Induction of memory CD8 T cells is important for controlling infections such as malaria HIV/AIDS, and for cancer immunotherapy. Accurate assessment of antigen (Ag)-specific CD8 T-cells is critical for vaccine optimization and defining correlates of protection. However, conditions for determining Ag-specific CD8 T-cell responses ex-vivo using ICS may be variable, especially in humans with complex antigens. Here, we used an attenuated whole parasite malaria vaccine model in humans and various experimental infections in mice to show that the duration of antigenic stimulation and timing of brefeldin A (BFA) addition influences the magnitude of Ag-specific and bystander T cell responses. Indeed, following immunization with an attenuated whole sporozoite malaria vaccine in humans, significantly higher numbers of IFN-γ producing memory CD8 T-cells comprised of antigen specific and bystander responses were detected by increasing the duration of Ag-stimulation prior to addition of BFA. Mechanistic analyses of virus-specific CD8 T-cells in mice revealed that the increase in IFNg producing CD8 T-cells was due to bystander activation of Ag-experienced memory CD8 T-cells, and correlated with the proportion of Ag-experienced CD8 T-cells in the stimulated populations. Incubation with anti-cytokine antibodies (ex. IL-12) improved accuracy in detecting bona-fide memory CD8 T-cell responses suggesting this as the mechanism for the bystander activation. These data have important implications for accurate assessment of immune responses generated by vaccines intended to elicit protective memory CD8 T-cells.
Matthew D. Martin, Isaac J. Jensen, Andrew S. Ishizuka, Mitchell Lefebvre, Qiang Shan, Hai-Hui Xue, John T. Harty, Robert A. Seder, Vladimir P. Badovinac
Shwachman-Diamond Syndrome (SDS) is a rare and clinically-heterogeneous bone marrow (BM) failure syndrome caused by mutations in the Shwachman-Bodian-Diamond Syndrome (SBDS) gene. Although SDS was described over 50 years ago, the molecular pathogenesis is poorly understood due, in part, to the rarity and heterogeneity of the affected hematopoietic progenitors. To address this, we used single cell RNA sequencing to profile scant hematopoietic stem and progenitor cells from SDS patients. We generated a single cell map of early lineage commitment and found that SDS hematopoiesis was left-shifted with selective loss of granulocyte-monocyte progenitors. Transcriptional targets of transforming growth factor-beta (TGFβ) were dysregulated in SDS hematopoietic stem cells and multipotent progenitors, but not in lineage-committed progenitors. TGFβ inhibitors (AVID200 and SD208) increased hematopoietic colony formation of SDS patient BM. Finally, TGFβ3 and other TGFβ pathway members were elevated in SDS patient blood plasma. These data establish the TGFβ pathway as a novel candidate biomarker and therapeutic target in SDS and translate insights from single cell biology into a potential therapy.
Cailin E. Joyce, Assieh Saadatpour, Melisa Ruiz-Gutierrez, Ozge Vargel Bolukbasi, Lan Jiang, Dolly D. Thomas, Sarah Young, Inga Hofmann, Colin Sieff, Kasiani C. Myers, Jennifer Whangbo, Towia A. Libermann, Chad Nusbaum, Guo-Cheng Yuan, Akiko Shimamura, Carl D. Novina
A population of Natural Killer (NK) cells expressing the activating receptor NKG2C and the maturation marker CD57 expands in response to human cytomegalovirus (HCMV) infection. CD3–CD56dimCD57+NKG2C+ NK cells are similar to CD8+ memory T cells with rapid and robust effector function upon re-stimulation, persistence, and epigenetic remodeling of the IFNG locus. Chronic antigen stimulation drives CD8+ memory T cell proliferation while also inducing genome-wide epigenetic reprograming and dysfunction. We hypothesized that chronic stimulation could similarly induce epigenetic reprograming and dysfunction in NK cells. Here we show that chronic stimulation of adaptive NK cells through NKG2C using plate-bound agonistic antibodies in combination with IL-15 drove robust proliferation and activation of CD3–CD56dimCD57+NKG2C+ NK cells while simultaneously inducing high expression of the checkpoint inhibitory receptors LAG-3 and PD-1. Marked induction of checkpoint inhibitory receptors was also observed on the surface of adaptive NK cells co-cultured with HCMV-infected endothelial cells. Chronically stimulated adaptive NK cells were dysfunctional when challenged with tumor targets. These cells exhibited a pattern of epigenetic reprograming, with genome-wide alterations in DNA methylation. Our study has important implications for cancer immunotherapy and suggest that exhausted NK cells could be targeted with inhibitory checkpoint receptor blockade.
