Search

A defining feature of adaptive immunity is the development of long-lived memory T cells to curtail infection. Recent studies have identified a unique stem-like T-cell subset amongst exhausted CD8-positive T cells in chronic infection.sup.1-3, but it remains unclear whether CD4-positive T-cell subsets with similar features exist in chronic inflammatory conditions. Amongst helper T cells, T.sub.H17 cells have prominent roles in autoimmunity and tissue inflammation and are characterized by inherent plasticity.sup.4-7, although how such plasticity is regulated is poorly understood. Here we demonstrate that T.sub.H17 cells in a mouse model of autoimmune disease are functionally and metabolically heterogeneous; they contain a subset with stemness-associated features but lower anabolic metabolism, and a reciprocal subset with higher metabolic activity that supports transdifferentiation into T.sub.H1-like cells. These two T.sub.H17-cell subsets are defined by selective expression of the transcription factors TCF-1 and T-bet, and by discrete levels of CD27 expression. We also identify signalling via the kinase complex mTORC1 as a central regulator of T.sub.H17-cell fate decisions by coordinating metabolic and transcriptional programmes. T.sub.H17 cells with disrupted mTORC1 signalling or anabolic metabolism fail to induce autoimmune neuroinflammation or to develop into T.sub.H1-like cells, but instead upregulate TCF-1 expression and acquire stemness-associated features. Single-cell RNA sequencing and experimental validation reveal heterogeneity in fate-mapped T.sub.H17 cells, and a developmental arrest in the T.sub.H1 transdifferentiation trajectory upon loss of mTORC1 activity or metabolic perturbation. Our results establish that the dichotomy of stemness and effector function underlies the heterogeneous T.sub.H17 responses and autoimmune pathogenesis, and point to previously unappreciated metabolic control of plasticity in helper T cells.Phenotypically, transcriptionally and metabolically diverse subsets of T.sub.H17 cells develop in a chronic autoimmune disease: one subset has inferred stemness features and low anabolic metabolism, while a reciprocal subset has higher metabolic activity that supports transdifferentiation into T.sub.H1 cells., Author(s): Peer W. F. Karmaus [sup.1] , Xiang Chen [sup.2] , Seon Ah Lim [sup.1] , Andrés A. Herrada [sup.1] , Thanh-Long M. Nguyen [sup.1] , Beisi Xu [sup.2] , [...]

The article employs single-cell Ribonucleic acid (RNA) sequencing (scRNA-Seq) meta-analysis of COVID-19 bronchoalveolar lavage samples. Topics include clustering methods, cell type identification through supervised learning, inference of transcription factor activity, cell-cell communication analysis, differential expression analysis, gene set variability analysis, pseudotime analysis, and the integration of scRNA-Seq data from lung COVID-19 samples.

