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Invasive fungal infections are a major cause of disease and death in immunocompromised hosts, including patients undergoing allogeneic hematopoietic stem cell transplant (HSCT). Recovery of adaptive immunity after HSCT correlates strongly with recovery from fungal infection. Using initial selection of lymphocytes expressing the activation marker CD137 after fungal stimulation, we rapidly expanded a population of mainly CD4+ T cells with potent antifungal characteristics, including production of tumor necrosis factor α, interferon γ, interleukin-17, and granulocyte-macrophage colony stimulating factor. Cells were manufactured using a fully good manufacturing practice–compliant process. In vitro, the T cells responded to fungal antigens presented on fully and partially HLA-DRB1 antigen–matched presenting cells, including when the single common DRB1 antigen was allelically mismatched. Administration of antifungal T cells lead to reduction in the severity of pulmonary and cerebral infection in an experimental mouse model of Aspergillus. These data support the establishment of a bank of cryopreserved fungus-specific T cells using normal donors with common HLA DRB1 molecules and testing of partially HLA-matched third-party donor fungus-specific T cells as a potential therapeutic in patients with invasive fungal infection after HSCT.

The ability to predict invasive fungal infections (IFI) in patients with hematological malignancies is fundamental for successful therapy. Although gut dysbiosis is known to occur in hematological patients, whether airway dysbiosis also contributes to the risk of IFI has not been investigated. Nasal and oropharyngeal swabs were collected for functional microbiota characterization in 173 patients with hematological malignancies recruited in a multicenter, prospective, observational study and stratified according to the risk of developing IFI. A lower microbial richness and evenness were found in the pharyngeal microbiota of high-risk patients that were associated with a distinct taxonomic and metabolic profile. A murine model of IFI provided biologic plausibility for the finding that loss of protective anaerobes, such as Clostridiales and Bacteroidetes, along with an apparent restricted availability of tryptophan, is causally linked to the risk of IFI in hematologic patients and indicates avenues for antimicrobial stewardship and metabolic reequilibrium in IFI. This work was supported by FunMeta Project (ERC-2011-AdG 293714), MicroTher(ERC-2018-PoC-813099), and Gilead (IN-IT-131-4525-518872.9) to L.R. and the GrantAgency of the Czech Republic (GACR No 17-24592Y) and the Czech Ministry ofEducation, Youth and Sports (CETOCOEN PLUS CZ.02.1.01/0.0/0.0/15_003/0000469;LM2015051 and CETOCOEN EXCELLENCE Teaming 2 project CZ.02.1.01/0.0/0.0/18_046/0015975; and Horizon2020 project 857560) to Z.S.

Primary sclerosing cholangitis (PSC) is a long-term liver disease characterized by a progressive course of cholestasis with liver inflammation and fibrosis. Intestinal barrier dysfunction has been implicated in the pathogenesis of PSC. According to the “leaky gut” hypothesis, gut inflammation alters the permeability of the intestinal mucosa, with the translocation of gut-derived products that enter the enterohepatic circulation and cause hepatic inflammation. Thus, the administration of molecules that preserve epithelial barrier integrity would represent a promising therapeutic strategy. Indole-3-carboxaldehyde (3-IAld) is a microbial-derived product working at the interface between the host and the microbiota and is able to promote mucosal immune homeostasis in a variety of preclinical settings. Herein, by resorting to a murine model of PSC, we found that 3-IAld formulated for localized delivery in the gut alleviates hepatic inflammation and fibrosis by modulating the intestinal microbiota and activating the aryl hydrocarbon receptor-IL-22 axis to restore mucosal integrity. This study points to the therapeutic potential of 3-IAld in liver pathology.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene encoding for the ion channel Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Long considered a lung disease for the devastating impact on the respiratory function, the recent diagnostic and therapeutic advances have shed the light on the extra-pulmonary manifestations of CF, including gastrointestinal, hepatobiliary and pancreatic symptoms. We have previously demonstrated that thymosin alpha1 (Tα1), a naturally occurring immunomodulatory peptide, displays multi-sided beneficial effects in CF that concur in ameliorating the lung inflammatory pathology. In the present study, by resorting to murine models of gut inflammation with clinical relevance for CF patients, we demonstrate that Tα1 can also have beneficial effects in extrapulmonary pathology. Specifically, Tα1 restored barrier integrity and immune homeostasis in the inflamed gut of CF mice as well as in mice with the metabolic syndrome, a disorder that may arise in CF patients with high caloric intake despite pancreatic sufficiency. The protective effects of Tα1 also extended to pancreas and liver, further emphasizing the beneficial effects of Tα1 in extra-pulmonary complications of CF. By performing wide-ranging multi-organ anti-inflammatory effects, Tα1 could potentially integrate current therapeutic approaches to tackle the complex symptomatology of CF disease.

