The facilitative GLUT1 and GLUT3 hexose transporters are expressed abundantly in macrophages, but whether they have distinct functions remains unclear. We confirmed that GLUT1 expression increased after M1 polarization stimuli and found that GLUT3 expression increased after M2 stimulation in macrophages. Conditional deletion of Glut3 (LysM-Cre Glut3fl/fl) impaired M2 polarization of bone marrow derived macrophages. Alternatively activated macrophages from the skin of atopic dermatitis patients showed increased GLUT3 expression, and a calcipotriol-induced model of atopic dermatitis was rescued LysM-Cre Glut3fl/fl mice. M2-like macrophages expressed GLUT3 in human wound tissues as assessed by transcriptomics and co-staining, and GLUT3 expression was significantly decreased in non-healing, compared with healing, diabetic foot ulcers. In an excisional wound healing model, LysM-Cre Glut3fl/fl mice showed significantly impaired M2 macrophage polarization and delayed wound healing. GLUT3 promoted IL-4/STAT6 signaling, independent from its glucose transport activity. Unlike plasma membrane-localized GLUT1, GLUT3 was localized primarily to endosomes and was required for the efficient endocytosis of IL4Ra subunits. GLUT3 interacted directly with GTP-bound RAS in vitro and in vivo through its intracytoplasmic loop domain (ICH), and this interaction was required for efficient STAT6 activation and M2 polarization. PAK activation and macropinocytosis were also impaired without GLUT3, suggesting broader roles for GLUT3 in the regulation of endocytosis. Thus, GLUT3 is required for efficient alternative macrophage polarization and function, through a glucose transport-independent, RAS-mediated role in the regulation of endocytosis and IL-4/STAT6 activation.
Dong-Min Yu, Jiawei Zhao, Eunice E. Lee, Dohun Kim, Ruchika Mahapatra, Elysha K. Rose, Zhiwei Zhou, Calvin R. Hosler, Abdullah El-Kurdi, Jun-yong Choe, E. Dale Abel, Gerta Hoxhaj, Kenneth D. Westover, Raymond J. Cho, Jeffrey B. Cheng, Richard C. Wang
The metastasis of cancer cells is the main cause of death for patients with gastric cancer (GC). Mounting evidence has demonstrated the vital importance of tumor-associated macrophages in promoting tumor invasion and metastasis; however, the interaction between tumor cells and macrophages in GC is largely unknown. In this study, we demonstrated that cyclase-associated protein 2 (CAP2) was upregulated in GC, especially in cases with lymph node metastasis, and was correlated with a poorer prognosis. The transcription factor JUN directly bound to the promoter region of CAP2 and activated CAP2 transcription. The N-terminal domain of CAP2 bound to the WD5-7 domain of receptor for activated C kinase 1 (RACK1) and induced M2 macrophage polarization by activating the SRC/focal adhesion kinase (FAK)/ ERK signaling pathway, which resulted in interleukin-4 (IL4) and IL10 secretion. Polarized M2 macrophages induced premetastatic niche formation and promoted GC metastasis by secreting transforming growth factor beta (TGFB1), which created a TGFB1/JUN/CAP2-positive feedback loop to activate CAP2 expression continuously. Furthermore, we identified Salvianolic acid B as an inhibitor of CAP2, which effectively inhibited GC cell invasion capabilities by suppressing the SRC/FAK/ERK signaling pathway. Our data suggest that CAP2, a key molecule mediating the interaction between GC cells and tumor-associated macrophages, may be a promising therapeutic target for suppressing tumor metastasis in GC.
