Indoleamine 2,3-dioxygenase 1 activation in mature cDC1 promotes tolerogenic education of inflammatory cDC2 via metabolic communication.

Conventional dendritic cells (cDCs), cDC1 and cDC2, act both to initiate immunity and maintain self-tolerance. The tryptophan metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is used by cDCs in maintaining tolerance, but its role in different subsets remains unclear. At homeostasis, only mature CCR7+ cDC1 expressed IDO1 that was dependent on IRF8. Lipopolysaccharide treatment induced maturation and IDO1-dependent tolerogenic activity in isolated immature cDC1, but not isolated cDC2. However, both human and mouse cDC2 could induce IDO1 and acquire tolerogenic function when co-cultured with mature cDC1 through the action of cDC1-derived l-kynurenine. Accordingly, cDC1-specific inactivation of IDO1 in vivo exacerbated disease in experimental autoimmune encephalomyelitis. This study identifies a previously unrecognized metabolic communication in which IDO1-expressing cDC1 cells extend their immunoregulatory capacity to the cDC2 subset through their production of tryptophan metabolite l-kynurenine. This metabolic axis represents a potential therapeutic target in treating autoimmune demyelinating diseases.

Amniotic fluid stem cell-derived extracellular vesicles are independent metabolic units capable of modulating inflammasome activation in THP-1 cells.

An immunoregulatory role of stem cells, often mediated by their secretome, has been claimed by several studies. Stem cell-derived extracellular vesicles (EVs) are crucial components of the secretome. EVs, a heterogeneous group of membranous vesicles released by many cell types into the extracellular space, are now considered as an additional mechanism for intercellular communication. In this study, we aimed at investigating whether human amniotic stem cell-derived extracellular vesicles (HASC-EVs) were able to interfere with inflammasome activation in the THP-1 cell line. Two subsets of HASC-EVs were collected by sequential centrifugation, namely HASC-P10 and HASC-P100. We demonstrated that HASC-EVs were neither internalized into nor undertake a direct interaction with THP-1 cells. We showed that HASC-P10 and P100 were able to intrinsically produce ATP, which was further converted to adenosine by 5'-nucleotidase (CD73) and ectonucleoside triphosphate diphosphohydrolase-1 (CD39). We found that THP-1 cells conditioned with both types of HASC-EVs failed to activate the NLRP3/caspase-1/inflammasome platform in response to LPS and ATP treatment by a mechanism involving A2a adenosine receptor activation. These results support a role for HASC-EVs as independent metabolic units capable of modifying the cellular functions, leading to anti-inflammatory effects in monocytic cells.

Positive allosteric modulation of indoleamine 2,3-dioxygenase 1 restrains neuroinflammation.

l-tryptophan (Trp), an essential amino acid for mammals, is the precursor of a wide array of immunomodulatory metabolites produced by the kynurenine and serotonin pathways. The kynurenine pathway is a paramount source of several immunoregulatory metabolites, including l-kynurenine (Kyn), the main product of indoleamine 2,3-dioxygenase 1 (IDO1) that catalyzes the rate-limiting step of the pathway. In the serotonin pathway, the metabolite N-acetylserotonin (NAS) has been shown to possess antioxidant, antiinflammatory, and neuroprotective properties in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). However, little is known about the exact mode of action of the serotonin metabolite and the possible interplay between the 2 Trp metabolic pathways. Prompted by the discovery that NAS neuroprotective effects in EAE are abrogated in mice lacking IDO1 expression, we investigated the NAS mode of action in neuroinflammation. We found that NAS directly binds IDO1 and acts as a positive allosteric modulator (PAM) of the IDO1 enzyme in vitro and in vivo. As a result, increased Kyn will activate the ligand-activated transcription factor aryl hydrocarbon receptor and, consequently, antiinflammatory and immunoregulatory effects. Because NAS also increased IDO1 activity in peripheral blood mononuclear cells of a significant proportion of MS patients, our data may set the basis for the development of IDO1 PAMs as first-in-class drugs in autoimmune/neuroinflammatory diseases.