Dendritic cells orchestrate the crosstalk between innate and adaptive immunity. CD8[alpha].sup.+ dendritic cells present antigens to CD8.sup.+ T cells and elicit cytotoxic T cell responses to viruses, bacteria and tumours.sup.1. Although lineage-specific transcriptional regulators of CD8[alpha].sup.+ dendritic cell development have been identified.sup.2, the molecular pathways that selectively orchestrate CD8[alpha].sup.+ dendritic cell function remain elusive. Moreover, metabolic reprogramming is important for dendritic cell development and activation.sup.3,4, but metabolic dependence and regulation of dendritic cell subsets are largely uncharacterized. Here we use a data-driven systems biology algorithm (NetBID) to identify a role of the Hippo pathway kinases Mst1 and Mst2 (Mst1/2) in selectively programming CD8[alpha].sup.+ dendritic cell function and metabolism. Our NetBID analysis reveals a marked enrichment of the activities of Hippo pathway kinases in CD8[alpha].sup.+ dendritic cells relative to CD8[alpha].sup.- dendritic cells. Dendritic cell-specific deletion of Mst1/2--but not Lats1 and Lats2 (Lats1/2) or Yap and Taz (Yap/Taz), which mediate canonical Hippo signalling--disrupts homeostasis and function of CD8.sup.+ T cells and anti-tumour immunity. Mst1/2-deficient CD8[alpha].sup.+ dendritic cells are impaired in presentation of extracellular proteins and cognate peptides to prime CD8.sup.+ T cells, while CD8[alpha].sup.- dendritic cells that lack Mst1/2 have largely normal function. Mechanistically, compared to CD8[alpha].sup.- dendritic cells, CD8[alpha].sup.+ dendritic cells exhibit much stronger oxidative metabolism and critically depend on Mst1/2 signalling to maintain bioenergetic activities and mitochondrial dynamics for their functional capacities. Further, selective expression of IL-12 by CD8[alpha].sup.+ dendritic cells depends on Mst1/2 and the crosstalk with non-canonical NF-[kappa]B signalling. Our findings identify Mst1/2 as selective drivers of CD8[alpha].sup.+ dendritic cell function by integrating metabolic activity and cytokine signalling, and highlight that the interplay between immune signalling and metabolic reprogramming underlies the unique functions of dendritic cell subsets.A data-driven analysis helps to identify specific roles of the Hippo signalling kinases Mst1 and Mst2 in integrating metabolic activity and cytokine signalling in dendritic cells, and thereby orchestrating immune cell function., Author(s): Xingrong Du [sup.1] , Jing Wen [sup.1] , Yanyan Wang [sup.1] , Peer W. F. Karmaus [sup.1] , Alireza Khatamian [sup.2] , Haiyan Tan [sup.3] [sup.4] , Yuxin Li [...]

A defining feature of adaptive immunity is the development of long-lived memory T cells to curtail infection. Recent studies have identified a unique stem-like T-cell subset amongst exhausted CD8-positive T cells in chronic infection1-3, but it remains unclear whether CD4-positive T-cell subsets with similar features exist in chronic inflammatory conditions. Amongst helper T cells, TH17 cells have prominent roles in autoimmunity and tissue inflammation and are characterized by inherent plasticity4-7, although how such plasticity is regulated is poorly understood. Here we demonstrate that TH17 cells in a mouse model of autoimmune disease are functionally and metabolically heterogeneous; they contain a subset with stemness-associated features but lower anabolic metabolism, and a reciprocal subset with higher metabolic activity that supports transdifferentiation into TH1-like cells. These two TH17-cell subsets are defined by selective expression of the transcription factors TCF-1 and T-bet, and by discrete levels of CD27 expression. We also identify signalling via the kinase complex mTORC1 as a central regulator of TH17-cell fate decisions by coordinating metabolic and transcriptional programmes. TH17 cells with disrupted mTORC1 signalling or anabolic metabolism fail to induce autoimmune neuroinflammation or to develop into TH1-like cells, but instead upregulate TCF-1 expression and acquire stemness-associated features. Single-cell RNA sequencing and experimental validation reveal heterogeneity in fate-mapped TH17 cells, and a developmental arrest in the TH1 transdifferentiation trajectory upon loss of mTORC1 activity or metabolic perturbation. Our results establish that the dichotomy of stemness and effector function underlies the heterogeneous TH17 responses and autoimmune pathogenesis, and point to previously unappreciated metabolic control of plasticity in helper T cells. Phenotypically, transcriptionally and metabolically diverse subsets of TH17 cells develop in a chronic autoimmune disease: one subset has inferred stemness features and low anabolic metabolism, while a reciprocal subset has higher metabolic activity that supports transdifferentiation into TH1 cells. [ABSTRACT FROM AUTHOR]

