Cytotoxic Effect Induced by Sicilian Oregano Essential Oil in Human Breast Cancer Cells.

Origanum vulgare L. is an aromatic plant that exerts antibacterial, antioxidant, anti-inflammatory, and antitumor activities, mainly due to its essential oil (EO) content. In this study, we investigated the possible mechanism underlying the in vitro antitumor activity of EO extracted by hydrodistillation of dried flowers and leaves of Origanum vulgare L. grown in Sicily (Italy) in MDA-MB-231 and MCF-7 breast cancer cell lines. Gas chromatography-mass spectrometry analysis of Oregano essential oil (OEO) composition highlighted the presence of twenty-six major phytocompounds, such as p-cymene, γ-terpinene, and thymoquinone p-acetanisole. OEO possesses strong antioxidant capacity, as demonstrated by the DPPH test. Our studies provided evidence that OEO reduces the viability of both MCF-7 and MDA-MB-231 cells. The cytotoxic effect of OEO on breast cancer cells was partially counteracted by the addition of z-VAD-fmk, a general caspase inhibitor. Caspases and mitochondrial dysfunction appeared to be involved in the OEO-induced death mechanism. Western blotting analysis showed that OEO-induced activation of pro-caspases-9 and -3 and fragmentation of PARP decreased the levels of Bcl-2 and Bcl-xL while increasing those of Bax and VDAC. In addition, fluorescence microscopy and cytofluorimetric analysis showed that OEO induces a loss of mitochondrial membrane potential in both cell lines. Furthermore, we tested the effects of p-cymene, γ-terpinene, thymoquinone, and p-acetanisole, which are the main components of OEO. Our findings highlighted that the effect of OEO on MDA-MB-231 and MCF-7 cells appears to be mainly due to the combination of different constituents of OEO, providing evidence of the potential use of OEO for breast cancer treatment.

Long non-coding RNA H19 enhances the pro-apoptotic activity of ITF2357 (a histone deacetylase inhibitor) in colorectal cancer cells.

Introduction: Long non-coding RNA H19 (lncH19) is highly expressed in colorectal cancer (CRC) and plays critical roles in tumor development, proliferation, metastasis, and drug resistance. Indeed, the expression of lncH19 usually affects the outcomes of chemo-, endocrine, and targeted therapies. ITF2357 (givinostat) is a histone deacetylase inhibitor (HDACi) that revealed a significant anti-tumor action by inducing apoptosis in different tumor models, including leukemia, melanoma, and glioblastoma. However, no data are present in the literature regarding the use of this compound for CRC treatment. Here, we investigate the role of lncH19 in ITF2357-induced apoptosis in CRC cells. Methods: The HCT-116 CRC cell line was stably silenced for H19 to investigate the role of this lncRNA in ITF2357-induced cell death. Cell viability assays and flow cytometric analyses were performed to assess the anti-proliferative and pro-apoptotic effects of ITF2357 in CRC cell lines that are silenced or not for lncH19. RT-PCR and Western blot were used to study the effects of ITF2357 on autophagy and apoptosis markers. Finally, bioinformatics analyses were used to identify miRNAs targeting pro-apoptotic factors that can be sponged by lncH19. Results: ITF2357 increased the expression levels of H19 and reduced HCT-116 cell viability, inducing apoptosis, as demonstrated by the increase in annexin-V positivity, caspase 3 cleavage, and poly (ADP-ribose) polymerase (PARP-1) degradation. Interestingly, the apoptotic effect of ITF2357 was much less evident in lncH19-silenced cells. We showed that lncH19 plays a functional role in the pro-apoptotic activity of the drug by stabilizing TP53 and its transcriptional targets, NOXA and PUMA. ITF2357 also induced autophagy in CRC cells, which was interpreted as a pro-survival response not correlated with lncH19 expression. Furthermore, ITF2357 induced apoptosis in 5-fluorouracil-resistant HCT-116 cells that express high levels of lncH19. Conclusion: This study shows that lncH19 expression contributes to ITF2357-induced apoptosis by stabilizing TP53. Overall, we suggest that lncH19 expression may be exploited to favor HDACi-induced cell death and overcome 5-fluorouracil chemoresistance.

