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In-Press Preview

Articles in this category appear as authors submitted them for publication, prior to copyediting and publication layout.
Targeting gliovascular connexins prevents inflammatory blood-brain barrier leakage and astrogliosis
The blood-brain barrier is formed by capillary endothelial cells expressing Cx37, Cx40 and Cx43, and is joined by closely apposed astrocytes expressing Cx43 and Cx30. We investigated whether...
Published July 26, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.135263.
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Research In-Press Preview Inflammation Neuroscience

Targeting gliovascular connexins prevents inflammatory blood-brain barrier leakage and astrogliosis

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Abstract

The blood-brain barrier is formed by capillary endothelial cells expressing Cx37, Cx40 and Cx43, and is joined by closely apposed astrocytes expressing Cx43 and Cx30. We investigated whether connexin-targeting peptides could limit barrier leakage triggered by LPS-induced systemic inflammation in mice. Intraperitoneal LPS increased endothelial and astrocytic Cx43 expression, elevated TNFα, IL1β, IFNγ and IL6 in plasma and IL6 in the brain, and induced barrier leakage recorded over 24h. Barrier leakage was largely prevented by global Cx43 knockdown and Cx43/Cx30 double-knockout in astrocytes, slightly diminished by endothelial Cx43 knockout and not protected by global Cx30 knockout. Intravenous administration of Gap27 or Tat-Gap19 just before LPS also prevented barrier leakage, and intravenous BAPTA-AM to chelate intracellular calcium was equally effective. Patch-clamp experiments demonstrated LPS-induced Cx43 hemichannel opening in endothelial cells, which was suppressed by Gap27, Gap19 and BAPTA. LPS additionally triggered astrogliosis that was prevented by intravenous Tat-Gap19 or BAPTA-AM. Cortically applied Tat-Gap19 or BAPTA-AM to primarily target astrocytes, also strongly diminished barrier leakage. In vivo dye uptake and in vitro patch-clamp showed Cx43 hemichannel opening in astrocytes that was induced by IL6 in a calcium-dependent manner. We conclude that targeting endothelial and astrocytic connexins is a powerful approach to limit barrier failure and astrogliosis.

Authors

Marijke De Bock, Maarten A.J. De Smet, Stijn Verwaerde, Hanane Tahiri, Steffi Schumacher, Valérie Van Haver, Katja Witschas, Christian Steinhäuser, Nathalie Rouach, Roosmarijn E. Vandenbroucke, Luc Leybaert

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Targeting ESR1 mutation-Induced transcriptional addiction in breast cancer with BET inhibition
Acquired mutations in the ligand-binding domain (LBD) of the gene encoding Estrogen Receptor alpha (ESR1) are a common mechanism of endocrine therapy resistance in metastatic ER-positive breast...
Published July 26, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.151851.
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Research In-Press Preview Oncology

Targeting ESR1 mutation-Induced transcriptional addiction in breast cancer with BET inhibition

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Abstract

Acquired mutations in the ligand-binding domain (LBD) of the gene encoding Estrogen Receptor alpha (ESR1) are a common mechanism of endocrine therapy resistance in metastatic ER-positive breast cancer patients. ESR1 Y537S mutation, in particular, is associated with development of resistance to most endocrine therapies used to treat breast cancer. Employing a high-throughput screen of nearly 1200 Federal Drug Administration (FDA)-approved drugs, we show that OTX015, a bromodomain and extraterminal domain (BET) inhibitor, is one of the top suppressors of ESR1 mutant cell growth. OTX015 was more efficacious than fulvestrant, a selective ER degrader, in inhibiting ESR1 mutant xenograft growth. When combined with abemaciclib, a CDK4/6 inhibitor, OTX015 induced more potent tumor regression than current standard-of-care treatment of abemaciclib+fulvestrant. OTX015 has preferential activity against Y537S mutant breast cancer cells and blocks their clonal selection in competition studies with wild-type cells. Thus, BET inhibition has the potential to both prevent and overcome ESR1 mutant-induced endocrine therapy resistance in breast cancer.