The chemical processes taking place in humans intersects the myriad of metabolic pathways occurring in commensal microorganisms that colonize the body to generate a complex biochemical network that regulates multiple aspects of human life. The role of tryptophan (Trp) metabolism at the intersection between the host and microbes is increasingly being recognized, and multiple pathways of Trp utilization in either direction have been identified with the production of a wide range of bioactive products. It comes that a dysregulation of Trp metabolism in either the host or the microbes may unbalance the production of metabolites with potential pathological consequences. The ability to redirect the Trp flux to restore a homeostatic production of Trp metabolites may represent a valid therapeutic strategy for a variety of pathological conditions, but identifying metabolic checkpoints that could be exploited to manipulate the Trp metabolic network is still an unmet need. In this review, we put forward the hypothesis that pyridoxal 5′-phosphate (PLP)-dependent enzymes, which regulate multiple pathways of Trp metabolism in both the host and in microbes, might represent critical nodes and that modulating the levels of vitamin B6, from which PLP is derived, might represent a metabolic checkpoint to re-orienteer Trp flux for therapeutic purposes.

The organisms of most domains of life have adapted to circadian changes of the environment and regulate their behavior and physiology accordingly. A particular case of such paradigm is represented by some types of host-pathogen interaction during infection. Indeed, not only some hosts and pathogens are each endowed with their own circadian clock, but they are also influenced by the circadian changes of the other with profound consequences on the outcome of the infection. It comes that daily fluctuations in the availability of resources and the nature of the immune response, coupled with circadian changes of the pathogen, may influence microbial virulence, level of colonization and damage to the host, and alter the equilibrium between commensal and invading microorganisms. In the present review, we discuss the potential relevance of circadian rhythms in human bacterial and fungal pathogens, and the consequences of circadian changes of the host immune system and microbiome on the onset and development of infection. By looking from the perspective of the interplay between host and microbes circadian rhythms, these concepts are expected to change the way we approach human infections, not only by predicting the outcome of the host-pathogen interaction, but also by indicating the best time for intervention to potentiate the anti-microbial activities of the immune system and to weaken the pathogen when its susceptibility is higher.

Cystic fibrosis (CF) is a rare genetic disorder caused by a defect in the ion channel Cystic Fibrosis Transmembrane conductance Regulator (CFTR), resulting in ionic imbalance of surface fluid. Although affecting multiple organs, the progressive deterioration of respiratory function by recurrent infections and chronic inflammation represents the main cause of morbidity and mortality in CF patients. The development of modulators targeting the basic defect of CFTR has represented a major breakthrough in CF therapy, but the impact on inflammation has remained enigmatic. The emerging scenario taking hold in the field points to inflammation as a major, somehow missed, therapeutic target for prevention of lung decline. Not surprisingly, the development of anti-inflammatory drugs is taking its share in the drug development pipeline. But the path is not straightforward and targeting inflammation should be balanced with the increased risk of infection. The strategy to restore the homeostatic regulation of inflammation to efficiently respond to infection while preventing lung damage needs to be based on identifying and targeting endogenous immunoregulatory pathways that are defective in CF. We herein provide an overview of anti-inflammatory drugs currently approved or under investigation in CF patients, and present our recent studies on how the knowledge on defective immune pathways in CF may translate into innovative and selective anti-inflammatory therapeutics. Through the discovery of naturally occurring molecules or their synthetic mimics, this review emphasizes the critical importance of selectively targeting key inflammatory pathways to preserve immunocompetence in CF patients.

