Release of IL-1ß and IL-18 in human primary bronchial epithelial cells exposed to cigarette smoke is independent of NLRP3.

Cigarette smoke (CS) is a major risk factor for chronic lung diseases and promotes activation of pattern recognition receptors in the bronchial epithelium. NOD-like receptor family, pyrin domain-containing 3 (NLRP3) is a pattern recognition receptor whose activation leads to caspase-1 cleavage, maturation/release of IL-1ß and IL-18, and eventually pyroptosis. Whether the NLRP3 inflammasome participates in CS-induced inflammation in bronchial epithelial cells is still unclear. Herein, we evaluated the involvement of NLRP3 in CS-induced inflammatory responses in human primary bronchial epithelial cells. To this purpose, human primary bronchial epithelial cells were stimulated with CS extracts (CSE) and lytic cell death, caspase activation (-1, -8, -3/7), cytokine release (IL-1ß, IL-18, and IL-8), NLRP3, pro-IL-1ß/pro-IL-18 mRNA, and protein expression were measured. The impact of inhibitors of NLRP3 (MCC950), caspases, and the effect of the antioxidant N-acetyl cysteine were evaluated. We found that CSE increased pro-IL-1ß expression and induced activation of caspase-1 and release of IL-1ß and IL-18. These events were independent of NLRP3 and we found that NLRP3 was not expressed. N-acetyl cysteine reverted CSE-induced caspase-1 activation. Overall, our findings support that the bronchial epithelium may play a central role in the release of IL-1 family cytokines independently of NLRP3 in the lungs of smokers.

Gut Microbiota Manipulation as a Tool for Colorectal Cancer Management: Recent Advances in Its Use for Therapeutic Purposes.

Colorectal cancer (CRC) is a multifaceted disease influenced by both environmental and genetic factors. A large body of literature has demonstrated the role of gut microbes in promoting inflammatory responses, creating a suitable microenvironment for the development of skewed interactions between the host and the gut microbiota and cancer initiation. Even if surgery is the primary therapeutic strategy, patients with advanced disease or cancer recurrence after surgery remain difficult to cure. Therefore, the gut microbiota has been proposed as a novel therapeutic target in light of recent promising data in which it seems to modulate the response to cancer immunotherapy. The use of microbe-targeted therapies, including antibiotics, prebiotics, live biotherapeutics, and fecal microbiota transplantation, is therefore considered to support current therapies in CRC management. In this review, we will discuss the importance of host-microbe interactions in CRC and how promoting homeostatic immune responses through microbe-targeted therapies may be useful in preventing/treating CRC development.

Gold nanowires-based sensor for quantification of H2O2 released by human airway epithelial cells.

Hydrogen peroxide (H2O2) is a biomarker relevant for oxidative stress monitoring. Most chronic airway diseases are characterized by increased oxidative stress. To date, the main methods for the detection of this analyte are expensive and time-consuming laboratory techniques such as fluorometric and colorimetric assays. There is a growing interest in the development of electrochemical sensors for H2O2 detection due to their low cost, ease of use, sensitivity and rapid response. In this work, an electrochemical sensor based on gold nanowire arrays has been developed. Thanks to the catalytic activity of gold against hydrogen peroxide reduction and the high surface area of nanowires, this sensor allows the quantification of this analyte in a fast, efficient and selective way. The sensor was obtained by template electrodeposition and consists of gold nanowires about 5 µm high and with an average diameter of about 200 nm. The high active surface area of this electrode, about 7 times larger than a planar gold electrode, ensured a high sensitivity of the sensor (0.98 µA µM-1cm-2). The sensor allows the quantification of hydrogen peroxide in the range from 10 µM to 10 mM with a limit of detection of 3.2 µM. The sensor has excellent properties in terms of reproducibility, repeatability and selectivity. The sensor was validated by quantifying the hydrogen peroxide released by human airways A549 cells exposed or not to the pro-oxidant compound rotenone. The obtained results were validated by comparing them with those obtained by flow cytometry after staining the cells with the fluorescent superoxide-sensitive Mitosox Red probe giving a very good concordance.