Aimee M. Merino, Bin Zhang, Phillip R. Dougherty, Xianghua Luo, Jinhua Wang, Bruce R. Blazar, Jeffrey S. Miller, Frank Cichocki
Resident microbiota activate regulatory cells that modulate intestinal inflammation and promote and maintain intestinal homeostasis. IL-10 is a key mediator of immune regulatory function. Our studies described the functional importance and mechanisms by which gut microbiota and specific microbial components influenced the development of intestinal IL-10-producing B cells. We used fecal transplant to germ-free (GF) Il10+/EGFP reporter and Il10-/- mice to demonstrate that microbiota from specific pathogen-free mice primarily stimulated IL-10-producing colon-specific B cells and T regulatory-1 cells in ex-GF mice. IL-10 in turn down-regulated microbiota-activated mucosal inflammatory cytokines. TLR2/9 ligands and enteric bacterial lysates preferentially induced IL-10 production and regulatory capacity of intestinal B cells. Analysis of Il10+/EGFP mice crossed with additional gene-deficient strains and B cell co-transfer studies demonstrated that microbiota-induced IL-10-producing intestinal B cells ameliorated chronic T cell-mediated colitis in a TLR2, MyD88 and PI3K-dependent fashion. In vitro studies implicated PI3Kp110δ and AKT downstream signaling. These studies demonstrated that resident enteric bacteria activated intestinal IL-10-producing B cells through TLR2, MyD88 and PI3K pathways. These B cells reduced colonic T cell activation and maintained mucosal homeostasis in response to intestinal microbiota.
Yoshiyuki Mishima, Akihiko Oka, Bo Liu, Jeremy W. Herzog, Chang Soo Eun, Ting-Jia Fan, Emily Bulik-Sullivan, Ian M. Carroll, Jonathan J. Hansen, Liang Chen, Justin E. Wilson, Nancy C. Fisher, Jenny P. Y. Ting, Tomonori Nochi, Angela Wahl, J. Victor Garcia, Christopher L. Karp, R. Balfour Sartor
Although joint pain in rheumatoid arthritis (RA) is conventionally thought to result from inflammation, arthritis pain and joint inflammation are at least partially uncoupled. This suggests that additional pain mechanisms in RA remain to be explored. Here we show that FcγRI, an immune receptor for IgG immune complex (IgG-IC), is expressed in a subpopulation of joint sensory neurons and that, under naïve conditions, FcγRI crosslinking by IgG-IC directly activates the somata and peripheral terminals of these neurons to evoke acute joint hypernociception without obvious concurrent joint inflammation. These effects were diminished in both global and sensory neuron-specific Fcgr1 knockout mice. In murine models of inflammatory arthritis, FcγRI signaling was upregulated in joint sensory neurons. Acute blockade or global genetic deletion of Fcgr1 significantly attenuated arthritis pain and hyperactivity of joint sensory neurons without measurably altering joint inflammation. Conditional deletion of Fcgr1 in sensory neurons produced similar analgesic effects in these models. We therefore suggest that FcγRI expressed in sensory neurons contributes to arthritis pain independently of its functions in inflammatory cells. These findings expand our understanding of the immunosensory capabilities of sensory neurons and imply that neuronal FcγRI merits consideration as a target for treating RA pain.
