Sphingosine-1-Phosphate Shapes Healthy Monocytes into An Immunosuppressive Phenotype.

BACKGROUND/AIMS: The physiological phenotype of individuals can influence and shape real-life phenomena in that it can contribute to the development of specific characteristics that can affect the immune response to specific stimuli. In this study we aimed to understand whether the sphingosine/sphingosine-1-phoshate (S1P) axis can modulate the immunotype of circulating cells. METHODS: To pursue this goal, we performed bioinformatic analyses of public datasets. RESULTS: The transcriptomic profile of healthy subjects of GSE192829 dataset identified two clusters with different transcriptional repertoire. Cluster 1 expressed higher levels of enzymes for S1P formation than cluster 0 which was characterized by enzymes that lead to ceramide formation, which represent the opposite metabolic direction. Inference analysis showed that cluster 1 was higher populated by monocytes, CD4+ T and B cells than cluster 0. Of particular interest was the phenotype of the monocytes in cluster 1 which showed an immunosuppressive nature compared to those in cluster 0. The role of S1P signature in healthy PBMCs was confirmed with other dataset analyses, supporting that circulating monocytes positive to the ceramidase, unlike the negative ones, had an immunosuppressive phenotype characterized by hub immunosuppressive markers (i.e. TYROBP, FCER1G, SYK, SIRPA, CSF1R, AIF1, FCGR2A, CLEC7A, LYN, PLCG2, LILRs, HCK, GAB2). This hub genes well discriminated the immunotype of healthy subjects. CONCLUSION: In conclusion this study highlights that S1P-associated hub markers can be useful to discriminate subjects with pronounced immunosuppression.

Sex Differences in Sphingosine-1-Phosphate Levels Are Dependent on Ceramide Synthase 1 and Ceramidase in Lung Physiology and Tumor Conditions.

Sex is a biological variable that can reflect clinical outcomes in terms of quality of life, therapy effectiveness, responsiveness and/or toxicity. Sphingosine-1-phosphate (S1P) is a lipidic mediator whose activity can be influenced by sex. To evaluate whether the S1P axis underlies sex 'instructions' in the lung during physiological and oncological lung conditions, sphingosine and S1P were quantified in the blood of healthy (H) volunteers, lung adenocarcinoma (ADK) and squamous cell carcinoma (SCC) patients of both sexes. S1P receptors and their metabolic enzymes were evaluated in the tissues. Circulating levels of S1P were similar among H female and male subjects and female SCC patients. Instead, male and female ADK patients had lower circulating S1P levels. S1P receptor 3 (S1PR3) was physiologically expressed in the lung, but it was overexpressed in male SCC, and female and male ADK, but not in female SCC patients, who showed a significantly reduced ceramide synthase 1 (CERS1) mRNA and an overexpression of the ceramidase (ASAH1) precursor in lung tumor tissues, compared to male SCC and both male and female ADK patients. These findings highlighted sex differences in S1P rheostat in pathological conditions, but not in physiological conditions, identifying S1P as a prognostic mediator depending on lung cancer histotype.

Induction of Inflammation Disrupts the Negative Interplay between STING and S1P Axis That Is Observed during Physiological Conditions in the Lung.

The stimulator of interferon genes (STING) is a master regulator of innate immunity, involved in several inflammatory diseases. Our previous data showed that sphingosine-1-phosphate (S1P) is released during inflammatory conditions in the lung. The aim of this study was to understand the interplay between S1P and STING during both physiological and pathological conditions. The mRNA levels of ceramidase (ASAH1), S1P precursor enzyme, and STING were inversely correlated in healthy lung tissues, but positively correlated in tumor tissues. The activation of STING induced higher expression of ASAH1 and was accompanied by IFN-ß and IL-6 release. ASAH1 and sphingosine kinases (SPHK I/II) blockade significantly reduced IL-6, but not IFNß, after STING activation. In support of this, taking advantage of a mouse model, we found that inflamed lungs had higher levels of inactive ASAH1 when STING was inhibited. This confirmed the human data, where higher levels of STING promoted the activation of ASAH1. Lung cancer patients positive to STING and ASAH1 mRNA levels had a dismal prognosis in that the overall survival was reduced compared to STING/ASAH1 negative patients. These data highlight that during physiological conditions, STING and the S1P axis do not interfere, whereas in lung cancer patients their interplay is associated to poor prognosis.