Foodomics-Based Approaches Shed Light on the Potential Protective Effects of Polyphenols in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic and progressive inflammatory disorder affecting the gastrointestinal tract (GT) caused by a wide range of genetic, microbial, and environmental factors. IBD is characterized by chronic inflammation and decreased gut microbial diversity, dysbiosis, with a lower number of beneficial bacteria and a concomitant increase in pathogenic species. It is well known that dysbiosis is closely related to the induction of inflammation and oxidative stress, the latter caused by an imbalance between reactive oxygen species (ROS) production and cellular antioxidant capacity, leading to cellular ROS accumulation. ROS are responsible for intestinal epithelium oxidative damage and the increased intestinal permeability found in IBD patients, and their reduction could represent a potential therapeutic strategy to limit IBD progression and alleviate its symptoms. Recent evidence has highlighted that dietary polyphenols, the natural antioxidants, can maintain redox equilibrium in the GT, preventing gut dysbiosis, intestinal epithelium damage, and radical inflammatory responses. Here, we suggest that the relatively new foodomics approaches, together with new technologies for promoting the antioxidative properties of dietary polyphenols, including novel delivery systems, chemical modifications, and combination strategies, may provide critical insights to determine the clinical value of polyphenols for IBD therapy and a comprehensive perspective for implementing natural antioxidants as potential IBD candidate treatment.

MCL1 Inhibition Overcomes the Aggressiveness Features of Triple-Negative Breast Cancer MDA-MB-231 Cells.

Triple-Negative Breast Cancer (TNBC) is a particularly aggressive subtype among breast cancers (BCs), characterized by anoikis resistance, high invasiveness, and metastatic potential as well as Epithelial-Mesenchymal Transition (EMT) and stemness features. In the last few years, our research focused on the function of MCL1, an antiapoptotic protein frequently deregulated in TNBC. Here, we demonstrate that MCL1 inhibition by A-1210477, a specific BH3-mimetic, promotes anoikis/apoptosis in the MDA-MB-231 cell line, as shown via an increase in proapoptotic markers and caspase activation. Our evidence also shows A-1210477 effects on Focal Adhesions (FAs) impairing the integrin trim and survival signaling pathways, such as FAK, AKT, ERK, NF-κB, and GSK3ß-inducing anoikis, thus suggesting a putative role of MCL1 in regulation of FA dynamics. Interestingly, in accordance with these results, we observed a reduction in migratory and invasiveness capabilities as confirmed by a decrease in metalloproteinases (MMPs) levels following A-1210477 treatment. Moreover, MCL1 inhibition promotes a reduction in EMT characteristics as demonstrated by the downregulation of Vimentin, MUC1, DNMT1, and a surprising re-expression of E-Cadherin, suggesting a possible mesenchymal-like phenotype reversion. In addition, we also observed the downregulation of stemness makers such as OCT3/4, SOX2, NANOG, as well as CD133, EpCAM, and CD49f. Our findings support the idea that MCL1 inhibition in MDA-MB-231 could be crucial to reduce anoikis resistance, aggressiveness, and metastatic potential and to minimize EMT and stemness features that distinguish TNBC.

A Deadly Liaison between Oxidative Injury and p53 Drives Methyl-Gallate-Induced Autophagy and Apoptosis in HCT116 Colon Cancer Cells.

Methyl gallate (MG), which is a gallotannin widely found in plants, is a polyphenol used in traditional Chinese phytotherapy to alleviate several cancer symptoms. Our studies provided evidence that MG is capable of reducing the viability of HCT116 colon cancer cells, while it was found to be ineffective on differentiated Caco-2 cells, which is a model of polarized colon cells. In the first phase of treatment, MG promoted both early ROS generation and endoplasmic reticulum (ER) stress, sustained by elevated PERK, Grp78 and CHOP expression levels, as well as an upregulation in intracellular calcium content. Such events were accompanied by an autophagic process (16-24 h), where prolonging the time (48 h) of MG exposure led to cellular homeostasis collapse and apoptotic cell death with DNA fragmentation and p53 and γH2Ax activation. Our data demonstrated that a crucial role in the MG-induced mechanism is played by p53. Its level, which increased precociously (4 h) in MG-treated cells, was tightly intertwined with oxidative injury. Indeed, the addition of N-acetylcysteine (NAC), which is a ROS scavenger, counteracted the p53 increase, as well as the MG effect on cell viability. Moreover, MG promoted p53 accumulation into the nucleus and its inhibition by pifithrin-α (PFT-α), which is a negative modulator of p53 transcriptional activity, enhanced autophagy, increased the LC3-II level and inhibited apoptotic cell death. These findings provide new clues to the potential action of MG as a possible anti-tumor phytomolecule for colon cancer treatment.