Li Wang, Xiaohua Jiang, Qin Zheng, Sang-Min Jeon, Tiane Chen, Yan Liu, Heather Kulaga, Randall Reed, Xinzhong Dong, Michael J. Caterina, Lintao Qu
Deep brain stimulation (DBS) is used to treat multiple neuropsychiatric disorders, including Parkinson’s Disease (PD). Despite widespread clinical use, its therapeutic mechanisms are unknown. Here, we developed a mouse model of subthalamic nucleus (STN) DBS for PD, to permit investigation using cell type-specific tools available in mice. We found that electrical STN DBS relieved bradykinesia, as measured by movement velocity. In addition, our model recapitulated several hallmarks of human STN DBS, including rapid onset and offset, frequency dependence, dyskinesia at higher stimulation intensity, and associations between electrode location, therapeutic benefit, and side effects. We used this model to assess whether high frequency stimulation is necessary for effective STN DBS, or if low frequency stimulation can be effective when paired with compensatory adjustments in other parameters. We found that low frequency stimulation, paired with greater pulse width and amplitude, relieved bradykinesia. Moreover, a composite metric incorporating pulse width, amplitude, and frequency predicted therapeutic efficacy better than frequency alone. We found a similar relationship between this composite metric and movement speed in a retrospective analysis of human data, suggesting correlations observed in the mouse model may extend to human patients. Together, these data establish a mouse model for elucidating mechanisms of DBS.
Jonathan S. Schor, Alexandra B. Nelson
Specific neuronal populations display high vulnerability to pathological processes in Parkinson’s disease (PD). The dorsal motor nucleus of the vagus nerve (DMnX) is a primary site of pathological α-synuclein deposition and may play a key role in the spreading of α-synuclein lesions within and outside the CNS. Using in vivo models, we show that cholinergic neurons forming this nucleus are particularly susceptible to oxidative challenges and accumulation of reactive oxidative species (ROS). Targeted α-synuclein overexpression within these neurons triggered an oxidative stress that became significantly more pronounced after exposure to the ROS-generating agent paraquat. A more severe oxidative stress resulted in enhanced production of oxidatively modified forms of α-synuclein, increased α-synuclein aggregation into oligomeric species and marked degeneration of DMnX neurons. Enhanced oxidative stress also affected neuron-to-neuron protein transfer, causing an increased spreading of α-synuclein from the DMnX toward more rostral brain regions. In vitro experiments confirmed a greater propensity of α-synuclein to pass from cell to cell under pro-oxidant conditions, and identified nitrated α-synuclein forms as highly transferable protein species. These findings substantiate the relevance of oxidative injury in PD pathogenetic processes, establish a relationship between oxidative stress and vulnerability to α-synuclein pathology and define a new mechanism, enhanced cell-to-cell α-synuclein transmission, by which oxidative stress could promote PD development and progression.
Ruth E. Musgrove, Michael Helwig, Eun-Jin Bae, Helia Aboutalebi, Seung-Jae Lee, Ayse Ulusoy, Donato A. Di Monte
Beta-arrestin-1 and -2 (Barr1 and Barr2, respectively) are intracellular signaling molecules that regulate many important metabolic functions. We previously demonstrated that mice lacking Barr2 selectively in pancreatic beta-cells showed pronounced metabolic impairments. Here we investigated whether Barr1 plays a similar role in regulating beta-cell function and whole body glucose homeostasis. Initially, we inactivated the Barr1 gene in beta-cells of adult mice (beta-barr1-KO mice). Beta-barr1-KO mice did not display any obvious phenotypes in a series of in vivo and in vitro metabolic tests. However, glibenclamide and tolbutamide, two widely used antidiabetic drugs of the sulfonylurea (SU) family, showed greatly reduced efficacy in stimulating insulin secretion in the KO mice in vivo and in perifused KO islets in vitro. Additional in vivo and in vitro studies demonstrated that Barr1 enhanced SU-stimulated insulin secretion by promoting SU-mediated activation of Epac2. Pull-down and co-immunoprecipitation experiments showed that Barr1 can directly interact with Epac2 and that SUs such as glibenclamide promote Barr1/Epac2 complex formation, triggering enhanced Rap1 signaling and insulin secretion. These findings suggest that strategies aimed at promoting Barr1 signaling in beta-cells may prove useful for the development of efficacious antidiabetic drugs.
