M1 polarization of hepatic macrophages in cows with subclinical ketosis is an important cause of liver injury.

Subclinical ketosis (SCK) is highly prevalent and easily overlooked, with insidious and slow progression of hepatic injury, often characterized by an imbalance in immune homeostasis. In nonruminants, macrophage polarization plays an important regulatory role in hepatic lipid accumulation, fibrosis, and inflammatory processes. Thus, we aimed to investigate the status of hepatic macrophage polarization in SCK cows and to corroborate its association with liver injury and inflammation. Twelve Holstein dairy cows (parity 2-4) were selected, and liver biopsy and blood were collected on the second week postpartum (10-14 d DIM). On the basis of serum beta-hydroxybutyric acid (BHBA) concentrations, selected cows were categorized into healthy (n = 6; BHBA <1.0 mM) and SCK (n = 6; 1.2 mM ≤ BHBA < 3.0 mM) groups. Serum biochemical parameters were measured using an automatic biochemical analyzer, which indicated higher serum levels of BHBA and nonesterified fatty acids and an upregulation of liver injury indicators (aspartate aminotransferase [AST], alanine aminotransferase [ALT], total protein, globulin) in SCK cows compared with healthy cows. The ELISA assays revealed that SCK cows displayed systemic low-grade inflammation, as demonstrated by increased serum levels of haptoglobin, serum amyloid A, TGF-β, IFN-γ, and IL-1β. Liver biopsies revealed pathological histological alterations, hepatic inflammation, and macrophage polarization status. Oil Red staining indicated steatosis, whereas Sirius red staining demonstrated mild extracellular matrix deposition in the liver of SCK cows. The expression of inflammatory response-related proteins (TLR4, p-NFκB, p-I-κB, NLRP3, and Caspase 1) was elevated in the liver of SCK cows, with the increased mean fluorescence intensity of NFκB further confirming the activation of the inflammatory pathway. Furthermore, the levels of pro-inflammatory factors, TNF-α and IFN-γ, were elevated in the tissue homogenate. Macrophage phenotypic changes in SCK cows were further explored based on the results of liver injury and inflammation. Compared with healthy cows, the protein and mRNA abundance of the macrophage marker CD68 in the liver of SCK cows was higher, along with an increased mean fluorescence intensity of CD68. The SCK cows also exhibited reduced mRNA expression of the Kupffer cell marker CLEC4F and elevated chemokine levels (CXCL1 and CCL2). As evidenced by greater protein and mRNA abundance of macrophage M1 polarization markers (iNOS, IL-1β, CD86, IL-6, IL-12b, and CCL3), higher fluorescence intensity of iNOS and CD86, and an increased number of CD68/CD86+-positive cells observed via immunofluorescence, the macrophage polarization phenotype in the liver of SCK cows was predominantly M1. In contrast, the protein and mRNA abundances of M2 polarization markers (CD206, IL-10, and Arg1) were lower in SCK cows, accompanied by a reduced fluorescence intensity of CD206 and a lower number of CD68/CD206-positive cells. Overall, the present study revealed that the number of macrophages in liver is enhanced during subclinical ketosis and is dominated by pro-inflammatory macrophages (M1 macrophages). This could partly explain the increased risk of steatosis, fibrosis, and inflammatory response processes in these cows.