Oncogenic BRAF and p53 Interplay in Melanoma Cells and the Effects of the HDAC Inhibitor ITF2357 (Givinostat).

Oncogenic BRAF mutations have been widely described in melanomas and promote tumour progression and chemoresistance. We previously provided evidence that the HDAC inhibitor ITF2357 (Givinostat) targets oncogenic BRAF in SK-MEL-28 and A375 melanoma cells. Here, we show that oncogenic BRAF localises to the nucleus of these cells, and the compound decreases BRAF levels in both the nuclear and cytosolic compartments. Although mutations in the tumour suppressor p53 gene are not equally frequent in melanomas compared to BRAF, the functional impairment of the p53 pathway may also contribute to melanoma development and aggressiveness. To understand whether oncogenic BRAF and p53 may cooperate, a possible interplay was considered in the two cell lines displaying a different p53 status, being p53 mutated into an oncogenic form in SK-MEL-28 and wild-type in A375 cells. Immunoprecipitation revealed that BRAF seems to preferentially interact with oncogenic p53. Interestingly, ITF2357 not only reduced BRAF levels but also oncogenic p53 levels in SK-MEL-28 cells. ITF2357 also targeted BRAF in A375 cells but not wild-type p53, which increased, most likely favouring apoptosis. Silencing experiments confirmed that the response to ITF2357 in BRAF-mutated cells depends on p53 status, thus providing a rationale for melanoma-targeted therapy.

Possible role for IL-40 and IL-40-producing cells in the lymphocytic infiltrated salivary glands of patients with primary Sjögren's syndrome.

OBJECTIVES: Aim of this study was to investigate the expression of interleukin (IL)-40, a new cytokine associated with B cells homoeostasis and immune response, in primary Sjögren syndrome (pSS) and in pSS-associated lymphomas. METHODS: 29 patients with pSS and 24 controls were enrolled. Minor salivary gland (MSG) biopsies from patients, controls and parotid gland biopsies from pSS-associated lymphoma were obtained. Quantitative gene expression analysis by TaqMan real-time PCR and immunohistochemistry for IL-40 were performed on MSG. MSG cellular sources of IL-40 were determined by flow-cytometry and immunofluorescence. Serum concentration of IL-40 was assessed by ELISA and cellular sources of IL-40 were determined by flow-cytometry. An in vitro assay with recombinant IL-40 (rIL-40) was performed to detect the effect on cytokine production from peripheral blood mononuclear cells (PBMCs). RESULTS: IL-40 was significantly increased in the lymphocytic infiltrated MSG of patients with pSS and correlated with focus score and with IL-4 and transforming growth factor-ß expression. In addition, IL-40 was increased in the serum of pSS and its levels correlated with the EULAR Sjögren's Syndrome Disease Activity Index score. B cells from patients were shown to be the major source of IL-40 at both tissue and peripheral level. PBMCs from patients, exposed to rIL-40 in vitro, released proinflammatory cytokines, specifically interferon-γ from B cells and T-CD8+ and tumour necrosis factor-α and IL-17 from both T-CD4+ and T-CD8+. IL-40 expression in parotid glands of pSS-associated lymphomas was also increased. Moreover, IL-40-driven NETosis was evidenced in neutrophils obtained from pSS. CONCLUSION: Our results suggest that IL-40 may play a role in pSS pathogenesis and pSS-associated lymphomas.

Interleukin 9 neutralisation reduces collagen-induced arthritis severity in mouse models.

