Sodium ferrous citrate in 5-Aminolevulinic acid supplements suppresses the effector function of feline lymphocytes by reducing the mitochondrial membrane potential

5-Aminolevulinic acid (5-ALA) is an endogenous amino acid in mammalian cells; it is the first amino acid in the heme biosynthesis pathway occurring in the mitochondria. 5-ALA with sodium ferrous citrate (SFC) possesses anti-inflammatory properties by inducing heme oxygenase (HO)-1 expression and releasing heme metabolites in humans and mice. Supplements containing 5-ALA and divalent iron is available in veterinary medicine. We previously showed that 5-ALA with SFC enhances the production of interferon-gamma (IFN-gamma) in concanavalin A (ConA)-stimulated canine lymphocytes. However, the effects of 5-ALA/SFC on feline lymphocytes remain to be investigated. This study demonstrated that 5-ALA/SFC-induced HO-1 expression and decreased IFN-gamma production in ConA-stimulated feline lymphocytes. Comprehensive RNA sequencing analysis revealed that the activating transcription factor 4 (ATF4) signaling pathway was inhibited by adding 5-ALA/SFC. Moreover, we confirmed that 5-ALA/SFC decreased ATF4 protein expression. Furthermore, separate analyses of the effects of 5-ALA and SFC on feline lymphocytes revealed that SFC, but not 5-ALA, induced AKT dephosphorylation and mitochondrial dysfunction in activated lymphocytes. Thus, SFC in 5-ALA supplements may suppress the effector function of feline lymphocytes via mitochondrial metabolism, thereby representing a novel mechanism in 5-ALA/SFC research.