Caspase-8 activation by cigarette smoke induces pro-inflammatory cell death of human macrophages exposed to lipopolysaccharide.

Cigarette smoking impairs the lung innate immune response making smokers more susceptible to infections and severe symptoms. Dysregulation of cell death is emerging as a key player in chronic inflammatory conditions. We have recently reported that short exposure of human monocyte-derived macrophages (hMDMs) to cigarette smoke extract (CSE) altered the TLR4-dependent response to lipopolysaccharide (LPS). CSE caused inhibition of the MyD88-dependent inflammatory response and activation of TRIF/caspase-8/caspase-1 pathway leading to Gasdermin D (GSDMD) cleavage and increased cell permeability. Herein, we tested the hypothesis that activation of caspase-8 by CSE increased pro-inflammatory cell death of LPS-stimulated macrophages. To this purpose, we measured apoptotic and pyroptotic markers as well as the expression/release of pro-inflammatory mediators in hMDMs exposed to LPS and CSE, alone or in combination, for 6 and 24 h. We show that LPS/CSE-treated hMDMs, but not cells treated with CSE or LPS alone, underwent lytic cell death (LDH release) and displayed apoptotic features (activation of caspase-8 and -3/7, nuclear condensation, and mitochondrial membrane depolarization). Moreover, the negative regulator of caspase-8, coded by CFLAR gene, was downregulated by CSE. Activation of caspase-3 led to Gasdermin E (GSDME) cleavage. Notably, lytic cell death caused the release of the damage-associated molecular patterns (DAMPs) heat shock protein-60 (HSP60) and S100A8/A9. This was accompanied by an impaired inflammatory response resulting in inhibited and delayed release of IL6 and TNF. Of note, increased cleaved caspase-3, higher levels of GSDME and altered expression of cell death-associated genes were found in alveolar macrophages of smoker subjects compared to non-smoking controls. Overall, our findings show that CSE sensitizes human macrophages to cell death by promoting pyroptotic and apoptotic pathways upon encountering LPS. We propose that while the delayed inflammatory response may result in ineffective defenses against infections, the observed cell death associated with DAMP release may contribute to establish chronic inflammation. CS exposure sensitizes human macrophages to pro-inflammatory cell death. Upon exposure to LPS, CS inhibits the TLR4/MyD88 inflammatory response, downregulating the pro-inflammatory genes TNF and IL6 and the anti-apoptotic gene CFLAR, known to counteract caspase-8 activity. CS enhances caspase-8 activation through TLR4/TRIF, with a partial involvement of RIPK1, resulting on the activation of caspase-1/GSDMD axis leading to increased cell permeability and DAMP release through gasdermin pores [19]. At later timepoints caspase-3 becomes strongly activated by caspase-8 triggering apoptotic events which are associated with mitochondrial membrane depolarization, gasdermin E cleavage and secondary necrosis with consequent massive DAMP release.

Electrochemical Quantification of H2O2 Released by Airway Cells Growing in Different Culture Media.

Quantification of oxidative stress is a challenging task that can help in monitoring chronic inflammatory respiratory airway diseases. Different studies can be found in the literature regarding the development of electrochemical sensors for H2O2 in cell culture medium to quantify oxidative stress. However, there are very limited data regarding the impact of the cell culture medium on the electrochemical quantification of H2O2. In this work, we studied the effect of different media (RPMI, MEM, DMEM, Ham's F12 and BEGM/DMEM) on the electrochemical quantification of H2O2. The used electrode is based on reduced graphene oxide (rGO) and gold nanoparticles (AuNPs) and was obtained by co-electrodeposition. To reduce the electrode fouling by the medium, the effect of dilution was investigated using diluted (50% v/v in PBS) and undiluted media. With the same aim, two electrochemical techniques were employed, chronoamperometry (CH) and linear scan voltammetry (LSV). The influence of different interfering species and the effect of the operating temperature of 37 °C were also studied in order to simulate the operation of the sensor in the culture plate. The LSV technique made the sensor adaptable to undiluted media because the test time is short, compared with the CH technique, reducing the electrode fouling. The long-term stability of the sensors was also evaluated by testing different storage conditions. By storing the electrode at 4 °C, the sensor performance was not reduced for up to 21 days. The sensors were validated measuring H2O2 released by two different human bronchial epithelial cell lines (A549, 16HBE) and human primary bronchial epithelial cells (PBEC) grown in RPMI, MEM and BEGM/DMEM media. To confirm the results obtained with the sensor, the release of reactive oxygen species was also evaluated with a standard flow cytometry technique. The results obtained with the two techniques were very similar. Thus, the LSV technique permits using the proposed sensor for an effective oxidative stress quantification in different culture media and without dilution.

