Giorgia Renga

IL-37 inhibits inflammasome activation and disease severity in murine aspergillosis

Since IL-37 transgenic mice possesses broad anti-inflammatory properties, we assessed whether recombinant IL-37 affects inflammation in a murine model of invasive pulmonary aspergillosis. Recombinant human IL-37 was injected intraperitoneally into mice prior to infection and the effects on lung inflammation and inflammasome activation were evaluated. IL-37 markedly reduced NLRP3-dependent neutrophil recruitment and steady state mRNA levels of IL-1β production and mitigated lung inflammation and damage in a relevant clinical model, namely aspergillosis in mice with cystic fibrosis. The anti-inflammatory activity of IL-37 requires the IL-1 family decoy receptor TIR-8/SIGIRR. Thus, by preventing activation of the NLRP3 inflammasome and reducing IL-1β secretion, IL-37 functions as a broad spectrum inhibitor of the innate response to infection-mediated inflammation, and could be considered to be therapeutic in reducing the pulmonary damage due to non-resolving Aspergillus infection and disease.

A mast cell-ILC2-Th9 pathway promotes lung inflammation in cystic fibrosis

T helper 9 (Th9) cells contribute to lung inflammation and allergy as sources of interleukin-9 (IL-9). However, the mechanisms by which IL-9/Th9 mediate immunopathology in the lung are unknown. Here we report an IL-9-driven positive feedback loop that reinforces allergic inflammation. We show that IL-9 increases IL-2 production by mast cells, which leads to expansion of CD25+ type 2 innate lymphoid cells (ILC2) and subsequent activation of Th9 cells. Blocking IL-9 or inhibiting CD117 (c-Kit) signalling counteracts the pathogenic effect of the described IL-9-mast cell-IL-2 signalling axis. Overproduction of IL-9 is observed in expectorates from cystic fibrosis (CF) patients, and a sex-specific variant of IL-9 is predictive of allergic reactions in female patients. Our results suggest that blocking IL-9 may be a therapeutic strategy to ameliorate inflammation associated with microbial colonization in the lung, and offers a plausible explanation for gender differences in clinical outcomes of patients with CF.

Fungal chitin induces trained immunity in human monocytes during cross-talk of the host with Saccharomyces cerevisiae

The immune system is essential to maintain the mutualistic homeostatic interaction between the host and its micro- and mycobiota. Living as a commensal, Saccharomyces cerevisiae could potentially shape the immune response in a significant way. We observed that S. cerevisiae cells induce trained immunity in monocytes in a strain-dependent manner through enhanced TNFα and IL-6 production upon secondary stimulation with TLR ligands, as well as bacterial and fungal commensals. Differential chitin content accounts for the differences in training properties observed among strains, driving induction of trained immunity by increasing cytokine production and direct antimicrobial activity both in vitro and in vivo. These chitin-induced protective properties are intimately associated with its internalization, identifying a critical role of phagosome acidification to facilitate microbial digestion. This study reveals how commensal and passenger microorganisms could be important in promoting health and preventing mucosal diseases by modulating host defense toward pathogens and thus influencing the host microbiota-immune system interactions.

Antifungal Th Immunity: Growing up in Family.

Fungal diseases represent an important paradigm in immunology since they can result from either the lack of recognition or over-activation of the inflammatory response. Current understanding of the pathophysiology underlying fungal infections and diseases highlights the multiple cell populations and cell-signaling pathways involved in these conditions. A systems biology approach that integrates investigations of immunity at the systems-level is required to generate novel insights into this complexity and to decipher the dynamics of the host–fungus interaction. It is becoming clear that a three-way interaction between the host, microbiota, and fungi dictates the types of host–fungus relationship. Tryptophan metabolism helps support this interaction, being exploited by the mammalian host and commensals to increase fitness in response to fungi via resistance and tolerance mechanisms of antifungal immunity. The cellular and molecular mechanisms that provide immune homeostasis with the fungal biota and its possible rupture in fungal infections and diseases will be discussed within the expanding role of antifungal Th cell responses.