Guohao Zhang, Zhaoxin Gao, Xiangyu Guo, Ranran Ma, Xiaojie Wang, Pan Zhou, Chunlan Li, Zhiyuan Tang, Ruinan Zhao, Peng Gao
Expansion of CAG and CTG (CWG) triplet repeats causes several inherited neurological diseases. The CWG repeat diseases are thought to involve complex pathogenic mechanisms through expanded CWG repeat-derived RNAs in a non-coding and polypeptides in a coding region, respectively. However, an effective therapeutic approach has not been established for the CWG repeat diseases. Here, we show that a CWG repeat DNA-targeting compound, cyclic pyrrole¬–imidazole polyamide (CWG-cPIP), suppresses the pathogenesis of coding and non-coding CWG repeat diseases. CWG-cPIP binds to the hairpin form of mismatched CWG DNA, interfering with transcription elongation by RNA polymerase through a preferential activity towards repeat-expanded DNA. We found that CWG-cPIP selectively inhibits pathogenic mRNA transcripts from expanded CWG repeats, reducing CUG RNA foci and polyglutamine accumulation in cells from patients with myotonic dystrophy type-1 (DM1) and Huntington’s disease (HD). Treatment with CWG-cPIP ameliorated behavioral deficits in adeno-associated virus-mediated CWG repeat-expressing mice and a genetic mouse model of HD, without cytotoxicity or off-target effects. Together, we present a novel candidate compound that targets expanded CWG repeat DNA independent of its genomic location and reduces both pathogenic RNA and protein levels. CWG-cPIP may be used for the treatment of CWG repeat diseases and for improving clinical outcomes.
Susumu Ikenoshita, Kazuya Matsuo, Yasushi Yabuki, Kosuke Kawakubo, Sefan Asamitsu, Karin Hori, Shingo Usuki, Yuki Hirose, Toshikazu Bando, Kimi Araki, Mitsuharu Ueda, Hiroshi Sugiyama, Norifumi Shioda
BACKGROUND. HIV-1-infected CD4+ T cells contribute to latent reservoir persistence by proliferating while avoiding immune recognition. Integration features of intact proviruses in elite controllers (EC) and people on long-term therapy suggests that proviruses in specific chromosomal locations can evade immune surveillance. However, direct evidence of this mechanism is missing. METHODS. In this case report, we characterized integration sites and full genome sequences of expanded T cell clones in an EC before and after chemoradiation. We identified the cognate peptide of infected clones to investigate cell proliferation and virus production induced by T cell activation, and susceptibility to autologous CD8+ T cells. RESULTS. The proviral landscape was dominated by two large clones with replication-competent proviruses integrated into Zinc Finger genes (ZNF470 and ZNF721) in locations previously associated with deeper latency. A third nearly intact provirus, with a stop codon in Pol, was integrated into an intergenic site. Upon stimulation with cognate Gag peptides, infected clones proliferated extensively and produced virus, but the provirus in ZNF721 was 200-folds less inducible. While autologous CD8+ T cells decreased the proliferation of cells carrying the intergenic provirus, they had no effect on cells with the provirus in the ZNF721 gene. CONCLUSION. We provide direct evidence that upon activation of infected clones by cognate antigen, the lower inducibility of intact proviruses in ZNF genes can result in immune evasion and persistence. FUNDING. Office of the NIH Director and National Institute of Dental & Craniofacial Research; NIAID, NIH; Johns Hopkins University Center for AIDS Research.
Filippo Dragoni, Abena Kwaa, Caroline C.G. Traut, Rebecca T. Veenhuis, Bezawit A. Woldemeskel, Angelica Camilo-Contreras, Hayley E. Raymond, Arbor G. Dykema, Eileen P. Scully, Amanda M. Rosecrans, Kellie N. Smith, Frederic D. Bushman, Francesco R. Simonetti, Joel N. Blankson
Prashant Rai, Martin Sharpe, Charan K. Ganta, Paul J. Baker, Katrin D. Mayer-Barber, Brian E. Fee, Gregory A. Taylor, Michael B. Fessler
Unabated activation of NLRP3 inflammasome activation is linked with the pathogenesis of various inflammatory disorders. PLK1 has been widely studied for its role in mitosis. Here, employing both pharmacological and genetic approaches, we demonstrated that PLK1 promoted NLRP3 inflammasome activation at cell interphase. Using an unbiased Bio-ID screen for PLK1 interactome in macrophages, we showed an enhanced proximal association of NLRP3 with PLK1 upon NLRP3 inflammasome activation. We further confirmed the interaction between PLK1 and NLRP3, and identified the interacting domains. Mechanistically, we showed that PLK1 orchestrated microtubule organizing center (MTOC) structure and NLRP3 subcellular positioning upon inflammasome activation. Treatment with a selective PLK1 kinase inhibitor suppressed IL1B production in in-vivo inflammatory models, including lipopolysaccharide-induced endotoxemia and monosodium urate-induced peritonitis in mice. Our results uncover an unprecedented role of PLK1 in regulating NLRP3 inflammasome activation during interphase, and identify pharmacological inhibition of PLK1 as a potential therapeutic strategy for inflammatory diseases with excessive NLRP3 inflammasome activation.
