Extracellular adenosine triphosphate (eATP) released by damaged cells, and its purinergic receptors, comprise a crucial signaling network after injury. Purinergic receptor P2X7 (P2RX7), a major driver of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and IL-1β processing, has been shown to play a role in liver injury in murine diet-and chemically-induced liver injury models. It is unclear, however, whether P2RX7 plays a role in non-alcoholic steatohepatitis (NASH) and which cell type is the main target of P2RX7 pharmacological inhibition. Here, we report that P2RX7 is expressed by infiltrating monocytes and resident Kupffer cells in livers from NASH-affected individuals. Using primary isolated human cells, we demonstrate that P2RX7 expression in CD14 + monocytes and Kupffer cells primarily mediates IL-1β release. In addition, we show that pharmacological inhibition of P2RX7 in monocytes and Kupffer cells, blocks IL-1β release, reducing hepatocyte caspase 3/7 activity, IL-1β-mediated CCL2 and CCL5 chemokine gene expression and secretion, and hepatic stellate cell (HSC) procollagen secretion. Consequently, in a chemically-induced nonhuman primate model of liver fibrosis, treatment with a P2RX7 inhibitor improved histological characteristics of NASH, protecting from liver inflammation and fibrosis. Taken together, these findings underscore the critical role of P2RX7 in the pathogenesis of NASH and implicate P2RX7 as a promising therapeutic target for the management of this disease.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a soluble protein that directs membrane-bound receptors to lysosomes for degradation. In the most studied example of this, PCSK9 binding leads to the degradation of low density lipoprotein receptor (LDLR), significantly affecting circulating LDL-C levels. The mechanism mediating this degradation, however, is not completely understood. We show here that LDLR facilitates PCSK9 interactions with amyloid precursor like protein 2 (APLP2) at neutral pH leading to PCSK9 internalization, although direct binding between PCSK9 and LDLR is not required. Moreover, binding to APLP2 or LDLR is independently sufficient for PCSK9 endocytosis in hepatocytes, while LDL can compete with APLP2 for PCSK9 binding to indirectly mediate PCSK9 endocytosis. Finally, we show that APLP2 and LDLR are also required for the degradation of another PCSK9 target, APOER2, necessitating a general role for LDLR and APLP2 in PCSK9 function. Together, these findings provide evidence that PCSK9 has at least two endocytic epitopes that are utilized by a variety of internalization mechanisms and clarifies how PCSK9 may direct proteins to lysosomes.

Factors determining the precision and variability of the Ames Salmonella test for mutagenicity were investigated. The most important source of variability in the agar-overlay method was nonuniformity in the soft-agar layer thickness. Solution of this problem resulted from application of an agar-leveling table described in this paper. Several other procedural elements also contribute to improved precision, including temperature uniformity during incubation, incubation interval, consistency of plate agar volume, completeness of mixing the soft-agar overlay, peculiarities in the interaction of mutagens and mammalian liver microsomal extract (S9), and methods of storage and controls for tester strains. When these and other effects were well-controlled, variability of the test results was reduced from 200 or 300% to only +/- 10% or less. The significance of the factors affecting precision are discussed and an improved experimental protocol is presented.

No abstract

The gut microbiota has emerged as an important player in cancer pathology, and increasing evidence supports its role in clinical response to immune checkpoint inhibitor (ICI) therapy. However, the specific microbiome-derived factors responsible for the improved response to ICI therapy remain unknown. Second Genome has developed a unique discovery platform to identify, screen, and validate microbiome-derived peptides that promote response to cancer immunotherapy. Using our multitechnology meta-analysis of published datasets and characterizing the baseline microbiome of melanoma patients treated with anti-PD-1, we have identified gut microbiome strains differentially abundant in responders versus nonresponders that are concordant across multiple cohorts. Next, peptides from strains associated with responder signatures were predicted from their genome sequences. In addition, we predicted peptides from assembled metagenomes that were associated with responders. The predicted peptides were screened using phage display technology to identify binders to immune cells known to play a role in the tumor microenvironment (TME). Peptides that bound to specific immune cells were then evaluated for activity in cell-based assays using isolated primary human T cells, dendritic cells (DCs), and macrophages. We have demonstrated that several microbiome-derived peptides induce secretion of proinflammatory cytokines and chemokines such as CXCL10 and TNF-α by primary human monocyte-derived dendritic cells (moDCs), as well as secretion of effector cytokines such as IFNγ and IL-2 by primary human T cells. We have also identified microbiome-derived peptides with the capacity to inhibit an M2-like phenotype in macrophages (decreased LPS-induced IL-10 secretion). These effects were dose dependent and evident across immune cells derived from multiple human blood donors. In a coculture assay using allogeneic moDCs and T cells from human donors, combination of our DC-activating peptides with CD40 agonistic antibody and/or anti-PD-L1 induced secretion of proinflammatory cytokines such as IFNγ and TNF-α. In vivo, peritumoral administration of a candidate DC-activating peptide into RENCA tumor-bearing mice led to a significant reduction in tumor volume as compared to the control-treated mice. Collectively, these data demonstrate the potential of the microbiome-derived peptides identified by Second Genome’s discovery platform to modulate immune-cell effector functions in vitro and promote antitumor immunity in vivo. These results validate the unique approach of Second Genome’s discovery platform to identify novel microbiome-derived agents with potential for use as therapeutics in cancer immunotherapy. This abstract is also being presented as Poster B19. Citation Format: Dhwani D. Haria, Jayamary Divya Ravichandar, Lynn Yamamoto, Bernat Baeza-Raja, Ashil Bans, Cheryl-Emiliane Chow, Jill Desnoyer, Joanna Dreux, Shoko Iwai, Sabina Lau, Jina Lee, Michelle Lin, Paul Loriaux, Nicole Narayan, Eskedar Nigatu, Erica Rutherford, Michi Wilcoxon, Yonggan Wu, Todd DeSantis, Toshihiko Takeuchi, Karim Dabbagh, Helena Kiefel. Novel microbiome-derived peptides modulate immune cell activity and the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr PR08.