A vitamin E long-chain metabolite and the inspired drug candidate α-amplexichromanol relieve asthma features in an experimental model of allergen sensitization.

Benefits for vitamin E intake in diseases with inflammatory components have been described and related in part, to endogenously formed metabolites (long-chain metabolites, LCM). Here, we have evaluated the role of LCM in relieving asthma features. To this aim, the endogenous vitamin E metabolite α-13'-carboxychromanol (α-T-13'-COOH) that acts as potent 5-lipoxygenase inhibitor has been administered either intraperitoneally or by oral gavage to BALB/c mice sensitized by subcutaneous injection of ovalbumin (OVA). We also have taken advantage of the metabolically stable α-T-13'-COOH derivative α-amplexichromanol (α-AC). Intraperitoneal treatment with α-T-13'-COOH reduced OVA-induced airway hyperreactivity (AHR) as well as peri-bronchial inflammatory cell infiltration. α-AC was more efficacious than α-T-13'-COOH, as demonstrated by better control of AHR and in reducing subepithelial. Both compounds exerted their protective function by reducing pulmonary leukotriene C4 levels. Beneficial effects of α-AC were coupled to inhibition of the sensitization process, as indicated by a reduction of IgE plasma levels, lung mast cell infiltration and Th2 immune response. Metabololipidomics analysis revealed that α-AC raises the pulmonary levels of prostanoids, their degradation products, and 12/15-lipoxygenase metabolites. Following oral administration, the pharmacodynamically different profile in α-T-13'-COOH and α-AC was abrogated as demonstrated by a similar and improved efficacy in controlling asthma features as well as by metabololipidomics analysis. In conclusion, this study highlights a role for LCM and of vitamin E derivatives as pharmacologically active compounds that ameliorate asthmatic features and defines an important role for endogenous vitamin E metabolites in regulating immune response underlying the sensitization process.

Intracellular Sphingosine-1-Phosphate Receptor 3 Contributes to Lung Tumor Cell Proliferation.

BACKGROUND/AIMS: The pleiotropic lipid mediator sphingosine-1-phosphate (S1P) exerts a multitude of effects on respiratory cell physiology and pathology through five S1P receptors (S1PR1-5). Epidemiological studies proved high levels of circulating S1P in non-small cell lung cancer (NSCLC) patients. Studies in literature suggest that high levels of S1P support carcinogenesis but the exact mechanism is still elusive. The aim of this study was to understand the mechanism/s underlying S1P-mediated lung tumor cell proliferation. METHODS: We used human samples of NSCLC, a mouse model of first-hand smoking and of Benzo(a)pyrene (BaP)-induced tumor-bearing mice and A549 lung adenocarcinoma cells. RESULTS: We found that the expression of S1PR3 was also into the nucleus of lung cells in vitro, data that were confirmed in lung tissues of NSCLC patients, smoking and tumor bearing BaP-exposed mice. The intranuclear, but not the membrane, localization of S1PR3 was associated to S1P-mediated proliferation of lung adenocarcinoma cells. Indeed, the inhibition of the membrane S1PR3 did not alter tumor cell proliferation after Toll Like Receptor (TLR) 9 activation. Instead, according to the nuclear localization of sphingosine kinase (SPHK) II, the inhibition of the kinase completely blocked the endogenous S1P-induced tumor cell proliferation. CONCLUSION: These results prove that the nuclear S1PR3/SPHK II axis is involved in lung tumor cell proliferation, highlighting a novel molecular mechanism which could provide differential therapeutic approaches especially in non-responsive lung cancer patients.

Sphingosine-1-Phosphate Contributes to TLR9-Induced TNF-α Release in Lung Tumor Cells.