Dendritic cells orchestrate the crosstalk between innate and adaptive immunity. CD8α+ dendritic cells present antigens to CD8+ T cells and elicit cytotoxic T cell responses to viruses, bacteria and tumours [1]. Although lineage-specific transcriptional regulators of CD8α+ dendritic cell development have been identified [2], the molecular pathways that selectively orchestrate CD8α+ dendritic cell function remain elusive. Moreover, metabolic reprogramming is important for dendritic cell development and activation [3], [4], but metabolic dependence and regulation of dendritic cell subsets are largely uncharacterized. Here we use a data-driven systems biology algorithm (NetBID) to identify a role of the Hippo pathway kinases Mst1 and Mst2 (Mst1/2) in selectively programming CD8α+ dendritic cell function and metabolism. Our NetBID analysis reveals a marked enrichment of the activities of Hippo pathway kinases in CD8α+ dendritic cells relative to CD8α− dendritic cells. Dendritic cell-specific deletion of Mst1/2—but not Lats1 and Lats2 (Lats1/2) or Yap and Taz (Yap/Taz), which mediate canonical Hippo signalling—disrupts homeostasis and function of CD8+ T cells and anti-tumour immunity. Mst1/2-deficient CD8α+ dendritic cells are impaired in presentation of extracellular proteins and cognate peptides to prime CD8+ T cells, while CD8α− dendritic cells that lack Mst1/2 have largely normal function. Mechanistically, compared to CD8α− dendritic cells, CD8α+ dendritic cells exhibit much stronger oxidative metabolism and critically depend on Mst1/2 signalling to maintain bioenergetic activities and mitochondrial dynamics for their functional capacities. Further, selective expression of IL-12 by CD8α+ dendritic cells depends on Mst1/2 and the crosstalk with non-canonical NF-κB signalling. Our findings identify Mst1/2 as selective drivers of CD8α+ dendritic cell function by integrating metabolic activity and cytokine signalling, and highlight that the interplay between immune signalling and metabolic reprogramming underlies the unique functions of dendritic cell subsets. A data-driven analysis helps to identify specific roles of the Hippo signalling kinases Mst1 and Mst2 in integrating metabolic activity and cytokine signalling in dendritic cells, and thereby orchestrating immune cell function. [ABSTRACT FROM AUTHOR]

Memory CD8+ T cells are an essential component of protective immunity. Signaling via mechanistic target of rapamycin (mTOR) has been implicated in the regulation of the differentiation of effector and memory T cells. However, little is understood about the mechanisms that control mTOR activity, or the effector pathways regulated by mTOR. We describe here that tuberous sclerosis 1 (Tsc1), a regulator of mTOR signaling, plays a crucial role in promoting the differentiation and function of memory CD8+ T cells in response to Listeria monocytogenes infection. Mice with specific deletion of Tsc1 in antigen-experienced CD8+ T cells evoked normal effector responses, but were markedly impaired in the generation of memory T cells and their recall responses to antigen reexposure in a cell-intrinsic manner. Tsc1 deficiency suppressed the generation of memory-precursor effector cells while promoting short-lived effector cell differentiation. Transcriptome analysis indicated that Tsc1 coordinated gene expression programs underlying immune function, transcriptional regulation, and cell metabolism. Furthermore, Tsc1 deletion led to excessive mTORC1 activity and dysregulated glycolytic and oxidative metabolism in response to IL-15 stimulation. These findings establish a Tsc1-mediated checkpoint in linking immune signaling and cell metabolism to orchestrate memory CD8+ T-cell development and function. [ABSTRACT FROM AUTHOR]

Cannabidiol (CBD) is a plant-derived cannabinoid that has been predominantly characterized as anti-inflammatory. However, it is clear that immune effects of cannabinoids can vary with cannabinoid concentration, or type or magnitude of immune stimulus. The present studies demonstrate that oral administration of CBD enhanced lipopolysaccharide (LPS)-induced pulmonary inflammation in C57BL/6 mice. The enhanced inflammatory cell infiltrate as observed in bronchoalveolar lavage fluid (BALF) was comprised mainly of neutrophils, with some monocytes. Concomitantly, CBD enhanced pro-inflammatory cytokine mRNA production, including tumor necrosis factor- α ( Tnfa), interleukins (IL)-5 and -23 ( Il6, Il23), and granulocyte colony stimulating factor ( Gcsf). These results demonstrate that the CBD-mediated enhancement of LPS-induced pulmonary inflammation is mediated at the level of transcription of a variety of pro-inflammatory genes. The significance of these studies is that CBD is part of a therapeutic currently in use for spasticity and pain in multiple sclerosis patients, and therefore it is important to further understand mechanisms by which CBD alters immune function. [ABSTRACT FROM AUTHOR]