The microbiome, i.e., the communities of microbes that inhabit the surfaces exposed to the external environment, participates in the regulation of host physiology, including the immune response against pathogens. At the same time, the immune response shapes the microbiome to regulate its composition and function. How the crosstalk between the immune system and the microbiome regulates the response to fungal infection has remained relatively unexplored. We have previously shown that strict anaerobes protect from infection with the opportunistic fungus Aspergillus fumigatus by counteracting the expansion of pathogenic Proteobacteria. By resorting to immunodeficient mouse strains, we found that the lung microbiota could compensate for the lack of B and T lymphocytes in Rag1–/– mice by skewing the composition towards an increased abundance of protective anaerobes such as Clostridia and Bacteroidota. Conversely, NSG mice, with major defects in both the innate and adaptive immune response, showed an increased susceptibility to infection associated with a low abundance of strict anaerobes and the expansion of Proteobacteria. Further exploration in a murine model of chronic granulomatous disease, a primary form of immunodeficiency characterized by defective phagocyte NADPH oxidase, confirms the association of lung unbalance between anaerobes and Proteobacteria and the susceptibility to aspergillosis. Consistent changes in the lung levels of short-chain fatty acids between the different strains support the conclusion that the immune system and the microbiota are functionally intertwined during Aspergillus infection and determine the outcome of the infection.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that compromise its chloride-channel activity. The most common mutation, p.Phe508del, results in the production of a misfolded CFTR protein, which has residual channel activity but is prematurely degraded. Because of the inherent complexity of the pathogenetic mechanisms involved in CF —which include impaired chloride permeability and persistent lung inflammation—a multidrug approach is required for efficacious CF therapy. To date, no individual, drug with pleiotropic beneficial effects for CF is available. Here we report on the ability of thymosin alpha 1 (Tα1)—a naturally occurring polypeptide with an excellent safety profile in the clinic when used as an adjuvant or an immunotherapeutic agent—to rectify the multiple tissue defects in CF mice as well as in cells from subjects with the p.Phe508del mutation. Tα1 displayed two combined properties that favorably opposed CF symptomatology; namely, it reduced inflammation and increased CFTR maturation, stability and activity. By virtue of this two-pronged action, Tα1 offers a strong potential to be an efficacious single molecule-based therapeutic agent in CF.

This study demonstrates that thymosin β4 stabilizes HIF-1a to promote autophagy and up-regulate genes involved in tissue and mucosal barrier protection in chronic granulomatous disease., Chronic granulomatous disease (CGD) is a genetic disorder of the NADPH oxidase characterized by increased susceptibility to infections and hyperinflammation associated with defective autophagy and increased inflammasome activation. Herein, we demonstrate that thymosin β4 (Tβ4), a g-actin sequestering peptide with multiple and diverse intracellular and extracellular activities affecting inflammation, wound healing, fibrosis, and tissue regeneration, promoted in human and murine cells noncanonical autophagy, a form of autophagy associated with phagocytosis and limited inflammation via the death-associated protein kinase 1. We further show that the hypoxia inducible factor-1 (HIF-1)α was underexpressed in CGD but normalized by Tβ4 to promote autophagy and up-regulate genes involved in mucosal barrier protection. Accordingly, inflammation and granuloma formation were impaired and survival increased in CGD mice with colitis or aspergillosis upon Tβ4 treatment or HIF-1α stabilization. Thus, the promotion of endogenous pathways of inflammation resolution through HIF-1α stabilization is druggable in CGD by Tβ4.