BACKGROUND/AIMS: Sphingosine-1-phosphate (S1P) is a membrane-derived bioactive phospholipid involved in many lung physiological and pathological processes. Higher levels of S1P have been registered in a broad range of respiratory diseases, including inflammatory disorders and cancer. The aim of our study was to understand the role of S1P in healthy versus tumor cells after Toll-Like Receptors (TLRs) activation, well-known modulators of sphingolipid metabolism. METHODS: Lung adenocarcinoma cells and non-pathological human fibroblasts were stimulated with unmethylated Cytosine phosphate Guanosine (CpG), the TLR9 ligand, and S1P-dependent TNF-α release was evaluated by means of ELISA. Immunofluorescence and LC-MS/MS analysis were performed to evaluate/quantify S1P generation following TLR9 activation. RESULTS: We found that S1P was involved in TLR9-induced TNF-α release in that the inhibition of both ceramidase and sphingosine kinase I/II (SPHK I/II) significantly reduced the levels of TNF-α after TLR9 triggering in lung adenocarcinoma cells. These results were not observed in healthy fibroblasts, implying that this pathway was mainly involved in pathological conditions. Moreover, the activation of TLR4 by means of LPS did not have similar effects as in the case of CpG-stimulated TLR9. Importantly, the activation of TLR9 induced S1P generation and allowed it to interact on the outside membrane receptor S1P1 and S1P3 via the efflux through its membrane transporter SPNS2. Indeed, both the blockade of S1P3 and the transporter SPNS2 significantly reduced the activity of S1P on TNF-α release from lung adenocarcinoma cells. CONCLUSION: Our study identifies a novel inflammatory pathway in that TLR9 increases the pro-inflammatory cytokine release, such as TNF-α, via the induction of a ceramide/S1P imbalance in favor of S1P, adding a novel puzzle piece in TLR9-orchestrated inflammatory pathway and shedding more light on the role of the higher levels of S1P during inflammatory conditions.

5α-dihydrotestosterone abrogates sex bias in asthma like features in the mouse.

Androgen levels inversely correlate with the incidence, susceptibility and severity of asthma. However, whether male sex hormones such as 5α-dihydrotestosterone (DHT) have beneficial effects on asthma symptoms and/or could affect asthma susceptibility have not been investigated. DHT administration to female mice, during the sensitization phase, abrogates the sex bias in bronchial hyperreactivity. This effect correlates with inhibition of leukotriene biosynthesis in the lung. DHT significantly inhibits also other asthma-like features such as airway hyperplasia and mucus production in sensitized female mice. Conversely, DHT does not affect plasma IgE levels as well as CD3+CD4+ IL-4+ cell and IgE+c-Kit+ cell infiltration within the lung but prevents pulmonary mast cell activation. The in vitro study on RBL-2H3 cells confirms that DHT inhibits mast cell degranulation. In conclusion, our data demonstrate that immunomodulatory effects of DHT on mast cell activation prevent the translation of allergen sensitization into clinical manifestation of asthma.

Leukotriene-mediated sex dimorphism in murine asthma-like features during allergen sensitization.

The incidence and severity of asthma preponderate in women versus men. Leukotrienes (LTs) are lipid mediators involved in asthma pathogenesis, and sex disparities in LT biosynthesis and anti-LT pharmacology in inflammation have recently emerged. Here, we report on sex dimorphism in LT production during allergen sensitization and its correlation to lung function. While high plasma levels of IgE, as sensitization index, were elevated in both sexes, LT levels increased only in lungs of female ovalbumin-sensitized BALB/c mice. Sex-dependent elevated LT levels strictly correlated to an enhanced airway hyperreactivity, pulmonary inflammation and mast cell infiltration/activation in female mice. Importantly, this sex bias was coupled to superior therapeutic efficacy of different types of clinically used LT modifiers like zileuton, MK886 and montelukast in female animals. Our findings reveal sex-dependent LT production as a basic mechanism of sex dimorphism in allergic asthma, and suggest that women might benefit more from anti-LT asthma therapy.

Human lung cancer-derived immunosuppressive plasmacytoid dendritic cells release IL-1α in an AIM2 inflammasome-dependent manner.