OBJECTIVES: Interleukin 9 (IL-9) is a mediator of tissue damage in several inflammatory diseases. In this study we aimed to evaluate the effects of in vivo IL-9 neutralisation in mice developing collagen induced arthritis (CIA). METHODS: DBA/1 were immunised with collagen in Freund's complete adjuvant (CFA) to induce arthritis. Anti-IL-9 mAb was injected in mice after the onset of arthritis (Group A) or on the same day as sensitisation and again on the day of the challenge (Group B). Histological analysis was performed in joints of mice and spleen cells were also analysed by flow cytometry. A geneset analysis was carried out on whole tarsal joint tissue transcriptomes. RESULTS: IL-9 was over-expressed in swollen joints of mice developing arthritis. Treatment with anti-IL-9 mAb after arthritis onset efficiently down-modulated the severity of joint inflammation. Similarly, anti-IL-9 mAb administered on the same day as sensitisation and on the day of challenge also delayed the onset of arthritis. Anti-IL-9 mAb injection after the onset of arthritis was associated with a decrease of CD4+ TNF-α+ cells and an increase of CD4+ FoxP3+ IL-10+ cells. Geneset analysis in CIA showed an up-regulation of GATA3 with no significant direct interactions between IL-9 and GATA3, which instead was mediated by IL-5 through STAT6. CONCLUSIONS: Our results suggest that IL-9 is involved in the immunopathogenesis of CIA. Further implications for the clinical translation of our findings are discussed.

The Abundance of Tumor-Infiltrating CD8+ Tissue Resident Memory T Lymphocytes Correlates with Patient Survival in Glioblastoma.

Glial tumors alone account for 40% of all CNS tumors and present a low survival rate. The tumor microenvironment is a critical regulator of tumor progression and therapeutic effectiveness in glioma. Growing evidence from numerous studies of human solid tumor-infiltrating CD8+ T cells indicates that tissue-resident memory T cells (TRM) represent a substantial subpopulation of tumor-infiltrating lymphocytes (TILs). Although it is reported that some types of cancer patients with high immune infiltration tend to have better outcomes than patients with low immune infiltration, it seems this does not happen in gliomas. This study aimed to characterize TRMs cells in the glioma tumor microenvironment to identify their potential predictive and prognostic role and the possible therapeutic applications. Fluorescence activated cell sorting (FACS) analysis and immunofluorescence staining highlighted a statistically significant increase in CD8+ TRM cells (CD103+ and CD69+ CD8+ T cells) in gliomas compared to control samples (meningioma). In-silico analysis of a dataset of n = 153 stage IV glioma patients confirmed our data. Moreover, the gene expression analysis showed an increase in the expression of TRM-related genes in tumor tissues compared to normal tissues. This analysis also highlighted the positive correlation between genes associated with CD8+ TRM and TILs, indicating that CD8+ TRMs cells are present among the infiltrating T cells. Finally, high expression of Integrin subunit alpha E (ITGAE), the gene coding for the integrin CD103, and high CD8+ TILs abundance were associated with more prolonged survival, whereas high ITGAE expression but low CD8+ TILs abundance were associated with lower survival.

Recent Advances on the Innate Immune Response to Coxiella burnetii.

Coxiella burnetii is an obligate intracellular Gram-negative bacterium and the causative agent of a worldwide zoonosis known as Q fever. The pathogen invades monocytes and macrophages, replicating within acidic phagolysosomes and evading host defenses through different immune evasion strategies that are mainly associated with the structure of its lipopolysaccharide. The main transmission routes are aerosols and ingestion of fomites from infected animals. The innate immune system provides the first host defense against the microorganism, and it is crucial to direct the infection towards a self-limiting respiratory disease or the chronic form. This review reports the advances in understanding the mechanisms of innate immunity acting during C. burnetii infection and the strategies that pathogen put in place to infect the host cells and to modify the expression of specific host cell genes in order to subvert cellular processes. The mechanisms through which different cell types with different genetic backgrounds are differently susceptible to C. burnetii intracellular growth are discussed. The subsets of cytokines induced following C. burnetii infection as well as the pathogen influence on an inflammasome-mediated response are also described. Finally, we discuss the use of animal experimental systems for studying the innate immune response against C. burnetii and discovering novel methods for prevention and treatment of disease in humans and livestock.