Microbiota-Associated HAF-EVs Regulate Monocytes by Triggering or Inhibiting Inflammasome Activation.

In pregnancy, human amniotic fluid extracellular vesicles (HAF-EVs) exert anti-inflammatory effects on T cells and on monocytes, supporting their immunoregulatory roles. The specific mechanisms are still not completely defined. The aim of this study was to investigate the ability of HAF-EVs, isolated from pregnant women who underwent amniocentesis and purified by gradient ultracentrifugation, to affect inflammasome activation in the human monocytes. Proteomic studies revealed that HAF-EV samples expressed several immunoregulatory molecules as well as small amounts of endotoxin. Surprisingly, metagenomic analysis shows the presence of specific bacterial strain variants associated with HAF-EVs as potential sources of the endotoxin. Remarkably, we showed that a single treatment of THP-1 cells with HAF-EVs triggered inflammasome activation, whereas the same treatment followed by LPS and ATP sensitization prevented inflammasome activation, a pathway resembling monocyte refractories. A bioinformatics analysis of microbiota-HAF-EVs functional pathways confirmed the presence of enzymes for endotoxin biosynthesis as well as others associated with immunoregulatory functions. Overall, these data suggest that HAF-EVs could serve as a source of the isolation of a specific microbiota during early pregnancy. Moreover, HAF-EVs could act as a novel system to balance immune training and tolerance by modulating the inflammasome in monocytes or other cells.

The Landscape of AhR Regulators and Coregulators to Fine-Tune AhR Functions.

The aryl-hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates numerous cellular responses. Originally investigated in toxicology because of its ability to bind environmental contaminants, AhR has attracted enormous attention in the field of immunology in the last 20 years. In addition, the discovery of endogenous and plant-derived ligands points to AhR also having a crucial role in normal cell physiology. Thus, AhR is emerging as a promiscuous receptor that can mediate either toxic or physiologic effects upon sensing multiple exogenous and endogenous molecules. Within this scenario, several factors appear to contribute to the outcome of gene transcriptional regulation by AhR, including the nature of the ligand as such and its further metabolism by AhR-induced enzymes, the local tissue microenvironment, and the presence of coregulators or specific transcription factors in the cell. Here, we review the current knowledge on the array of transcription factors and coregulators that, by interacting with AhR, tune its transcriptional activity in response to endogenous and exogenous ligands.

Tryptophan Metabolites at the Crossroad of Immune-Cell Interaction via the Aryl Hydrocarbon Receptor: Implications for Tumor Immunotherapy.

The Aryl hydrocarbon receptor (AhR) is a critical regulator of both innate and adaptive immune responses, with potent immunomodulatory effects that makes this receptor an attractive molecular target for novel therapeutics. Accumulating evidence indicates that diverse-both host's and microbial-tryptophan metabolites profoundly regulate the immune system in the host via AhR, promoting either tolerance or immunity, largely as a function of the qualitative and quantitative nature of the metabolites being contributed by either source. Additional findings indicate that host and microbiota-derived tryptophan metabolic pathways can influence the outcome of immune responses to tumors. Here, we review recent studies on the role and modalities of AhR activation by various ligands, derived from either host-cell or microbial-cell tryptophan metabolic pathways, in the regulation of immune responses. Moreover, we highlight potential implications of those ligands and pathways in tumor immunotherapy, with particular relevance to checkpoint-blockade immune intervention strategies.

Hops/Tmub1 Heterozygous Mouse Shows Haploinsufficiency Effect in Influencing p53-Mediated Apoptosis.