Plasmacytoid dendritic cells (pDCs) highly populate lung tumor masses and are strictly correlated to bad prognosis, yet their role in lung cancer is controversial. To understand their role in lung cancer, we isolated pDCs from human samples of lung obtained from non-small cell lung cancer patients undergoing thoracic surgery. Tumor masses presented a higher percentage of pDCs than healthy tissues; pDCs were in the immunosuppressive phenotype, as determined by higher levels of CD33 and PD-L1. Despite higher HLA-A and HLA-D expression, cancerous pDCs did not exert cytotoxic activity against tumor cells but instead promoted their proliferation. In this scenario, cancerous pDCs were able to produce high levels of IL-1α. This effect was observed on the specific activation of the inflammasome absent in melanoma 2 (AIM2), which led to higher cytoplasmic calcium release responsible for calpain activation underlying IL-1α release. The blockade of type I interferon receptor and of AIM2 via the addition of LL-37 significantly reduced the release of IL-1α, which was still high after Nod-like receptor P3 inhibition via glibenclamide. More important, mitochondrial-derived reactive oxygen species sequester diminished AIM2-dependent IL-1α release. Our data demonstrate that lung tumor-associated pDCs are responsive to the activation of AIM2 that promotes calcium efflux and reactive oxygen species from mitochondria, leading to calpain activation and high levels of IL-1α, which facilitate tumor cell proliferation in the lung.

Inflammatory mediators in a short-time mouse model of doxorubicin-induced cardiotoxicity.

Doxorubicin (DOXO) is commonly used to treat a wide range of malignant tumors, but its clinical use is limited by acute and chronic cardiotoxicity. The precise mechanism underlying DOXO-induced cardiotoxicity is still not completely elucidated, but cardiac inflammation seems to be involved. Effects of DOXO on proinflammatory cytokines, inflammatory cell infiltration, and necrosis have been proven only when a functional impairment has already occurred, so this study aimed to investigate the acute effect of DOXO administration in mouse heart. The results of our study demonstrated alterations in cardiac function parameters assessed by ultrasound within 24h after a single injection of DOXO, with a cumulative effect along the increase of the dose and the number of DOXO administrations. At the same time, DOXO causes a significant production of proinflammatory cytokines (such as TNF-α and IL-6) with a concomitant reduction of IL-10, a well-known antiinflammatory cytokine. Furthermore, overexpression of inducible nitric oxide synthase (iNOS) in heart tissue and increased levels of serum nitrite in DOXO-treated mice were detected. Notably, DOXO administration significantly increased nitrotyrosine expression in mouse heart. Our data support the hypothesis that these early events, could be responsible for the later onset of more severe deleterious remodeling leading to DOXO induced cardiomyopathy.

Disodium cromoglycate inhibits asthma-like features induced by sphingosine-1-phosphate.

Compelling evidence suggests the involvement of sphingosine-1-phosphate (S1P) in the pathogenesis of asthma. The systemic administration of S1P causes asthma like features in the mouse involving mast cells. In this study we investigated whether disodium cromoglycate (DSCG), administered as a preventative treatment as in human therapy, could affect S1P effects on airways. BALB/c mice, treated with DSCG, received subcutaneous administration of S1P. Bronchi and pulmonary tissues were collected and functional, molecular and cellular studies were performed. DSCG inhibited S1P-induced airway hyper-reactivity as well as pulmonary inflammation. DSCG decreased the recruitment of solely mast cells and B cells in the lung. IgE serum levels, prostaglandin D2, mucus production and IL-13 were also reduced when mice were pretreated with DSCG. S1P induced pulmonary expression of CD23 on T and B cells, that was reversed by DSCG. Conversely, S1P failed to upregulate CD23 in mast cell-deficient Kit W-sh/W-sh mice. In conclusion we have shown that DSCG inhibits S1P-induced asthma like features in the mouse. This beneficial effect is due to a regulatory action on mast cell activity, and in turn to an inhibition of IgE-dependent T and B cells responses.

B cell depletion increases sphingosine-1-phosphate-dependent airway inflammation in mice.

