bims-nastce 2021-12-19 papers

JHEP Rep. 2022 Jan;4(1): 100386

ROCK2 inhibition attenuates profibrogenic immune cell function to reverse thioacetamide-induced liver fibrosis.

Christina Nalkurthi, Wayne A Schroder, Michelle Melino, Katharine M Irvine, Melanie Nyuydzefe, Wei Chen, Jing Liu, Michele W L Teng, Geoffrey R Hill, Patrick Bertolino, Bruce R Blazar, Gregory C Miller, Andrew D Clouston, Alexandra Zanin-Zhorov, Kelli P A MacDonald.

Background & Aims: Fibrosis, the primary cause of morbidity in chronic liver disease, is induced by pro-inflammatory cytokines, immune cell infiltrates, and tissue resident cells that drive excessive myofibroblast activation, collagen production, and tissue scarring. Rho-associated kinase 2 (ROCK2) regulates key pro-fibrotic pathways involved in both inflammatory reactions and altered extracellular matrix remodelling, implicating this pathway as a potential therapeutic target.
Methods: We used the thioacetamide-induced liver fibrosis model to examine the efficacy of administration of the selective ROCK2 inhibitor KD025 to prevent or treat liver fibrosis and its impact on immune composition and function.
Results: Prophylactic and therapeutic administration of KD025 effectively attenuated thioacetamide-induced liver fibrosis and promoted fibrotic regression. KD025 treatment inhibited liver macrophage tumour necrosis factor production and disrupted the macrophage niche within fibrotic septae. ROCK2 targeting in vitro directly regulated macrophage function through disruption of signal transducer and activator of transcription 3 (STAT3)/cofilin signalling pathways leading to the inhibition of pro-inflammatory cytokine production and macrophage migration. In vivo, KDO25 administration significantly reduced STAT3 phosphorylation and cofilin levels in the liver. Additionally, livers exhibited robust downregulation of immune cell infiltrates and diminished levels of retinoic acid receptor-related orphan receptor gamma (RORγt) and B-cell lymphoma 6 (Bcl6) transcription factors that correlated with a significant reduction in liver IL-17, splenic germinal centre numbers and serum IgG.
Conclusions: As IL-17 and IgG-Fc binding promote pathogenic macrophage differentiation, together our data demonstrate that ROCK2 inhibition prevents and reverses liver fibrosis through direct and indirect effects on macrophage function and highlight the therapeutic potential of ROCK2 inhibition in liver fibrosis.
Lay summary: By using a clinic-ready small-molecule inhibitor, we demonstrate that selective ROCK2 inhibition prevents and reverses hepatic fibrosis through its pleiotropic effects on pro-inflammatory immune cell function. We show that ROCK2 mediates increased IL-17 production, antibody production, and macrophage dysregulation, which together drive fibrogenesis in a model of chemical-induced liver fibrosis. Therefore, in this study, we not only highlight the therapeutic potential of ROCK2 targeting in chronic liver disease but also provide previously undocumented insights into our understanding of cellular and molecular pathways driving the liver fibrosis pathology.

Keywords: ALT, alanine aminotransferase; AST, aspartate aminotransferase; B cells; BMDM, bone marrow-derived macrophages; Bcl6, B-cell lymphoma 6; CLD, chronic liver disease; Col1a2, collagen type α1; DR, ductular reaction; ECM, extracellular matrix; GC, germinal centre; HCC, hepatocellular carcinoma; HSC, hepatic stellate cell; IHC, immunohistochemical; IL-17; Inflammation; LPS, lipopolysaccharide; Liver fibrosis; MMP, matrix metalloproteinase; Macrophages; NASH, non-alcoholic steatohepatitis; RAR, retinoic acid receptor; ROCK, Rho-associated coiled-coil forming protein kinases; ROCK2; ROCK2, Rho-associated kinase 2; RORγt, RAR-related orphan receptor gamma; SR, Sirius red; STAT3, signal transducer and activator of transcription 3; TAA, thioacetamide; TGF-β, transforming growth factor-beta; TNF, tumour necrosis factor; Tfh, T follicular helper; Th17, T helper 17; Therapy; cGVHD, chronic graft-vs-host disease; pCofilin, phosphorylated cofilin; pMac, peritoneal macrophages; pSTAT3, phosphorylated signal transducer and activator of transcription; qRT-PCR, quantitative real-time PCR; α-SMA, alpha smooth muscle actin

DOI: https://doi.org/10.1016/j.jhepr.2021.100386

J Intern Med. 2021 Dec 16.

