Figure 6. CoH mice have increased frequency of “classical” Ly6C^hi CD115⁺ monocytes at steady state that produce more TNF-α.

(A–D) CD11b⁺CD115⁺ monocytes were flow sort-purified from spleens of steady-state SPF and CoH mice, RNA was isolated, bulk RNA-seq analysis was performed, and comparison of gene expression was conducted.

(A) Principal-component analysis from RNA-seq data showing unique clustering of CD115⁺ monocytes from steady-state SPF and CoH mice.

(B) Volcano plot of log false discovery rate (FDR, y axis) by log fold change (x axis). Genes with an FDR < 0.01 with increased expression in CD115⁺ monocytes from spleens of steady-state CoH mice compared with SPF mice are shown in green, while genes with an FDR < 0.01 with decreased expression in CD115⁺ monocytes from spleens of steady-state CoH mice compared with SPF mice are shown in red.

(C) Heatmap showing relative gene expression in CD115⁺ monocytes from spleens of steady-state SPF and CoH mice. Seven hundred and twenty genes were differentially expressed with an FDR < 0.01.

(D) GO term analysis for pathways differentially regulated between CD115⁺ monocytes from spleens of steady-state CoH and SPF mice.

(E) Representative histogram of MPO expression in SPF and CoH CD115⁺ monocyte/Mφ (left) and frequency of myeloperoxidase (MPO)⁺ cells among CD115⁺ monocytes in the spleen as measured by flow cytometry (right).

(F) Number of Ly6c1 and Ly6c2 mRNA transcripts in CD115⁺ monocytes from steady-state SPF and CoH mice (left), representative histogram of Ly6C protein expression on SPF and CoH CD115⁺ monocytes (middle), and frequency of Ly6C⁺ cells among CD115⁺ monocytes in the spleens of CoH mice (right).

(G) Frequency of TNF-α⁺ cells within CD11b⁻CD64⁺ resident macrophages, Ly6C^hiCD64⁺ monocytes, and Ly6C^loCD64⁺ monocytes in the spleens of SPF and CoH at steady-state and after in vitro LPS stimulation (100 ng/mL).

(H) Geometric mean fluorescence intensity (gMFI) of TNF-α expression by unstimulated and LPS-stimulated TNF-a⁺CD11b⁻CD64⁺ resident macrophages (green), CD11b⁺Ly6C^hiCD64⁺ classical (red), and CD11b⁺Ly6C^loCD64⁺ non-classical (black) monocytes was determined by flow cytometry (left). Dashed lines, SPF; solid lines, CoH. TNF-α gMFI within TNF-α⁺CD11b⁻CD64⁺ resident macrophages, Ly6C^hiCD64⁺ classical monocytes, and Ly6C^loCD64⁺ non-classical monocytes in the spleens of SPF and CoH at steady-state and after in vitro LPS stimulation (right). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005, and ****p < 0.0001 as determined by nonparametric Mann-Whitney test. Transcript (normalized copies per million [nCPM]) data in (F) were obtained from 3 to 4 mice per group. Flow data in (E) were combined from two experiments using a total of 9 mice per group, in (F) were combined from three experiments using a total of 25–26 mice per group, in (G) were combined from three experiments using a total of 8–9 mice per group, in (H) were combined from two experiments using 3 mice per group. Each symbol in (A and E–H) represents a mouse and bars indicate means with SEM.