Sphingosine-1-phosphate (S1P) has been widely associated with inflammation-based lung pathologies. Because B cells play a critical role as antigen-presenting and/or Ig-producing cells during asthmatic conditions, we wanted to dissect the role of these cells in S1P-dependent airway hyperreactivity and inflammation. Mice were sensitized to ovalbumin or exposed to S1P. Ovalbumin sensitization caused airway hyperreactivity coupled to an increased lung infiltration of B cells, which was significantly reduced after the inhibition of sphingosine kinases I/II. Similarly, the sole administration of S1P increased bronchial reactivity compared with vehicle and was accompanied by a higher influx of B cells in a time-dependent manner. This effect was associated with higher levels of IL-13, transforming growth factor-ß, IL-10, and T regulatory cells. In addition, isolated S1P-derived lung B cells increased CD4(+) and CD8(+) T cell proliferation in vitro, and their suppressive nature at Day 14 was associated with the higher release of transforming growth factor-ß and IL-10 when they were cocultured. Therefore, to prove the role of B cells in S1P-mediated airway inflammation, and because CD20 expression, contrary to major hystocompatibility complex I and major hystocompatibility complex II, was up-regulated at Day 14, CD20(+) B cells were depleted by means of a specific monoclonal antibody. The absence of CD20(+) B cells increased airway reactivity and inflammation in S1P-treated mice compared with control mice. These data imply that sphingosine kinase/S1P-mediated airway inflammation is countered by B cells via the induction of an immune-suppressive environment to reduce asthma-like outcomes in mice.

A single infection with Chlamydia pneumoniae is sufficient to exacerbate atherosclerosis in ApoE deficient mice.

Several studies have demonstrated a strong link between Chlamydia pneumoniae (Cp) infection and atherosclerosis progression/exacerbation. Here, we try to understand whether a single administration of Cp could exacerbate atherosclerosis. Apoe(-/-) mice were intranasally infected with Cp followed by a high fat diet. Mice were sacrificed at different time points after Cp infection to monitor the development of the atheroma. Cp infection increased lipid content in the aortic sinus of Apoe(-/-) mice starting from 8 weeks. This was associated with increased numbers of active myeloid dendritic cells and plasmacytoid DCs which were co-localized with T-cells in the atherosclerotic plaque. The serum levels of IFN-γ showed a Th1-like environment typical of atherosclerosis. In conclusion, we demonstrate that one dose of Cp could exacerbate atherosclerotic lesion development, triggering innate immune cell accumulation early on that allowed the involvement of Th1-like cells in the exacerbation of the atherosclerotic plaque at later time points.

Hydrogen sulfide inhalation ameliorates allergen induced airway hypereactivity by modulating mast cell activation.

Compelling evidence suggests that hydrogen sulfide represents an important gaseous transmitter in the mammalian respiratory system. In the present study, we have evaluated the role of mast cells in hydrogen sulfide-induced effects on airways in a mouse model of asthma. Mice were sensitized to ovalbumin and received aerosol of a hydrogen sulfide donor (NaHS; 100 ppm) starting at day 7 after ovalbumin challenge. Exposure to hydrogen sulfide abrogated ovalbumin-induced bronchial hypereactivity as well as the increase in lung resistance. Concomitantly, hydrogen sulfide prevented mast cell activity as well as FGF-2 and IL-13 upregulation. Conversely, pulmonary inflammation and the increase in plasmatic IgE levels were not affected by hydrogen sulfide. A lack of hydrogen sulfide effects in mast cell deficient mice occurred. Primary fibroblasts harvested from ovalbumin-sensitized mice showed an increased proliferation rate that was inhibited by hydrogen sulfide aerosol. Furthermore, ovalbumin-induced transdifferentiation of pulmonary fibroblasts into myofibroblasts was reversed. Finally, hydrogen sulfide did abrogate in vitro the degranulation of the mast cell-like RBL-2H3 cell line. Similarly to the in vivo experiments the inhibitory effect was present only when the cells were activated by antigen exposure. In conclusion, inhaled hydrogen sulfide improves lung function and inhibits bronchial hyper-reactivity by modulating mast cells and in turn fibroblast activation.

Plasmacytoid dendritic cells play a key role in tumor progression in lipopolysaccharide-stimulated lung tumor-bearing mice.