Redox Imbalance and Mitochondrial Release of Apoptogenic Factors at the Forefront of the Antitumor Action of Mango Peel Extract.

Today, an improved understanding of cancer cell response to cellular stress has become more necessary. Indeed, targeting the intracellular pro-oxidant/antioxidant balance triggering the tumor commitment to cell demise could represent an advantageous strategy to develop cancer-tailored therapies. In this scenario, the present study shows how the peel extract of mango-a tropical fruit rich in phytochemicals with nutraceutical properties-can affect the cell viability of three colon cancer cell lines (HT29, Caco-2 and HCT116), inducing an imbalance of cellular redox responses. By using hydro-alcoholic mango peel extract (MPE), we observed a consistent decline in thiol group content, which was accompanied by upregulation of MnSOD-a mitochondrial scavenger enzyme that modulates the cellular response against oxidative damage. Such an effect was the consequence of an early production of mitochondrial superoxide anions that appeared after just 30 min of exposure of colon cancer cells to MPE. The effect was accompanied by mitochondrial injury, consisting of the dissipation of mitochondrial membrane potential and a decrease in the level of proteins localized in the mitochondrial membrane-such as voltage-dependent anion-selective channel (VDAC1), mitofilin, and some members of Bcl-2 family proteins (Mcl-1, Bcl-2 and Bcl-XL)-with the mitochondrial release of apoptogenic factors (cytochrome C and AIF). The analysis of the cytotoxic effects exerted by the different constituents of MPE (gallic acid, mangiferin, citric acid, quinic acid, pentagalloyl glucose, and methyl gallate) allowed us to identify those phytochemicals responsible for the observed anticancer effects, sustaining their future employment as chemopreventive or therapeutic agents.

Innate Immune Response to Tick-Borne Pathogens: Cellular and Molecular Mechanisms Induced in the Hosts.

Many pathogens are transmitted by tick bites, including Anaplasma spp., Ehrlichia spp., Rickettsia spp., Babesia and Theileria sensu stricto species. These pathogens cause infectious diseases both in animals and humans. Different types of immune effector mechanisms could be induced in hosts by these microorganisms, triggered either directly by pathogen-derived antigens or indirectly by molecules released by host cells binding to these antigens. The components of innate immunity, such as natural killer cells, complement proteins, macrophages, dendritic cells and tumor necrosis factor alpha, cause a rapid and intense protection for the acute phase of infectious diseases. Moreover, the onset of a pro-inflammatory state occurs upon the activation of the inflammasome, a protein scaffold with a key-role in host defense mechanism, regulating the action of caspase-1 and the maturation of interleukin-1ß and IL-18 into bioactive molecules. During the infection caused by different microbial agents, very similar profiles of the human innate immune response are observed including secretion of IL-1α, IL-8, and IFN-α, and suppression of superoxide dismutase, IL-1Ra and IL-17A release. Innate immunity is activated immediately after the infection and inflammasome-mediated changes in the pro-inflammatory cytokines at systemic and intracellular levels can be detected as early as on days 2-5 after tick bite. The ongoing research field of "inflammasome biology" focuses on the interactions among molecules and cells of innate immune response that could be responsible for triggering a protective adaptive immunity. The knowledge of the innate immunity mechanisms, as well as the new targets of investigation arising by bioinformatics analysis, could lead to the development of new methods of emergency diagnosis and prevention of tick-borne infections.

p62: Friend or Foe? Evidences for OncoJanus and NeuroJanus Roles.

p62 is a versatile protein involved in the delicate balance between cell death and survival, which is fundamental for cell fate decision in the context of both cancer and neurodegenerative diseases. As an autophagy adaptor, p62 recognizes polyubiquitin chains and interacts with LC3, thereby targeting the selected cargo to the autophagosome with consequent autophagic degradation. Beside this function, p62 behaves as an interactive hub in multiple signalling including those mediated by Nrf2, NF-κB, caspase-8, and mTORC1. The protein is thus crucial for the control of oxidative stress, inflammation and cell survival, apoptosis, and metabolic reprogramming, respectively. As a multifunctional protein, p62 falls into the category of those factors that can exert opposite roles in the cells. Chronic p62 accumulation was found in many types of tumors as well as in stress granules present in different forms of neurodegenerative diseases. However, the protein seems to have a Janus behaviour since it may also serve protective functions against tumorigenesis or neurodegeneration. This review describes the diversified roles of p62 through its multiple domains and interactors and specifically focuses on its oncoJanus and neuroJanus roles.