Marta Baldrighi, Christian Doreth, Yang Li, Xiaohui Zhao, Emily F. Warner, Hannah Chenoweth, Kamal Kishore, Yagnesh Umrania, David-Paul Minde, Sarah Winkler, Xian Yu, Yuning Lu, Alice Knapton, James Harrison, Murray C.H. Clarke, Eicke Latz, Guillermo de Cárcer, Marcos Malumbres, Bernhard Ryffel, Clare E. Bryant, Jinping Liu, Kathryn S. Lilley, Ziad Mallat, Xuan Li
CD8+ T cells outnumber CD4+ cells in multiple sclerosis lesions associated with disease progression, but the pathogenic role and antigenic targets of these clonally expanded effectors are unknown. Based on evidence that demyelination is necessary but not sufficient for disease progression in multiple sclerosis (MS), we previously hypothesized that CNS-infiltrating CD8+ T cells specific for neuronal antigens directly drive the axon and neuron injury that leads to cumulative neurologic disability in MS patients. We now show that demyelination induced expression of MHC class I on neurons and axons and resulted in presentation of a neuron-specific neoantigen (synapsin promoter-driven chicken ovalbumin) to antigen-specific CD8+ T cells (anti-ovalbumin OT-I transgenic T cells). These neuroantigen-specific effectors surveilled the CNS in the absence of demyelination but were not retained. However, upon induction of demyelination via cuprizone intoxication, neuroantigen-specific CD8+ T cells proliferated, accumulated in the CNS, and damaged neoantigen-expressing neurons and axons. We further report elevated neuronal expression of MHC class I and β2-microglobulin transcripts and protein in gray matter and white matter tracts in tissue from patients with MS. These findings support a pathogenic role for autoreactive anti-axonal and anti-neuronal CD8+ T cells in MS progression.
Benjamin D.S. Clarkson, Ethan M. Grund, Miranda M. Standiford, Kanish Mirchia, Maria S. Westphal, Elizabeth S. Muschler, Charles L. Howe
Even when successfully induced, immunological tolerance to solid organ remains vulnerable to inflammatory insults, which can trigger rejection. In a mouse model of cardiac allograft tolerance in which infection with Listeria monocytogenes (Lm) precipitates rejection of previously accepted grafts, we showed that recipient CD4+ TCR75 cells reactive to a donor MHC Class I-derived peptide become hypofunctional if the allograft is accepted for > 3 weeks. Paradoxically, infection-induced transplant rejection was not associated with transcriptional or functional reinvigoration of TCR75 cells. We hypothesized that there is heterogeneity in the level of dysfunction of different allospecific T cells, depending on duration of their cognate antigen expression. Unlike CD4+ TCR75 cells, CD4+ TEa cells specific for a peptide derived from donor MHC Class II, an alloantigen whose expression declines post-transplantation but remains inducible in settings of inflammation, retained function in tolerant mice and expanded during Lm-induced rejection. Repeated injections of alloantigens drove hypofunction in TEa cells and rendered grafts resistant to Lm-dependent rejection. Our results uncover a functional heterogeneity in allospecific T cells of distinct specificities post-tolerance induction and reveal a strategy to defunctionalize a greater repertoire of allospecific T cells, thereby mitigating a critical vulnerability of tolerance.
