC)-derived multicellular liver culture comprising hepatocytes, hepatic stellate cells (HSCs), and isogenic macrophages that differed only at PNPLA3 (148M vs. 148I). Cultures were exposed to lipotoxic conditions to induce MASLD. We quantified inflammatory cytokines, oxidative stress, hepatocyte lipid accumulation, and HSC activation. Macrophage death pathways were profiled (apoptosis, pyroptosis, necroptosis), with emphasis on RIPK3 expression and phosphorylation. Downstream effects on hepatocytes and HSCs were assessed.
Results:
Under lipotoxic stress, 148M macrophages amplified MASLD-like features. PNPLA3 transcripts were induced in macrophages-rising in iPSC-derived macrophages from lipotoxic cultures, and in Kupffer cells isolated from murine MASLD. Single-cell RNA-seq further confirmed PNPLA3 expression in human liver macrophage clusters, in addition to hepatocytes and HSCs. Although PNPLA3 mRNA was comparable between genotypes, 148M macrophages displayed higher PNPLA3 protein and increased necroptosis, evidenced by elevated RIPK3 expression and phosphorylation without changes in apoptosis or pyroptosis. Integrative analyses identified NACC1 as a key transcriptional regulator of RIPK3, with NF-κB-linked upregulation of NACC1 in 148M macrophages. NACC1 inhibition (genetic or NIC3) reduced RIPK3, suppressed necroptosis, and lowered pro-inflammatory cytokine secretion. NIC3 additionally decreased hepatocyte lipid accumulation and ATP and diminished HSC activation markers.
Conclusions:
The PNPLA3 148M variant promotes MASLD through a macrophage-specific NF-κB-NACC1-RIPK3 axis that enhances necroptosis and inflammatory signaling, thereby exacerbating hepatocyte steatosis and HSC activation. NACC1 emerges as a tractable therapeutic target for genetically at-risk individuals.
Insights
The PNPLA3 148M variant drives metabolic dysfunction-associated steatotic liver disease (MASLD) by promoting macrophage necroptosis and inflammation. Targeting NACC1 may offer a therapeutic strategy for individuals genetically predisposed to MASLD.
Area of Science:
- Hepatology
- Genetics
- Immunology
Background:
- The PNPLA3 148M variant is a significant genetic risk factor for metabolic dysfunction-associated steatotic liver disease (MASLD).
- The specific role of this variant in macrophage behavior and its contribution to liver pathology under lipotoxic conditions remain largely unexplored.
Purpose of the Study:
- To investigate how the PNPLA3-148M variant influences macrophage behavior and multicellular liver pathology in the context of lipotoxic stress.
- To elucidate the molecular mechanisms by which PNPLA3-148M exacerbates MASLD-related liver damage.
Main Methods:
- Utilized human induced pluripotent stem cell (iPSC)-derived multicellular liver cultures (hepatocytes, hepatic stellate cells, isogenic macrophages) exposed to lipotoxic conditions.
- Quantified inflammatory cytokines, oxidative stress, lipid accumulation, and hepatic stellate cell activation.
- Profiled macrophage death pathways, focusing on RIPK3 expression and phosphorylation, and performed integrative analyses to identify transcriptional regulators.
Main Results:
- PNPLA3-148M macrophages amplified MASLD-like features, including increased necroptosis via elevated RIPK3 expression and phosphorylation.
- Identified NACC1 as a key transcriptional regulator of RIPK3, upregulated in 148M macrophages through an NF-κB-dependent pathway.
- NACC1 inhibition reduced RIPK3, suppressed necroptosis, decreased pro-inflammatory cytokine secretion, and ameliorated hepatocyte steatosis and hepatic stellate cell activation.
Conclusions:
- The PNPLA3 148M variant promotes MASLD through a macrophage-specific NF-κB-NACC1-RIPK3 axis, enhancing necroptosis and inflammation.
- This axis exacerbates hepatocyte steatosis and hepatic stellate cell activation, highlighting a novel mechanism in MASLD pathogenesis.
- NACC1 inhibition presents a potential therapeutic strategy for individuals with a genetic predisposition to MASLD.
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