The antitumor activity of LPS was first described by Dr. William Coley. However, its role in lung cancer remains unclear. The aim of our study was to elucidate the dose-dependent effects of LPS (0.1-10 µg/mouse) in a mouse model of B16-F10-induced metastatic lung cancer. Lung tumor growth increased at 3 and 7 d after the administration of low-dose LPS (0.1 µg/mouse) compared with control mice. This was associated with an influx of plasmacytoid dendritic cells (pDCs), regulatory T cells, myeloid-derived suppressor cells, and CD8(+) regulatory T cells. In contrast, high-dose LPS (10 µg/mouse) reduced lung tumor burden and was associated with a greater influx of pDCs, as well as a stronger Th1 and Th17 polarization. Depletion of pDCs during low-dose LPS administration resulted in a decreased lung tumor burden. Depletion of pDCs during high-dose LPS treatment resulted in an increased tumor burden. The dichotomy in LPS effects was due to the phenotype of pDCs, which were immunosuppressive after the low-dose LPS, and Th1- and T cytotoxic-polarizing cells after the high-dose LPS. Adoptive transfer of T cells into nude mice demonstrated that CD8(+) T cells were responsible for pDC recruitment following low-dose LPS administration, whereas CD4(+) T cells were required for pDC influx after the high-dose LPS. In conclusion, our data suggest differential effects of low-dose versus high-dose LPS on pDC phenotype and tumor progression or regression in the lungs of mice.

Polyinosinic-polycytidylic acid limits tumor outgrowth in a mouse model of metastatic lung cancer.

Polyinosinic-polycytidylic acid (poly I:C), a TLR3 ligand, is currently being tested in human clinical trials as an adjuvant to anti-cancer vaccines and in combination with other therapies. However, little is known about its activity in established pulmonary metastasis. The aim of our study was to elucidate the effect of poly I:C (1, 10, or 100 µg/mouse) in a mouse model of B16-F10-induced metastatic lung cancer. Lung tumor growth was arrested after a single administration of poly I:C. This was associated with higher influx of mature dendritic cells (DCs), which drove toward a Th1-like, Th17-like, and cytotoxic immune environment. The interference with IFN type I receptor signaling by means of a specific mAb reversed poly I:C-mediated tumor regression due to lower presence of myeloid DCs, cytotoxic DCs (CD11c(+)CD8(+)), NKT cells, CD8(+) T cells, and Th1-like cytokines. Moreover, the adoptive transfer of poly I:C-activated bone marrow-derived DCs into tumor-bearing mice resulted in activities similar to those of the systemic administration of poly I:C on lung tumor burden. In conclusion, our data prove that poly I:C has potential anti-tumor activity in a mouse model of established pulmonary metastasis. The activation of DCs and the production of IFN type I are responsible for an effective T cytotoxic immune response against metastatic lung cancer progression after poly I:C treatment.

Inhibition of CD73 improves B cell-mediated anti-tumor immunity in a mouse model of melanoma.

CD73 is a cell surface enzyme that suppresses T cell-mediated immune responses by producing extracellular adenosine. Growing evidence suggests that targeting CD73 in cancer may be useful for an effective therapeutic outcome. In this study, we demonstrate that administration of a specific CD73 inhibitor, adenosine 5'-(α,ß-methylene)diphosphate (APCP), to melanoma-bearing mice induced a significant tumor regression by promoting the release of Th1- and Th17-associated cytokines in the tumor microenvironment. CD8+ T cells were increased in melanoma tissue of APCP-treated mice. Accordingly, in nude mice APCP failed to reduce tumor growth. Importantly, we observed that after APCP administration, the presence of B cells in the melanoma tissue was greater than that observed in control mice. This was associated with production of IgG2b within the melanoma. Depletion of CD20+ B cells partially blocked the anti-tumor effect of APCP and significantly reduced the production of IgG2b induced by APCP, implying a critical role for B cells in the anti-tumor activity of APCP. Our results also suggest that APCP could influence B cell activity to produce IgG through IL-17A, which significantly increased in the tumor tissue of APCP-treated mice. In support of this, we found that in melanoma-bearing mice receiving anti-IL-17A mAb, the anti-tumor effect of APCP was ablated. This correlated with a reduced capacity of APCP-treated mice to mount an effective immune response against melanoma, as neutralization of this cytokine significantly affected both the CD8+ T cell- and B cell-mediated responses. In conclusion, we demonstrate that both T cells and B cells play a pivotal role in the APCP-induced anti-tumor immune response.

The activation of the AIM2 inflammasome after cigarette smoke exposure leads to an immunosuppressive lung microenvironment.