Christine M. McIntosh, Jennifer B. Allocco, Peter Wang, Michelle L. McKeague, Alexandra Cassano, Ying Wang, Stephen Z. Xie, Grace E. Hynes, Ricardo Mora-Cartín, Domenic Abbondanza, Luqiu Chen, Husain Sattar, Dengping Yin, Zheng J. Zhang, Anita S. Chong, Maria-Luisa Alegre
Donor-recipient (D-R) mismatches outside of human leukocyte antigens (HLA) contribute to kidney allograft loss, but mechanisms remain unclear, specifically for intronic mismatches. We quantified non-HLA mismatches at variant-, gene-, and genome-wide scales from SNP data of D- Rs from two well-phenotyped transplant cohorts: Genomics of Chronic Allograft Rejection (GoCAR; n=385) and Clinical Trials in Organ Transplantation-01/17 (CTOT-01/17; n=146). Unbiased gene-level screening in GoCAR uncovered the LIMS1 locus as the top-ranked gene where D-R mismatches associated with death-censored graft loss (DCGL). A previously unreported, intronic, LIMS1 haplotype of 30 SNPs independently associated with DCGL in both cohorts. Haplotype mismatches showed a dosage effect, and minor-allele introduction to major- allele-carrying recipients showed greater hazard of DCGL. The LIMS1 haplotype and the previously reported LIMS1 SNP rs893403 are expression quantitative trait loci (eQTL) in immune cells for GCC2 (not LIMS1), which encodes a protein involved in mannose-6-phosphase receptor (M6PR) recycling. Peripheral blood and T-cell transcriptome analyses associated GCC2 gene and LIMS1 SNPs with the TGFB1-SMAD pathway, suggesting a regulatory effect. In vitro GCC2 modulation impacted M6PR-dependent regulation of active TGFB1 and downstream signaling in T-cells. Together, our data link LIMS1 locus D-R mismatches to DCGL via GCC2 eQTLs that modulate TGFB1-dependent effects on T-cells.
Zeguo Sun, Zhongyang Zhang, Khadija Banu, Ian W. Gibson, Robert B. Colvin, Zhengzi Yi, Weijia Zhang, Bony De Kumar, Anand Reghuvaran, John Pell, Thomas D. Manes, Arjang Djamali, Lorenzo Gallon, Philip J. O'Connell, John He, Jordan S. Pober, Peter S. Heeger, Madhav C. Menon
Consumption of low dietary potassium, common with ultra-processed foods, activates the thiazide-sensitive sodium chloride cotransporter (NCC) via the WNK-SPAK kinase pathway to induce salt retention and elevate blood pressure (BP). However, it remains unclear how high potassium “DASH-like” diets inactivate the cotransporter and whether this decreases BP. A transcriptomic screen identified Ppp1C⍺, encoding PP1A, as a potassium up-regulated gene, and its negative regulator, Ppp1r1a, as a potassium-suppressed gene in the kidney. PP1A directly binds to and dephosphorylates NCC when extracellular potassium is elevated. Using mice genetically engineered to constitutively activate the NCC-regulatory kinase SPAK and thereby eliminate the effects of the WNK-SPAK kinase cascade, we confirmed that PP1A dephosphorylates NCC directly in a potassium-regulated manner. Prior adaptation to a high potassium diet was required to maximally dephosphorylate NCC and lower BP in the constitutively active SPAK mice, and this was associated with potassium-dependent suppression of Ppp1r1a, and dephosphorylation of its cognate protein, Inhibitory Subunit 1 (I1). In conclusion, potassium-dependent activation of PP1A and inhibition of I1 drives NCC dephosphorylation, providing a mechanism to explain how high dietary K+ lowers BP. Shifting signaling of PP1A in favor of activation of WNK-SPAK may provide an improved therapeutic approach for treating salt-sensitive hypertension.