Authors

Sm N. Udden, Qian Wang, Sunil Kumar, Venkat S. Malladi, Shwu-Yuan Wu, Shuguang Wei, Bruce A. Posner, Sophie Geboers, Noelle S. Williams, Yu-Lun Liu, Jayesh K. Sharma, Ram S. Mani, Srinivas Malladi, Karla Parra, Mia Hofstad, Ganesh V. Raj, Jose M. Larios, Reshma Jagsi, Max S. Wicha, Ben Ho Park, Gaorav P. Gupta, Arul M. Chinnaiyan, Cheng-Ming Chiang, Prasanna G. Alluri

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Succinate dehydrogenase inversely regulates red cell distribution width and healthy lifespan in chronically hypoxic mice
Increased red cell distribution width (RDW), which measures erythrocyte volume (MCV) variability (anisocytosis), has been linked to early mortality in many diseases and in older adults through...
Published July 26, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.158737.
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Research In-Press Preview Hematology Pulmonology

Succinate dehydrogenase inversely regulates red cell distribution width and healthy lifespan in chronically hypoxic mice

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Increased red cell distribution width (RDW), which measures erythrocyte volume (MCV) variability (anisocytosis), has been linked to early mortality in many diseases and in older adults through unknown mechanisms. Hypoxic stress has been proposed as a potential mechanism. However, experimental models to investigate the link between increased RDW and reduced survival are lacking. Here, we show that lifelong hypobaric hypoxia (~10% O2) increases erythrocyte numbers, hemoglobin and RDW, while reducing longevity in male mice. Compound heterozygous knockout (chKO) mutations in succinate dehydrogenase (Sdh; mitochondrial complex II) genes Sdhb, Sdhc and Sdhd reduce Sdh subunit protein levels, RDW, and increase healthy lifespan compared to wild-type (WT) mice in chronic hypoxia. RDW-SD, a direct measure of MCV variability, and the standard deviation of MCV (1SD-RDW) show the most statistically significant reductions in Sdh hKO mice. Tissue metabolomic profiling of 147 common metabolites shows the largest increase in succinate with elevated succinate to fumarate and succinate to oxoglutarate (2-ketoglutarate) ratios in Sdh hKO mice. These results demonstrate that mitochondrial complex II level is an underlying determinant of both RDW and healthy lifespan in hypoxia, and suggest that therapeutic targeting of Sdh might reduce high RDW-associated clinical mortality in hypoxic diseases.

Authors

Bora E. Baysal, Abdulrahman A. Alahmari, Tori C. Rodrick, Debra Tabaczynski, Leslie Curtin, Mukund Seshadri, Drew R. Jones, Sandra Sexton

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Treg suppression of immunity within inflamed allogeneic grafts
Regulatory CD4+Foxp3+ T cells (Treg) restrain inflammation and immunity. However, the mechanisms underlying Treg suppressor function in inflamed non-lymphoid tissues remain largely unexplored....
Published July 26, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.160579.
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Research In-Press Preview Immunology Transplantation

Treg suppression of immunity within inflamed allogeneic grafts

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Regulatory CD4+Foxp3+ T cells (Treg) restrain inflammation and immunity. However, the mechanisms underlying Treg suppressor function in inflamed non-lymphoid tissues remain largely unexplored. Here, we restricted immune responses to non-lymphoid tissues and used intravital microscopy to visualize Treg suppression of rejection by effector T cells (Teff) within inflamed allogeneic islet transplants. Despite their elevated motility, Treg preferentially contact antigen-presenting cells (APCs) over Teff. Interestingly, Treg specifically target APCs that are extensively and simultaneously contacted by Teff. In turn, Treg decrease MHC-II expression on APCs and hinder Teff function. Lastly, we demonstrate that Treg suppressor function within inflamed allografts requires ecto-nucleotidase CD73 activity, which generates the anti-inflammatory adenosine. Consequently, CD73-/- Treg exhibit reduced contacts with APCs within inflamed allografts compared to wt Treg, but not in spleen. Overall, our findings demonstrate that Treg suppress immunity within inflamed grafts through CD73 activity and suggest that Treg-APC direct contacts are central to this process.