Thymosin alpha1 (Tα1), an endogenous peptide first isolated from the thymic tissue in the mid-sixties, has gained considerable attention for its immunostimulatory activity that led to its application to diverse pathological conditions, including cancer. Studies in animal models and human patients have shown promising results in different types of malignancies, especially when Tα1 was used in combination with other chemo- and immune therapies. For this reason, the advancements in our knowledge on the adjuvant role of Tα1 have moved in parallel with the development of novel cancer therapies in a way that Tα1 was integrated to changing paradigms and protocols, and tested for increased efficacy and safety. Cancer immunotherapy has recently experienced a tremendous boost following the development and clinical application of immune checkpoint inhibitors. By unleashing the full potential of the adaptive immune response, checkpoint inhibitors were expected to be very effective against tumors, but it soon became clear that a widespread and successful application was not straightforward and shortcomings in efficacy and safety clearly emerged. This scenario led to the development of novel concepts in immunotherapy and the design of combination protocols to overcome these limitations, thus opening up novel opportunities for Tα1 application. Herein, we summarize in a historical perspective the use of Tα1 in cancer, with particular reference to melanoma, hepatocellular carcinoma and lung cancer. We will discuss the current limitations of checkpoint inhibitors in clinical practice and the mechanisms at the basis of a potential application of Tα1 in combination protocols.

Vulvovaginal candidiasis (VVC) is a common mucosal infection caused by Candida spp., most frequently by Candida albicans, which may become recurrent and severely impacting the quality of life of susceptible women. Although it is increasingly being recognized that mucosal damage is mediated by an exaggerated inflammatory response, current therapeutic approaches are only based on antifungals that may relieve the symptomatology, but fail to definitely prevent recurrences. The unrestrained activation of the NLRP3 inflammasome with continuous production of IL-1β and recruitment of neutrophils is recognized as a pathogenic factor in VVC. We have previously shown that IL-22 is required to dampen pathogenic inflammasome activation in VVC via the NLRC4/IL-1Ra axis. However, IL-22 also regulates IL-18, a product of the inflammasome activity that regulates IL-22 expression. Here we describe a cross-regulatory circuit between IL-18 and IL-22 in murine VVC that is therapeutically druggable. We found that IL-18 production was dependent on IL-22 and NLRC4, and that IL-18, in turn, contributes to IL-22 activity. Like in IL-22 deficiency, IL-18 deficiency was associated with an increased susceptibility to VVC and unbalanced Th17/Treg response, suggesting that IL-18 can regulate both the innate and the adaptive responses to the fungus. Administration of the microbial metabolite indole-3-aldehyde, known to stimulate the production of IL-22 via the aryl hydrocarbon receptor (AhR), promoted IL-18 expression and protection against Candida infection. Should low levels of IL-18 be demonstrated in the vaginal fluids of women with recurrent VVC, targeting the AhR/IL-22/IL-18 pathway could be exploited for future therapeutic approaches in VVC. This study suggests that a deeper understanding of the mechanisms regulating inflammasome activity may lead to the identification of novel targets for intervention in VVC.

Celiac disease (CD) is an immune-mediated disorder triggered by the ingestion of gluten and characterized by reversible small-bowel mucosal atrophy in genetically predisposed subjects. Although the prevalence of CD has increased, many aspects of this pathology are still unrecognized. Candida albicans, a commensal of the human gastrointestinal tract, has been linked to CD for a long time based, among others, upon the observation of similarity between the fungal wall component, hyphal wall protein 1, and CD-related gliadin T-cell epitopes. We have recently demonstrated that Candida may switch from commensal to pathogen contingent upon several players, including mast cells, key sentinels of the immune system at the interface between the environment and the host, and the pleiotropic cytokine IL-9. However, other factors are likely to play a role by altering the balance between inflammation and tolerance. In this regard, tryptophan and its metabolites are increasingly being recognized in promoting mucosal homeostasis by balancing the immune response to external cues. Based on these premises, we will discuss how the output of Candida colonization in the gut is highly contextual, being determined at the intersection of many immunological (IL-9/mast cells) and metabolic (tryptophan) pathways that ultimately dictate the Candida commensalism vs. pathogenicity in CD, thus paving the way for novel therapeutic opportunities in CD.