IL10 Secretion Endows Intestinal Human iNKT Cells with Regulatory Functions Towards Pathogenic T Lymphocytes.

BACKGROUND AND AIMS: Invariant natural killer T [iNKT] cells perform pleiotropic functions in different tissues by secreting a vast array of pro-inflammatory and cytotoxic molecules. However, the presence and function of human intestinal iNKT cells capable of secreting immunomodulatory molecules such as IL-10 has never been reported so far. Here we describe for the first time the presence of IL10-producing iNKT cells [NKT10 cells] in the intestinal lamina propria of healthy individuals and of Crohn's disease [CD] patients. METHODS: Frequency and phenotype of NKT10 cells were analysed ex vivo from intestinal specimens of Crohn's disease [n = 17] and controls [n = 7]. Stable CD-derived intestinal NKT10 cell lines were used to perform in vitro suppression assays and co-cultures with patient-derived mucosa-associated microbiota. Experimental colitis models were performed by adoptive cell transfer of splenic naïve CD4+ T cells in the presence or absence of IL10-sufficient or -deficient iNKT cells. In vivo induction of NKT10 cells was performed by administration of short chain fatty acids [SCFA] by oral gavage. RESULTS: Patient-derived intestinal NKT10 cells demonstrated suppressive capabilities towards pathogenic CD4+ T cells. The presence of increased proportions of mucosal NKT10 cells associated with better clinical outcomes in CD patients. Moreover, an intestinal microbial community enriched in SCFA-producing bacteria sustained the production of IL10 by iNKT cells. Finally, IL10-deficient iNKT cells failed to control the pathogenic activity of adoptively transferred CD4+ T cells in an experimental colitis model. CONCLUSIONS: These results describe an unprecedentd IL10-mediated immunoregulatory role of intestinal iNKT cells in controlling the pathogenic functions of mucosal T helper subsets and in maintaining the intestinal immune homeostasis.

Antibiotic-associated dysbiosis affects the ability of the gut microbiota to control intestinal inflammation upon fecal microbiota transplantation in experimental colitis models.

BACKGROUND: The gut microbiota plays a central role in host physiology and in several pathological mechanisms in humans. Antibiotics compromise the composition and functions of the gut microbiota inducing long-lasting detrimental effects on the host. Recent studies suggest that the efficacy of different clinical therapies depends on the action of the gut microbiota. Here, we investigated how different antibiotic treatments affect the ability of the gut microbiota to control intestinal inflammation upon fecal microbiota transplantation in an experimental colitis model and in ex vivo experiments with human intestinal biopsies. RESULTS: Murine fecal donors were pre-treated with different antibiotics, i.e., vancomycin, streptomycin, and metronidazole before FMT administration to colitic animals. The analysis of the gut microbiome, fecal metabolome, and the immunophenotyping of colonic lamina propria immune cells revealed that antibiotic pre-treatment significantly influences the capability of the microbiota to control intestinal inflammation. Streptomycin and vancomycin-treated microbiota failed to control intestinal inflammation and were characterized by the blooming of pathobionts previously associated with IBD as well as with metabolites related to the presence of oxidative stress and metabolism of simple sugars. On the contrary, the metronidazole-treated microbiota retained its ability to control inflammation co-occurring with the enrichment of Lactobacillus and of innate immune responses involving iNKT cells. Furthermore, ex vivo cultures of human intestinal lamina propria mononuclear cells and iNKT cell clones from IBD patients with vancomycin pre-treated sterile fecal water showed a Th1/Th17 skewing in CD4+ T-cell populations; metronidazole, on the other hand, induced the polarization of iNKT cells toward the production of IL10. CONCLUSIONS: Diverse antibiotic regimens affect the ability of the gut microbiota to control intestinal inflammation in experimental colitis by altering the microbial community structure and microbiota-derived metabolites. Video Abstract.

