Lipid antigen presentation and thymic selection of iNKT cells

The previous introductory part has reviewed the current knowledge on CD1 and lipid immunology. Although the last 20 years witnessed a dramatic expansion of the field, many aspects remain elusive and require additional studies. Little is known about the mechanisms by which stimulatory CD1:lipid antigen complexes are generated and how is the influence of CD1d lipid antigen presentation on thymic selection and peripheral activation of iNKT cells. The aim of this study is to investigate the mechanisms contributing to generation of lipid-specific complexes. In particular, I aim to investigate the impact of LTP residing in different cellular compartments on lipid antigen generation, loading on CD1 molecules, presentation to T cells, positive selection in the thymus as well as stimulation of peripheral CD1-restricted T cells. These studies are divided in three parts: 1. Impact of the absence of the lysosomal LTP NPC2 or of the enzyme β- Galactosidase on lipid antigen presentation and on thymic selection of iNKT cells. Recent studies demonstrate that different lysosomal lipid transfer proteins, like saposins and GM2-A, are implicated in loading of endogenous and exogenous lipid antigens onto CD1d and thus in CD1d-restricted presentation of lipid antigens important for iNKT cells thymic selection [84, 87]. Development of iNKT cells is also completely abolished in mice deficient in saposins [84]. Deficiencies in the lysosomal enzyme Hexb, which is involved in the lipid degradation pathway, or in saposins cause severe imbalances in lipid metabolism. It has never been addressed whether lipid metabolism alteration, and thus accumulation of storage lipids, could contribute to the impairment of lipid antigen presentation and iNKT cell thymic selection. This study has directly assessed the contribution of imbalance in lipid metabolism, consequence of ablation of the LTP NPC2 and of the enzyme β-Galactosidase, on generation of stimulatory CD1d:lipid antigen complexes, on their presentation to iNKT cells, and thus on thymic selection of iNKT cells. 2. Investigation of the functional interaction of CD1e with the other CD1 molecules CD1e is the fifth member of the CD1 family. It is the only member of the CD1 family that does not reach the plasma membrane and is soluble in lysosomes, therefore it might have functions going beyond presentation of lipid antigen to T cells. CD1e binds lipid molecules in the lysosomal compartment and facilitates processing of complex glycolipid antigens, thus participating in lipid editing of CD1b-presented antigens [16]. Nothing is known about the role of CD1e in editing CD1 restricted lipids, either of self or of microbial origin. This study has demonstrated the capacity of CD1e to interact with all lysosomal CD1 molecules and to influence the response of lipid specific-T cells, including that of iNKT cells. Generation of transgenic mice expressing CD1e in professional APC allowed us to study the contribution of CD1e in the formation of antigenic CD1d:lipid complexes and their capacity to activate iNKT cells. 3. Impact of the absence of the peroxisomal and cytoplasmic LTP SCP-2 on lipid antigen presentation and on thymic selection of iNKT cells. All the LTP influencing lipid antigenicity and thus iNKT cell thymic selection that have been investigated so far are localised within the lysosome. The role of LTP residing in other subcellular compartments, like the cytoplasm and the peroxisomes, on presentation of lipid antigens has never been investigated. Furthermore, whether cytoplasmic LTP are involved in generation and intracellular trafficking of endogenous-lipid antigens remains unknown. This study has directly investigated the role of a cytoplasmic/peroxisomal LTP in the generation of the repertoire of selecting lipids, and its impact on thymic selection of iNKT cells

Implementation of an automated inclusion system for the histological analysis of murine tissue samples: A feasibility study in DSS-induced chronic colitis:

Animal models are powerful tools to expand our understanding of human diseases. Histopathological evaluation of murine experimental models is often required to support further research; thus, a more rigorous evaluation of murine histological samples is strongly advocated. Indeed, the overall quality of tissue sections is critical to draw reliable and accurate conclusions. As several methodological variables may reduce the reliability of the pathological analysis, a standardization of the procedural steps required for the processing of histological murine tissues is advisable. Here, we describe a method to standardize the technical procedure from the initial preparation to the paraffin embedding of murine samples. Specifically, we have implemented an automated inclusion system, that is, the SAKURA Tissue-Tek inclusion instrument, which is routinely used for paraffin inclusion of human samples, to process murine specimens of intestinal inflammation. Colitis severity was assessed in chronically Dextran Sodium Sulphate (DSS)–treated mice by cytofluorimetric analysis of colonic cellular infiltrates, expression of inflammatory genes and histopathological analysis of tissue samples, comparing manual and automated tissue preparation systems. We here conclude that implementation of this technique can significantly increase the quality and the reliability of histopathological examination of murine tissues

