A population of NK cells expressing the activating receptor NKG2C and the maturation marker CD57 expands in response to human CMV (HCMV) infection. CD3–CD56dimCD57+NKG2C+ NK cells are similar to CD8+ memory T cells with rapid and robust effector function upon restimulation, 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 Abs 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 cocultured 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. We believe our study has important implications for cancer immunotherapy and propose that exhausted NK cells could be targeted with inhibitory checkpoint receptor blockade.
Aimee Merino, Bin Zhang, Philip Dougherty, Xianghua Luo, Jinhua Wang, Bruce R. Blazar, Jeffrey S. Miller, Frank Cichocki
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 naive conditions, FcγRI cross-linking 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
We previously generated 32 rotavirus-specific (RV-specific) recombinant monoclonal antibodies (mAbs) derived from B cells isolated from human intestinal resections. Twenty-four of these mAbs were specific for the VP8* fragment of RV VP4, and most (20 of 24) were non-neutralizing when tested in the conventional MA104 cell–based assay. We reexamined the ability of these mAbs to neutralize RVs in human intestinal epithelial cells including ileal enteroids and HT-29 cells. Most (18 of 20) of the “non-neutralizing” VP8* mAbs efficiently neutralized human RV in HT-29 cells or enteroids. Serum RV neutralization titers in adults and infants were significantly higher in HT-29 than MA104 cells and adsorption of these sera with recombinant VP8* lowered the neutralization titers in HT-29 but not MA104 cells. VP8* mAbs also protected suckling mice from diarrhea in an in vivo challenge model. X-ray crystallographic analysis of one VP8* mAb (mAb9) in complex with human RV VP8* revealed that the mAb interaction site was distinct from the human histo-blood group antigen binding site. Since MA104 cells are the most commonly used cell line to detect anti-RV neutralization activity, these findings suggest that prior vaccine and other studies of human RV neutralization responses may have underestimated the contribution of VP8* antibodies to the overall neutralization titer.
Ningguo Feng, Liya Hu, Siyuan Ding, Mrinmoy Sanyal, Boyang Zhao, Banumathi Sankaran, Sasirekha Ramani, Monica McNeal, Linda L. Yasukawa, Yanhua Song, B.V. Venkatar Prasad, Harry B. Greenberg
Vascular development in the mammalian retina is a paradigm for CNS vascular development in general, and its study is revealing fundamental mechanisms that explain the efficacy of antiangiogenic therapies in retinal vascular disease. During development of the mammalian retina, hypoxic astrocytes are hypothesized to secrete VEGF, which attracts growing endothelial cells as they migrate radially from the optic disc. However, published tests of this model using astrocyte-specific deletion of Vegf in the developing mouse retina appear to contradict this theory. Here, we report that selectively eliminating Vegf in neonatal retinal astrocytes with a Gfap-Cre line that recombines with approximately 100% efficiency had no effect on proliferation or radial migration of astrocytes, but completely blocked radial migration of endothelial cells, strongly supporting the hypoxic astrocyte model. Using additional Cre driver lines, we found evidence for essential and partially redundant actions of retina-derived (paracrine) and astrocyte-derived (autocrine) VEGF in controlling astrocyte proliferation and migration. We also extended previous studies by showing that HIF-1α in retinal neurons and HIF-2α in Müller glia play distinct roles in retinal vascular development and disease, adding to a growing body of data that point to the specialization of these 2 hypoxia-sensing transcription factors.
