H may contribute to the innate immune defense against a variety of bacteria and protozoans [10]. As IL4I1 expression is induced in mononuclear phagocytes by pathogenderived signals, such as Toll-like receptor ligands [2], it may participate in the innate immune defense against pathogens in mammals, in addition to its regulatory effects on the specific immune response.In this work, we thus evaluated the effect of recombinant IL4I1 on Gram-positive and Gram-negative bacteria growth. We show that IL4I1 can act as an antibiotic, which could potentially contribute to the arsenal used by activated macrophages to generate an extracellular environment hostile to bacterial growth.ResultsWe tested the antibacterial effect of conditioned media from IL4I1-expressing THP1 monocytes on four bacterial strains, get Castanospermine representative of Gram-negative bacilli and Gram-positive cocci, i.e. Escherichia coli DH5a (E. coli), an Escherichia coli strain auxotrophic for Phe (B2599) and 2 Staphylococcus strains, a methicillin-susceptible Staphylococcus aureus (MSSA) and a coagulase-IL4I1 Antibacterial Propertiesnegative Staphylococcus (CNS). IL4I1 activity in these media was 340.88 pmol/H2O2/h/ml 666.27 (Figure 1A). The IL4I1containing medium was then diluted in control-conditioned medium from THP1 cells at increasing ratios (Figure 1B). We observed that the growth of all four bacterial strains was blocked in an IL4I1 dose-dependent fashion, indicating an antibacterial effect of the enzyme. IL4I1 degrades L-Phe and to a lesser extent L-tryptophan (Trp) to the corresponding a-keto-aminoacid, NH3 and H2O2. The activity of IL4I1 is usually measured based on H2O2 production in the presence of Phe (Figure 1A). Since all the catabolites of the reaction are theoretically produced in equimolar amounts, we expected ammonia accumulation in the presence of IL4I1. Although IL4I1-mediated NH3 production could not be detected in THP1 and THP1-IL4I1 conditioned media, because of a strong basal level mainly due to glutamine instability, we confirmed the production of ammonia in Phe-containing conditioned PBS (Figure S1). The 24 h accumulation of ammonia in THP1 cultures (74.0616.2 mM) doubled in the presence of IL4I1 (153.8627.6 mM). Moreover, the presence of IL4I1 induced a 0.5 pH increase in the medium (THP1: pH = 7.5; THP1-IL4I1: pH = 8) that was not modified by bacterial growth. The Phe, Trp and phenylpyruvate content were measured using an HPLC technique. Similar to NH3, phenylpyruvate was significantly increased in the presence of IL4I1 (Figure S2). Phe and Trp content were at the limit of sensitivity of the method. Two samples were transferred to a laboratory specialized in Phe quantification in human serum and urine. No significant variation in Phe content was measured, as expected from the low rate of Phe consumption (less then 10 mM/24 h). We sought to determine which mechanisms associated with this reaction could be involved in the observed antibacterial effect (Figure 2). We first explored the role of amino acid consumption in the bacterial microenvironment. For this purpose, bacteria were grown in DMEM/F12 supplemented with glutathione but SIS 3 lacking Phe and Trp. Both amino acids were alternatively added to the medium, alone or in combination (Figure 2A). E. coli growth was not affected by amino acid deprivation. In contrast, the growth of the three other strains was blocked in Phe- and Trp-deprived media. As expected, the growth of B2599 was restored by Phe, but not by Trp.H may contribute to the innate immune defense against a variety of bacteria and protozoans [10]. As IL4I1 expression is induced in mononuclear phagocytes by pathogenderived signals, such as Toll-like receptor ligands [2], it may participate in the innate immune defense against pathogens in mammals, in addition to its regulatory effects on the specific immune response.In this work, we thus evaluated the effect of recombinant IL4I1 on Gram-positive and Gram-negative bacteria growth. We show that IL4I1 can act as an antibiotic, which could potentially contribute to the arsenal used by activated macrophages to generate an extracellular environment hostile to bacterial growth.ResultsWe tested the antibacterial effect of conditioned media from IL4I1-expressing THP1 monocytes on four bacterial strains, representative of Gram-negative bacilli and Gram-positive cocci, i.e. Escherichia coli DH5a (E. coli), an Escherichia coli strain auxotrophic for Phe (B2599) and 2 Staphylococcus strains, a methicillin-susceptible Staphylococcus aureus (MSSA) and a coagulase-IL4I1 Antibacterial Propertiesnegative Staphylococcus (CNS). IL4I1 activity in these media was 340.88 pmol/H2O2/h/ml 666.27 (Figure 1A). The IL4I1containing medium was then diluted in control-conditioned medium from THP1 cells at increasing ratios (Figure 1B). We observed that the growth of all four bacterial strains was blocked in an IL4I1 dose-dependent fashion, indicating an antibacterial effect of the enzyme. IL4I1 degrades L-Phe and to a lesser extent L-tryptophan (Trp) to the corresponding a-keto-aminoacid, NH3 and H2O2. The activity of IL4I1 is usually measured based on H2O2 production in the presence of Phe (Figure 1A). Since all the catabolites of the reaction are theoretically produced in equimolar amounts, we expected ammonia accumulation in the presence of IL4I1. Although IL4I1-mediated NH3 production could not be detected in THP1 and THP1-IL4I1 conditioned media, because of a strong basal level mainly due to glutamine instability, we confirmed the production of ammonia in Phe-containing conditioned PBS (Figure S1). The 24 h accumulation of ammonia in THP1 cultures (74.0616.2 mM) doubled in the presence of IL4I1 (153.8627.6 mM). Moreover, the presence of IL4I1 induced a 0.5 pH increase in the medium (THP1: pH = 7.5; THP1-IL4I1: pH = 8) that was not modified by bacterial growth. The Phe, Trp and phenylpyruvate content were measured using an HPLC technique. Similar to NH3, phenylpyruvate was significantly increased in the presence of IL4I1 (Figure S2). Phe and Trp content were at the limit of sensitivity of the method. Two samples were transferred to a laboratory specialized in Phe quantification in human serum and urine. No significant variation in Phe content was measured, as expected from the low rate of Phe consumption (less then 10 mM/24 h). We sought to determine which mechanisms associated with this reaction could be involved in the observed antibacterial effect (Figure 2). We first explored the role of amino acid consumption in the bacterial microenvironment. For this purpose, bacteria were grown in DMEM/F12 supplemented with glutathione but lacking Phe and Trp. Both amino acids were alternatively added to the medium, alone or in combination (Figure 2A). E. coli growth was not affected by amino acid deprivation. In contrast, the growth of the three other strains was blocked in Phe- and Trp-deprived media. As expected, the growth of B2599 was restored by Phe, but not by Trp.