HOPS is a ubiquitin-like protein implicated in many aspects of cellular function including the regulation of mitotic activity, proliferation, and cellular stress responses. In this study, we focused on the complex relationship between HOPS and the tumor suppressor p53, investigating both transcriptional and non-transcriptional p53 responses. Here, we demonstrated that Hops heterozygous mice and mouse embryonic fibroblasts exhibit an impaired DNA-damage response to etoposide-induced double-strand breaks when compared to wild-type genes. Specifically, alterations in HOPS levels caused significant defects in the induction of apoptosis, including a reduction in p53 protein level and percentage of apoptotic cells. We also analyzed the effect of reduced HOPS levels on the DNA-damage response by examining the transcript profiles of p53-dependent genes, showing a suggestive deregulation of the mRNA levels for a number of p53-dependent genes. Taken together, these results show an interesting haploinsufficiency effect mediated by Hops monoallelic deletion, which appears to be enough to destabilize the p53 protein and its functions. Finally, these data indicate a novel role for Hops as a tumor-suppressor gene in DNA damage repair in mammalian cells.

Toll-like receptors as novel therapeutic targets for herpes simplex virus infection.

Seropositivity for HSV reaches more than 70% within the world population, and yet no approved vaccine exists. While HSV1 is responsible for keratitis, encephalitis, and labialis, HSV2 carriers have a high susceptibility to other STD infections, such as HIV. Induction of antiviral innate immune responses upon infection depends on a family of pattern recognition receptors called Toll-like receptors (TLR). TLRs bridge innate and adaptive immunity by sensing virus infection and activating antiviral immune responses. HSV adopts smart tricks to evade innate immunity and can also manipulate TLR signaling to evade the immune system or even confer destructive effects in favor of virus replication. Here, we review mechanisms by which HSV can trick TLR signaling to impair innate immunity. Then, we analyze the role of HSV-mediated molecular cues, in particular, NF-κB signaling, in promoting protective versus destructive effects of TLRs. Finally, TLR-based therapeutic opportunities with the goal of preventing or treating HSV infection will be discussed.

RhoA signaling through platelet P2Y1 receptor controls leukocyte recruitment in allergic mice.

BACKGROUND: Clinical studies reveal platelet activation in patients with asthma, allergic rhinitis, and eczema. This is distinct from platelet aggregation, which is critical for the maintenance of hemostasis and in which a role for platelet purinergic receptors is well documented. However, purines are also essential for inflammatory cell trafficking in animal models of allergic lung inflammation, which are known to be platelet dependent, yet the role of purines in the platelet activation accompanying inflammation is unknown. OBJECTIVES: We investigated whether the involvement of purine activation of platelets during allergic inflammation is distinct from purine involvement in platelet aggregation. METHODS: BALB/c mice were sensitized to ovalbumin and subsequent airway ovalbumin challenge. Bronchoalveolar lavage fluid was analyzed for inflammatory cells, and blood samples were assessed for platelet activation. The role of platelet purinergic receptors and associated signaling mechanisms (RhoA) were assessed. RESULTS: P2Y1, but not P2Y12 or P2X1, antagonism inhibited pulmonary leukocyte recruitment. The formation of platelet-leukocyte complexes in vivo and platelet/P-selectin-dependent polymorphonuclear cell migration in vitro were exclusively platelet P2Y1 receptor dependent. Furthermore, platelet P2Y1 activation resulted in RhoA activity in vivo after allergen challenge, and RhoA signaling in platelets through P2Y1 stimulation was required for platelet-dependent leukocyte chemotaxis in vitro. Leukocyte recruitment in thrombocytopenic mice remained suppressed after reinfusion of platelets pretreated with a P2Y1 antagonist or a Rho-associated kinase 1 inhibitor, confirming the crucial role of platelet P2Y1 receptor and subsequent activation of RhoA. CONCLUSION: RhoA signaling downstream of platelet P2Y1, but not P2Y12, represents a clear dichotomy in platelet activation during allergic inflammation versus hemostasis.

Amniotic fluid stem cell-derived extracellular vesicles educate type 2 conventional dendritic cells to rescue autoimmune disorders in a multiple sclerosis mouse model.

