# Investigation of HCAR2 antagonists as a potential strategy to modulate bovine leukocytes

## TL;DR

This study explores how HCAR2 antagonists can modulate immune cell activity in dairy cows, potentially improving health during lactation.

## Contribution

The study identifies and tests HCAR2 antagonists to counteract BHBA-induced immunosuppression in bovine leukocytes.

## Key findings

- HCAR2 is expressed in bovine leukocytes, with higher expression in mid-lactation cows.
- Niacin-derived antagonists NA-1 and NA-5 reduce BHBA-induced Ca2+ mobilization in immune cells.
- NA-5 prevents BHBA-related decreases in cyclic AMP in monocytes.

## Abstract

Dairy cows experiencing ketosis after calving suffer greater disease incidence and are at greater risk of leaving the herd. In vitro administration of beta-hydroxybutyric acid (BHBA; the primary blood ketone) has inhibitory effects on the function of bovine leukocytes. BHBA is a ligand of HCAR2 and the activation of these receptors promotes an anti-inflammatory response which may be related with immunosuppression observed in transition dairy cattle. The objective of this study was to identify and test antagonists for HCAR2 in bovine immune cells cultured with BHBA.

We observed expression of HCAR2 at the protein level within lymphocytes, monocytes, and granulocytes. The proportion of cells expressing HCAR2 tended to be greater in mid-lactation compared to early lactation cows; the increase was a result of increased proportion of T and B cells expressing HCAR2. Stimulation of HCAR2 with niacin or BHBA promoted Ca2+ mobilization in neutrophils and mononuclear cells. Mononuclear cells treated with BHBA had diminished intracellular Ca2+ responses when HCAR2 was knocked down by siRNA silencing, indicating Ca2+ mobilization was mediated by HCAR2 signaling. Two candidate antagonists for HCAR2, synthesized from niacin (NA-1 and NA-5), were tested; monocytes and neutrophils pre-treated with NA-1 and NA-5 had reduced Ca2+ mobilization after incubation with BHBA. Furthermore, NA-5 but not NA-1 prevented BHBA-associated reductions in cyclic AMP.

We demonstrated that HCAR2 is present on bovine leukocytes and has greater expression later in lactation. We confirmed that BHBA and niacin derived HCAR2 antagonists alter bovine leukocyte activity. Our results demonstrate that both BHBA and niacin affect bovine leukocyte Ca2+ mobilization in a HCAR2-dependent manner.

The online version contains supplementary material available at 10.1186/s40104-024-00999-5.