Luiz F. Barella, Mario Rossi, Lu Zhu, Yinghong Cui, Fang C. Mei, Xiaodong Cheng, Wei Chen, Vsevolod V. Gurevich, Jürgen Wess
Cyclooxygenase 2 (Cox2) total knockout and myeloid knockout (MKO) mice develop Crohn’s-like intestinal inflammation when fed cholate-containing high fat diet (CCHF). We demonstrated that CCHF impaired intestinal barrier function and increased translocation of endotoxin, initiating TLR/MyD88-dependent inflammation in Cox2 KO but not WT mice. Cox2 MKO increased pro-inflammatory mediators in LPS-activated macrophages, and in the intestinal tissue and plasma upon CCHF challenge. Cox2 MKO also reduced inflammation resolving lipoxin A4 (LXA4) in intestinal tissue, while administration of an LXA4 analog rescued disease in Cox2 MKO mice fed CCHF. The apolipoprotein A-I (APOA1) mimetic 4F mitigated disease in both the Cox2 MKO/CCHF and piroxicam-accelerated Il10-/- models of inflammatory bowel disease (IBD) and reduced elevated levels of pro-inflammatory mediators in tissue and plasma. APOA1 mimetic Tg6F therapy was also effective in reducing intestinal inflammation in the Cox2 MKO/CCHF model. We further demonstrated that APOA1 mimetic peptides: i) inhibited LPS and oxidized 1-palmitoyl-2-arachidonoyl-sn-phosphatidylcholine (oxPAPC) dependent pro-inflammatory responses in human macrophages and intestinal epithelium; and ii) directly cleared pro-inflammatory lipids from mouse intestinal tissue and plasma. Our results support a causal role for pro-inflammatory and inflammation resolving lipids in IBD pathology and a translational potential for APOA1 mimetic peptides for the treatment of IBD.
David Meriwether, Dawoud Sulaiman, Carmen Volpe, Anna Dorfman, Victor Grijalva, Nasrin Dorreh, R. Sergio Solorzano-Vargas, Jiafang Wang, Ellen O’Connor, Jeremy Papesh, Muriel Larauche, Hannah Trost, Mayakonda N. Palgunachari, G.M. Anantharamaiah, Harvey R. Herschman, Martin G. Martin, Alan M. Fogelman, Srinivasa T. Reddy
Combined germline and somatic second hit inactivating mutations of the RASA1 gene, which encodes a negative regulator of the Ras signaling pathway, cause blood and lymphatic vascular lesions in the human autosomal dominant vascular disorder capillary malformation-arteriovenous malformation (CM-AVM). How RASA1 mutations in endothelial cells (EC) result in vascular lesions in CM-AVM is unknown. Here, using different murine models of RASA1-deficiency, we found that RASA1 was essential for the survival of EC during developmental angiogenesis in which primitive vascular plexuses are remodeled into hierarchical vascular networks. RASA1 was required for EC survival during developmental angiogenesis because it was necessary for export of collagen IV from EC and deposition in vascular basement membranes. In the absence of RASA1, dysregulated Ras mitogen-activated protein kinase (MAPK) signal transduction in EC resulted in impaired folding of collagen IV and its retention in the endoplasmic reticulum (ER) leading to EC death. Remarkably, the chemical chaperone, 4-phenylbutyric acid, and small molecule inhibitors of MAPK and 2-oxoglutarate dependent collagen IV modifying enzymes rescued ER retention of collagen IV and EC apoptosis and resulted in normal developmental angiogenesis. These findings have important implications with regards an understanding of the molecular pathogenesis of CM-AVM and possible means of treatment.
Di Chen, Joyce Teng, Paula North, Philip E. Lapinski, Philip D. King
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