NOX2-derived reactive oxygen species are crucial for CD29-induced pro-survival signalling in cardiomyocytes

Aims The highly expressed cell adhesion receptor CD29 (β1-integrin) is essential for cardiomyocyte growth and survival, and its loss of function causes severe heart disease. However, CD29-induced signalling in cardiomyocytes is ill defined and may involve reactive oxygen species (ROS). A decisive source of cardiac ROS is the abundant NADPH oxidase (NOX) isoform NOX2. Because understanding of NOX-derived ROS in the heart is still poor, we sought to test the role of ROS and NOX in CD29-induced survival signalling in cardiomyocytes. Methods and results In neonatal rat ventricular myocytes, CD29 activation induced intracellular ROS formation (oxidative burst) as assessed by flow cytometry using the redox-sensitive fluorescent dye dichlorodihydrofluorescein diacetate. This burst was inhibited by apocynin and diphenylene iodonium. Further, activation of CD29 enhanced NOX activity (lucigenin-enhanced chemiluminescence) and activated the MEK/ERK and PI3K/Akt survival pathways. CD29 also induced phosphorylation of the inhibitory Ser9 on the pro-apoptotic kinase glycogen synthase kinase-3β in a PI3K/Akt- and MEK-dependent manner, and improved cardiomyocyte viability under conditions of oxidative stress. The ROS scavenger MnTMPyP or adenoviral co-overexpression of the antioxidant enzymes superoxide dismutase and catalase inhibited CD29-induced pro-survival signalling. Further, CD29-induced protective pathways were lost in mouse cardiomyocytes deficient for NOX2 or functional p47phox, a regulatory subunit of NOX. Conclusion p47phox-dependent, NOX2-derived ROS are mandatory for CD29-induced pro-survival signalling in cardiomyocytes. These findings go in line with a growing body of evidence suggesting that ROS can be beneficial to the cell and support a crucial role for NOX2-derived ROS in cell survival in the hear

Short-term Oral Antibiotics Treatment Promotes Inflammatory Activation of Colonic Invariant Natural Killer T and Conventional CD4+T Cells

The gut mucosa is continuously exposed to a vast community of microorganisms, collectively defined as microbiota, establishing a mutualistic relationship with the host and contributing to shape the immune system. Gut microbiota is acquired at birth, and its composition is relatively stable during the entire adult life. Intestinal dysbiosis, defined as a microbial imbalance of gut bacterial communities, can be caused by several factors, including bacterial infections and antibiotic use, and has been associated with an increased risk to develop or exacerbate immune-mediated pathologies, such as allergic reactions, asthma, and inflammatory bowel diseases. Still, the mechanisms by which antibiotic-induced gut dysbiosis may lead to development of mucosal inflammation are still matter of debate. To this end, we aimed to evaluate the impact of antibiotic treatment on phenotype and functions of intestinal immune cell populations, including invariant natural killer T (iNKT) cells, a subset of lipid-specific T cells profoundly influenced by alterations on the commensal microbiota. To this aim, a cocktail of broad-spectrum antibiotics was administered for 2\u2009weeks to otherwise healthy mice before re-colonization of the intestinal microbial community with oral gavage of eubiotic or dysbiotic mucosa-associated bacteria and luminal colonic content, followed or not by intestinal inflammation induction. Here. we showed that short-term antibiotic treatment alters frequency and functions of intestinal iNKT cells, even in the absence of intestinal inflammation. The presence of a dysbiotic microbiota after antibiotic treatment imprints colonic iNKT and CD4+T cells toward a pro-inflammatory phenotype that collectively contributes to aggravate intestinal inflammation. Nonetheless, the inflammatory potential of the dysbiotic microbiota decreases over time opening the possibility to temporally intervene on the microbial composition to re-equilibrate dysbiosis, thus controlling concomitantly mucosal immune T cell activations

Reduced humoral response to two doses of COVID-19 vaccine in patients with inflammatory bowel disease: Data from ESCAPE-IBD, an IG-IBD study