Liver damage promotes pro-inflammatory T-cell responses against apolipoprotein B-100.

Bastian F J Plochg, Hanna Englert, Chandini Rangaswamy, Sandra Konrath, Mandy Malle, Sibylle Lampalzer, Claudia Beisel, Salma Wollin, Maike Frye, Jens Aberle, Johannes Kluwe, Thomas Renné, Reiner K Mailer.

OBJECTIVES: Liver-derived apolipoprotein B-100 (ApoB100) is an autoantigen that is recognized by atherogenic CD4+ T cells in cardiovascular disease (CVD). CVD is a major mortality risk for patients with chronic inflammatory liver diseases. However, the impact of liver damage for ApoB100-specific T-cell responses is unknown.
METHODS: We identified ApoB100-specific T cells in blood from healthy controls, non-alcoholic fatty liver disease (NAFLD) patients and CVD patients by activation-induced marker expression and analyzed their differentiation pattern in correlation to the lipid profile and liver damage parameters in a cross-sectional study. To assess the induction of extrahepatic ApoB100-specific T cells upon transient liver damage in vivo, we performed hydrodynamic tail vein injections with diphtheria toxin A (DTA)-encoding plasmid in human ApoB100-transgenic mice.
RESULTS: Utilizing immunodominant ApoB100-derived peptides, we found increased ApoB100-specific T-cell populations in NAFLD and CVD patients compared to healthy controls. In a peptide-specific manner, ApoB100 reactivity in healthy controls was accompanied by expression of the regulatory T (Treg)-cell transcription factor FOXP3. In contrast, FOXP3 expression decreased, whereas expression of pro-inflammatory cytokine interleukin (IL)-17A increased in ApoB100-specific T cells from NAFLD and CVD patients. Dyslipidemia and liver damage parameters in blood correlated with reduced FOXP3 expression and elevated IL-17A production in ApoB100-specific T-cell populations, respectively. Moreover, DTA-mediated transient liver damage in human ApoB100-transgenic mice accumulated IL-17a-expressing ApoB100-specific T cells in the periphery.
CONCLUSION: Our results show that liver damage promotes pro-inflammatory ApoB100-specific T-cell populations, thereby providing a cellular mechanism for the increased CVD risk in liver disease patients. This article is protected by copyright. All rights reserved.

Keywords: apolipoproteins; atherosclerosis; cardiovascular clinical research; immunology; liver disease

DOI: https://doi.org/10.1111/joim.13434

Immunity. 2021 Dec 14. pii: S1074-7613(21)00507-0. [Epub ahead of print]54(12): 2701-2711

Cytotoxic CD4+ T cells in cancer: Expanding the immune effector toolbox.

David Y Oh, Lawrence Fong.

Cytotoxic T cells are important effectors of anti-tumor immunity. While tumor killing is ascribed to CD8+ T cell function, pre-clinical and clinical studies have identified intra-tumoral CD4+ T cells that possess cytotoxic programs and can directly kill cancer cells. Cytotoxic CD4+ T cells are found in other disease settings including infection and autoimmunity. Here, we review the phenotypic and functional characteristics of cytotoxic CD4+ T cells in non-cancer and cancer contexts. We conduct a comparative examination of cytolytic mechanisms of cytotoxic CD4+ T cells across disease states and synthesize features that define these cells independent of context. We discuss regulatory mechanisms driving ontogeny and effector function and evidence for the clinical relevance of cytotoxic CD4+ T cells in cancer. In this context, we highlight important gaps in understanding in the biology of cytotoxic CD4+ T cells as well as the potential use of these cells in immunotherapies for specific cancers.

DOI: https://doi.org/10.1016/j.immuni.2021.11.015

Nat Metab. 2021 Dec 13.

Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis.

Lianggong Ding, Wenfei Sun, Miroslav Balaz, Anyuan He, Manuel Klug, Stefan Wieland, Robert Caiazzo, Violeta Raverdy, Francois Pattou, Philippe Lefebvre, Irfan J Lodhi, Bart Staels, Markus Heim, Christian Wolfrum.