TNF-α, IL-17, and IL-22 production in the rectal mucosa of nonceliac wheat sensitivity patients: role of adaptive immunity.

In recent years, a new gluten- or wheat-related disease has emerged, a condition labeled "nonceliac gluten sensitivity" (NCGS) or "nonceliac wheat sensitivity" (NCWS). NCWS pathogenesis is still uncertain and attributed to very different mechanisms. We aimed to study the different T-lymphocyte subsets in the rectal mucosa of NCWS patients to demonstrate the possible contribution of adaptative immune response. Twelve patients (11 women, 1 man, age range 23-61 yr, median 32 yr) with a definitive diagnosis of NCWS were recruited at random for the present study. They underwent rectal endoscopy with multiple mucosal biopsies at the end of a double-blind placebo-controlled (DBPC) wheat challenge when they reported the reappearance of the symptoms. As controls we included 11 "healthy patients", sex- and age-matched with the patients who underwent colonoscopy evaluation for rectal bleeding due to hemorrhoids. Cells freshly obtained from rectal tissue were stained to detect anti-CD45, anti-CD3, anti-CD4, and anti-CD8. Furthermore, intracellular staining was performed with anti-tumor necrosis factor (TNF)-α, anti-interleukin (IL)-17, and anti-IL-22. Production of TNF-α by CD45+, CD3+, CD4+, and CD8+ cells, as well as of IL-17 by CD4+ cells, was higher in the rectal tissue of NCWS patients than in controls. On the contrary, IL-22 production by CD8+ cells was lower in NCWS patients than in the controls. In NCWS patients diagnosed by DBPC wheat challenge, there is a complex immunological activation, with a significant role for the adaptive response.NEW & NOTEWORTHY Nonceliac wheat sensitivity (NCWS) is a syndrome characterized by symptoms triggered by gluten intake. The pathogenesis is still uncertain. Studies have shown a role for innate immunity. We demonstrated that production of TNF-α by CD45+, CD3+, CD4+, and CD8+ cells and of IL-17 by CD4+ cells is higher in the rectal tissue of NCWS patients than in controls. We clearly demonstrated that in patients with NCWS there is a significant role for the adaptive response.

Wheat Consumption Leads to Immune Activation and Symptom Worsening in Patients with Familial Mediterranean Fever: A Pilot Randomized Trial.

We have identified a clinical association between self-reported non-celiac wheat sensitivity (NCWS) and Familial Mediterranean Fever (FMF). Objectives: A) To determine whether a 2-week double-blind placebo-controlled (DBPC) cross-over wheat vs. rice challenge exacerbates the clinical manifestations of FMF; B) to evaluate innate immune responses in NCWS/FMF patients challenged with wheat vs. rice. The study was conducted at the Department of Internal Medicine of the University Hospital of Palermo and the Hospital of Sciacca, Italy. Six female volunteers with FMF/NCWS (mean age 36 ± 6 years) were enrolled, 12 age-matched non-FMF, NCWS females, and 8 sex- and age-matched healthy subjects served as controls. We evaluated: 1. clinical symptoms by the FMF-specific AIDAI (Auto-Inflammatory Diseases Activity Index) score; 2. serum soluble CD14 (sCD14), C-reactive protein (CRP), and serum amyloid A (SSA); 3. circulating CD14+ monocytes expressing interleukin (IL)-1ß and tumor necrosis factor (TNF)-α. The AIDAI score significantly increased in FMF patients during DBPC with wheat, but not with rice (19 ± 6.3 vs. 7 ± 1.6; p = 0.028). sCD14 values did not differ in FMF patients before and after the challenge, but were higher in FMF patients than in healthy controls (median values 11357 vs. 8710 pg/ml; p = 0.002). The percentage of circulating CD14+/IL-1ß+ and of CD14+/TNF-α+ monocytes increased significantly after DBPC with wheat vs. baseline or rice challenge. Self-reported NCWS can hide an FMF diagnosis. Wheat ingestion exacerbated clinical and immunological features of FMF. Future studies performed on consecutive FMF patients recruited in centers for auto-inflammatory diseases will determine the real frequency and relevance of this association.