Background Patients on immunosuppressive drugs have been excluded from COVID-19 vaccines trials, creating concerns regarding their efficacy. Aims To explore the humoral response to COVID-19 vaccines in patients with inflammatory bowel disease (IBD) Methods Effectiveness and Safety of COVID-19 Vaccine in Patients with Inflammatory Bowel Disease (IBD) Treated with Immunomodulatory or Biological Drugs (ESCAPE-IBD) is a prospective, multicentre study promoted by the Italian Group for the study of Inflammatory Bowel Disease. We present data on serological response eight weeks after the second dose of COVID-19 vaccination in IBD patients and healthy controls (HCs). Results 1076 patients with IBD and 1126 HCs were analyzed. Seropositivity for anti-SARS-CoV-2 IgG was reported for most IBD patients, even if with a lesser rate compared with HCs (92.1% vs. 97.9%; p<0.001). HCs had higher antibody concentrations (median OD 8.72 [IQR 5.2-14-2]) compared to the whole cohort of IBD patients (median OD 1.54 [IQR 0.8-3.6]; p<0.001) and the subgroup of IBD patients (n=280) without any treatment or on aminosalicylates only (median OD 1.72 [IQR 1.0–4.1]; p<0.001). Conclusions Although most IBD patients showed seropositivity after COVID-19 vaccines, the magnitude of the humoral response was significantly lower than in HCs. Differently from other studies, these findings seem to be mostly unrelated to the use of immune-modifying treatments (ClinicalTrials.govID:NCT04769258)

Diagnóstico de la producción de plantas forestales en los viveros de la Municipalidad de Guatemala; evaluación del enraizamiento de matilisguate (Tabebuia rosea bertol) y servicios realizados en el Vivero Municipal Acatán, Zona 16, Ciudad De Guatemala, Guatemala, C.A.

El presente trabajo de investigación se orientó hacia la producción de plantas forestales de los viveros de la Municipalidad de Guatemala, referente a distintos criterios de selección del material al momento de plantarse en campo, así como principales modos de propagación que se utilizan en los viveros para abastecer a los distintos proyectos de jardinización y arborización urbana. Mediante el diagnóstico se puedo conoce la situación actual de los sistemas de producción de plantas forestales en los viveros, se determinaron las principales características de las especies forestales que se utilizan para arborización urbana, jardinización, criterios de selección y modos de propagación, encontrándose como principal medio de propagación la semilla y no otras alternativas que agilicen la obtención masal de plántulas de mayor tamaño en el menor tiempo posible. La investigación se dirigió a establecer el un método alternativo para la propagación vegetativa de una especie ampliamente utilizada para arborización urbana: Tabebuia rosea bertol. Se evaluó la respuesta al enraizamiento en vástago, en función de tres concentraciones del ácido Indol3Butírico y tres diámetros de estaca, en plantas en fase de vivero con material juvenil para propagar. La investigación tuvo una duración de 4 meses. El estudio se realizó en el Huerto y Vivero Urbano Municipal Acatán zona 16, ciudad de Guatemala. El resultado de la investigación, demostró que existe respuesta a la propagación vegetativa por la especie bajo estudio, la respuesta fue mayor para el caso de propagación por vástago de 2.5cm. de diámetro y 2500ppm del regulador. Los resultados se midieron en presencia de callo, de raíces, y de brotes; además, peso seco de raíces, altura de la planta. Este resultado es de importancia ya que permite obtener plantas de matilisguate de mayor tamaño en menos tiempo y esto se vería reflejado en el arbolado urbano

The Role of Gut Microbiota Biomodulators on Mucosal Immunity and Intestinal Inflammation

Alterations of the gut microbiota may cause dysregulated mucosal immune responses leading to the onset of inflammatory bowel diseases (IBD) in genetically susceptible hosts. Restoring immune homeostasis through the normalization of the gut microbiota is now considered a valuable therapeutic approach to treat IBD patients. The customization of microbe-targeted therapies, including antibiotics, prebiotics, live biotherapeutics and faecal microbiota transplantation, is therefore considered to support current therapies in IBD management. In this review, we will discuss recent advancements in the understanding of host−microbe interactions in IBD and the basis to promote homeostatic immune responses through microbe-targeted therapies. By considering gut microbiota dysbiosis as a key feature for the establishment of chronic inflammatory events, in the near future it will be suitable to design new cost-effective, physiologic, and patient-oriented therapeutic strategies for the treatment of IBD that can be applied in a personalized manner

Implementation of an automated inclusion system for the histological analysis of murine tissue samples: A feasibility study in DSS-induced chronic colitis