To liberate fatty acids (FAs) from intracellular stores, lipolysis is regulated by the activity of the lipases adipose triglyceride lipase (ATGL), hormone-sensitive lipase and monoacylglycerol lipase. Excessive FA release as a result of uncontrolled lipolysis results in lipotoxicity, which can in turn promote the progression of metabolic disorders. However, whether cells can directly sense FAs to maintain cellular lipid homeostasis is unknown. Here we report a sensing mechanism for cellular FAs based on peroxisomal degradation of FAs and coupled with reactive oxygen species (ROS) production, which in turn regulates FA release by modulating lipolysis. Changes in ROS levels are sensed by PEX2, which modulates ATGL levels through post-translational ubiquitination. We demonstrate the importance of this pathway for non-alcoholic fatty liver disease progression using genetic and pharmacological approaches to alter ROS levels in vivo, which can be utilized to increase hepatic ATGL levels and ameliorate hepatic steatosis. The discovery of this peroxisomal β-oxidation-mediated feedback mechanism, which is conserved in multiple organs, couples the functions of peroxisomes and lipid droplets and might serve as a new way to manipulate lipolysis to treat metabolic disorders.

DOI: https://doi.org/10.1038/s42255-021-00489-2

J Immunol. 2021 Dec 15. pii: ji2100581. [Epub ahead of print]

Reinvestigation of Classic T Cell Subsets and Identification of Novel Cell Subpopulations by Single-Cell RNA Sequencing.

Xuefei Wang, Xiangru Shen, Shan Chen, Hongyi Liu, Ni Hong, Hanbing Zhong, Xi Chen, Wenfei Jin.

Classic T cell subsets are defined by a small set of cell surface markers, while single-cell RNA sequencing (scRNA-seq) clusters cells using genome-wide gene expression profiles. The relationship between scRNA-seq clustered populations (scCPops) and cell surface marker-defined classic T cell subsets remains unclear. In this article, we integrated six bead-enriched T cell subsets with 62,235 single-cell transcriptomes from human PBMCs and clustered them into nine scCPops. Bead-enriched CD4+/CD45RA+/CD25- naive T and CD8+/CD45RA+ naive T cells were mainly clustered into their scCPop counterparts, while cells from the other T cell subsets were assigned to multiple scCPops, including mucosal-associated invariant T cells and NKT cells. The multiple T cell subsets forming one scCPop exhibit similar expression patterns, but not vice versa, indicating scCPop is a more homogeneous cell population with similar cell states. Interestingly, we discovered and named IFN signaling-associated gene (ISAG) high T (ISAGhi T) cells, a T cell subpopulation that highly expressed ISAGs. We further enriched ISAGhi T cells from human PBMCs by FACS of BST2 for scRNA-seq analyses. The ISAGhi T cell cluster disappeared on t-distributed stochastic neighbor embedding plot after removing ISAGs, whereas the ISAGhi T cell cluster showed up by analysis of ISAGs alone, indicating ISAGs are the major contributor of the ISAGhi T cell cluster. BST2+ and BST2- T cells showing different efficiencies of T cell activation indicate that a high level of ISAGs may contribute to quick immune responses.

DOI: https://doi.org/10.4049/jimmunol.2100581

World J Hepatol. 2021 Nov 27. 13(11): 1534-1551

Liver involvement in inflammatory bowel disease: What should the clinician know?

Giuseppe Losurdo, Irene Vita Brescia, Chiara Lillo, Martino Mezzapesa, Michele Barone, Mariabeatrice Principi, Enzo Ierardi, Alfredo Di Leo, Maria Rendina.

Inflammatory bowel disease (IBD) may show a wide range of extraintestinal manifestations. In this context, liver involvement is a focal point for both an adequate management of the disease and its prognosis, due to possible serious comorbidity. The association between IBD and primary sclerosing cholangitis is the most known example. This association is relevant because it implies an increased risk of both colorectal cancer and cholangiocarcinoma. Additionally, drugs such as thiopurines or biologic agents can cause drug-induced liver damage; therefore, this event should be considered when planning IBD treatment. Additionally, particular consideration should be given to the evidence that IBD patients may have concomitant chronic viral hepatitis, such as hepatitis B and hepatitis C. Chronic immunosuppressive regimens may cause a hepatitis flare or reactivation of a healthy carrier state, therefore careful monitoring of these patients is necessary. Finally, the spread of obesity has involved even IBD patients, thus increasing the risk of non-alcoholic fatty liver disease, which has already proven to be more common in IBD patients than in the non-IBD population. This phenomenon is considered an emerging issue, as it will become the leading cause of liver cirrhosis.

Keywords: Immunosuppression; Inflammatory bowel disease; Liver; Non-alcoholic fatty liver disease; Primary sclerosing cholangitis; Viral hepatitis

DOI: https://doi.org/10.4254/wjh.v13.i11.1534

Int Immunol. 2021 Dec 13. pii: dxab108. [Epub ahead of print]

Memory-phenotype CD4 + T cells: a naturally arising T lymphocyte population possessing innate immune function.