Recent developments of novel single-cell analysis techniques have rapidly advanced the fields of immunotoxicology and immunometabolism. Single-cell analyses enable the characterization of immune cells, unraveling heterogeneity, and population dynamics in response to cellular perturbations, including toxicant insults and changes in cellular metabolism. This review provides an overview of current technologies and recent discoveries, illustrating an emerging role of single-cell analyses in the field of immunotoxicology and immunometabolism. Various single-cell techniques, including flow cytometry, mass cytometry, multiplexed imaging, and sequencing, together with their applications to studying immunotoxicology and immunometabolism are discussed. This review emphasizes the potential for single-cell analyses to revolutionize our understanding of immune cell heterogeneity, uncover novel cellular therapeutic targets, and pave the way for novel mechanistic insights., Competing Interests: Disclosures The author declares no conflicts of interest.

Histone crotonylation is a non-acetyl histone lysine modification that is as widespread as acetylation. However, physiological functions associated with histone crotonylation remain almost completely unknown. Here we report that histone crotonylation is crucial for endoderm differentiation. We demonstrate that key crotonyl-coenzyme A (CoA)-producing enzymes are specifically induced in endodermal cells during differentiation of human embryonic stem cells (hESCs) in vitro and in mouse embryos, where they function to increase histone crotonylation and enhance endodermal gene expression. Chemical enhancement of histone crotonylation promotes endoderm differentiation of hESCs, whereas deletion of crotonyl-CoA-producing enzymes reduces histone crotonylation and impairs meso/endoderm differentiation in vitro and in vivo. Our study uncovers a histone crotonylation-mediated mechanism that promotes endodermal commitment of pluripotent stem cells, which may have important implications for therapeutic strategies against a number of human diseases., Competing Interests: Declaration of interests Y.Z. is on the science advisory board of PTM Biolabs., (Published by Elsevier Inc.)

An emerging emphasis on mechanism-focused and human-relevant alternatives to animal use in toxicology underlies the toxicology testing in the twenty-first-century initiative. Herein we describe in vitro high-throughput screening programs seeking to address this goal, as well as strategies established to integrate assay results to build weight of evidence in support of hazard assessment. Furthermore, we discuss unique challenges facing the application of such alternatives for assessing immunotoxicity given the complexity of immune responses. Addressing these challenges will require the development of novel in vitro assays that evaluate well-characterized biochemical processes involved in immune response to help inform on putative adverse outcomes in vivo.

Myelopoiesis is necessary for the generation of mature myeloid cells during homeostatic turnover and immunological insults; however, the metabolic requirements for this process remain poorly defined. Here, we demonstrate that myelopoiesis, including monocyte and macrophage differentiation, requires mechanistic target of rapamycin complex 1 (mTORC1) signaling and anabolic metabolism. Loss of mTORC1 impaired myelopoiesis under steady state and dampened innate immune responses against Listeria monocytogenes infection. Stimulation of hematopoietic progenitors with macrophage colony-stimulating factor (M-CSF) resulted in mTORC1-dependent anabolic metabolism, which in turn promoted expression of M-CSF receptor and transcription factors PU.1 and IRF8, thereby constituting a feed-forward loop for myelopoiesis. Mechanistically, mTORC1 engaged glucose metabolism and initiated a transcriptional program involving Myc activation and sterol biosynthesis after M-CSF stimulation. Perturbation of glucose metabolism or disruption of Myc function or sterol biosynthesis impaired myeloid differentiation. Integrative metabolomic and genomic profiling further identified one-carbon metabolism as a central node in mTORC1-dependent myelopoiesis. Therefore, the interplay between mTORC1 signaling and metabolic reprogramming underlies M-CSF-induced myelopoiesis., (© 2017 Karmaus et al.)

