Alban Bessede

Aryl hydrocarbon receptor control of a disease tolerance defence pathway

Disease tolerance is the ability of the host to reduce the effect of infection on host fitness. Analysis of disease tolerance pathways could provide new approaches for treating infections and other inflammatory diseases. Typically, an initial exposure to bacterial lipopolysaccharide (LPS) induces a state of refractoriness to further LPS challenge (endotoxin tolerance). We found that a first exposure of mice to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase, which provided an activating ligand to the former, to downregulate early inflammatory gene expression. However, on LPS rechallenge, AhR engaged in long-term regulation of systemic inflammation only in the presence of indoleamine 2,3-dioxygenase 1 (IDO1). AhR-complex-associated Src kinase activity promoted IDO1 phosphorylation and signalling ability. The resulting endotoxin-tolerant state was found to protect mice against immunopathology in Gram-negative and Gram-positive infections, pointing to a role for AhR in contributing to host fitness.

Tryptophan metabolism activation by indoleamine 2,3-dioxygenase in adipose tissue of obese women: an attempt to maintain immune homeostasis and vascular tone.

Human obesity is characterized by chronic low-grade inflammation in white adipose tissue and is often associated with hypertension. The potential induction of indoleamine 2,3-dioxygenase-1 (IDO1), the rate-limiting enzyme in tryptophan/kynurenine degradation pathway, by proinflammatory cytokines, could be associated with these disorders but has remained unexplored in obesity. Using immunohistochemistry, we detected IDO1 expression in white adipose tissue of obese patients, and we focused on its contribution in the regulation of vascular tone and on its immunoregulatory effects. Concentrations of tryptophan and kynurenine were measured in sera of 36 obese and 15 lean women. The expression of IDO1 in corresponding omental and subcutaneous adipose tissues and liver was evaluated. Proinflammatory markers and T-cell subsets were analyzed in adipose tissue via the expression of CD14, IL-18, CD68, TNFα, CD3ε, FOXP3 [a regulatory T-cell (Treg) marker] and RORC (a Th17 marker). In obese subjects, the ratio of kynurenine to tryptophan, which reflects IDO1 activation, is higher than in lean subjects. Furthermore, IDO1 expression in both adipose tissues and liver is increased and is inversely correlated with arterial blood pressure. Inflammation is associated with a T-cell infiltration in obese adipose tissue, with predominance of Th17 in the omental compartment and of Treg in the subcutaneous depot. The Th17/Treg balance is decreased in subcutaneous fat and correlates with IDO1 activation. In contrast, in the omental compartment, despite IDO1 activation, the Th17/Treg balance control is impaired. Taken together, our results suggest that IDO1 activation represents a local compensatory mechanism to limit obesity-induced inflammation and hypertension.

The Kynurenine pathway is activated in human obesity and shifted toward kynurenine monooxygenase activation

This study characterized the kynurenine pathway (KP) in human obesity by evaluating circulating levels of kynurenines and the expression of KP enzymes in adipose tissue.

CD95L Cell Surface Cleavage Triggers a Prometastatic Signaling Pathway in Triple-Negative Breast Cancer

Triple-negative breast cancers (TNBC) lacking estrogen and progesterone receptors and HER2 amplification have a relatively high risk of metastatic dissemination, but the mechanistic basis for this risk is not understood. Here, we report that serum levels of CD95 ligand (CD95L) are higher in patients with TNBC than in other patients with breast cancer. Metalloprotease-mediated cleavage of CD95L expressed by endothelial cells surrounding tumors generates a gradient that promotes cell motility due to the formation of an unconventional CD95-containing receptosome called the motility-inducing signaling complex. The formation of this complex was instrumental for Nox3-driven reactive oxygen species generation. Mechanistic investigations revealed a Yes-Orai1-EGFR-PI3K pathway that triggered migration of TNBC cells exposed to CD95L. Our findings establish a prometastatic function for metalloprotease-cleaved CD95L in TNBCs, revisiting its role in carcinogenesis.