Dendritic cells (DCs) are essential orchestrators of immune responses and represent potential targets for immunomodulation in autoimmune diseases. Human amniotic fluid secretome is abundant in immunoregulatory factors, with extracellular vesicles (EVs) being a significant component. However, the impact of these EVs on dendritic cells subsets remain unexplored. In this study, we investigated the interaction between highly purified dendritic cell subsets and EVs derived from amniotic fluid stem cell lines (HAFSC-EVs). Our results suggest that HAFSC-EVs are preferentially taken up by conventional dendritic cell type 2 (cDC2) through CD29 receptor-mediated internalization, resulting in a tolerogenic DC phenotype characterized by reduced expression and production of pro-inflammatory mediators. Furthermore, treatment of cDC2 cells with HAFSC-EVs in coculture systems resulted in a higher proportion of T cells expressing the regulatory T cell marker Foxp3 compared to vehicle-treated control cells. Moreover, transfer of HAFSC-EV-treated cDC2s into an EAE mouse model resulted in the suppression of autoimmune responses and clinical improvement. These results suggest that HAFSC-EVs may serve as a promising tool for reprogramming inflammatory cDC2s towards a tolerogenic phenotype and for controlling autoimmune responses in the central nervous system, representing a potential platform for the study of the effects of EVs in DC subsets.

Endotoxin-Tolerance Mimicking to Study TLR in Promotion of Tolerogenic DCs and Tr1 Cells.

Dendritic cells (DCs) are key regulators of immunogenic and tolerogenic immune responses. Both these immune responses require DCs respectively to activate effector T cells or to induce their anergy and T regulatory activity. Modifications of DCs in the laboratory and several pharmacological agents can enhance and stabilize their tolerogenic properties. Recent evidences demonstrate that activation of specific toll-like receptors (TLRs) can be involved in induction of DCs with tolerogenic properties able to initiate T regulatory cell responses.In the present chapter, we show a detail protocol to obtain in vitro regulatory conventional DCs (cDCs) in response to repeated exposure to lipopolysaccharide (LPS), a ligand of TLR4, by mimicking the mechanism of endotoxin tolerance. Subsequently, the protective effect of cDCs' conditionate with LPS will be describe in in vivo inflammatory model of endotoxemia. Finally, we illustrate the method to study the ability of LPS-conditionate cDCs to promote T regulatory cells in ex vivo system.

Extracellular Vesicles in Aging: An Emerging Hallmark?

Extracellular vesicles (EVs) are membrane-enclosed particles secreted by cells and circulating in body fluids. Initially considered as a tool to dispose of unnecessary material, they are now considered an additional method to transmit cell signals. Aging is characterized by a progressive impairment of the physiological functions of tissues and organs. The causes of aging are complex and interconnected, but there is consensus that genomic instability, telomere erosion, epigenetic alteration, and defective proteostasis are primary hallmarks of the aging process. Recent studies have provided evidence that many of these primary stresses are associated with an increased release of EVs in cell models, able to spread senescence signals in the recipient cell. Additional investigations on the role of EVs during aging also demonstrated the great potential of EVs for the modulation of age-related phenotypes and for pro-rejuvenation therapies, potentially beneficial for many diseases associated with aging. Here we reviewed the current literature on EV secretion in senescent cell models and in old vs. young individual body fluids, as well as recent studies addressing the potential of EVs from different sources as an anti-aging tool. Although this is a recent field, the robust consensus on the altered EV release in aging suggests that altered EV secretion could be considered an emerging hallmark of aging.

A tryptophan metabolite prevents depletion of circulating endothelial progenitor cells in systemic low-grade inflammation.

