Research
Abstract
Mutations in mitochondrial DNA (mtDNA) cause various mitochondrial diseases that are currently incurable. Allotopic expression of nuclear-recoded mitochondrial genes represents a promising therapeutic strategy, given its demonstrated capacity to restore mitochondrial function in human cell models harboring mtDNA mutations. However, the in vivo evaluation of allotopic gene therapy has been hindered by optimization challenges and the lack of appropriate animal models. Here, we overcome these limitations by utilizing an optimized AAV2-ND6 construct with codon optimization and mitochondrial targeting sequence in a mouse model bearing the homoplasmic ND6P25L mutation, which recapitulates Leber hereditary optic neuropathy (LHON). High-dose administration of the AAV2-ND6 construct resulted in robust, sustained expression within the retina and optic nerve without apparent systemic toxicity. Strikingly, We compared the therapeutic efficacy in mutant mice at different ages and pre-symptomatic intervention with AAV2-ND6 effectively attenuated disease progression, mitigated retinal cellular deficiencies and optic nerve damage, and restored visual function in ND6P25L mice. Mechanistically, allotopic ND6 expression markedly rescued the mitochondrial dysfunction, corrected dysregulated retinol metabolism and phototransduction pathways, and suppressed apoptotic processes in the mutant retina. Our study validates the safety and therapeutic potential of allotopic expression in vivo and provide critical mechanistic insights into its role in treating LHON and other mitochondrial diseases.
Authors
Cheng Ai, Huiying Li, Jing Wu, Tianwei Zhou, Jing Wang, Shao-Hui Pan, Jun Yu, Douglas C. Wallace, Min-Xin Guan
Abstract
Inflammatory cytokines reprogram keratinocyte metabolism, but the metabolic pathways that couple immune signals to pathological epidermal growth remain incompletely defined. Here, we identify GLS1-mediated glutaminolysis as a metabolic program preferentially induced in keratinocytes under type 3 inflammatory conditions. Integrated transcriptomic, metabolomic, genetic, and functional analyses showed that IL-17A induced GLS1 expression and glutaminolysis in keratinocytes. Keratinocyte-specific Gls1 deletion reduced the intracellular availability of arginine, proline, and methionine, impaired amino acid-dependent mTORC1 activation, disrupted redox homeostasis, and limited keratinocyte proliferation. Amino acid or antioxidant supplementation partially rescued these defects, whereas rapamycin blocked the amino acid-mediated proliferative rescue. Gls1 deletion did not impair steady-state skin development or homeostasis and did not alter MC903-induced type 2 dermatitis, but it delayed wound re-epithelialization and attenuated IMQ-induced psoriasiform inflammation. Loss of keratinocyte GLS1 also reduced epidermal chemokine expression and the accumulation of neutrophils and IL-17A-producing γδ T cells, revealing a role for glutaminolysis in amplifying epithelial-immune crosstalk. These findings define GLS1-mediated glutaminolysis as a context-specific metabolic checkpoint linking type 3 inflammation to keratinocyte proliferation and cutaneous immune amplification, and support locally or temporally controlled GLS1 inhibition as a potential therapeutic strategy for psoriasis.
Authors
Yongfei Hu, Hai Yu, Kaiyu Liang, Liyan Yuan, Wenjun Zhang, Zhe Zhuang, Suyun Ji, Xichun Xia
Abstract
Clinical immunity to malaria develops after repeated malaria episodes. In this process, the inflammatory response is modulated to respond less vigorously upon reinfection. Monocytes are a major source of pro-inflammatory mediators during blood-stage infection and are known to adapt to repeated pathogen exposure. Here, we investigated the impact of previous malaria exposure on monocytes during blood-stage malaria by comparing the response in previously exposed and primary infected individuals. We observed reduced levels of several proinflammatory chemokines in previously exposed individuals, linked to changes in monocytes. Similarly, BAFF levels were lower in these individuals and associated with modulation of monocyte and dendritic cells. This affected the BAFF-BAFF-R axis, crucial for B cell responses, correlating with increasing parasite-specific antibody levels. Collectively, we present insights into how previous malaria exposure shapes monocyte responses during acute malaria and how these in turn correlate with modulation of the B cell compartment and humoral immune response.
