Significance: Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunoregulatory enzyme that transforms tryptophan into kynurenine, an endogenous agonist of the aryl hydrocarbon receptor (AhR) whose activation sustains IDO1 expression and activity over the long term in dendritic cells (DCs). Here we found that N -acetylserotonin (NAS), a tryptophan metabolite produced along the serotonin pathway, acts as a positive allosteric modulator (PAM) of IDO1 and thus increases kynurenine-mediated AhR activation in DCs. NAS effects on IDO1 translated into protection of mice from neuroinflammation and reinstallment of physiologic IDO1 activity in peripheral blood mononuclear cells from patients with relapsing-remitting multiple sclerosis.

Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the first step in the kynurenine pathway of tryptophan (Trp) degradation that produces several biologically active Trp metabolites. L-kynurenine (Kyn), the first byproduct by IDO1, promotes immunoregulatory effects via activation of the Aryl hydrocarbon Receptor (AhR) in dendritic cells (DCs) and T lymphocytes. We here identified the nuclear coactivator 7 (NCOA7) as a molecular target of 3-hydroxyanthranilic acid (3-HAA), a Trp metabolite produced downstream of Kyn along the kynurenine pathway. In cells overexpressing NCOA7 and AhR, the presence of 3-HAA increased the association of the two molecules and enhanced Kyn-driven, AhR-dependent gene transcription. Physiologically, conventional (cDCs) but not plasmacytoid DCs or other immune cells expressed high levels of NCOA7. In cocultures of CD4⁺ T cells with cDCs, the co-addition of Kyn and 3-HAA significantly increased the induction of Foxp3⁺ regulatory T cells and the production of immunosuppressive transforming growth factor β in an NCOA7-dependent fashion. Thus, the co-presence of NCOA7 and the Trp metabolite 3-HAA can selectively enhance the activation of ubiquitary AhR in cDCs and consequent immunoregulatory effects. Because NCOA7 is often overexpressed and/or mutated in tumor microenvironments, our current data may provide evidence for a new immune check-point mechanism based on Trp metabolism and AhR.

In mammals, amino acid metabolism has evolved to act as a critical regulator of innate and adaptive immune responses. Rheumatoid arthritis (RA) is the most common form of inflammatory arthropathy sustained by autoimmune responses. We examine here the current knowledge of tryptophan and arginine metabolisms and the main immunoregulatory pathways in amino acid catabolism, in both RA patients and experimental models of arthritis. We found that l-tryptophan (Trp) metabolism and, in particular, the kynurenine pathway would exert protective effects in all experimental models and in some, but not all, RA patients, possibly due to single nucleotide polymorphisms in the gene coding for indoleamine 2,3-dioxygenase 1 (IDO1; the enzyme catalyzing the rate-limiting step of the kynurenine pathway). The function, i.e., either protective or pathogenetic, of the l-arginine (Arg) metabolism in RA was less clear. In fact, although immunoregulatory arginase 1 (ARG1) was highly induced at the synovial level in RA patients, its true functional role is still unknown, possibly because of few available preclinical data. Therefore, our analysis would indicate that amino acid metabolism represents a fruitful area of research for new drug targets for a more effective and safe therapy of RA and that further studies are demanding to pursue such an important objective.

Mutations in the WFS1 gene, encoding wolframin (WFS1), cause endoplasmic reticulum (ER) stress and are associated with a rare autosomal-recessive disorder known as Wolfram syndrome (WS). WS is clinically characterized by childhood-onset diabetes mellitus, optic atrophy, deafness, diabetes insipidus and neurological signs. We identified two novel WFS1 mutations in a patient with WS, namely, c.316-1G > A (in intron 3) and c.757A > T (in exon 7). Both mutations, located in the N-terminal region of the protein, were predicted to generate a truncated and inactive form of WFS1. We found that although the WFS1 protein was not expressed in peripheral blood mononuclear cells (PBMCs) of the proband, no constitutive ER stress activation could be detected in those cells. In contrast, WS proband’s PBMCs produced very high levels of proinflammatory cytokines (i.e. TNF-α, IL-1β, and IL-6) in the absence of any stimulus. WFS1 silencing in PBMCs from control subjects by means of small RNA interference also induced a pronounced proinflammatory cytokine profile. The same cytokines were also significantly higher in sera from the WS patient as compared to matched healthy controls. Moreover, the chronic inflammatory state was associated with a dominance of proinflammatory T helper 17 (Th17)-type cells over regulatory T (Treg) lymphocytes in the WS PBMCs. The identification of a state of systemic chronic inflammation associated with WFS1 deficiency may pave the way to innovative and personalized therapeutic interventions in WS.

The dried stigmas of Crocus sativus L. (Iridaceae) are traditionally processed to produce saffron, a spice widely used as a food coloring and flavoring agent, which is important in the pharmaceutical and textile dye-producing industries. The labor-intensive by-hand harvesting and the use of only a small amount of each flower cause saffron to be the most expensive spice in the world. Crocus sp. petals are by-products of saffron production and represent an interesting raw material for the preparation of extracts intended for health protection in the perspective of a circular economy. In the present study, ethanolic extract from Crocus sativus L. petals (Crocus sativus L. petal extract, CsPE) was tested on macrophages by in vitro models of inflammation and osteoclastogenesis. The extract was found to be endowed with anti-inflammatory activity, significantly reducing the nitric oxide production and IL-6 release by RAW 264.7 murine cells. Moreover, CsPE demonstrated an anti-osteoclastogenic effect, as revealed by a complete inhibition of tartrate-resistant acid phosphatase (TRAP)-positive osteoclast formation and a decreased expression of key osteoclast-related genes. This study, which focuses on the macrophage as the target cell of the bioactive extract from Crocus sativus L. petals, suggests that the petal by-product of saffron processing can usefully be part of a circular economy network aimed at producing an extract that potentially prevents bone disruption.

Genetic engineering of plants has turned out to be an attractive approach to produce various secondary metabolites. Here, we attempted to produce kynurenine, a health-promoting metabolite, in plants of Nicotiana tabacum (tobacco) transformed by Agrobacterium tumefaciens with the gene, coding for human indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme responsible for the kynurenine production because of tryptophan degradation. The presence of IDO1 gene in transgenic plants was confirmed by PCR, but the protein failed to be detected. To confer higher stability to the heterologous human IDO1 protein and to provide a more sensitive method to detect the protein of interest, we cloned a gene construct coding for IDO1-GFP. Analysis of transiently transfected tobacco protoplasts demonstrated that the IDO1-GFP gene led to the expression of a detectable protein and to the production of kynurenine in the protoplast medium. Interestingly, the intracellular localisation of human IDO1 in plant cells is similar to that found in mammal cells, mainly in cytosol, but in early endosomes as well. To the best of our knowledge, this is the first report on the expression of human IDO1 enzyme capable of secreting kynurenines in plant cells.