Porphyromonas gingivalis fuels colorectal cancer through CHI3L1-mediated iNKT cell-driven immune evasion.

The interaction between the gut microbiota and invariant Natural Killer T (iNKT) cells plays a pivotal role in colorectal cancer (CRC). The pathobiont Fusobacterium nucleatum influences the anti-tumor functions of CRC-infiltrating iNKT cells. However, the impact of other bacteria associated with CRC, like Porphyromonas gingivalis, on their activation status remains unexplored. In this study, we demonstrate that mucosa-associated P. gingivalis induces a protumour phenotype in iNKT cells, subsequently influencing the composition of mononuclear-phagocyte cells within the tumor microenvironment. Mechanistically, in vivo and in vitro experiments showed that P. gingivalis reduces the cytotoxic functions of iNKT cells, hampering the iNKT cell lytic machinery through increased expression of chitinase 3-like-1 protein (CHI3L1). Neutralization of CHI3L1 effectively restores iNKT cell cytotoxic functions suggesting a therapeutic potential to reactivate iNKT cell-mediated antitumour immunity. In conclusion, our data demonstrate how P. gingivalis accelerates CRC progression by inducing the upregulation of CHI3L1 in iNKT cells, thus impairing their cytotoxic functions and promoting host tumor immune evasion.

iNKT cell-neutrophil crosstalk promotes colorectal cancer pathogenesis.

iNKT cells account for a relevant fraction of effector T-cells in the intestine and are considered an attractive platform for cancer immunotherapy. Although iNKT cells are cytotoxic lymphocytes, their functional role in colorectal cancer (CRC) is still controversial, limiting their therapeutic use. Thus, we examined the immune cell composition and iNKT cell phenotype of CRC lesions in patients (n = 118) and different murine models. High-dimensional single-cell flow-cytometry, metagenomics, and RNA sequencing experiments revealed that iNKT cells are enriched in tumor lesions. The tumor-associated pathobiont Fusobacterium nucleatum induces IL-17 and Granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in iNKT cells without affecting their cytotoxic capability but promoting iNKT-mediated recruitment of neutrophils with polymorphonuclear myeloid-derived suppressor cells-like phenotype and functions. The lack of iNKT cells reduced the tumor burden and recruitment of immune suppressive neutrophils. iNKT cells in-vivo activation with α-galactosylceramide restored their anti-tumor function, suggesting that iNKT cells can be modulated to overcome CRC-associated immune evasion. Tumor co-infiltration by iNKT cells and neutrophils correlates with negative clinical outcomes, highlighting the importance of iNKT cells in the pathophysiology of CRC. Our results reveal a functional plasticity of iNKT cells in CRC, suggesting a pivotal role of iNKT cells in shaping the tumor microenvironment, with relevant implications for treatment.

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.

Human intestinal and circulating invariant natural killer T cells are cytotoxic against colorectal cancer cells via the perforin-granzyme pathway.

Invariant natural killer T (iNKT) cells are lipid-specific T lymphocytes endowed with cytotoxic activities and are thus considered important in antitumor immunity. While several studies have demonstrated iNKT cell cytotoxicity against different tumors, very little is known about their cell-killing activities in human colorectal cancer (CRC). Our aim was to assess whether human iNKT cells are cytotoxic against colon cancer cells and the mechanisms underlying this activity. For this purpose, we generated stable iNKT cell lines from peripheral blood and colon specimens and used NK-92 and peripheral blood natural killer cells as cell-mediated cytotoxicity controls. In vitro cytotoxicity was assessed using a panel of well-characterized human CRC cell lines, and the cellular requirements for iNKT cell cytotoxic functions were evaluated. We demonstrated that both intestinal and circulating iNKT cells were cytotoxic against the entire panel of CRC lines, as well as against freshly isolated patient-derived colonic epithelial cancer cells. Perforin and/or granzyme inhibition impaired iNKT cell cytotoxicity, whereas T-cell receptor (TCR) signaling was a less stringent requirement for efficient killing. This study is the first evidence of tissue-derived iNKT cell cytotoxic activity in humans, as it shows that iNKT cells depend on the perforin-granzyme pathway and both adaptive and innate signal recognition for proper elimination of colon cancer cells.

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.

Persistence of Anti-SARS-CoV-2 Antibodies in Non-Hospitalized COVID-19 Convalescent Health Care Workers.

