Metagenomic analysis of gut microbiome reveals a dynamic change in Alistipes onderdonkii in the preclinical model of pancreatic cancer, suppressing its proliferation.

Pancreatic cancer is a lethal cancer with aggressive and invasive characteristics. By the time it is diagnosed, patients already have tumors extended to other organs and show extremely low survival rates. The gut microbiome is known to be associated with many diseases and its imbalance affects the pathogenesis of pancreatic cancer. In this study, we established an orthotopic, patient-derived xenograft model to identify how the gut microbiome is linked to pancreatic ductal adenocarcinoma (PDAC). Using the 16S rDNA metagenomic sequencing, we revealed that the levels of Alistipes onderdonkii and Roseburia hominis decreased in the gut microbiome of the PDAC model. To explore the crosstalk between the two bacteria and PDAC cells, we collected the supernatant of the bacteria or cancer cell culture medium and treated it in a cross manner. While the cancer cell medium did not affect bacterial growth, we observed that the A. onderdonkii medium suppressed the growth of the pancreatic primary cancer cells. Using the bromodeoxyuridine/7-amino-actinomycin D (BrdU/7-AAD) staining assay, we confirmed that the A. onderdonkii medium inhibited the proliferation of the pancreatic primary cancer cells. Furthermore, RNA-seq analysis revealed that the A. onderdonkii medium induced unique transcriptomic alterations in the PDAC cells, compared to the normal pancreatic cells. Altogether, our data suggest that the reduction in the A. onderdonkii in the gut microbiome provides a proliferation advantage to the pancreatic cancer cells. KEY POINTS: • Metagenome analysis of pancreatic cancer model reveals A. onderdonkii downregulation. • A. onderdonkii culture supernatant suppressed the proliferation of pancreatic cancer cells. • RNA seq data reveals typical gene expression changes induced by A. onderdonkii.

Demonstration of functional similarity of a biosimilar adalimumab SB5 to Humira®.

A biosimilar is a biological medicinal product that is highly similar to an authorized biological product in terms of quality, biological activity, safety and efficacy. SB5 was developed by Samsung Bioepis as a biosimilar referencing adalimumab, and was authorized by the European Commission (EC) in August 2017 (Imraldi®). Extensive characterization studies were performed to demonstrate functional similarity of SB5 to reference adalimumab (Humira®, AbbVie Inc. and AbbVie Deutschland GmbH & Co. KG). SB5 and Humira® showed highly similar soluble TNF-α binding and neutralizing activity, as well as transmembrane TNF-α binding activity and reverse signaling induced in the membrane TNF-α expressing cell line. Both products exhibited similar binding of the Fc gamma receptors and Fc-related effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In addition, additional mechanisms of action induced by TNF-α, such as cytokine release and expression of adhesion molecules, were analyzed and shown to be similar between SB5 and Humira®. Taken together, our results demonstrate that SB5 and Humira® are highly similar in terms of their functional characteristics.

IFN-γ-producing NKT cells exacerbate sepsis by enhancing C5a generation via IL-10-mediated inhibition of CD55 expression on neutrophils.

A role for NKT cells has been implicated in sepsis, but the mechanism by which NKT cells contribute to sepsis remains unclear. Here, we examined WT and NKT-cell-deficient mice of C57BL/6 background during cecal ligation and puncture-induced sepsis. The levels of C5a, IFN-γ, and IL-10 were higher in the serum and peritoneal fluid of WT mice than in those of CD1d(-/-) mice, while the mortality rate was lower in CD1d(-/-) mice than in WT mice. C5a blockade decreased mortality of WT mice during sepsis, whereas it did not alter that of CD1d(-/-) mice. As assessed by intracellular staining, NKT cells expressed IFN-γ, while neutrophils expressed IL-10. Upon coculture, IL-10-deficient NKT cells enhanced IL-10 production by WT, but not IFN-γR-deficient, neutrophils. Meanwhile, CD1d(-/-) mice exhibited high CD55 expression on neutrophils during sepsis, whereas those cells from WT mice expressed minimal levels of CD55. Recombinant IL-10 administration into CD1d(-/-) mice reduced CD55 expression on neutrophils. Furthermore, adoptive transfer of sorted WT, but not IFN-γ-deficient, NKT cells into CD1d(-/-) mice suppressed CD55 expression on neutrophils, but increased IL-10 and C5a levels. Taken together, IFN-γ-producing NKT cells enhance C5a generation via IL-10-mediated inhibition of CD55 expression on neutrophils, thereby exacerbating sepsis.

GM-CSF and IL-4 produced by NKT cells inversely regulate IL-1ß production by macrophages.

Natural Killer T (NKT) cells are distinct T cell subset that link innate and adaptive immune responses. IL-1ß, produced by various immune cells, plays a key role in the regulation of innate immunity in vivo. However, it is unclear whether NKT cells regulate IL-1ß production by macrophages. To address this, we co-cultured NKT cells and peritoneal macrophages in the presence of TCR stimulation and inflammasome activators. Among cytokines secreted from NKT cells, GM-CSF enhanced IL-1ß production by macrophages via regulating LPS-mediated pro-IL-1ß expression and NLRP3-dependent inflammasome activation, whereas IL-4 enhanced M2-differentiation of macrophages and decreased IL-1ß production. Together, our findings suggest the NKT cells have double-sided effects on IL-1ß-mediated innate immune responses by producing IL-4 and GM-CSF. These findings may be helpful for a comprehensive understanding of NKT cell-mediated regulatory mechanisms of the pro-inflammatory effects of IL-1ß in inflammatory diseases in vivo.

Notch 1 and Notch 2 synergistically regulate the differentiation and function of invariant NKT cells.

Invariant natural killer T cells are a distinct subset of T cells that exert Janus-like functions. Moreover, Notch signaling is known to have critical roles in the development and functions of T cells. However, it is not known whether Notch signaling contributes to the development or functions of invariant natural killer T cells. Here, we found that CD4-specific gene ablation of Notch 1 and Notch 2 (N1N2(-/-)) increased the number of invariant natural killer T cells in the thymus but decreased them in the liver. N1N2(-/-) mice showed impaired thymic maturation of invariant natural killer T cells from the NK1.1(-)CD44(+) to the NK1.1(+)CD44(+) stage, resulting in accumulation of NK1.1(-)CD44(+) invariant natural killer T cells in the thymus. Upon activation, hepatic invariant natural killer T cells from N1N2(-/-) mice produced lower cytokine levels and increased apoptosis versus wild-type invariant natural killer T cells. Furthermore, Notch 1/Notch 2-deficient, but not wild type, invariant natural killer T cells failed to promote antibody-induced arthritis in CD1d(-/-) mice. Unlike N1N2(-/-) mice, RBP-j(lox) (/) (lox) CD4-Cre mice showed similar percentages and numbers of thymic invariant natural killer T cells to wild-type mice but had defects in their homeostasis, maturation, and cytokine production in the liver. Taken together, our data indicate distinct effects of Notch signaling on invariant natural killer T cells in the thymus and liver, which are at least partly independent of RBP-j in the thymus.