G1 and G2 ApolipoproteinL1 modulate macrophage inflammation and lipid accumulation through the polyamine pathway

Reviewer #1 (Public review):

Summary:

Liu et al. investigated the mechanisms by which apolipoprotein L1 (APOL1) G1 and G2 variants cause inflammation and lipid accumulation in macrophages by bone-marrow-derived macrophages from transgenic mice and human iPS cells. Although these findings are not novel, this work provides solid evidence to prove enhanced inflammation and lipid accumulation in macrophages by APOL1 G1 and G2 variants by a variety of in vitro assays and metabolomics measurements. Further, metabolomics measurements identified that the spermidine synthesis pathway was altered by APOL1 G1 and G2 variants, and the polyamine inhibitor reversed the variants-induced phenotypes.

Strengths:

Their hypothesis and choice of experiments in each section were clear and mostly solid. Mitochondrial morphological quantification by transmission electron microscopy images was convincing. The authors confirmed APOL1 localization inside macrophages and built stories based on their findings. Showing relevant positive and negative findings in line with current knowledge of APOL1-variants-driven pathologies, such as cation flux, cGAS-STING pathways, indicates a good rigor.

Weaknesses:

Although most methods in this work were solid, the choice of α-difluoromethylornithine (DFMO) as an inhibitor of spermidine synthesis was not direct. It was still unclear if DFMO was reversing the phenotypes by lowering spermidine levels. Seahorse assay results would have avoided potential variabilities in cell densities by normalization. Heatmaps showing RNA-seq results would be appreciated better with a clear description of how the color is defined and calculated.

Reviewer #2 (Public review):

Summary:

The G1 and G2 variants of the Apolipoprotein L1 (APOL1) gene are well-established risk factors for chronic kidney disease. While macrophages have been implicated in the pathogenesis of APOL1-mediated kidney diseases (AMKD), the precise impact of the G1 and G2 APOL1 variants on macrophage function and the underlying molecular mechanisms remains insufficiently characterized. In this manuscript, the authors investigate pathological phenotypes in macrophages carrying the G1 and G2 APOL1 variants. They report an accumulation of neutral lipids and activation of pro-inflammatory pathways, which appear to be at least partly driven by an accumulation of the polyamine spermidine and upregulation of the spermidine synthesis pathway. These findings reveal a pro-inflammatory role for G1 and G2 APOL1 in macrophages and identify the spermidine synthesis pathway as a potential therapeutic target.

Strengths:

The authors employ a comprehensive set of approaches to characterize macrophage phenotypes, including assessments of lipid accumulation, pro-inflammatory cytokine release, responses to M2-polarizing cytokines, autophagy, mitochondrial function, and metabolic profiling. The reversal of pathological phenotypes in G1 and G2 APOL1 macrophages by the polyamine synthesis inhibitor α-difluoromethylornithine provides compelling evidence supporting a causal role for spermidine in mediating APOL1 variant-associated dysfunction. Furthermore, the inclusion of both mouse and human models strengthens the translational relevance of the findings.

Weaknesses:

The manuscript would benefit from a clearer articulation of the specific role macrophages play in the pathogenesis of APOL1-associated kidney diseases to better emphasize the significance of the study. Additionally, the experimental design lacks a clear, logical progression, and the rationale behind some experiments is insufficiently justified, making certain conclusions difficult to fully support based on the presented data. Given the availability of established animal models of APOL1-associated kidney diseases, it is unclear why the authors chose to derive macrophages from the bone marrow of G1 and G2 APOL1 mice for in vitro assays rather than isolating and testing macrophages in vivo within these models. In vitro assays may exaggerate macrophage responses compared to physiological conditions, which could affect the interpretation of the data. Addressing this point would strengthen the manuscript.

Reviewer #3 (Public review):

Summary:

Liu et al investigate the impact of G1 and G2 variants of the gene encoding Apolipoprotein L1 (APOL1) on macrophage inflammation. The authors have used bone marrow-derived macrophages and human induced pluripotent stem cell-derived macrophages as their model to identify altered immune signaling caused by G1 and G2 APOL1. The unbiased metabolite analysis indicates the possible involvement of altered polyamine metabolism in the regulation of inflammatory response in G1 and G2 macrophages. This study shows that targeting polyamine metabolism can limit macrophage inflammation and lipid accumulation in vitro conditions.

Strengths:

This study shows the importance of polyamine metabolism in the regulation of macrophage inflammatory response. The authors showed that spermidine synthesis is closely associated with altered macrophage functions with two risk-variant forms of APOL1 (G1 and G2). The altered macrophage lipid metabolism is known to be associated with macrophage dysfunction in G1 and G2 APOL1. However, the involvement of polyamine in the regulation of lipid accumulation and inflammation in macrophages in G1 and G2 variants is interesting and could be explored as a novel therapeutic approach for chronic inflammation.

Weaknesses:

The novelty of this study lies in the association of polyamine metabolism with lipid metabolism dysregulation in macrophages. The weakness of the manuscript is that insufficient experiments to support the claim of involvement of polyamine metabolism in the regulation of macrophage inflammation, which undermines the novelty of this study. The authors performed in vitro experiments targeting spermidine synthesis to show reduced inflammation and lipid accumulation, but have not performed any in vivo analysis of chronic kidney inflammation progression in G1 and G2 mice, which they have used to generate bone-marrow-derived macrophages. They have not shown any data that supports the specificity of DFMO in targeting spermidine synthesis.