Authors
Maximilian Julius Lautenbach, Pengjun Xi, Linn Kleberg, Alan-Dine Courey-Ghaouzi, Maia Serene Gower, Carolina Sousa Silva, Felicia Chammas, Anna Färnert, Christopher Sundling
Abstract
Menin is a scaffolding protein that interacts with context-specific partners to regulate gene expression. In MLL-rearranged leukemias, Menin:MLL interactions drive leukemogenesis and Menin inhibitors have been FDA approved for these cancers. We previously reported that Menin promotes oncogenic phenotypes in Ewing sarcoma (EwS). Here, we sought to define EwS-specific functions of Menin and determine if Menin inhibitors could be therapeutically leveraged for these tumors. Genetic knockout of Menin had no impact on EwS cell proliferation in vitro, but metastatic potential of Menin-depleted cells in vivo was impaired. Transcriptional profiling of Menin knockout cells in vitro showed reproducible downregulation of MYC signature genes and upregulation of developmental programs. Conversely, transcriptional rewiring of developmental genes and restoration of MYC target gene expression were evident in tumors that arose from Menin knockout cells. Exposing EwS cells to the Menin inhibitor VTP50469 (revumenib) inhibited expression of MYC targets and co-immunoprecipitation studies detected Menin:MYC interactions that were partially disrupted by the drug. Metastatic colonization of disseminated EwS cells in vivo was significantly inhibited in mice fed VTP50469 chow. Together these findings implicate Menin as a mediator of EwS metastasis and suggest that Menin inhibitors warrant investigation as novel therapeutics for patients with high-risk disease.
Authors
Katherine A. Braun, Nicolas M. Garcia, Mohamed A. Ahmed, Darleen S. Tu, Stephanie I. Walter, Emma D. Wrenn, Megan E. B. Dean, Neerja Katiyar, Elizabeth R. Lawlor
Abstract
Autoimmune kidney diseases can cause glomerulonephritis and tubulointerstitial nephritis, which if unresolved, lead to progressive glomerulosclerosis and tubulointerstitial fibrosis. The IL-1 receptor (IL-1R1) is known to have divergent and cell-specific effects in kidney injury. We hypothesized that IL-1R1 would dampen pro-inflammatory activation of myeloid cells such that deletion of myeloid cell IL-1R1 would exacerbate autoimmune nephritis. Mice with myeloid cell-specific deletion of IL-1R1 (LysMCre(+) / Il1r1fl/fl - MKO) and littermate controls (LysMCre(-) / Il1r1fl/fl - MWT) were subjected to nephrotoxic serum (NTS) nephritis. MKO mice demonstrated worsened glomerular and tubular injury as indicated by increased albuminuria, glomerular injury scores, and kidney mRNA levels of kidney injury molecule (KIM)-1 (Havcr1) and neutrophil gelatinase-associated lipocalin (NGAL/Lcn2). We further found that myeloid IL-1R1 deficiency resulted in increased myeloid cell ER stress and expression of the heterodimeric cytokine Ebi3/Il27a (IL-27). IL-27 then induced increased type I IFN expression by kidney endothelial cells. In turn, anti-IL-27 limited type I IFN expression in endothelial cells and NTS nephritis, and anti-IFNAR1 therapy ameliorated glomerular and tubular injury in MKO mice. Thus, we demonstrated a myeloid cell-endothelial cell immunoregulatory axis whereby myeloid IL-1R1 activity constrained endothelial type I IFN generation to limit chronic kidney damage.
Authors
Yanting Chen, Yu Li, Jiafa Ren, Chia-Chun Wu, Xiaohan Lu, Achintya Inumarty, Steven D. Crowley, Jamie R. Privratsky
Abstract
Identifying factors that govern retinal ganglion cells’ (RGCs) ability to extend axons is an important step in developing therapies to achieve recovery after optic nerve injury. Here we report that the intracellular domain of the leukemia inhibitory factor receptor (LIFR/CD118) is essential for mature RGCs’ ability to regenerate injured axons independent of the cognate ligand (LIF) and other therapies. Overexpression of LIFR in adult RGCs induces neurite outgrowth in cultured RGCs and axon regeneration in vivo while strongly amplifying RGCs’ response to LIF itself and to unrelated growth factors. Conversely, downregulation of LIFR strongly suppresses the pro-regenerative effects of Pten deletion and other potent stimuli. LIFR modulation alters the constitutive activity of the MAP kinase pathway, in contrast to LIF itself, which primarily activates pSTAT3. The extracellular-domain-truncated LIFR construct retains substantial pro-regenerative activity, whereas mutation of intracellular signaling motifs reduces the full regenerative effect of LIFR. Together, these findings identify LIFR as a key cell-autonomous regulator of optic nerve regeneration in mature RGCs.