Naive T cells respond to antigen stimulation by exiting from quiescence and initiating clonal expansion and functional differentiation, but the control mechanism is elusive. Here we describe that Raptor-mTORC1-dependent metabolic reprogramming is a central determinant of this transitional process. Loss of Raptor abrogated T cell priming and T helper 2 (Th2) cell differentiation, although Raptor function is less important for continuous proliferation of actively cycling cells. mTORC1 coordinated multiple metabolic programs in T cells including glycolysis, lipid synthesis, and oxidative phosphorylation to mediate antigen-triggered exit from quiescence. mTORC1 further linked glucose metabolism to the initiation of Th2 cell differentiation by orchestrating cytokine receptor expression and cytokine responsiveness. Activation of Raptor-mTORC1 integrated T cell receptor and CD28 costimulatory signals in antigen-stimulated T cells. Our studies identify a Raptor-mTORC1-dependent pathway linking signal-dependent metabolic reprogramming to quiescence exit, and this in turn coordinates lymphocyte activation and fate decisions in adaptive immunity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The role of cannabinoid receptors, CB1 and CB2, in immune competence and modulation by Delta9-tetrahydrocannabinol (Delta9-THC) was investigated in CB1(-/-)/CB2(-/-) mice. Immunofluorescence analysis of splenic leukocytes showed no significant differences in the percentage of T cell subsets, B cells, or macrophages between wild-type and CB1(-/-)/CB2(-/-) mice. Lymphoproliferative control responses to PHA, phorbol ester plus ionomycin, or LPS and sensitivity to suppression by Delta9-THC showed no profound differences between the two genotypes, although some differences were observed in control baseline responses. Likewise, similar control responses and sensitivity to Delta9-THC were observed in mixed lymphocyte responses (MLR) and in IL-2 and IFN-gamma production in both genotypes. Conversely, humoral immune responses showed a markedly different profile of activity. Delta9-THC suppressed the in vivo T cell-dependent, anti-sheep RBC (anti-sRBC) IgM antibody-forming cell (AFC) response in wild-type but not in CB1(-/-)/CB2(-/-) mice, and the in vitro anti-sRBC IgM response in CB1(-/-)/CB2(-/-) splenocytes was too low to rigorously assess CB1/CB2 involvement in modulation by Delta9-THC. Conversely, comparable in vitro IgM AFC control responses to LPS and CD40 ligand (CD40L) activation were observed in the two genotypes. Interestingly, LPS-induced IgM responses were refractory to suppression by Delta9-THC, regardless of genotype, and CD40L-induced IgM responses were only suppressed by Delta9-THC in wild-type but not in CB1(-/-)/CB2(-/-) B cells. Collectively, we demonstrate differential involvement of CB1 and/or CB2 in immune modulation by Delta9-THC and in some control responses. Moreover, CB1/CB2 involvement was observed in humoral responses requiring CD40-initiated signaling for suppression by Delta9-THC.

We have previously reported that Delta-9-tetrahydrocannabinol (Delta(9)-THC)-treated mice challenged with influenza virus A/PR/8/34 (PR8) developed increased viral hemagglutinin 1 (H1) mRNA levels and decreased monocyte and lymphocyte recruitment to the pulmonary airways when compared with mice challenged with PR8 alone. The objective of the present study was to examine the role of cannabinoid (CB(1)/CB(2)) receptors in mediating the effects of Delta(9)-THC on immune and epithelial cell responses to PR8. In the current study, Delta(9)-THC-treated CB(1)/CB(2) receptor null (CB(1)-/-/CB(2)-/-) and wild-type mice infected with PR8 had marked increases in viral H1 mRNA when compared with CB(1)-/-/CB(2)-/- and wild-type mice challenged with PR8 alone. However, the magnitude of the H1 mRNA levels was greatly reduced in CB(1)-/-/CB(2)-/- mice as compared with wild-type mice. In addition, Delta(9)-THC-treated CB(1)-/-/CB(2)-/- mice infected with PR8 had increased CD4+ T cells and IFN-gamma in bronchoalveolar lavage fluid with greater pulmonary inflammation when compared with Delta(9)-THC-treated wild-type mice infected with PR8. Delta(9)-THC treatment of CB(1)-/-/CB(2)-/- mice in the presence or absence of PR8 challenge also developed greater amounts of mucous cell metaplasia in the affected bronchiolar epithelium. Collectively, the immune and airway epithelial cell responses to PR8 challenge in Delta(9)-THC-treated CB(1)-/-/CB(2)-/- and wild-type mice indicated the involvement of CB(1)/CB(2) receptor-dependent and -independent mechanisms.