Fecal Microbiota Transplantation Controls Murine Chronic Intestinal Inflammation by Modulating Immune Cell Functions and Gut Microbiota Composition.

Different gastrointestinal disorders, including inflammatory bowel diseases (IBD), have been linked to alterations of the gut microbiota composition, namely dysbiosis. Fecal microbiota transplantation (FMT) is considered an encouraging therapeutic approach for ulcerative colitis patients, mostly as a consequence of normobiosis restoration. We recently showed that therapeutic effects of FMT during acute experimental colitis are linked to functional modulation of the mucosal immune system and of the gut microbiota composition. Here we analysed the effects of therapeutic FMT administration during chronic experimental colitis, a condition more similar to that of IBD patients, on immune-mediated mucosal inflammatory pathways. Mucus and feces from normobiotic donors were orally administered to mice with established chronic Dextran Sodium Sulphate (DSS)-induced colitis. Immunophenotypes and functions of infiltrating colonic immune cells were evaluated by cytofluorimetric analysis. Compositional differences in the intestinal microbiome were analyzed by 16S rRNA sequencing. Therapeutic FMT in mice undergoing chronic intestinal inflammation was capable to decrease colonic inflammation by modulating the expression of pro-inflammatory genes, antimicrobial peptides, and mucins. Innate and adaptive mucosal immune cells manifested a reduced pro-inflammatory profile in FMT-treated mice. Finally, restoration of a normobiotic core ecology contributed to the resolution of inflammation. Thus, FMT is capable of controlling chronic intestinal experimental colitis by inducing a concerted activation of anti-inflammatory immune pathways, mechanistically supporting the positive results of FMT treatment reported in ulcerative colitis patients.

Mucosa-associated microbiota drives pathogenic functions in IBD-derived intestinal iNKT cells.

Inflammatory bowel disease (IBD) pathogenesis has been linked to the aberrant activation of the Gut-associated lymphoid tissues against components of the intestinal microbiota. Although the contribution of CD4+ T helper cells to inflammatory processes is being increasingly acknowledged, the functional engagement of human invariant natural killer T (iNKT) cells is still poorly defined. Here, we evaluated the functional characteristics of intestinal iNKT cells during IBD pathogenesis and to exploit the role of mucosa-associated microbiota recognition in triggering iNKT cells' pro-inflammatory responses in vivo. Lamina propria iNKT cells, isolated from surgical specimens of active ulcerative colitis and Crohn's disease patients and non-IBD donors, were phenotypically and functionally analyzed ex vivo, and stable cell lines and clones were generated for in vitro functional assays. iNKT cells expressing a pro-inflammatory cytokine profile were enriched in the lamina propria of IBD patients, and their exposure to the mucosa-associated microbiota drives pro-inflammatory activation, inducing direct pathogenic activities against the epithelial barrier integrity. These observations suggest that iNKT cell pro-inflammatory functions may contribute to the fuelling of intestinal inflammation in IBD patients.

Pathogenicity of In Vivo Generated Intestinal Th17 Lymphocytes is IFNγ Dependent.

