Abstract Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease, in which infections are considered to play a pathogenic role, but the cellular and molecular culprits remain unknown. Bacterial biofilms are multicellular bacterial communities important in the establishment of chronic infection by pathogens. Bacteria produce amyloids, complex proteins with a conserved beta sheet structure that strengthen the extracellular matrix of their biofilms. We have reported that bacterial and eukaryotic DNA is incorporated into curli fibers, functional bacterial amyloids present in Salmonella and E. coli biofilms. I.P. infections with curli-expressing S. Typhimurium or E. coli Nissle, and also systemic administration of curli/DNA complexes purified from Salmonella biofilms accelerated onset in lupus-prone NZBxW/F1 mice and triggered autoantibodies production in non-predisposed mice, suggesting curli/DNA complexes as novel players in SLE pathogenesis. We present here evidence that curli/DNA complexes can activate multiple B cell subsets in vivo. In addition, curli/DNA complexes are capable of directly inducing in vitro polyclonal activation and proliferation of B cells from wildtype mice and from lupus-prone mice and also from 3H9 mice, which are knocked-in for a rearranged anti-DNA Ig heavy chain and whose B cells are normally tolerized for the self-Antigen. Our results suggest that curli/DNA complexes may induce autoantibody production by directly breaking tolerance in autoreactive B cell.
Liver cancer remains the third leading cause of worldwide cancer-related deaths.1 Despite clinical advances, established immunotherapies largely fail patients due to poor immune responses.1 2 Peripheral nerves influence tumors, but brain-liver interactions remain largely unstudied.3 A recent study reported a decreased risk of liver cancers in patients that underwent truncal vagotomy (vagus snip) compared to simple suture procedure.4 Here, we identify a vagal-immune arc regulating hepatic cancer.
Methods
C57BL/6 or BALB/c mice (9–12 weeks old) underwent a surgical hepatic vagotomy (HV) or sham procedure (SV). Following established protocols,5 HV and SV mice received orthotopic tumors via intrahepatic injection (2.5x105 RIL-175 or B16-F10 cells) or tail vein/flank injection (1.0x106 A20, RIL-175 cells) to model primary and metastatic liver cancer. Growth of luciferase-labeled cells was measured via in vivo imaging and immune profiling was conducted via flow cytometry and scRNASEQ. Open field test, Y maze, and phenotyper cages assessed mouse behavior. Highly-multiplexed immunofluorescence (CODEX platform) revealed peripheral nerves in clinical resection samples.
Results
Precise liver denervation reduced tumor growth in three models of primary (RIL-175) and metastatic (B16-F10, A20) tumors (figure 1A). Outcomes remained organ specific as HV mice exhibited reduced tumor burden of intrahepatic, but not subcutaneous, models. HV livers exhibited decreased levels of vagal neurotransmitter acetylcholine (ACh). As immunofluorescent analyses revealed colocalization of peripheral nerves and lymphocytes in clinical liver cancer, we profiled HV immunity. HV livers exhibited broad anti-tumor immunity, notably increased CD8+ T cells and higher expression of intracellular cytokines (IFNγ, TNFα) (figure 1B). We then examined whether immune alterations were a cause or consequence of HV tumor burden. ACh exposure reduced intracellular cytokine levels in ex vivo CD8+ T cells following anti-CD3/CD8 activation. Treatment with bethanechol (ACh receptor agonist, 400 μg/mL drinking water) increased tumor burden and reduced CD8+TNFα+ subsets. Bethanechol failed to promote tumor growth in Rag1KO mice lacking mature B and T cells, and targeted depletion of CD8+ T cells abrogated the effects of vagotomy (figure 1C). Finally, as the vagus nerve is largely comprised of afferent fibers, we assessed murine behavior and ambulation. Tumor-bearing HV mice displayed decreased anxiety-life features and fatigue compared to sham controls.
Conclusions
Our findings highlight a vagal-CD8+ T cell axis modulating hepatic tumor burden and behavior. This work furthers the emerging field of cancer neuroscience and identifies ACh signaling targets to alter hepatic immunosuppression and cancer outcomes.
References
Kim E, Viatour P. Hepatocellular carcinoma: old friends and new tricks. Exp Mol Med. 2020;52:1898–1907. Greten T, et al. Society for Immunotherapy of Cancer (SITC) clinical practice guideline on immunotherapy for the treatment of hepatocellular carcinoma. 2021. JITC. 2021;9:e002794. Monje M, et al. Roadmap for the Emerging Field of Cancer Neuroscience. Cell. 2020;181:219–222. Wu S, et al. Decreased risk of liver and intrahepatic cancer in non-H. pylori-infected perforated peptic ulcer patients with truncal vagotomy: a nationwide study. Sci Rep. 2021;11:e15594. Brown Z, Heinrich B, Greten T. Establishment of orthotopic liver tumors by surgical intrahepatic tumor injection in mice with underlying non-alcoholic fatty liver disease. Methods protoc, 2018;2:e21.
Ethics Approval
This research was approved by the NCI Division of Intramural Research Animal Care and Use Committee, proposal numbers: MOB-028 and TGOB-015. Patients provided informed consent for clinical tissue acquisition: Institutional Review Board protocol #2017–0365.