T helper 1 response is correlated with widespread pain, fatigue, sleeping disorders and the quality of life in patients with fibromyalgia and is modulated by hyperbaric oxygen therapy.

OBJECTIVES: Hyperbaric oxygen therapy (HBOT) has been used as treatment for different clinical conditions, including fibromyalgia (FM). HBOT modulates brain activity, ameliorates chronic pain and modifies the ratio of immune cells. Clinical studies have provided evidence that FM is associated with immune system dysregulation. In the present study we aimed to evaluate the effect of HBOT on immune system and on the quality of life-style of FM patients. METHODS: Patients with primary FM and controls were treated with HBOT. Physical, emotional and social assessment, quality of sleep, tender points, intensity score, WPI and symptom severity were evaluated before and after HBOT. Furthermore, a characterisation of CD4 T lymphocytes and their cytokine production was performed by flow cytometry. The expression of TNF-α, IFN-γ, IL-17, IL-9 and IL-22 was also assessed by RT-PCR. Finally, the serum levels of serotonin were evaluated by ELISA. RESULTS: Our results confirm the participation of immune system in the pathogenesis of FM and highlight the impact of HBOT treatment, with particular regard to the changes on proinflammatory cytokines production by CD4 T cells subsets. CONCLUSIONS: FM patients show a Th1 signature and the activation of this subset is modulated by HBOT.

Proinflammatory CX3CR1+CD59+Tumor Necrosis Factor-Like Molecule 1A+Interleukin-23+ Monocytes Are Expanded in Patients With Ankylosing Spondylitis and Modulate Innate Lymphoid Cell 3 Immune Functions.

OBJECTIVE: Gut-derived innate lymphoid cell 3 (ILC3) has been shown to participate in the pathogenesis of ankylosing spondylitis (AS). CX3 CR1+ mononuclear phagocytes (MNPs) have been demonstrated to modulate ILC3 function in the gut. This study was undertaken to investigate the role of proinflammatory CX3 CR1+CD59+ MNPs in modulating ILC3 function in AS patients. METHODS: MNP subsets in the blood of AS patients and controls were analyzed by flow cytometry. The presence of CX3 CR1+CD59+ cells in tissue was confirmed by confocal microscopy. Expression of the proinflammatory chemokines CX3 CL1 and CCL2 and decoy receptor 6 (DcR-6) was analyzed. Peripheral CX3 CR1+CD59+ cells were cocultured with ILC3, and changes in their frequency were evaluated by flow cytometry. Transcriptome analysis of circulating CX3 CR1+ monocytes was also performed. RESULTS: DcR-6 deficiency and CCL2 overexpression were observed in inflamed tissues from AS patients. In the gut, the proinflammatory CX3 CR1+CD59+ MNP population was expanded, correlated with the presence of bacteria, and produced high levels of tumor necrosis factor-like molecule 1A (TL1A) and interleukin-23 (IL-23). MNPs positive for CD11b, CD11c, and major histocompatibility complex class II, predominantly expressing CX3 CR1, were also expanded in the small intestines of treatment-naive SKG relative to BALB/c mice. The frequency of gut-derived CX3 CR1+CD59+CCR9+TL1A+IL-23+ MNPs was significantly higher in the peripheral blood and synovial fluid of AS patients than controls. CCR9+CX3 CR1+CD59+ monocytes were also expanded in AS synovial and bone marrow samples. Transcriptome analysis of isolated CX3 CR1+CD59+ monocytes demonstrated a specific proinflammatory profile in AS. Isolated proinflammatory CX3 CR1+CD59+ MNPs from AS patients induced the expansion and activation of ILC3. CONCLUSION: Proinflammatory CX3 CR1+CD59+TL1A+IL-23+ MNPs are expanded in AS patients and display a specific proinflammatory transcriptome profile. Given the ability of these cells to support ILC3 expansion, they may promote a sustained proinflammatory status in AS.