Authors
Qian Jiang, Cong Wang, Yuerong Ren, Peiyun Duan, Ke Tian, Xiangwei Duan, Binghan Cai, Changzhong Xu, Ke Liu, Jian Li, Larry Benowitz, Ningli Wang, Bing Jiang, Lili Xie
Abstract
Identifying and monitoring autoreactive T cells that drive beta cell destruction remains a major obstacle to developing effective immunotherapies for type 1 diabetes (T1D). These cells are extremely rare in peripheral blood and cannot be accessed directly from the pancreas. We used intradermal injection of Glutamic Acid Decarboxylase (GAD)-Alum to recruit GAD-specific T cells to accessible sites in the skin and skin-draining lymph nodes (LNs), sampled by skin suction blisters and ultrasound-guided LN aspiration. Peripheral blood samples obtained before GAD injection were restimulated with GAD in vitro to detect reactive CD4+ T cells. Single-cell RNA sequencing (scRNAseq) followed by re-expression of selected T cell receptors (TCRs) confirmed antigen specificity. Up to 70% of T cells at the skin injection site were clonally-expanded and 4 of 14 (28%) re-expressed TCRs were GAD-reactive. In LNs 1 of 14 (4%) clonally-expanded TCRs was GAD-reactive, representing ~0.08% of all T-cells. GAD-reactive cells across compartments displayed Th1 and Th17-associated transcription signatures. These results demonstrate the intradermal autoantigen challenge and scRNAseq, enable direct identification and molecular profiling of autoreactive T cells in vivo. This minimally invasive approach provides a powerful platform for tracking antigen-specific T cells to monitor disease activity and evaluate immune interventions in T1D.
Authors
Stephanie J. Hanna, Emma J.S. Robinson, Terri C. Thayer, Maki Nakayama, Laurie Landry, Robert Andrews, Garry Dolton, Joanne Davies, Evangelia Williams, James A. Pearson, Andrew K. Sewell, Parth Narendran, David Wraith, Alexandra Howell, Philippa Young, Mary Hart, Anton Lindqvist, F. Susan Wong, Tim I.M. Tree, Colin M. Dayan, Danijela Tatovic
Abstract
Vascular plasticity is a crucial biological asset enabling our bodies to rapidly adapt to infections and acute inflammation. However, repeated insults during chronic disease can result in these vascular adaptations becoming irreversible, thereby driving disease progression and fibrosis. This study aimed to understand if phenotypic changes in endothelial cell (EC) identity could be indicative of progressive fibrosis and thereby offer diagnostic and therapeutic opportunities for patients with metabolic dysfunction-associated steatotic liver disease (MASLD). We integrated high-resolution imaging, proteomic and transcriptomic analysis which collectively highlighted a central role for endothelial-to-mesenchymal transition (EndMT)-induced EC plasticity in the derivation of ‘fibrosis-associated’ EC (FAEC). We demonstrated that: 1) full spectrum flow cytometry can provide new opportunities to categorize and phenotype EC subpopulations, 2) two distinct EndMT-derived FAEC subpopulations expanded during fibrogenesis; THY1.2+ICAM1+ and TAGLN+MCAM+ EC that displayed unique immunomodulatory and metabolic phenotypes, 3) TAGLN+ FAEC are a conserved, pro-fibrotic cell type that arose at early stages of MASLD, and 4) increased hepatic expression of TAGLN was significantly associated with detrimental patient outcomes at all stages of liver disease. This study paves the way for the development of FAEC-specific diagnostic and therapeutic approaches to tackle progressive fibrotic disease.