Abstract Liver cancer ranks amongst the deadliest cancers. Nerves have emerged as an understudied regulator of tumor progression. The parasympathetic vagus nerve influences systemic immunity via acetylcholine (ACh). Whether cholinergic neuroimmune interactions influence hepatocellular carcinoma (HCC) remains uncertain. Liver denervation via hepatic vagotomy (HV) significantly reduced liver tumor burden, while pharmacological enhancement of parasympathetic tone promoted tumor growth. Cholinergic disruption in Rag1KO mice revealed that cholinergic regulation requires adaptive immunity. Further scRNA-seq and in vitro studies indicated that vagal ACh dampens CD8+ T cell activity via muscarinic ACh receptor (AChR) CHRM3. Depletion of CD8+ T cells abrogated HV outcomes and selective deletion of Chrm3 on CD8 + T cells inhibited liver tumor growth. Beyond tumor-specific outcomes, vagotomy improved cancer-associated fatigue and anxiety-like behavior. As microbiota transplantation from HCC donors was sufficient to impair behavior, we investigated putative microbiota-neuroimmune crosstalk. Tumor, rather than vagotomy, robustly altered fecal bacterial composition, increasing Desulfovibrionales and Clostridial taxa. Strikingly, in tumor-free mice, vagotomy permitted HCC-associated microbiota to activate hepatic CD8+ T cells. These findings reveal that gut bacteria influence behavior and liver anti-tumor immunity via a dynamic and pharmaceutically targetable, vagus-liver axis.
Background & AimsIntrahepatic cholangiocarcinoma (iCCA) accounts for a fraction of primary liver cancers but has a 5-year survival rate of only 10%. Immune checkpoint inhibitors are effective in treating many solid cancers, but immune checkpoint inhibitor monotherapy has no clear benefit in iCCA. Mitogen-activated kinase (MEK) inhibitors, such as trametinib, have shown promising results in preclinical studies for iCCA by inhibiting cell proliferation and modifying the tumor microenvironment. This study aimed to show the potential benefit of combining trametinib with anti–programmed cell death protein 1 (PD-1) therapy in different iCCA mouse models.MethodsHere, we assessed the in vitro cytotoxicity of trametinib in mouse (SB1 and LD-1) and human (EGI-1) cholangiocarcinoma cell lines. We examined the efficacy of single-agent trametinib, anti–PD-1, and a combination of both in subcutaneous, orthotopic, and plasmid-induced iCCA mouse models. Flow cytometry analysis was used to elucidate changes in the tumor immune microenvironment upon treatment. Whole-exome sequencing (WES) was performed on the SB1 tumor cell line to correlate this preclinical model with iCCAs in patients.ResultsTrametinib reduced tumor cell growth of SB1, LD-1, and EGI-1 tumor cells in vitro. Trametinib treatment led to up-regulation of major histocompatibility complex (MHC-I) and programmed cell death ligand 1 (PD-L-1) (programmed cell death ligand 1) on tumor cells in vitro. The combination of trametinib and anti–PD-1 reduced tumor burden in several iCCA tumor models and improved survival in SB1 tumor-bearing mice compared with either agent alone. Immunoprofiling of tumor-bearing mice showed an increase of hepatic effector memory CD8+ and CD4+ T cells, as well as an increased degranulation of CD8+ T cells, indicating enhanced cytotoxicity. WES and somatic mutational analysis showed no mutations of KRAS, BRAF, and ERK in SB1 tumor cells, and showed a similar genetic signature of SB1 found in a cohort of patients with iCCA.ConclusionsAltogether, our study shows that trametinib improves the immunogenicity of tumor cells by up-regulating MHC-I surface expression. The combination with anti–PD-1 results in optimal treatment efficacy for iCCA. WES of SB1 cells suggests that KRAS wild-type iCCAs also respond to this combination therapy. Intrahepatic cholangiocarcinoma (iCCA) accounts for a fraction of primary liver cancers but has a 5-year survival rate of only 10%. Immune checkpoint inhibitors are effective in treating many solid cancers, but immune checkpoint inhibitor monotherapy has no clear benefit in iCCA. Mitogen-activated kinase (MEK) inhibitors, such as trametinib, have shown promising results in preclinical studies for iCCA by inhibiting cell proliferation and modifying the tumor microenvironment. This study aimed to show the potential benefit of combining trametinib with anti–programmed cell death protein 1 (PD-1) therapy in different iCCA mouse models. Here, we assessed the in vitro cytotoxicity of trametinib in mouse (SB1 and LD-1) and human (EGI-1) cholangiocarcinoma cell lines. We examined the efficacy of single-agent trametinib, anti–PD-1, and a combination of both in subcutaneous, orthotopic, and plasmid-induced iCCA mouse models. Flow cytometry analysis was used to elucidate changes in the tumor immune microenvironment upon treatment. Whole-exome sequencing (WES) was performed on the SB1 tumor cell line to correlate this preclinical model with iCCAs in patients. Trametinib reduced tumor cell growth of SB1, LD-1, and EGI-1 tumor cells in vitro. Trametinib treatment led to up-regulation of major histocompatibility complex (MHC-I) and programmed cell death ligand 1 (PD-L-1) (programmed cell death ligand 1) on tumor cells in vitro. The combination of trametinib and anti–PD-1 reduced tumor burden in several iCCA tumor models and improved survival in SB1 tumor-bearing mice compared with either agent alone. Immunoprofiling of tumor-bearing mice showed an increase of hepatic effector memory CD8+ and CD4+ T cells, as well as an increased degranulation of CD8+ T cells, indicating enhanced cytotoxicity. WES and somatic mutational analysis showed no mutations of KRAS, BRAF, and ERK in SB1 tumor cells, and showed a similar genetic signature of SB1 found in a cohort of patients with iCCA. Altogether, our study shows that trametinib improves the immunogenicity of tumor cells by up-regulating MHC-I surface expression. The combination with anti–PD-1 results in optimal treatment efficacy for iCCA. WES of SB1 cells suggests that KRAS wild-type iCCAs also respond to this combination therapy.
