Proinflammatory polarization of adipose tissue macrophages in cows with subclinical ketosis constitutes a critical driver of adipose tissue remodeling and inflammation

Abstract

Sustained lipolysis exacerbates subclinical ketosis (SCK) in dairy cows and is associated with inflammation and adipose tissue macrophage (ATM) infiltration. While ATM involvement in adipose homeostasis and inflammation in early lactation is recognized, a comprehensive exploration of ATM polarization phenotypes in SCK cows is lacking. This study aimed to characterize ATM polarization and its link to lipolysis and inflammation in SCK cows. Subcutaneous adipose tissue samples were obtained from dairy cows to analyze protein expression and gene profiles. Compared with healthy cows, SCK cows had higher serum BHBA and NEFA, smaller adipocytes, and increased expression of lipolytic enzymes (LIPE, ATGL), indicating enhanced lipolysis. Decreased levels of FASN, PPARγ, p-SMAD3, and TGFβ suggested impaired adipogenesis. Inflammatory markers (TNF-α, IFN-γ, TLR4, Caspase1) and NFκB signaling activity were elevated. ATM infiltration was supported by increased CD9, CD68, TREM2, and CXCL1 expression. Protein abundance of M1 polarization markers (iNOS, CD86 and CCL2) in ATMs were associated with greater levels of NOS2, IL1B, CD86 and CCL2 mRNA expression in SCK cows; fluorescence intensity of NOS2 and CD86 also was elevated, alongside a higher proportion of CD68+/CD86+ immunopositive cells within adipose tissue. ELISA further quantified increased concentrations of IL-1β and CCL2. Conversely, the abundance of ATM M2 polarization markers, including CD206, IL-10, KLF4, and Arg1, at both the protein and mRNA levels demonstrated a decline. Meanwhile, the proportion of CD68+/CD206+ immune response cells was relatively low in SCK cows. Overall, the present study indicated an augmented macrophage presence within adipose tissue during subclinical ketosis, with a predominance of pro-inflammatory macrophages (M1 ATM). This observation suggested a vicious cycle wherein macrophage infiltration and pro-inflammatory polarization coincide with enhanced lipolysis and an amplified inflammatory cascade.