Interleukin-25 Axis Is Involved in the Pathogenesis of Human Primary and Experimental Murine Sjögren's Syndrome.

OBJECTIVE: To investigate the role of the interleukin-25 (IL-25)/IL-17 receptor B (IL-17RB) axis in experimental Sjögren's syndrome (SS) and in patients with primary SS and primary SS-associated lymphoma. METHODS: Expression of IL-25, IL-17RB, IL-17B, and tumor necrosis factor receptor-associated factor 6 (TRAF6) was analyzed on minor salivary gland (SG) samples from patients with primary SS and on parotid gland samples from patients with primary SS-associated B cell non-Hodgkin's lymphoma (NHL). IL-17RB expression and the frequencies of natural group 2 innate lymphoid cells (ILC2s), inflammatory ILC2s, and M2-polarized macrophages were assessed by flow cytometry in SG mononuclear cells and peripheral blood mononuclear cells (PBMCs). Tissue distribution of ILC2s was studied by confocal microscopy. The role of recombinant IL-25 and of rituximab in modulating IL-25 expression was investigated in in vitro studies. IL-25/IL-17RB and TRAF6 expression and the role of IL-25 inhibition were also studied in the experimental murine model of SS. RESULTS: Activation of the IL-25/IL-17RB/TRAF6 axis correlated with the focus score and was observed in patients with primary SS and in patients with primary SS-associated NHL. A significant increase in the frequency of inflammatory ILC2s was observed both in SG mononuclear cells and in PBMCs. IL-25 stimulation of isolated SG mononuclear cells and PBMCs from patients and controls resulted both in inflammatory ILC2 expansion and in increased autoantibody production. Rituximab modulated expression of inflammatory ILC2s and IL-25 in primary SS. SG protein-immunized mice developed overt SS symptoms with increased IL-25 expression and increased frequency of CD4+IL-17RB+TRAF6+ cells. IL-25 neutralization attenuated disease progression and tissue pathology in mice with experimental SS. CONCLUSION: IL-25 may promote the inflammatory state in primary SS and may be a potential target for novel disease-modifying therapeutic strategies in patients with primary SS.

Janus-faced liposomes enhance antimicrobial innate immune response in Mycobacterium tuberculosis infection.

We have generated unique asymmetric liposomes with phosphatidylserine (PS) distributed at the outer membrane surface to resemble apoptotic bodies and phosphatidic acid (PA) at the inner layer as a strategy to enhance innate antimycobacterial activity in phagocytes while limiting the inflammatory response. Results show that these apoptotic body-like liposomes carrying PA (ABL/PA) (i) are more efficiently internalized by human macrophages than by nonprofessional phagocytes, (ii) induce cytosolic Ca(2+) influx, (iii) promote Ca(2+)-dependent maturation of phagolysosomes containing Mycobacterium tuberculosis (MTB), (iv) induce Ca(2+)-dependent reactive oxygen species (ROS) production, (v) inhibit intracellular mycobacterial growth in differentiated THP-1 cells as well as in type-1 and -2 human macrophages, and (vi) down-regulate tumor necrosis factor (TNF)-α, interleukin (IL)-12, IL-1ß, IL-18, and IL-23 and up-regulate transforming growth factor (TGF)-ß without altering IL-10, IL-27, and IL-6 mRNA expression. Also, ABL/PA promoted intracellular killing of M. tuberculosis in bronchoalveolar lavage cells from patients with active pulmonary tuberculosis. Furthermore, the treatment of MTB-infected mice with ABL/PA, in combination or not with isoniazid (INH), dramatically reduced lung and, to a lesser extent, liver and spleen mycobacterial loads, with a concomitant 10-fold reduction of serum TNF-α, IL-1ß, and IFN-γ compared with that in untreated mice. Altogether, these results suggest that apoptotic body-like liposomes may be used as a Janus-faced immunotherapeutic platform to deliver polar secondary lipid messengers, such as PA, into phagocytes to improve and recover phagolysosome biogenesis and pathogen killing while limiting the inflammatory response.