Noncanonical Fungal Autophagy Inhibits Inflammation in Response to IFN-γ via DAPK1

Summary Defects in a form of noncanonical autophagy, known as LC3-associated phagocytosis (LAP), lead to increased inflammatory pathology during fungal infection. Although LAP contributes to fungal degradation, the molecular mechanisms underlying LAP-mediated modulation of inflammation are unknown. We describe a mechanism by which inflammation is regulated during LAP through the death-associated protein kinase 1 (DAPK1). The ATF6/C/EBP-β/DAPK1 axis activated by IFN-γ not only mediates LAP to Aspergillus fumigatus but also concomitantly inhibits Nod-like receptor protein 3 (NLRP3) activation and restrains pathogenic inflammation. In mouse models and patient samples of chronic granulomatous disease, which exhibit defective autophagy and increased inflammasome activity, IFN-γ restores reduced DAPK1 activity and dampens fungal growth. Additionally, in a cohort of hematopoietic stem cell-transplanted patients, a genetic DAPK1 deficiency is associated with increased inflammation and heightened aspergillosis susceptibility. Thus, DAPK1 is a potential drugable player in regulating the inflammatory response during fungal clearance initiated by IFN-γ.

Fine-tuning of Th17 Cytokines in Periodontal Disease by IL-10

Periodontitis (PD) is a chronic disease caused by the host inflammatory response to bacteria colonizing the oral cavity. In addition to tolerance to oral microbiome, a fine-tuned balance of IL-10 levels is critical to efficiently mount antimicrobial resistance without causing immunopathology. Clinical and animal studies support that adaptive T-helper (Th) cytokines are involved in the pathogenesis of alveolar bone destruction in PD. However, it remains unclear what type of Th response is related to human PD progression and what role IL-10 has on this process. We addressed the contribution of IL-10 in limiting Th1 and Th17 inflammatory response in murine and human PD. Through a combination of basic and translational approaches involving selected cytokine-deficient mice as well as human genetic epidemiology, our results demonstrate the requirement for IL-10 in fine-tuning the levels of Th17 (IL-17A and IL-17F) cytokines in experimental and human PD. Of novelty, we found that IL-17F correlated with protection in murine and human PD and was positively regulated by IL-10. To our knowledge, this is the first demonstration of the protective role for IL-17F in PD, its positive regulation by IL-10, and the potential differential role for IL-17A and IL-17F in periodontal disease.

IL-9 and Mast Cells Are Key Players of Candida albicans Commensalism and Pathogenesis in the Gut

Summary Candida albicans is implicated in intestinal diseases. Identifying host signatures that discriminate between the pathogenic versus commensal nature of this human commensal is clinically relevant. In the present study, we identify IL-9 and mast cells (MCs) as key players of Candida commensalism and pathogenicity. By inducing TGF-β in stromal MCs, IL-9 pivotally contributes to mucosal immune tolerance via the indoleamine 2,3-dioxygenase enzyme. However, Candida-driven IL-9 and mucosal MCs also contribute to barrier function loss, dissemination, and inflammation in experimental leaky gut models and are upregulated in patients with celiac disease. Inflammatory dysbiosis occurs with IL-9 and MC deficiency, indicating that the activity of IL-9 and MCs may go beyond host immunity to include regulation of the microbiota. Thus, the output of the IL-9/MC axis is highly contextual during Candida colonization and reveals how host immunity and the microbiota finely tune Candida behavior in the gut.

Reply to ‘F508del-CFTR is not corrected by thymosin α1’