Takeshi Kawabe, Alan Sher.

In conventional adaptive immune responses, upon recognition of foreign antigens, naive CD4 + T lymphocytes are activated to differentiate into effector / memory cells. In addition, emerging evidence suggests that in the steady state, naive CD4 + T cells spontaneously proliferate in response to self antigens to acquire a memory phenotype (MP) through homeostatic proliferation. This expansion is particularly profound in lymphopenic environments but also occurs in lymphoreplete, normal conditions. The "MP T lymphocytes" generated in this manner are maintained by rapid proliferation in the periphery and they tonically differentiate into T-bet-expressing "MP1" cells. Such MP1 CD4 + T lymphocytes can exert innate effector function, producing IFN-γin response to IL-12 in the absence of antigen recognition, thereby contributing to host defense. In this review, we will discuss our current understanding of how MP T lymphocytes are generated and persist in steady-state conditions, their populational heterogeneity as well as the evidence for their effector function. We will also compare these properties with those of a similar population of innate memory cells previously identified in the CD8 + T lymphocyte lineage.

Keywords: CD4 + T lymphocytes; Homeostasis; Memory

DOI: https://doi.org/10.1093/intimm/dxab108

Methods Mol Biol. 2022 ;2403 81-89

Single Cell RNA-Seq: Cell Isolation and Data Analysis.

Val Yianni, Paul T Sharpe.

Single-cell RNA-sequencing technologies have revolutionized the way that researchers can interrogate cellular relationships and the level of detail by which tissue architecture can be characterized. Multiple cell capturing methods have been developed that, when coupled to next-generation sequencing, can yield cell-to-cell specific information regarding gene expression profiles. One of the commonalities between all of the cell capturing techniques to succeed is the necessity to submit samples with a high cell viability. In addition, these cells should have undergone minimal processing to limit induced stress responses so that their transcriptomes, when sequenced, closely reflect their transcriptomes in vivo at the time of isolation. Below we present a streamlined protocol to isolate fresh cells from tissues in vivo. We also share extensive notes to highlight considerations researchers should take into account before beginning their cell isolation protocol.

Keywords: RNA sequencing; Single-cell RNA-seq; Transcriptomics

DOI: https://doi.org/10.1007/978-1-0716-1847-9_7

EBioMedicine. 2021 Dec 10. pii: S2352-3964(21)00540-5. [Epub ahead of print]74 103746

CD4+CCR6+ T cells dominate the BCG-induced transcriptional signature.

Akul Singhania, Paige Dubelko, Rebecca Kuan, William D Chronister, Kaylin Muskat, Jyotirmoy Das, Elizabeth J Phillips, Simon A Mallal, Grégory Seumois, Pandurangan Vijayanand, Alessandro Sette, Maria Lerm, Bjoern Peters, Cecilia Lindestam Arlehamn.

BACKGROUND: The century-old Mycobacterium bovis Bacillus Calmette-Guerin (BCG) remains the only licensed vaccine against tuberculosis (TB). Despite this, there is still a lot to learn about the immune response induced by BCG, both in terms of phenotype and specificity.
METHODS: We investigated immune responses in adult individuals pre and 8 months post BCG vaccination. We specifically determined changes in gene expression, cell subset composition, DNA methylome, and the TCR repertoire induced in PBMCs and CD4 memory T cells associated with antigen stimulation by either BCG or a Mycobacterium tuberculosis (Mtb)-derived peptide pool.
FINDINGS: Following BCG vaccination, we observed increased frequencies of CCR6+ CD4 T cells, which includes both Th1* (CXCR3+CCR6+) and Th17 subsets, and mucosal associated invariant T cells (MAITs). A large number of immune response genes and pathways were upregulated post BCG vaccination with similar patterns observed in both PBMCs and memory CD4 T cells, thus suggesting a substantial role for CD4 T cells in the cellular response to BCG. These upregulated genes and associated pathways were also reflected in the DNA methylome. We described both qualitative and quantitative changes in the BCG-specific TCR repertoire post vaccination, and importantly found evidence for similar TCR repertoires across different subjects.
INTERPRETATION: The immune signatures defined herein can be used to track and further characterize immune responses induced by BCG, and can serve as reference for benchmarking novel vaccination strategies.

Keywords: Adaptive immunity; BCG; CCR6+ T cells; T cell; transcriptomics; tuberculosis; vaccine

DOI: https://doi.org/10.1016/j.ebiom.2021.103746