HUMANIN From Efferocytic Macrophages Dials Down Inflammation
Human macrophages that clear dead cells release the mitochondrial peptide HUMANIN, which actively resolves inflammation in lab and animal models.
Summary
When macrophages engulf apoptotic neutrophils—a process called efferocytosis—they upregulate mitochondrial metabolism genes and release a small peptide called HUMANIN. Researchers performed RNA sequencing on three subtypes of human macrophages after efferocytosis, identifying HUMANIN (encoded by the mitochondrial gene MT-RNR2) as a key secreted factor. In a mouse peritonitis model, HUMANIN reduced leukocyte infiltration and pro-inflammatory cytokines, while promoting pro-resolving gene expression. It also suppressed cytokine production in human neutrophils. Notably, HUMANIN was detected in gingival fluid from periodontitis patients after inflammation onset, suggesting a physiological role. These findings position HUMANIN as a novel pro-resolving mediator with therapeutic potential in chronic inflammatory diseases.
Detailed Summary
Efferocytosis—the clearance of apoptotic neutrophils by macrophages—is a pivotal checkpoint in resolving inflammation. Defects in this process have been linked to atherosclerosis, lupus, rheumatoid arthritis, and Crohn's disease. Despite extensive rodent data, the transcriptional landscape of human efferocytic macrophages has remained poorly characterized, motivating this study.
The researchers developed a clean in vitro human model using monocyte-derived macrophages (M0, M1, and M2a subtypes) co-cultured with CFSE-labeled apoptotic neutrophils for 24 hours. Because neutrophil RNA degrades rapidly, only macrophage RNA was recovered for bulk RNA sequencing. GSEA analysis revealed that all three macrophage subtypes consistently upregulated oxidative phosphorylation (OXPHOS) genes and downregulated cholesterol biosynthesis genes after efferocytosis. Four standout genes—MTRNR2L12, MTRNR2L6, PLIN5, and MTLN—linked OXPHOS to fatty acid oxidation regulation and were most strongly induced in M1-like macrophages. Seahorse respirometry confirmed increased basal oxygen consumption in efferocytic M1 macrophages, validating the metabolic reprogramming at the functional level.
Among the upregulated genes, MT-RNR2 encoding the mitochondria-derived peptide HUMANIN (HN) emerged as a novel efferocytosis-associated factor. HUMANIN protein was confirmed to be secreted by efferocytic macrophages in vitro. In a zymosan-induced acute peritonitis mouse model, HUMANIN levels rose early during the resolution phase. Preventive intraperitoneal HUMANIN administration significantly reduced peritoneal leukocyte counts and pro-inflammatory cytokines (TNF-α, IL-6, IL-12), while promoting expression of pro-resolving markers ALOX15 and RETNLA. HUMANIN-treated mice also showed earlier acquisition of a CD11b-low non-efferocytic macrophage phenotype, indicative of accelerated resolution. In primary human neutrophils, HUMANIN dampened LPS-induced pro-inflammatory cytokine secretion, corroborating its anti-inflammatory capacity across species.
Critically, HUMANIN was detected in gingival crevicular fluid (GCF) from periodontitis patients after inflammation onset but not at baseline, suggesting it is naturally produced in human inflammatory contexts and may serve as an endogenous brake on excessive inflammation.
The study also identified two previously unreported efferocytosis-regulated genes in humans—PLIN5 (a lipid droplet protein promoting FAO) and MTLN (mitochondrial microprotein)—extending knowledge beyond rodent models and highlighting human-specific regulatory nodes. Collectively, the data support a model in which efferocytosis drives mitochondrial metabolic rewiring that culminates in HUMANIN secretion, linking organelle physiology to systemic inflammation resolution.
Key Findings
- All three human macrophage subtypes upregulate OXPHOS genes and downregulate cholesterol biosynthesis genes after efferocytosis.
- The mitochondrial peptide HUMANIN is produced by efferocytic human macrophages and rises early during acute peritonitis resolution in mice.
- Preventive HUMANIN treatment reduced leukocyte infiltration and pro-inflammatory cytokines in a mouse peritonitis model.
- HUMANIN suppressed LPS-induced pro-inflammatory cytokine secretion in primary human neutrophils.
- HUMANIN was detected in gingival crevicular fluid of periodontitis patients after inflammation onset, suggesting endogenous pro-resolving activity.
Methodology
RNA sequencing was performed on CFSE-sorted efferocytic human M0, M1, and M2a monocyte-derived macrophages co-cultured with apoptotic neutrophils for 24 hours, exploiting neutrophil RNA degradation to eliminate contamination. Functional validation included Seahorse respirometry, Western blot, RT-qPCR, and flow cytometry, complemented by a zymosan-induced mouse peritonitis model and gingival crevicular fluid sampling from periodontitis patients.
Study Limitations
The mouse peritonitis model used preventive rather than therapeutic HUMANIN dosing, limiting direct clinical translation. In vitro human models used monocyte-derived macrophages, which may not fully recapitulate tissue-resident macrophage biology. The periodontitis cohort data are observational and cannot establish causality between HUMANIN levels and clinical outcomes.
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