Background: Chronic systemic inflammation reduces the bioavailability of circulating endothelial progenitor cells (EPCs). Indoleamine 2,3-dioxygenase 1 (IDO1), a key enzyme of immune tolerance catalyzing the initial step of tryptophan degradation along the so-called l-kynurenine (l-kyn) pathway, that is induced by inflammatory stimuli and exerts anti-inflammatory effects. A specific relationship between IDO1 activity and circulating EPC numbers has not yet been investigated. Methods: In this study, circulating EPCs were examined in mice treated with low doses of lipopolysaccharide (LPS) to mimic low-grade inflammation. Moreover, the association between IDO1 activity and circulating EPCs was studied in a cohort of 277 patients with variable systemic low-grade inflammation. Results: Repeated low doses of LPS caused a decrease in circulating EPCs and l-kyn supplementation, mimicking IDO1 activation, significantly increased EPC numbers under homeostatic conditions preventing EPC decline in low-grade endotoxemia. Accordingly, in patients with variable systemic low-grade inflammation, there was a significant interaction between IDO1 activity and high-sensitivity C-reactive protein (hs-CRP) in predicting circulating EPCs, with high hs-CRP associated with significantly lower EPCs at low IDO1 activity but not at high IDO1 activity. Interpretation: Overall, these findings demonstrate that systemic low-grade inflammation reduces circulating EPCs. However, high IDO1 activity and l-kyn supplementation limit circulating EPC loss in low-grade inflammation.

Liver gene therapy with intein-mediated F8 trans-splicing corrects mouse haemophilia A.

Liver gene therapy with adeno-associated viral (AAV) vectors is under clinical investigation for haemophilia A (HemA), the most common inherited X-linked bleeding disorder. Major limitations are the large size of the F8 transgene, which makes packaging in a single AAV vector a challenge, as well as the development of circulating anti-F8 antibodies which neutralise F8 activity. Taking advantage of split-intein-mediated protein trans-splicing, we divided the coding sequence of the large and highly secreted F8-N6 variant in two separate AAV-intein vectors whose co-administration to HemA mice results in the expression of therapeutic levels of F8 over time. This occurred without eliciting circulating anti-F8 antibodies unlike animals treated with the single oversized AAV-F8 vector under clinical development. Therefore, liver gene therapy with AAV-F8-N6 intein should be considered as a potential therapeutic strategy for HemA.

Anakinra restores cellular proteostasis by coupling mitochondrial redox balance to autophagy.

Autophagy selectively degrades aggregation-prone misfolded proteins caused by defective cellular proteostasis. However, the complexity of autophagy may prevent the full appreciation of how its modulation could be used as a therapeutic strategy in disease management. Here, we define a molecular pathway through which recombinant IL-1 receptor antagonist (IL-1Ra, anakinra) affects cellular proteostasis independently from the IL-1 receptor (IL-1R1). Anakinra promoted H2O2-driven autophagy through a xenobiotic sensing pathway involving the aryl hydrocarbon receptor that, activated through the indoleamine 2,3-dioxygenase 1-kynurenine pathway, transcriptionally activated NADPH oxidase 4 independent of the IL-1R1. By coupling the mitochondrial redox balance to autophagy, anakinra improved the dysregulated proteostasis network in murine and human cystic fibrosis. We anticipate that anakinra may represent a therapeutic option in addition to its IL-1R1-dependent antiinflammatory properties by acting at the intersection of mitochondrial oxidative stress and autophagy with the capacity to restore conditions in which defective proteostasis leads to human disease.

3-hydroxy-L-kynurenamine is an immunomodulatory biogenic amine.

Tryptophan catabolism is a major metabolic pathway utilized by several professional and non-professional antigen presenting cells to maintain immunological tolerance. Here we report that 3-hydroxy-L-kynurenamine (3-HKA) is a biogenic amine produced via an alternative pathway of tryptophan metabolism. In vitro, 3-HKA has an anti-inflammatory profile by inhibiting the IFN-γ mediated STAT1/NF-κΒ pathway in both mouse and human dendritic cells (DCs) with a consequent decrease in the release of pro-inflammatory chemokines and cytokines, most notably TNF, IL-6, and IL12p70. 3-HKA has protective effects in an experimental mouse model of psoriasis by decreasing skin thickness, erythema, scaling and fissuring, reducing TNF, IL-1ß, IFN-γ, and IL-17 production, and inhibiting generation of effector CD8+ T cells. Similarly, in a mouse model of nephrotoxic nephritis, besides reducing inflammatory cytokines, 3-HKA improves proteinuria and serum urea nitrogen, overall ameliorating immune-mediated glomerulonephritis and renal dysfunction. Overall, we propose that this biogenic amine is a crucial component of tryptophan-mediated immune tolerance.