Kynurenine pathway metabolomics predicts and provides mechanistic insight into multiple sclerosis progression

Activation of the kynurenine pathway (KP) of tryptophan metabolism results from chronic inflammation and is known to exacerbate progression of neurodegenerative disease. To gain insights into the links between inflammation, the KP and multiple sclerosis (MS) pathogenesis, we investigated the KP metabolomics profile of MS patients. Most significantly, we found aberrant levels of two key KP metabolites, kynurenic acid (KA) and quinolinic acid (QA). The balance between these metabolites is important as it determines overall excitotoxic activity at the N-methyl-D-Aspartate (NMDA) receptor. We also identified that serum KP metabolic signatures in patients can discriminate clinical MS subtypes with high sensitivity and specificity. A C5.0 Decision Tree classification model discriminated the clinical subtypes of MS with a sensitivity of 91%. After validation in another independent cohort, sensitivity was maintained at 85%. Collectively, our studies suggest that abnormalities in the KP may be associated with the switch from early-mild stage MS to debilitating progressive forms of MS and that analysis of KP metabolites in MS patient serum may have application as MS disease biomarkers.

Use of PD-1 Targeting, Macrophage Infiltration, and IDO Pathway Activation in Sarcomas: A Phase 2 Clinical Trial

Importance There is a strong rationale for treating sarcomas with immunotherapy. Objective To assess the efficacy and safety of programmed cell death protein 1 (PD-1) targeting in combination with metronomic chemotherapy in sarcomas. Design, Setting, and Participants This was an open-label, multicenter, phase 2 study of 4 cohorts of patients with advanced soft-tissue sarcoma (STS), including leiomyosarcoma (LMS), undifferentiated pleomorphic sarcoma (UPS), other sarcomas (others), and gastrointestinal stromal tumor (GIST). All patients received 50 mg twice daily cyclophosphamide 1 week on and 1 week off and 200 mg of intravenous pembrolizumab every 3 weeks. Intervention or Exposure Pembrolizumab in combination with metronomic cyclophosphamide. Main Outcomes and Measures There was a dual primary end point, encompassing both the nonprogression and objective responses at 6 months per Response Evaluation Criteria in Solid Tumours (RECIST) v1.1 for LMS, UPS, and others and 6-month nonprogression for GIST. An objective response rate of 20% and/or a 6-month nonprogression rate of 60% were determined as reasonable objectives for treatment with meaningful effect. Correlative studies of immune biomarkers were planned from patient tumor and plasma samples. Results Between June 2015 and July 2016, 57 patients were included (median [range] age, 59.5 [18.5-84.0] years; 24 women [42%]); 50 patients were assessable for the efficacy end point. Three patients experienced tumor shrinkage, resulting in a partial response in a single solitary fibrous tumor. The 6-month nonprogression rates were 0%, 0%, 14.3% (95% CI, 1.8%-42.8%) for LMS, UPS, and others, respectively, and 11.1% (95% CI, 2.8%-48.3%) for GIST. The most frequent adverse events were grade 1 or 2 fatigue, diarrhea, and anemia. The only patient who experienced partial response was the only one with strong programmed cell death 1 ligand 1–positive staining in immune cell. Strong infiltration by macrophage expressing the inhibitory enzyme indoleamine 2,3-dioxygenase 1 (IDO1) was observed in the majority of cases. Moreover, a significant increase in the kynurenine to tryptophan ratio was observed in patient plasma samples during the study treatment. Conclusions and Relevance We found that PD-1 inhibition has limited activity in selected STS and GIST. This may be explained by an immunosuppressive tumor microenvironment resulting from macrophage infiltration and IDO1 pathway activation. Trial Registration clinicaltrials.gov Identifier: NCT02406781

Understanding the role of the kynurenine pathway in human breast cancer immunobiology

Breast cancer (BrCa) is the leading cause of cancer related death in women. While current diagnostic modalities provide opportunities for early medical intervention, significant proportions of breast tumours escape treatment and metastasize. Gaining increasing recognition as a factor in tumour metastasis is the local immuno-surveillance environment. Following identification of the role played by the enzyme indoleamine dioxygenase 1 (IDO1) in mediating maternal foetal tolerance, the kynurenine pathway (KP) of tryptophan metabolism has emerged as a key metabolic pathway contributing to immune escape. In inflammatory conditions activation of the KP leads to the production of several immune-modulating metabolites including kynurenine, kynurenic acid, 3-hydroxykynurenine, anthranilic acid, 3-hydroxyanthranilic acid, picolinic acid and quinolinic acid. KP over-activation was first described in BrCa patients in the early 1960s. More evidence has since emerged to suggest that the IDO1 is elevated in advanced BrCa patients and is associated with poor prognosis. Further, IDO1 positive breast tumours have a positive correlation with the density of immune suppressive Foxp3+ T regulatory cells and lymph node metastasis. The analysis of clinical microarray data in invasive BrCa compared to normal tissue showed, using two microarray databank (cBioportal and TCGA), that 86.3% and 91.4% BrCa patients have altered KP enzyme expression respectively. Collectively, these data highlight the key roles played by KP activation in BrCa, particularly in basal BrCa subtypes where expression of most KP enzymes was altered. Accordingly, the use of KP enzyme inhibitors in addition to standard chemotherapy regimens may present a viable therapeutic approach.