BACKGROUND AND AIMS: T helper 17 [Th17] cells are crucially involved in the immunopathogenesis of inflammatory bowel diseases in humans. Nevertheless, pharmacological blockade of interleukin 17A [IL17A], the Th17 signature cytokine, yielded negative results in patients with Crohn's disease [CD], and attempts to elucidate the determinants of Th17 cells' pathogenicity in the gut have so far proved unsuccessful. Here, we aimed to identify and functionally validate the pathogenic determinants of intestinal IL-17-producing T cells. METHODS: In vivo-generated murine intestinal IL-17-producing T cells were adoptively transferred into immunodeficient Rag1-/- recipients to test their pathogenicity. Human IL-17, IFNγ/IL-17, and IFNγ actively secreting T cell clones were generated from lamina propria lymphocytes of CD patients. The pathogenic activity of intestinal IL-17-producing T cells against the intestinal epithelium was evaluated. RESULTS: IL-17-producing cells with variable colitogenic activity can be generated in vivo using different experimental colitis models. The pathogenicity of IL-17-secreting cells was directly dependent on their IFNγ secretion capacity, as demonstrated by the reduced colitogenic activity of IL-17-secreting cells isolated from IFNγ-/- mice. Moreover, IFNγ production is a distinguished attribute of CD-derived lamina propria Th17 cells. IFNγ secretion by CD-derived IL-17-producing intestinal clones is directly implicated in the epithelial barrier disruption through the modulation of tight junction proteins. CONCLUSIONS: Intestinal Th17 cell pathogenicity is associated with IFNγ production, which directly affects intestinal permeability through the disruption of epithelial tight junctions.

APOA-1Milano muteins, orally delivered via genetically modified rice, show anti-atherogenic and anti-inflammatory properties in vitro and in Apoe-/- atherosclerotic mice.

BACKGROUND: Atherosclerosis is a slowly progressing, chronic multifactorial disease characterized by the accumulation of lipids, inflammatory cells, and fibrous tissue that drives to the formation of asymmetric focal thickenings in the tunica intima of large and mid-sized arteries. Despite the high therapeutic potential of ApoA-1 proteins, the purification and delivery into the disordered organisms of these drugs is still limited by low efficiency in these processes. METHODS AND RESULTS: We report here a novel production and delivery system of anti-atherogenic APOA-1Milano muteins (APOA-1M) by means of genetically modified rice plants. APOA-1M, delivered as protein extracts from transgenic rice seeds, significantly reduced macrophage activation and foam cell formation in vitro in oxLDL-loaded THP-1 model. The APOA-1M delivery method and therapeutic efficacy was tested in healthy mice and in Apoe-/- mice fed with high cholesterol diet (Western Diet, WD). APOA-1M rice milk significantly reduced atherosclerotic plaque size and lipids composition in aortic sinus and aortic arch of WD-fed Apoe-/- mice as compared to wild type rice milk-treated, WD-fed Apoe-/- mice. APOA-1M rice milk also significantly reduced macrophage number in liver of WD-fed Apoe-/- mice as compared to WT rice milk treated mice. TRANSLATIONAL IMPACT: The delivery of therapeutic APOA-1M full length proteins via oral administration of rice seeds protein extracts (the 'rice milk') to the disordered organism, without any need of purification, might overcome the main APOA1-based therapies' limitations and improve the use of this molecules as therapeutic agents for cardiovascular patients.

The TGF-ß pathway is activated by 5-fluorouracil treatment in drug resistant colorectal carcinoma cells.

TGF-ß pathway is generally associated with the processes of metastasis, angiogenesis and EMT in cancer. Very little is known, however, about the role of TGF-ß in cancer drug resistance. In this work, we show a specific activation of the TGF-ß pathway in consequence of chemotherapeutic treatment in in vivo and in vitro models of colorectal carcinoma. 5-Fluorouracil (5FU) was able to stimulate the activation of SMAD3 and the transcription of specific genes such as ACVRL1, FN1 and TGFB1. On the other hand, the specific inhibition of TGF-ßRI was able to repress the 5FU-induced genes transcription and to restore the sensitivity of chemoresistant cells to the toxic action of the drug, by decreasing the expression of BCL2L1 and ID1 genes. The role of the TGF-ß molecule in the chemoresistant colon carcinoma cells' response to 5FU was further demonstrated by conditioned medium (CM) experiments: CM from 5FU-treated chemoresistant cells was able to protect chemosensitive cells against the toxic action of 5FU. In conclusion, these findings showed the pivotal role of TGF-ß pathway in colon cancer mechanisms of drug resistance suggesting new possible approaches in diagnosis and treatment of colon cancer patients.