Liver regeneration offers a distinctive opportunity to study cell proliferation in vivo. Mammalian silent information regulator 1 (SIRT1), a NAD+-dependent histone deacetylase, is an important regulator of various cellular processes, including proliferation, metabolism, and circadian rhythms. In the liver, SIRT1 coordinates the circadian oscillation of clock-controlled genes, including genes that encode enzymes involved in metabolic pathways. We performed partial hepatectomy in WT and liver-specific Sirt1-deficient mice and analyzed the expression of cell cycle regulators in liver samples taken at different times during the regenerative process, by real time PCR, Western blotting analysis, and immunohistochemistry. Lipidomic analysis was performed in the same samples by MS/HPLC. We showed that G1/S progression was significantly affected by absence of SIRT1 in the liver, as well as circadian gene expression. This was associated to lipid accumulation due to defective fatty acid beta-oxidation. Our study revealed for the first time the importance of SIRT1 in the regulation of hepatocellular proliferation, circadian rhythms, and lipid metabolism during liver regeneration in mice. These results represent an additional step toward the characterization of SIRT1 function in the liver.

This study demonstrates that Tα1 protects mice from anti–CTLA-4–induced colitis and sustains its antitumor activity, thus suggesting that Tα1 may be used in combination protocols., The advent of immune checkpoint inhibitors has represented a major boost in cancer therapy, but safety concerns are increasingly being recognized. Indeed, although beneficial at the tumor site, unlocking a safeguard mechanism of the immune response may trigger autoimmune-like effects at the periphery, thus making the safety of immune checkpoint inhibitors a research priority. Herein, we demonstrate that thymosin α1 (Tα1), an endogenous peptide with immunomodulatory activities, can protect mice from intestinal toxicity in a murine model of immune checkpoint inhibitor–induced colitis. Specifically, Tα1 efficiently prevented immune adverse pathology in the gut by promoting the indoleamine 2,3-dioxygenase (IDO) 1–dependent tolerogenic immune pathway. Notably, Tα1 did not induce IDO1 in the tumor microenvironment, but rather modulated the infiltration of T-cell subsets by inverting the ratio between CD8+ and Treg cells, an effect that may depend on Tα1 ability to regulate the differentiation and chemokine expression profile of DCs. Thus, through distinct mechanisms that are contingent upon the context, Tα1 represents a plausible candidate to improve the safety/efficacy profile of immune checkpoint inhibitors.

Thymosin alpha 1 (Tα1) is a naturally occurring polypeptide of 28 amino acids, whose mechanism of action is thought to be related to its ability to signal through innate immune receptors. Tα1 (ZADAXIN®) is used worldwide for treating viral infections, immunodeficiencies, and malignancies. Owing to its ability to activate the tolerogenic pathway of tryptophan catabolism - via the immunoregulatory enzyme indoleamine 2,3-dioxygenase - Tα1 potentiates immune tolerance mechanisms, breaking the vicious circle that perpetuates chronic inflammation in response to a variety of infectious noxae.Tα1 has never been studied in Cystic fibrosis (CF) in which the hyperinflammatory state is associated with early and nonresolving activation of innate immunity, which impairs microbial clearance and promotes a self-sustaining condition of progressive lung damage. Optimal CF treatments should, indeed, not only rescue CF transmembrane conductance regulator protein localization and functionality but also alleviate the associated hyperinflammatory pathology. Because of the inherent complexity of the pathogenetic mechanisms, a multidrug approach is required.By providing a multipronged attack against CF, i.e. restraining inflammation and correcting the basic defect, Tα1 favorably opposed CF symptomatology in preclinical relevant disease settings, thus suggesting its possible exploitation for 'real-life' clinical efficacy in CF. This could represent a major conceptual advance in the CF field, namely the proposal of a drug with the unique activity to correct CFTR defects through regulation of inflammation.

The liver is the most important organ in cholesterol metabolism, which is instrumental in regulating cell proliferation and differentiation. The gene Tm7sf2 codifies for 3 β-hydroxysterol-Δ14-reductase (C14-SR), an endoplasmic reticulum resident protein catalyzing the reduction of C14-unsaturated sterols during cholesterol biosynthesis from lanosterol. In this study we analyzed the role of C14-SR in vivo during cell proliferation by evaluating liver regeneration in Tm7sf2 knockout (KO) and wild-type (WT) mice. Tm7sf2 KO mice showed no alteration in cholesterol content. However, accumulation and delayed catabolism of hepatic triglycerides was observed, resulting in persistent steatosis at all times post hepatectomy. Moreover, delayed cell cycle progression to the G1/S phase was observed in Tm7sf2 KO mice, resulting in reduced cell division at the time points examined. This was associated to abnormal ER stress response, leading to alteration in p53 content and, consequently, induction of p21 expression in Tm7sf2 KO mice. In conclusion, our results indicate that Tm7sf2 deficiency during liver regeneration alters lipid metabolism and generates a stress condition, which, in turn, transiently unbalances hepatocytes cell cycle progression.

