Identification of a cytoplasmic motif in the erythropoietin receptor required for receptor internalization

AbstractErythropoietin (EPO) promotes the viability, proliferation and differentiation of mammalian erythroid progenitor cells via its specific cell surface receptor. The EPO receptor (EPO-R) is a member of the cytokine receptor superfamily and is comprised of one identified subunit which homodimerizes upon ligand binding. To study the role of the intracellular domain of the EPO-R in the endocytosis of EPO, we compared the rate and extent of 125I-EPO endocytosis by wild type (wt) EPO-R and five cytoplasmically truncated EPO-Rs: 1–251 EPO-R, 1–257 EPO-R, 1–267 EPO-R, 1–276 EPO-R and 1–306 EPO-R which contain 4, 10, 20, 29 or 59 amino acids of the cytoplasmic domain, respectively. We also studied an EPO-R mutant (PB) which lacks amino acid residues 281–300 of the cytoplasmic domain. The experiments were conducted in COS 7 cells transfected with the EPO-R cDNAs and in Ba/F3 cells stably expressing the wt EPO-R, 1–251 or 1–257 EPO-R. Cells expressing wt EPO-R, PB EPO-R (Δ281–300), 1–276 EPO-R or 1–306 EPO-R internalized approximately 50% of 125I-EPO bound to the cell surface, while cells expressing 1–251, 1–257 or 1–267 EPO-R internalized only 25% of the bound 125I-EPO. The steady-state expression levels of these latter receptors on the cell surface were typically 2–5-fold higher than wt EPO-R. Our data indicate that amino acid residues 267–276 (FEGLFTTHK) of the EPO-R cytoplasmic domain may have a role in receptor internalization. Metabolic labeling experiments suggest that in transiently transfected COS 7 cells most of the wt EPO-R and 1–257 EPO-Rs do not exit the ER and may be degraded there. The half-life of both receptors was essentially similar and was in the range of 1 h. In Ba/F3 cells the mature Golgi processed 1–257 EPO-R was more stable than the corresponding form of the wt EPO-R, possibly contributing to its higher cell surface expression

Role of the Fractalkine Receptor in CNS Autoimmune Inflammation: New Approach Utilizing a Mouse Model Expressing the Human CX3CR1

Multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS) is the leading cause of non-traumatic neurological disability in young adults. Immune mediated destruction of myelin and oligodendrocytes is considered the primary pathology of MS, but progressive axonal loss is the major cause of neurological disability. In an effort to understand microglia function during CNS inflammation, our laboratory focuses on the fractalkine/CX3CR1 signaling as a regulator of microglia neurotoxicity in various models of neurodegeneration. Fractalkine (FKN) is a transmembrane chemokine expressed in the CNS by neurons and signals through its unique receptor CX3CR1 present in microglia. During experimental autoimmune encephalomyelitis (EAE), CX3CR1 deficiency confers exacerbated disease defined by severe inflammation and neuronal loss. The CX3CR1 human polymorphism I249/M280 present in ∼20% of the population exhibits reduced adhesion for FKN conferring defective signaling whose role in microglia function and influence on neurons during MS remains unsolved. The aim of this study is to assess the effect of weaker signaling through hCX3CR1I249/M280 during EAE. We hypothesize that dysregulated microglial responses due to impaired CX3CR1 signaling enhance neuronal/axonal damage. We generated an animal model replacing the mouse CX3CR1 locus for the hCX3CR1I249/M280 variant. Upon EAE induction, these mice exhibited exacerbated EAE correlating with severe inflammation and neuronal loss. We also observed that mice with aberrant CX3CR1 signaling are unable to produce FKN and ciliary neurotrophic factor during EAE in contrast to wild type mice. Our results provide validation of defective function of the hCX3CR1I249/M280 variant and the foundation to broaden the understanding of microglia dysfunction during neuroinflammation. © 2018 Cardona et al

Generation of T Follicular Helper Cells Is Mediated by Interleukin-21 but Independent of T Helper 1, 2, or 17 Cell Lineages

SummaryAfter activation, CD4+ helper T (Th) cells differentiate into distinct effector subsets. Although chemokine (C-X-C motif) receptor 5-expressing T follicular helper (Tfh) cells are important in humoral immunity, their developmental regulation is unclear. Here we show that Tfh cells had a distinct gene expression profile and developed in vivo independently of the Th1 or Th2 cell lineages. Tfh cell generation was regulated by ICOS ligand (ICOSL) expressed on B cells and was dependent on interleukin-21 (IL-21), IL-6, and signal transducer and activator of transcription 3 (STAT3). However, unlike Th17 cells, differentiation of Tfh cells did not require transforming growth factor β (TGF-β) or Th17-specific orphan nuclear receptors RORα and RORγ in vivo. Finally, naive T cells activated in vitro in the presence of IL-21 but not TGF-β signaling preferentially acquired Tfh gene expression and promoted germinal-center reactions in vivo. This study thus demonstrates that Tfh is a distinct Th cell lineage

Role of the Fractalkine Receptor in CNS Autoimmune Inflammation: New Approach Utilizing a Mouse Model Expressing the Human CX3CR1I249/M280 Variant

Multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS) is the leading cause of non-traumatic neurological disability in young adults. Immune mediated destruction of myelin and oligodendrocytes is considered the primary pathology of MS, but progressive axonal loss is the major cause of neurological disability. In an effort to understand microglia function during CNS inflammation, our laboratory focuses on the fractalkine/CX3CR1 signaling as a regulator of microglia neurotoxicity in various models of neurodegeneration. Fractalkine (FKN) is a transmembrane chemokine expressed in the CNS by neurons and signals through its unique receptor CX3CR1 present in microglia. During experimental autoimmune encephalomyelitis (EAE), CX3CR1 deficiency confers exacerbated disease defined by severe inflammation and neuronal loss. The CX3CR1 human polymorphism I249/M280 present in ∼20% of the population exhibits reduced adhesion for FKN conferring defective signaling whose role in microglia function and influence on neurons during MS remains unsolved. The aim of this study is to assess the effect of weaker signaling through hCX3CR1I249/M280 during EAE. We hypothesize that dysregulated microglial responses due to impaired CX3CR1 signaling enhance neuronal/axonal damage. We generated an animal model replacing the mouse CX3CR1 locus for the hCX3CR1I249/M280 variant. Upon EAE induction, these mice exhibited exacerbated EAE correlating with severe inflammation and neuronal loss. We also observed that mice with aberrant CX3CR1 signaling are unable to produce FKN and ciliary neurotrophic factor during EAE in contrast to wild type mice. Our results provide validation of defective function of the hCX3CR1I249/M280 variant and the foundation to broaden the understanding of microglia dysfunction during neuroinflammation

Intestinal toxicity to CTLA-4 blockade driven by IL-6 and myeloid infiltration

View full abstracthttps://openworks.mdanderson.org/leading-edge/1055/thumbnail.jp

Differences in In Vivo and In Vitro cDC1 Maturation After Stimulation with Different TLR Agonists

https://openworks.mdanderson.org/sumexp21/1083/thumbnail.jp

Effects of different TLR Agonists on in vivo cDC1 and in vivo cDC2 Activation

https://openworks.mdanderson.org/sumexp21/1075/thumbnail.jp