Accumulation of an endogenous tryptophan-derived metabolite in colorectal and breast cancers.

Tumor immune escape mechanisms are being regarded as suitable targets for tumor therapy. Among these, tryptophan catabolism plays a central role in creating an immunosuppressive environment, leading to tolerance to potentially immunogenic tumor antigens. Tryptophan catabolism is initiated by either indoleamine 2,3-dioxygenase (IDO-1/-2) or tryptophan 2,3-dioxygenase 2 (TDO2), resulting in biostatic tryptophan starvation and l-kynurenine production, which participates in shaping the dynamic relationship of the host’s immune system with tumor cells. Current immunotherapy strategies include blockade of IDO-1/-2 or TDO2, to restore efficient antitumor responses. Patients who might benefit from this approach are currently identified based on expression analyses of IDO-1/-2 or TDO2 in tumor tissue and/or enzymatic activity assessed by kynurenine/tryptophan ratios in the serum. We developed a monoclonal antibody targeting l-kynurenine as an in situ biomarker of IDO-1/-2 or TDO2 activity. Using Tissue Micro Array technology and immunostaining, colorectal and breast cancer patients were phenotyped based on l-kynurenine production. In colorectal cancer l-kynurenine was not unequivocally associated with IDO-1 expression, suggesting that the mere expression of tryptophan catabolic enzymes is not sufficiently informative for optimal immunotherapy.

Circulating Antibodies to IDO/THO Pathway Metabolites in Alzheimer's Disease

In Alzheimers disease, indoleamine 2,3-dioxygenase and tryptophan hydroxylase are known to induce an overproduction of neurotoxic compounds, such as quinolinic acid and 3-hydroxykynurenine from the former, and 5-hydroxytryptophol and 5-methoxytryptophol from the latter. Other compounds, such as kynurenic acid, serotonin, and melatonin are produced via the same pathways. An improved ELISA method identified circulating antibodies directed against these compounds, linked to proteins, as previously described for other chronic diseases. This describes how only the A isotype of circulating immunoglobulins recognized a pattern of conjugated tryptophan metabolites in the sera of Alzheimer patients. These data indirectly confirmed the involvement of tryptophan derivatives in the pathogenic processes of Alzheimers disease. Further studies are required to evaluate the relevance of these antibody patterns in monitoring this disease.

In vitro α-synuclein neurotoxicity and spreading among neurons and astrocytes using Lewy body extracts from Parkinson disease brains

Synucleinopathies are a group of diseases characterized by the presence of intracellular protein aggregates containing α-synuclein (α-syn). While α-syn aggregates have been shown to induce multimodal cellular dysfunctions, uptake and transport mechanisms remain unclear. Using high-content imaging on cortical neurons and astrocytes, we here define the kinetics of neuronal and astrocytic abnormalities induced by human-derived α-syn aggregates grounding the use of such system to identify and test putative therapeutic compounds. We then aimed at characterizing uptake and transport mechanisms using primary cultures of cortical neurons and astrocytes either in single well or in microfluidic chambers allowing connection between cells and cell-types. We report that astrocytes take up α-syn-aggregates far more efficiently than neurons through an endocytic event. We also highlight that active α-syn transport occurs between cells and any cell-types. Of special interest regarding the disease, we also show that uptake and spreading of α-syn from astrocytes to neurons can lead to neuronal death. Altogether, we here show that patients-derived α-synuclein aggregates, which are taken up by neurons and astrocytes, induce a differential endogenous response in the two cell types including a peculiar astrocytic toxic gain-of-function that leads to neuronal death.