Cigarette smoke is widely known as contributing to chronic inflammation underlying several airway diseases, such as chronic obstructive pulmonary disease (COPD) and lung cancer. In our previous studies we found that the lung of both COPD and cancer patients were characterized by the presence and activation of the AIM2 inflammasome. Here, we wanted to investigate the upstream step during the establishment of chronic lung inflammation after cigarette smoke exposure. We took advantage of a mouse model of smoking exposure and public scRNAseq data. We found that AIM2 mRNA was expressed in both alveolar type II, B cells, T regulatory (Treg) and macrophages detected in the lung of non-smokers (n = 4) and smokers (n = 3). The activation of AIM2 in smoking mice by using PolydA:dT did not alter cigarette-smoke-induced alveoli enlargement and mucus production, rather it induced higher recruitment of immunosuppressive cells, such as non-active dendritic cells (DCs), Arginase I+ macrophages, myeloid-derived suppressor cells (MDSC) and Tregs. In addition, the inflammatory environment after AIM2 activation in smoking mice was characterized by higher levels of IL-1α, IL-1ß, IL-33, TNFα, LDH, IL-10 and TGFß. This scenario was not altered after the pharmacological inhibition of both caspase-1 and STING pathway. In conclusion, these data suggest that chronic inflammation after cigarette smoke exposure is associated with AIM2 activation, which could lead towards cigarette smoke-associated lung diseases.

ATP-induced fibrogenic pathway in circulating cells obtained by idiopathic pulmonary fibrotic (IPF) patients is not blocked by nintedanib and pirfenidone.

Idiopathic pulmonary fibrosis (IPF) is a severe disability due to progressive lung dysfunction. IPF has long been viewed as a non-immune form of pulmonary fibrosis, but nowadays it is accepted that a chronic inflammatory response can exacerbate fibrotic patterns. IL-1-like cytokines and ATP are highly detected in the lung and broncho-alveolar lavage fluid of IPF patients. Because ATP binds the purinergic receptor P2RX7 involved in the release of IL-1-like cytokines, we aimed to understand the role of P2RX7 in IPF. PBMCs from IPF patients were treated with nintedanib or pirfenidone in the presence of ATP. Under these conditions, PBMCs still released IL-1-like cytokines and the pro-fibrotic TGFß. Bulk and scRNAseq demonstrated that lung tissues of IPF patients had higher levels of P2RX7, especially on macrophages, which were correlated to T cell activity and inflammatory response with a TGFBI and IL-10 signature. A subcluster of macrophages in IPF lung tissues had 2055 genes that were not in common with the other subclusters, and that were involved in metabolic and PDGF, FGF and VEGF associated pathways. These data confirmed what observed on circulating cells that, although treated with anti-fibrotic agents, nintedanib or pirfenidone, they were still able to release IL-1 cytokines and the fibrogenic TGFß. In conclusion, these data imply that because nintedanib and pirfenidone do not block ATP-induced IL-1-like cytokines and TGFß induced during P2RX7 activation, it is plausible to consider P2RX7 on circulating cells and/or tissue biopsies as potential pharmacological tool for IPF patients.

Absent in melanoma 2 (AIM2) positive profile identifies a poor prognosis of lung adenocarcinoma patients.

The absent in melanoma 2 (AIM2) inflammasome has been demonstrated as involved in tumor growth. In this study we used human samples of lung adenocarcinoma (LUAD) patients, taking advantage of a mouse model of smoking cessation. Human samples were stratified according to the smoking status, high-risk factor for this type of tumor. Both public transcriptomic and human samples obtained by a clinical trial proved that AIM2 was upregulated either in terms of mRNA or protein, respectively, in the tumor mass according to the TNM stage, but it did not relate to the smoking status, age and sex. The upregulation of AIM2 was correlated to an immunosuppressive environment according to resting/non-active dendritic cells (DCs) and T regulatory cells, as demonstrated in both human samples and by means of an experimental model of smoking mice. Computational analysis showed that AIM2 upregulation was correlated to both an inflammasome profile, responsible for the poor prognosis of non-smoker and smoker LUAD patients, and to a non-inflammasome profile for former smoker. In conclusion, our study demonstrated that AIM2 is involved in lung carcinogenesis either in a canonical and non-canonical manner due to an immunosuppressive microenvironment associated to a dismal prognosis of LUAD patients.