Paul Richard Grimm, Anamaria Tatomir, Lena L. Rosenbaek, Bo Young Kim, Dimin Li, Eric J. Delpire, Robert A. Fenton, Paul A. Welling
Christoph Strumann, Otavio T. Ranzani, Jeanne Moor, Reinhard Berner, Nicole Toepfner, Cho-Ming Chao, Matthias B. Moor
Identifying branched-chain amino acid (BCAA) oxidation enzymes in the nucleus led us to predict that they are a source of propionyl-CoA that are utilized for histone propionylation and, thereby, regulate gene expression. To investigate the effects of BCAA on the development of cardiac hypertrophy and failure, we applied pressure overload on the heart in mice maintained on a diet with standard levels of BCAA (BCAA-control) versus a BCAA-free diet. The former was associated with an increase in histone H3K23-propionyl (H3K23Pr) at the promoters of upregulated genes [e.g., cell signaling and extracellular matrix genes] and a decrease at the promoters of downregulated genes [e.g., electron transfer complex (ETC I-V) and metabolic genes]. Intriguingly, the BCAA-free diet tempered the increases in promoter-H3K23Pr, thus, reducing collagen gene expression and fibrosis during cardiac hypertrophy. Conversely, the BCAA-free diet inhibited the reductions in promoter-H3K23Pr and abolished the downregulation of ETC I-V subunits, enhanced mitochondrial respiration, and curbed progression of cardiac hypertrophy. Thus, lowering the intake of BCAA reduces pressure overload-induced changes in histone propionylation-dependent gene expression in the heart, which retards the development of cardiomyopathy.
Zhi Yang, Minzhen He, Julianne Austin, Danish Sayed, Maha Abdellatif
Lung cancer progression relies on angiogenesis, which is a response to hypoxia typically coordinated by hypoxia-inducible transcription factors (HIFs); but growing evidence indicate that transcriptional programs beyond HIFs control tumor angiogenesis. Here we show that the redox-sensitive transcription factor BTB and CNC homology 1 (BACH1) controls the transcription of a broad range of angiogenesis genes. BACH1 is stabilized by lowering reactive oxygen species levels; consequently, angiogenesis gene expression in lung cancer cells, tumor organoids, and xenograft tumors increased substantially following vitamin C and E and N-acetylcysteine administration in a BACH1-dependent fashion under normoxia. Moreover, angiogenesis gene expression increased in endogenous BACH1–overexpressing cells and decreased in BACH1-knockouts in the absence of antioxidants. BACH1 levels also increased upon hypoxia and following administration of prolyl hydroxylase inhibitors in both HIF1a-knockout and wild-type cells. BACH1 was found to be a transcriptional target of HIF1α but BACH1’s ability to stimulate angiogenesis gene expression was HIF1a independent. Antioxidants increased tumor vascularity in vivo in a BACH1-dependent fashion, and overexpressing BACH1 rendered tumors sensitive to anti-angiogenesis therapy. BACH1 expression in tumor sections from lung cancer patients correlates with angiogenesis gene and protein expression. We conclude that BACH1 is an oxygen- and redox-sensitive angiogenesis transcription factor.
Ting Wang, Yongqiang Dong, Zhiqiang Huang, Guoqing Zhang, Ying Zhao, Haidong Yao, Jianjiang Hu, Elin Tüksammel, Huan Cai, Ning Liang, Xiufeng Xu, Xijie Yang, Sarah Schmidt, Xi Qiao, Susanne Schlisio, Staffan Strömblad, Hong Qian, Changtao Jiang, Eckardt Treuter, Martin O. Bergo
Endothelial phospholipase Cγ (PLCγ) is essential for vascular development, however, its role in healthy, mature or pathological vessels is unexplored. PLCγ was prominently expressed in vessels of several human cancer forms, notably in renal cell carcinoma (RCC). High PLCγ expression in clear cell RCC correlated with angiogenic activity and poor prognosis, while low expression correlated with immune cell activation. PLCγ was induced downstream of vascular endothelial growth factor receptor 2 (VEGFR2) phosphosite Y1173 (pY1173). Heterozygous Vegfr2+/Y1173F mice or mice lacking endothelial PLCγ (Plcg1iECKO) exhibited a stabilized endothelial barrier and diminished vascular leakage. Barrier stabilization was accompanied by decreased expression of immunosuppressive cytokines, reduced infiltration of B-cells, CD4+ and regulatory T-cells, and improved response to chemo- and immunotherapy. Mechanistically, pY1173/PLCγ signaling induced Ca2+/protein kinase C dependent activation of endothelial nitric oxide synthase (eNOS), required for tyrosine nitration and activation of Src. Src-induced phosphorylation of VE-cadherin at Y685 was accompanied by disintegration of endothelial junctions. This pY1173/PLCγ/eNOS/Src pathway was detected in both healthy and tumor vessels in Vegfr2Y1173F/+ mice, which displayed decreased activation of PLCγ and eNOS, and suppressed vascular leakage. Thus, we have identified a clinically relevant endothelial PLCγ pathway downstream of VEGFR2 pY1173, which destabilizes the endothelial barrier resulting in loss of anti-tumor immunity.