Although antibody response to SARS-CoV-2 can be detected early during the infection, several outstanding questions remain to be addressed regarding the magnitude and persistence of antibody titer against different viral proteins and their correlation with the strength of the immune response. An ELISA assay has been developed by expressing and purifying the recombinant SARS-CoV-2 Spike Receptor Binding Domain (RBD), Soluble Ectodomain (Spike), and full length Nucleocapsid protein (N). Sera from healthcare workers affected by non-severe COVID-19 were longitudinally collected over four weeks, and compared to sera from patients hospitalized in Intensive Care Units (ICU) and SARS-CoV-2-negative subjects for the presence of IgM, IgG and IgA antibodies as well as soluble pro-inflammatory mediators in the sera. Non-hospitalized subjects showed lower antibody titers and blood pro-inflammatory cytokine profiles as compared to patients in Intensive Care Units (ICU), irrespective of the antibodies tested. Noteworthy, in non-severe COVID-19 infections, antibody titers against RBD and Spike, but not against the N protein, as well as pro-inflammatory cytokines decreased within a month after viral clearance. Thus, rapid decline in antibody titers and in pro-inflammatory cytokines may be a common feature of non-severe SARS-CoV-2 infection, suggesting that antibody-mediated protection against re-infection with SARS-CoV-2 is of short duration. These results suggest caution in using serological testing to estimate the prevalence of SARS-CoV-2 infection in the general population.

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.

License to Kill: When iNKT Cells Are Granted the Use of Lethal Cytotoxicity.

Invariant Natural Killer T (iNKT) cells are a non-conventional, innate-like, T cell population that recognize lipid antigens presented by the cluster of differentiation (CD)1d molecule. Although iNKT cells are mostly known for mediating several immune responses due to their massive and diverse cytokine release, these cells also work as effectors in various contexts thanks to their cytotoxic potential. In this Review, we focused on iNKT cell cytotoxicity; we provide an overview of iNKT cell subsets, their activation cues, the mechanisms of iNKT cell cytotoxicity, the specific roles and outcomes of this activity in various contexts, and how iNKT killing functions are currently activated in cancer immunotherapies. Finally, we discuss the future perspectives for the better understanding and potential uses of iNKT cell killing functions in tumor immunosurveillance.

Meta-analysis of diagnostic performance of serological tests for SARS-CoV-2 antibodies up to 25 April 2020 and public health implications.

We reviewed the diagnostic accuracy of SARS-CoV-2 serological tests. Random-effects models yielded a summary sensitivity of 82% for IgM, and 85% for IgG and total antibodies. For specificity, the pooled estimate were 98% for IgM and 99% for IgG and total antibodies. In populations with ≤ 5% of seroconverted individuals, unless the assays have perfect (i.e. 100%) specificity, the positive predictive value would be ≤ 88%. Serological tests should be used for prevalence surveys only in hard-hit areas.

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.

Effect of diabetes status and hyperglycemia on global DNA methylation and hydroxymethylation.

Type 2 diabetes mellitus (T2DM) is characterized by oxidative stress that could lead to chronic micro- and macrovascular complications. We hypothesized that some of the target organ damage is mediated by oxidative alterations in epigenetic mechanisms involving DNA methylation (5mC) and DNA hydroxymethylation (5hmC). We analyzed global DNA methylation and hydroxymethylation in peripheral blood cells in well-controlled and poorly controlled patients with T2DM and compared them with healthy controls. We also analyzed microarrays of DNA methylation and gene expression of other important tissues in the context of diabetes from the GEO database repository and then compared these results with our experimental gene expression data. DNA methylation and, more importantly, DNA hydroxymethylation levels were increased in poorly controlled patients compared to well-controlled and healthy individuals. Both 5mC and 5hmC measurements were correlated with the percentage of glycated hemoglobin, indicating a direct impact of hyperglycemia on changes over the epigenome. The analysis of methylation microarrays was concordant, and 5mC levels were increased in the peripheral blood of T2DM patients. However, the DNA methylation levels were the opposite of those in other tissues, such as the pancreas, adipose tissue and skeletal muscle. We hypothesize that a process of DNA oxidation associated with hyperglycemia may explain the DNA demethylation in which the activity of ten-eleven translocation (TET) proteins is not sufficient to complete the process. High levels of glucose lead to cellular oxidation, which triggers the process of DNA demethylation aided by TET enzymes, resulting in epigenetic dysregulation of the damaged tissues.