Romani et al. reply: We thank Matthes et al.1 for their comment on our article that reported on the potential therapeutic activity of thymosin alpha 1 (Tα 1) in cystic fibrosis (CF)2. Their reported failure to detect any increase in cystic fibrosis transmembrane conductance regulator (CFTR)-mediated current in primary human bronchial epithelial (HBE) cells from four patients and in CFBE41ocells treated with Tα 1 is a highly relevant issue, as it questions the molecule’s corrector activity that we reported in our recent paper2. Nevertheless, careful examination of their data reveals that this issue might not be due to biologic variability but rather to differences in the handling of the peptide. Before discussing the handling issue, we first want to note that the ability of Tα 1 to activate the enzyme indoleamine 2,3-dioxygenase 1 (IDO1)2, which promotes the tolerogenic pathway of tryptophan catabolism2 as well as autophagy3, may explain both the anti-inflammatory and the corrector activity of Tα 1 in CF2, in accordance with a potential for autophagy in rescuing CFTR function4. IDO1 and the deubiquitinating enzyme ubiquitin-specific peptidase 36 (USP36)—whose epigenetic modifications are linked to lung function and disease5—are similarly deficient in CF2,6. Thus, the extent of the residual activity of both enzymes in individual patients likely impacts the corrector activity conferred by Tα 1. Indeed, person-to-person genetic and epigenetic differences may account for biological variability in response to Tα 1. But in addition to biological variability, there might be issues regarding the modalities of Tα 1’s solubilization. Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy techniques have indicated that Tα 1 is substantially unstructured in a water solution. But, upon interaction with negative phospholipids on cell membranes, it assumes a definite conformation with two helical domains and, in between, a short flexible tract7,8. Because Tα 1’s insertion of the hydrophobic region occurs at residues 1–5 of the N-terminus end8, any perturbing environment and/or condition preventing the helical folding and the N-terminus-mediated insertion may result in the abrogation of Tα 1’s biological activity. DMSO has been used by Matthes et al.1 and others9 to dissolve Tα 1 when exploring its activity in vitro as a corrector. In addition, Tα 1 from two different sources (CRIBI and Abcam) was assessed. The difference between the two was lack of N-terminal acetylation in the latter. Because of DMSO’s ability to abolish the α -helix in favor of a β -sheet conformation in proteins10 as well as to trigger protein unfolding by hydrogen bonding with the backbone amides11 and the possible decrease in stability of nonacetylated Tα 1, the impact of such experimental conditions should not be underestimated. We assessed whether the improper solubilization in DMSO and the lack of N-terminal acetylation were both experimental conditions leading to loss of the peptide’s conformation, stability and function. Acetylated Tα 1 from CRIBI and nonacetylated Tα 1 from Abcam were initially dissolved in either DMSO, which was then diluted to 0.1% DMSO in water, or water, which was followed by scalar addition of DMSO before the assays. As freeze-thawing (FT) affects peptide stability, we also assessed peptide stability after FT. Experiments were carried out in order to evaluate: (i) peptide structural changes and potential aggregation by CD, Fourier-transform infrared spectroscopy (FT-IR) and dynamic light scattering (DLS); (ii) detection of bioactive peptide by specific ELISA; (iii) the ability to correct p.Phe508del-CFTR in p.Phe508del-CFTR-transfected CFBE41oor FRT cell lines by immunoblotting and whole-cell patch-clamp analysis; and (iv) the ability to promote IDO1 expression in those cells. The actual data will be published elsewhere given the space limitation afforded to this reply, but in summary, the results were:

Towards Targeting the Aryl Hydrocarbon Receptor in Cystic Fibrosis

Tryptophan (trp) metabolism is an important regulatory component of gut mucosal homeostasis and the microbiome. Metabolic pathways targeting the trp can lead to a myriad of metabolites, of both host and microbial origins, some of which act as endogenous low-affinity ligands for the aryl hydrocarbon receptor (AhR), a cytosolic, ligand-operated transcription factor that is involved in many biological processes, including development, cellular differentiation and proliferation, xenobiotic metabolism, and the immune response. Low-level activation of AhR by endogenous ligands is beneficial in the maintenance of immune health and intestinal homeostasis. We have defined a functional node whereby certain bacteria species contribute to host/microbial symbiosis and mucosal homeostasis. A microbial trp metabolic pathway leading to the production of indole-3-aldehyde (3-IAld) by lactobacilli provided epithelial protection while inducing antifungal resistance via the AhR/IL-22 axis. In this review, we highlight the role of AhR in inflammatory lung diseases and discuss the possible therapeutic use of AhR ligands in cystic fibrosis.

Autophagy and LAP in the Fight against Fungal Infections: Regulation and Therapeutics

Phagocytes fight fungi using canonical and noncanonical, also called LC3-associated phagocytosis (LAP), autophagy pathways. However, the outcomes of autophagy/LAP in shaping host immune responses appear to greatly vary depending on fungal species and cell types. By allowing efficient pathogen clearance and/or degradation of inflammatory mediators, autophagy proteins play a broad role in cellular and immune homeostasis during fungal infections. Indeed, defects in autophagic machinery have been linked with aberrant host defense and inflammatory states. Thus, understanding the molecular mechanisms underlying the relationship between the different forms of autophagy may offer a way to identify drugable molecular signatures discriminating between selective recognition of cargo and host protection. In this regard, IFN-γ and anakinra are teaching examples of successful antifungal agents that target the autophagy machinery. This article provides an overview of the role of autophagy/LAP in response to fungi and in their infections, regulation, and therapeutic exploitation.