Elin Sjöberg, Marit Melssen, Mark Richards, Yindi Ding, Catarina Chanoca, Dongying Chen, Emmanuel Nwadozi, Sagnik Pal, Dominic T. Love, Takeshi Ninchoji, Masabumi Shibuya, Michael Simons, Anna Dimberg, Lena Claesson-Welsh
Biofilms are structured communities of microbial cells embedded in a self-produced matrix of extracellular polymeric substances. Biofilms are associated with many health issues in humans, including chronic wound infections and tooth decay. Current antimicrobials are often incapable of disrupting the polymeric biofilm matrix and reaching the bacteria within. Alternative approaches are needed. Here, we describe a unique structure of dextran coated gold in a gold cage nanoparticle that enables photoacoustic and photothermal properties for biofilm detection and treatment. Activation of these nanoparticles with a near infrared laser can selectively detect and kill biofilm bacteria with precise spatial control and in a short timeframe. We observe a strong biocidal effect against both Streptococcus mutans and Staphylococcus aureus biofilms in mouse models of oral plaque and wound infections respectively. These effects were over 100 times greater than that seen with chlorhexidine, a conventional antimicrobial agent. Moreover, this approach did not adversely affect surrounding tissues. We conclude that photothermal ablation using theranostic nanoparticles is a rapid, precise, and non-toxic method to detect and treat biofilm-associated infections.
Maryam Hajfathalian, Christiaan R. de Vries, Jessica C. Hsu, Ahmad Amirshaghaghi, Yuxi C. Dong, Zhi Ren, Yuan Liu, Yue Huang, Yong Li, Simon A.B. Knight, Pallavi Jonnalagadda, Aimen Zlitni, Elizabeth A. Grice, Paul L. Bollyky, Hyun Koo, David P. Cormode
The triggering receptor expressed on myeloid cell 1 (TREM1) plays a critical role in development of chronic inflammatory disorders and the inflamed tumor microenvironment (TME) associated with most solid tumors. We examined whether loss of TREM1 signaling can abrogate immunosuppressive TME and enhance cancer immunity. To investigate the therapeutic potential of TREM1 in cancer, we used mice deficient in Trem1 and developed a novel small molecule TREM1 inhibitor, VJDT. We demonstrated that genetic or pharmacological TREM1 silencing significantly delayed tumor growth in murine melanoma (B16F10) and fibrosarcoma (MCA205) models. Single-cell RNA-seq combined with functional assays during TREM1 deficiency revealed decreased immunosuppressive capacity of myeloid-derived suppressor cells (MDSCs) accompanied by expansion in cytotoxic CD8+ T cells and increased PD-1 expression. Furthermore, TREM1 inhibition enhanced antitumorigenic effect of anti-PD-1 treatment in part by limiting MDSC frequency and abrogating T cell exhaustion. In melanoma patient-derived xenograft tumors, treatment with VJDT downregulated key oncogenic signaling pathways involved in cell proliferation, migration, and survival. Our work highlights the role in cancer progression of TREM1 expressed intrinsically in cancer cells and extrinsically in TME. Thus, targeting TREM1 to modify an immunosuppressive TME and improve efficacy of immune checkpoint therapy represents a promising therapeutic approach in cancer.