The cellular prion protein beyond prion diseases.

The cellular prion protein (PrPC), a cell surface glycoprotein originally identified for its central role in prion diseases (also called transmissible spongiform encephalopathies), has recently been implicated in the pathogenesis of other neurodegenerative disorders, such as Alzheimer’s and Parkinson’s diseases, by acting as a toxicity-transducing receptor for different misfolded protein isoforms, or in some case by exerting neuroprotective effects. Interestingly, PrPC has also been reported to play unexpected functions outside the nervous system, for example by contributing to myelin homeostasis, regulating specific processes of the immune system and participating in various aspects of cancer progression. Collectively, these observations point to a much broader role for PrPC in physiological and disease processes than originally assumed. In this manuscript, we provide an overview of what is known about the role of PrPC beyond prion disorders and discuss the potential implications of targeting this protein in different diseases.

Pharmacologic Induction of Endotoxin Tolerance in Dendritic Cells by L-Kynurenine.

Endotoxin tolerance aims at opposing hyperinflammatory responses to lipopolysaccharide (LPS) exposure. The aryl hydrocarbon receptor (AhR) participates in protection against LPS-mediated tissue damage, as it plays a necessary role in restraining the proinflammatory action of IL-1ß and TNF-α while fostering the expression of protective TGF-ß. TGF-ß, in turn, promotes durable expression of the immune regulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1). IDO1 degrades L-tryptophan to L-kynurenine-an activating ligand for AhR-thus establishing a feed-forward loop. In this study, we further demonstrate that L-kynurenine also promotes the dissociation of the Src kinase-AhR cytosolic complex, leading to the activation of both genomic and non-genomic events in conventional dendritic cells (cDCs) primed with LPS. Specifically, the Src kinase, by phosphorylating the downstream target IDO1, triggers IDO1's signaling ability, which results in enhanced production of TGF-ß, an event key to establishing full endotoxin tolerance. We demonstrated that exogenous L-kynurenine can substitute for the effects of continued or repeated LPS exposure and that the AhR-Src-IDO1 axis represents a critical step for the transition from endotoxin susceptibility to tolerance. Moreover, much like fully endotoxin-tolerant dendritic cells (DCs) (i.e., treated twice with LPS in vitro), DCs-treated once with LPS in vitro and then with kynurenine-confer resistance on naïve recipients to an otherwise lethal LPS challenge. This may have clinical implications under conditions in which pharmacologically induced onset of endotoxin tolerance is a therapeutically desirable event.

The role of platelets in mediating a response to human influenza infection.

Influenza infection increases the incidence of myocardial infarction but the reason is unknown. Platelets mediate vascular occlusion through thrombotic functions but are also recognized to have immunomodulatory activity. To determine if platelet processes are activated during influenza infection, we collected blood from 18 patients with acute influenza infection. Microscopy reveals activated platelets, many containing viral particles and extracellular-DNA associated with platelets. To understand the mechanism, we isolate human platelets and treat them with influenza A virus. Viral-engulfment leads to C3 release from platelets as a function of TLR7 and C3 leads to neutrophil-DNA release and aggregation. TLR7 specificity is confirmed in murine models lacking the receptor, and platelet depletion models support platelet-mediated C3 and neutrophil-DNA release post-influenza infection. These findings demonstrate that the initial intrinsic defense against influenza is mediated by platelet-neutrophil cross-communication that tightly regulates host immune and complement responses but can also lead to thrombotic vascular occlusion.