Empowered pro-inflammatory features in Ly6C monocytes and altered antigen-presenting capacity in Ly6C^high monocytes...

Inflammatory monocyte (MC) subset polarization is a hallmark of systemic and tissue inflammatory feature in diabetes. The underlying molecular mechanism remains unclear.

Blood pro-inflammatory Ly6C^high and anti-inflammatory Ly6C^low MC subsets were isolated from control (C57/BL6), type 1 diabetes mellitus (T1DM), and type 2 diabetes mellitus (T2DM) mice by flow cytometry sorting and subjected to bulk high-throughput RNA-sequencing. Intensive and integrative functional bioinformatic studies were performed by analyzing the transcriptome through seven pairs of comparison between Ly6C^high and Ly6C^low MC subsets and between different mouse groups. We examined molecular features of three key immune activation signals in innate and adaptive immune responses, including signal 1 antigen (Ag)-presenting, signal 2 immune checkpoint, and signal 3 cytokine, and their upstream transcription factors (TF). A total of 10 differentially expressed genes (DEG) of MHC-II molecules (signal 1) presented a low expression profile in Ly6C^high MCs from all mice and mostly further reduced in T2DM Ly6C^high MC. Ly6C^high MCs show high intercellular inflammatory propagation based on immune checkpoint/cytokine-ligand expression but low intracellular inflammatory capacity based on immune checkpoint/cytokine-receptor expression, both further enhanced in T1/T2DM. In contrast, Ly6C^low MCs exhibit low intercellular propagation but high intracellular inflammatory capacity; both also increased in T1/T2DM. Furthermore, 921 upstream DEG transcription factors and 17 transcriptional axes were recognized in diabetic Ly6C^high MCs by IPA upstream regulator analysis. Finally, three critical transcriptional axes that drove the altered immunological phenotype in Ly6C^high MCs were recognized: ↓PAX5-↓CIITA-↓Cd74/H2-Eb2 for reduced Ag-presenting power, ↓MYC-↑Sema4a for elevated intercellular inflammatory propagation capacity, and ↑CEBPA/E-↑Csf2ra/3r for enhanced intracellular inflammatory propagation capacity in MC.

We have three major discoveries: 1) Ly6C^high MCs exhibit lower Ag-presenting power, which was further suppressed in T2DM, mostly via ↓PAX5-↓CIITA-↓CD74 regulation. 2) Ly6C^high MCs display high intercellular inflammatory propagation but low intracellular inflammatory capacity, both further enhanced in T1/T2DM, mostly via ↓MYC-↑Sema4a and ↑CEBPA/E-↑Csf2ra/3r regulation. 3) Ly6C^low MCs acquired additional intercellular and intracellular inflammatory capacity in T1/T2DM.