Authors
Christina Gkantsinikoudi, Joshua P. Dignam, Raju Kumar, Elliot Jokl, Meenakshi Rana, Wenhao Li, Maryna Samus, Stephanie Landi, Varinder S. Athwal, Timothy J. Kendall, Antal Rot, Jonathan A. Fallowfield, Karen Piper Hanley, William Alazawi, Neil P. Dufton
Abstract
Allergic contact dermatitis (ACD), a recurrent inflammatory skin disorder, affects 21% of humans and is the second leading cause of occupational diseases in USA. ACD is initiated by the innate immune response to skin-contact sensitizers potentiated by the neuropeptide substance P (SP). Skin sensitizers stimulate SP-secreting sensory nerves and trigger proinflammatory functions of keratinocytes expressing the neurokinin 1 receptor (NK1R). Nevertheless, the neuroimmune regulation of hapten-initiated skin inflammation, remains incompletely elucidated. Using K14Cre/+NK1RKO mice skin-sensitized with 2,4-dinitrochlorobenzene (DNCB), we demonstrate that NK1R deletion exclusively in keratinocytes prevents hapten-initiated skin inflammation, impairs the mobilization of conventional dendritic cells (cDC) to draining lymph nodes (dLN) and blocks the elicitation of the contact hypersensitivity reaction (CHS) to the same extent observed in global Tac1KO (without SP) and NK1RKO mice. The DNCB effects were restored by skin co-administration of IL-1β and TNF-α. SP-NK1R signaling of mouse and human keratinocytes increased transcripts encoding proteins of the NLRP3 inflammasome. Although, DNCB and SP induced pro-IL-1β synthesis, only SP triggered intracellular Ca2+ increase, NFATc1 nuclear translocation and TNF-α synthesis, a cytokine mediating systemic inflammation in ACD. Our data identifying SP-NK1R-signaling of keratinocytes as a key mechanism for ACD provide relevant insight for therapies targeting skin neuroimmune interactions.
Authors
Sumeet Manandhar, Mohna Bandyopadhyay, Olga Tkacheva, William Shufesky, Greg Gibson, Simon C. Watkins, Adrian Morelli, Adriana T. Larregina
Abstract
DNA ligase IV (LIG4) is essential for DNA double-strand break (DSB) repair. Hypomorphic LIG4 variants cause LIG4 syndrome, characterized by growth disturbance, increased radiosensitivity, predisposition to malignancies, adaptive immunodeficiency and inflammatory conditions. Most of these manifestations are recapitulated in hypomorphic LIG4 mutant mice. However, no model mice with defective DSB repair have consistently exhibited inflammation. Here, we have generated mutant mice carrying the LIG4 missense variant, p.W447C, found in a patient with LIG4 syndrome. Lig4W447C/W447C mice showed functional defects of LIGIV and manifested growth retardation, increased radiosensitivity, and life-threatening intestinal inflammation under severe adaptive immunodeficiency. The inflammation was dependent on lymphocytes and characterized by marked infiltration of Th1 cells and macrophages, along with elevated expression of IFN-γ-inducible genes. When Ifng was deleted, Th2 and Th17 instead of Th1 cells drove the inflammation. Single-cell RNA-seq analyses with TCR repertoire revealed that T cells from Lig4W447C/W447C mice preferentially used proximal Vα and Jα segments in V regions of TCRα chains and exhibited expansion of several clonotypes, a substantial portion of which were CD4 T cells expressing IFN-γ. Thus, our hypomorphic Lig4 mutant mice represent a unique model for studying Th1-skewed intestinal inflammation under severe adaptive immunodeficiency.
Authors
Yusuke Yamashita, Hideki Kosako, Takashi Kato, Izumi Sasaki, Sadahiro Iwabuchi, Yuri Fukuda-Ohta, Tadashi Okamura, Misato Tane, Shotaro Tabata, Kazutaka Nakashima, Ken Tanaka, Kazunori Shiraishi, Yuki Uchihara, Daisuke Okuzaki, Kyoichi Isono, Atsushi Shibata, Tsunehiro Mizushima, Hiroaki Hemmi, Nobuo Kanazawa, Seiji Kodama, Hiroaki Miyoshi, Koichi Ohshima, Shinichi Hashimoto, Yoshio Fujitani, Takashi Sonoki, Shinobu Tamura, Tsuneyasu Kaisho
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