Ashwin Ajith, Kenza Mamouni, Daniel D. Horuzsko, Abu Musa, Amiran K. Dzutsev, Jennifer R. Fang, Ahmed Chadli, Xingguo Zhu, Iryna Lebedyeva, Giorgio Trinchieri, Anatolij Horuzsko
Multisystem inflammatory syndrome in children (MIS-C) is a rare but life-threatening hyperinflammatory condition induced by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes pediatric COVID-19 (pCOVID-19). The relationship of the systemic tissue injury to the pathophysiology of MIS-C is poorly defined. We leveraged the high sensitivity of epigenomic analyses of plasma cell-free DNA (cfDNA) and plasma cytokine measurements to identify the spectrum of tissue injury and glean mechanistic insights. Compared to pediatric healthy controls (pHC) and pCOVID-19, MIS-C patients had higher levels of cfDNA primarily derived from innate immune cells, megakaryocyte-erythroid precursor cells, and non-hematopoietic tissues such as hepatocytes, cardiac myocytes, and kidney cells. Non-hematopoietic tissue cfDNA levels demonstrated significant inter-individual variability, consistent with the heterogenous clinical presentation of MIS-C. In contrast, adaptive immune cell-derived cfDNA levels were comparable in MIS-C and pCOVID-19 patients. Indeed, the innate immune cells cfDNA in MIS-C correlated with levels of innate immune inflammatory cytokines and non-hematopoietic tissue-derived cfDNA, suggesting a primarily innate immunity-mediated response to account for multi-system pathology. These data provide insight into the pathogenesis of MIS-C and support the value of cfDNA as a sensitive biomarker to map tissue injury in MIS-C and likely other multi-organ inflammatory conditions.
Temesgen E. Andargie, Katerina Roznik, Neelam R. Redekar, Tom Hill, Weiqiang Zhou, Zainab Apalara, Hyesik Kong, Oren Gordon, Rohan Meda, Woojin Park, Trevor S. Johnston, Yi Wang, Sheila Brady, Hongkai Ji, Jack A. Yanovski, Moon Kyoo Jang, Clarence M. Lee, Andrew H. Karaba, Andrea L. Cox, Sean Agbor-Enoh
BACKGROUND. FXLEARN, the first-ever large multi-site trial of effects of disease-targeted pharmacotherapy on learning, was designed to explore a new paradigm for measuring effects of mechanism-targeted treatment in fragile X syndrome (FXS). In FXLEARN, the effects of mGluR5 negative allosteric modulator (NAM) AFQ056 on language learning were evaluated in 3-6 year-old children with FXS, expected to have more learning plasticity than adults, where prior trials of mGluR5 NAMs have failed. METHODS. After a 4-month single-blind placebo lead-in, participants were randomized 1:1 to AFQ056 or placebo, with 2 months of dose optimization to the maximum tolerated dose, then 6 months of treatment during which a language learning intervention was implemented for both groups. The primary outcome was a centrally scored videotaped communication measure, the Weighted Communication Scale (WCS). Secondary outcomes were objective performance-based and parent-report cognitive and language measures. RESULTS. FXLEARN enrolled 110 participants, randomized 99, and 91 completed the placebo-controlled period. Although both groups made language progress and there were no safety issues, the change in WCS score during the placebo-controlled period was not significantly different between the AFQ056 and placebo-treated groups, nor were there any significant between-group differences in change in any secondary measures. CONCLUSION. Despite the large body of evidence supporting use of mGluR5 NAMs in animal models of FXS, this study suggests that this mechanism of action does not translate into benefit for the human FXS population and that better strategies are needed to determine which mechanisms will translate from pre-clinical models to humans in genetic neurodevelopmental disorders. TRIAL REGISTRATION. ClincalTrials.gov NCT02920892 FUNDING. This study was supported by NeuroNEXT network NIH grants U01NS096767, U24NS107200, U24NS107209, U01NS077323, U24NS107183, U24NS107168, U24NS107128, U24NS107199, U24NS107198, U24NS107166, U10NS077368, U01NS077366, U24NS107205, U01NS077179, and U01NS077352, NIH grant P50HD103526 and Novartis IIT grant AFQ056X2201T for provision of AFQ056.