Animal models are powerful tools to expand our understanding of human diseases. Histopathological evaluation of murine experimental models is often required to support further research; thus, a more rigorous evaluation of murine histological samples is strongly advocated. Indeed, the overall quality of tissue sections is critical to draw reliable and accurate conclusions. As several methodological variables may reduce the reliability of the pathological analysis, a standardization of the procedural steps required for the processing of histological murine tissues is advisable. Here, we describe a method to standardize the technical procedure from the initial preparation to the paraffin embedding of murine samples. Specifically, we have implemented an automated inclusion system, that is, the SAKURA Tissue-Tek inclusion instrument, which is routinely used for paraffin inclusion of human samples, to process murine specimens of intestinal inflammation. Colitis severity was assessed in chronically Dextran Sodium Sulphate (DSS)–treated mice by cytofluorimetric analysis of colonic cellular infiltrates, expression of inflammatory genes and histopathological analysis of tissue samples, comparing manual and automated tissue preparation systems. We here conclude that implementation of this technique can significantly increase the quality and the reliability of histopathological examination of murine tissues

Intratumor Microbiome in Neuroendocrine Neoplasms: A New Partner of Tumor Microenvironment? A Pilot Study

Neuroendocrine neoplasms (NENs) are rare neoplasms with heterogeneous clinical behavior. Alteration in human microbiota was reported in association with carcinogenesis in different solid tumors. However, few studies addressed the role of microbiota in NEN. We here aimed at evaluating the presence of bacterial infiltration in neuroendocrine tumoral tissue. To assess the presence of bacteria, 20 specimens from pancreatic NEN (pan-NEN) and 20 from intestinal NEN (I-NEN) were evaluated through Fluorescent In situ Hybridization and confocal microscopy. Demographic data, pre-operative investigations, operative findings, pathological diagnosis, follow-up, and survival data were evaluated. Among I-NEN, bacteria were detected in 15/20 (75%) specimens, with high variability in microbial distribution. In eight patients, a high infiltration of microorganisms was observed. Among pan-NEN, 18/20 (90%) showed microorganisms’ infiltration, with a homogeneous microbial distribution. Bacterial localization in pan-NEN was observed in the proximity of blood vessels. A higher bacterial infiltration in the tumoral specimen as compared with non-tumoral tissue was reported in 10/20 pan-NEN (50%). No significant differences were observed in mean bacterial count according to age, sex, ki67%, site, tumor stage. Mean bacterial count did not result to be a predictor of disease-specific survival. This preliminary study demonstrates the presence of a significant microbiota in the NEN microenvironment. Further research is needed to investigate the potential etiological or clinical role of microbiota in NEN

Novel Odoribacter splanchnicus Strain and Its Outer Membrane Vesicles Exert Immunoregulatory Effects in vitro

Odoribacter splanchnicus, belonging to the order Bacteroidales, is a common, short-chain fatty acid producing member of the human intestinal microbiota. A decreased abundance of Odoribacter has been linked to different microbiota-associated diseases, such as non-alcoholic fatty liver disease, cystic fibrosis and inflammatory bowel disease (IBD). The type strain of O. splanchnicus has been genome-sequenced, but otherwise very little is known about this anaerobic bacterium. The species surfaces in many microbiota studies and, consequently, comprehension on its interactions with the host is needed. In this study, we isolated a novel strain of O. splanchnicus from a healthy fecal donor, identified it by genome sequencing and addressed its adhesive, epithelium reinforcing and immunoregulatory properties. Our results show that O. splanchnicus strain 57 is non-adherent to enterocytes or mucus, does not reinforce nor compromise Caco-2 monolayer integrity and most likely harbors penta-acylated, less endotoxic lipid A as part of its lipopolysaccharide (LPS) structure based on the lack of gene lpxM and in vitro results on low-level NF-κB activity. The studies by transmission electron microscopy revealed that O. splanchnicus produces outer membrane vesicles (OMV). O. splanchnicus cells, culture supernatant i.e., spent medium or OMVs did not induce interleukin-8 (IL-8) response in HT-29 enterocyte cells suggesting a very low proinflammatory capacity. On the contrary, the treatment of HT-29 cells with O. splanchnicus cells, spent medium or OMVs prior to exposure to Escherichia coli LPS elicited a significant decrease in IL-8 production as compared to E. coli LPS treatment alone. Moreover, O. splanchnicus spent supernatant induced IL-10 production by immune cells, suggesting anti-inflammatory activity. Our in vitro findings indicate that O. splanchnicus and its effector molecules transported in OMVs could potentially exert anti-inflammatory action in the gut epithelium. Taken together, O. splanchnicus seems to be a commensal with a primarily beneficial interaction with the host.Peer reviewe