Mammalian sirtuins and the circadian clock appear to be intimately linked. The circadian clock is able to modulate the activity of SIRT1 while SIRT1 also deacetylates specific elements of the clock machinery and modifies histones to regulate gene expression. In this chapter, we review the current understanding of the field on the interrelationship between SIRT1 and the circadian clock machinery, how this regulation occurs, and what other possible functions the sirtuins could have in circadian biology. This chapter also discusses the critical metabolic cues that are in place to regulate enzymes involved in histone modifications and subsequent regulation of gene expression. The role of the clock in regulating metabolic state of the cell is also a concept that will be explored, as the clock-dependent control over metabolism is far more extensive than originally thought. The concept that metabolism and epigenetics are closely linked opens new directions that need to be explored in terms of circadian metabolic regulation and how this modulates a host of epigenetic modifying enzymes and chromatin remodelers.

Immune parameters change with time of day and disruption of circadian rhythms has been linked to inflammatory pathologies. A circadian-clock-controlled immune system might allow an organism to anticipate daily changes in activity and feeding and the associated risk of infection or tissue damage to the host. Responses to bacteria have been shown to vary depending on time of infection, with mice being more at risk of sepsis when challenged ahead of their activity phase. Studies highlight the extent to which the molecular clock, most notably the core clock proteins BMAL1, CLOCK, and REV-ERBα, control fundamental aspects of the immune response. Examples include the BMAL1:CLOCK heterodimer regulating toll-like receptor 9 (TLR9) expression and repressing expression of the inflammatory monocyte chemokine ligand (CCL2) as well as REV-ERBα suppressing the induction of interleukin-6. Understanding the daily rhythm of the immune system could have implications for vaccinations and how we manage infectious and inflammatory diseases.

Accumulating evidence highlights intriguing interplays between circadian and metabolic pathways. We show that PER2 directly and specifically represses PPARγ, a nuclear receptor critical in adipogenesis, insulin sensitivity, and inflammatory response. PER2-deficient mice display altered lipid metabolism with drastic reduction of total triacylglycerol and nonesterified fatty acids. PER2 exerts its inhibitory function by blocking PPARγ recruitment to target promoters and thereby transcriptional activation. Whole-genome microarray profiling demonstrates that PER2 dictates the specificity of PPARγ transcriptional activity. Indeed, lack of PER2 results in enhanced adipocyte differentiation of cultured fibroblasts. PER2 targets S112 in PPARγ, a residue whose mutation has been associated with altered lipid metabolism. Lipidomic profiling demonstrates that PER2 is necessary for normal lipid metabolism in white adipocyte tissue. Our findings support a scenario in which PER2 controls the proadipogenic activity of PPARγ by operating as its natural modulator, thereby revealing potential avenues of pharmacological and therapeutic intervention. © 2010 Elsevier Inc.

Major mood disorders have been linked to abnormalities in circadian rhythms, leading to disturbances in sleep, mood, temperature, and hormonal levels. We provide evidence that ketamine, a drug with rapid antidepressant effects, influences the function of the circadian molecular machinery. Ketamine modulates CLOCK:BMAL1-mediated transcriptional activation when these regulators are ectopically expressed in NG108-15 neuronal cells. Inhibition occurs in a dose-dependent manner and is attenuated after treatment with the GSK3β antagonist SB21673. We analyzed the effect of ketamine on circadian gene expression and observed a dose-dependent reduction in the amplitude of circadian transcription of the Bmal1, Per2, and Cry1 genes. Finally, chromatin-immunoprecipitation analyses revealed that ketamine altered the recruitment of the CLOCK:BMAL1 complex on circadian promoters in a time-dependent manner. Our results reveal a yet unsuspected molecular mode of action of ketamine and thereby may suggest possible pharmacological antidepressant strategies.