Elizabeth Berry-Kravis, Leonard Abbeduto, Randi Hagerman, Christopher S. Coffey, Merit Cudkowicz, Craig A. Erickson, Andrea McDuffie, David Hessl, Lauren E. Ethridge, Flora Tassone, Walter E. Kaufmann, Katherine Friedmann, Lauren Bullard, Anne Hoffmann, Jeremy Veenstra-VanderWeele, Kevin Staley, David Klements, Michael Moshinsky, Brittney Harkey, Jeffrey D. Long, Janel Fedler, Elizabeth Klingner, Dixie J. Ecklund, Michele Costigan, Trevis Huff, Brenda Pearson
The NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome is a crucial component of the innate immune system that initiates inflammatory responses. Post-translational modifications (PTMs) of NLRP3, including ubiquitination and phosphorylation, control inflammasome activation and determine the intensity of inflammation. However, the role of other PTMs in controlling NLRP3 inflammasome activation remains unclear. This study founded that toll-like receptor (TLR) priming induced NLRP3 ISGylation (a type of PTM in which ISG15 covalently binds to the target protein) to stabilise the NLRP3 protein. Viral infection, represented by SARS-COV-2 infection, and type I IFNs induced the expression of ISG15 and the predominant E3 ISGylation ligases HECT domain- and RCC1-like domain-containing proteins (HERCs; HERC5 in humans and HERC6 in mice). HERCs promoted NLRP3 ISGylation and inhibited K48-linked ubiquitination and proteasomal degradation, resulting in the enhancement of NLRP3 inflammasome activation. Concordantly, Herc6 deficiency ameliorated NLRP3-dependent inflammation, and hyperinflammation caused by viral infection. These results illustrate the mechanism by which type I IFNs responses control inflammasome activation and viral infection-induced aberrant NLRP3 activation. This work identifies ISGylation as a PTM of NLRP3 and provides a priming target for modulating NLRP3-dependent immunopathology.
Ying Qin, Xintong Meng, Mengge Wang, Wenbo Liang, Rong Xu, Jingchunyu Chen, Hui Song, Yue Fu, Jingxin Li, Chengjiang Gao, Mutian Jia, Chunyuan Zhao, Wei Zhao
Microvillus Inclusion Disease (MVID), caused by loss-of-function mutations in the motor protein Myosin Vb (MYO5B), is a severe infantile disease characterized by diarrhea, malabsorption, and acid-base instability, requiring intensive parenteral support for nutritional and fluid management. Human patient-derived enteroids represent a model for investigation of monogenic epithelial disorders but are a rare resource from MVID patients. We developed human enteroids with different loss-of function MYO5B variants and showed that they recapitulated the structural changes found in native MVID enterocytes. Multiplex Immunofluorescence imaging of patient duodenal tissues revealed patient-specific changes in localization of brush border transporters. Functional analysis of electrolyte transport revealed profound loss of Na+/H+ exchange (NHE) activity in MVID patient enteroids with near-normal chloride secretion. The chloride channel-blocking anti-diarrheal drug, Crofelemer, dose-dependently inhibited agonist-mediated fluid secretion. MVID enteroids exhibited altered differentiation and maturation versus healthy enteroids. Gamma-secretase inhibition with DAPT recovered apical brush border structure and functional Na+/H+ exchange activity in MVID enteroids. Transcriptomic analysis revealed potential pathways involved in the rescue of MVID cells including serum- and glucocorticoid-induced protein kinase 2 (SGK2), and NHE regulatory factor 3 (NHERF3). These results demonstrate the utility of patient-derived enteroids for developing therapeutic approaches to MVID.
Meri Kalashyan, Krishnan Raghunathan, Haley Oller, Marie-Theres Bayer, Lissette Jimenez, Joseph T. Roland, Elena Kolobova, Susan J. Hagen, Jeffrey D. Goldsmith, Mitchell D. Shub, James R. Goldenring, Izumi Kaji, Jay R. Thiagarajah