Amir Rattner, John Williams, Jeremy Nathans
Multiple sclerosis (MS) is a putative T cell–mediated autoimmune disease. As with many autoimmune diseases, females are more susceptible than males. Sexual dimorphisms may be due to differences in sex hormones, sex chromosomes, or both. Regarding sex chromosome genes, a small percentage of X chromosome genes escape X inactivation and have higher expression in females (XX) compared with males (XY). Here, high-throughput gene expression analysis in CD4+ T cells showed that the top sexually dimorphic gene was Kdm6a, a histone demethylase on the X chromosome. There was higher expression of Kdm6a in females compared with males in humans and mice, and the four core genotypes (FCG) mouse model showed higher expression in XX compared with XY. Deletion of Kdm6a in CD4+ T cells ameliorated clinical disease and reduced neuropathology in the classic CD4+ T cell–mediated autoimmune disease experimental autoimmune encephalomyelitis (EAE). Global transcriptome analysis in CD4+ T cells from EAE mice with a specific deletion of Kdm6a showed upregulation of Th2 and Th1 activation pathways and downregulation of neuroinflammation signaling pathways. Together, these data demonstrate that the X escapee Kdm6a regulates multiple immune response genes, providing a mechanism for sex differences in autoimmune disease susceptibility.
Yuichiro Itoh, Lisa C. Golden, Noriko Itoh, Macy Akiyo Matsukawa, Emily Ren, Vincent Tse, Arthur P. Arnold, Rhonda R. Voskuhl
Clostridioides difficile infection (CDI) accounts for a substantial proportion of deaths attributable to antibiotic-resistant bacteria in the United States. Although C. difficile can be an asymptomatic colonizer, its pathogenic potential is most commonly manifested in patients with antibiotic-modified intestinal microbiomes. In a cohort of 186 hospitalized patients, we showed that host and microbe-associated shifts in fecal metabolomes had the potential to distinguish patients with CDI from those with non–C. difficile diarrhea and C. difficile colonization. Patients with CDI exhibited a chemical signature of Stickland amino acid fermentation that was distinct from those of uncolonized controls. This signature suggested that C. difficile preferentially catabolizes branched chain amino acids during CDI. Unexpectedly, we also identified a series of noncanonical, unsaturated bile acids that were depleted in patients with CDI. These bile acids may derive from an extended host-microbiome dehydroxylation network in uninfected patients. Bile acid composition and leucine fermentation defined a prototype metabolomic model with potential to distinguish clinical CDI from asymptomatic C. difficile colonization.
John I. Robinson, William H. Weir, Jan R. Crowley, Tiffany Hink, Kimberly A. Reske, Jennie H. Kwon, Carey-Ann D. Burnham, Erik R. Dubberke, Peter J. Mucha, Jeffrey P. Henderson
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 ROS. Targeted α-synuclein overexpression within these neurons triggered an oxidative stress that became 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 prooxidant 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 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
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 (ECs) 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 ECs 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 ECs and deposition in vascular basement membranes. In the absence of RASA1, dysregulated Ras/MAPK signal transduction in ECs 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 for a better understanding of the molecular pathogenesis of CM-AVM and possible means of treatment.
Di Chen, Joyce M. Teng, Paula E. North, Philip E. Lapinski, Philip D. King
Resident microbiota activates regulatory cells that modulate intestinal inflammation and promote and maintain intestinal homeostasis. IL-10 is a key mediator of immune regulatory function. Our studies describe the functional importance and mechanisms by which gut microbiota and specific microbial components influence the development of intestinal IL-10–producing B cells. Using fecal transplant into germ-free (GF) Il10+/EGFP reporter and Il10–/– mice, we demonstrated 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 downregulated microbiota-activated mucosal inflammatory cytokines. TLR2 and -9 ligands and enteric bacterial lysates preferentially induced IL-10 production and the regulatory capacity of intestinal B cells. Analysis of Il10+/EGFP mice crossed with additional gene-deficient strains and B cell cotransfer 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 downstream signaling of PI3Kp110δ and AKT. 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
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 proinflammatory 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, whereas 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 proinflammatory 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 (a) inhibited LPS and oxidized 1-palmitoyl-2-arachidonoyl-sn-phosphatidylcholine–dependent (oxPAPC-dependent) proinflammatory responses in human macrophages and intestinal epithelium, and (b) directly cleared proinflammatory lipids from mouse intestinal tissue and plasma. Our results support a causal role for proinflammatory 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, Jifang 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
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