Authors

Hehua Dai, Andressa Pena, Lynne Bauer, Amanda Williams, Simon c. Watkins, Geoffrey Camirand

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Dichloroacetate improves mitochondrial function, physiology, and morphology in FBXL4 disease models
Pathogenic variants in the human F Box and Leucine Rich Repeat Protein 4 (FBXL4) gene result in an autosomal recessive, multi-systemic, mitochondrial disorder involving variable mitochondrial...
Published July 26, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.156346.
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Research In-Press Preview Genetics Metabolism

Dichloroacetate improves mitochondrial function, physiology, and morphology in FBXL4 disease models

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Pathogenic variants in the human F Box and Leucine Rich Repeat Protein 4 (FBXL4) gene result in an autosomal recessive, multi-systemic, mitochondrial disorder involving variable mitochondrial depletion and respiratory chain (RC) complex deficiencies with lactic acidemia. As no FDA-approved effective therapies exist, we sought to characterize translational C. elegans and zebrafish animal models, as well as human fibroblasts, to study FBXL4-/- disease mechanisms and identify preclinical therapeutic leads. Developmental delay, impaired fecundity and neurologic and/or muscular activity, mitochondrial dysfunction, and altered lactate metabolism were identified in fbxl-1(ok3741) C. elegans. Detailed studies of a pyruvate dehydrogenase complex activator, dichloroacetate (DCA) in fbxl-1(ok3741) C. elegans demonstrated its beneficial effects on fecundity, neuromotor activity, and mitochondrial function. Validation studies were performed in fbxl4sa12470 zebrafish larvae and in FBXL4-/- human fibroblasts, which showed DCA efficacy in preventing brain damage, impairment of neurologic and/or muscular function, mitochondrial biochemical dysfunction, and stress-induced morphologic and ultrastructural mitochondrial defects. These data demonstrate that fbxl-1 (ok3741) C. elegans and fbxl4sa12470 zebrafish provide robust translational models to study mechanisms and identify preclinical therapeutic candidates for FBXL4-/- disease. Further, DCA is a lead therapeutic candidate with therapeutic benefit on diverse aspects of survival, neurologic and/or muscular function, and mitochondrial physiology that warrants rigorous clinical trial study in human subjects with FBXL4-/- disease.

Authors

Manuela Lavorato, Eiko Nakamaru-Ogiso, Neal D. Mathew, Elizabeth Herman, Nina K. Shah, Suraiya Haroon, Rui Xiao, Christoph Seiler, Marni J. Falk

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Ebola virus protein VP40 stimulates IL-12- and IL-18-dependent activation of human natural killer cells
Accumulation of activated natural killer (NK) cells in tissues during Ebola virus infection contributes to Ebola virus disease (EVD) pathogenesis. Yet, immunization with Ebola virus-like particles...
Published July 21, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.158902.
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Research In-Press Preview Immunology Infectious disease

Ebola virus protein VP40 stimulates IL-12- and IL-18-dependent activation of human natural killer cells

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Accumulation of activated natural killer (NK) cells in tissues during Ebola virus infection contributes to Ebola virus disease (EVD) pathogenesis. Yet, immunization with Ebola virus-like particles (VLPs) comprising glycoprotein (GP) and matrix protein VP40 provides rapid, NK cell-mediated protection against Ebola challenge. We used Ebola VLPs as the viral surrogates to elucidate the molecular mechanism by which Ebola virus triggers heightened NK cell activity. Incubation of human peripheral blood mononuclear cells (PBMCs) with Ebola VLPs or VP40 protein led to increased expression of IFN-γ, TNF-α, granzyme B, and perforin by CD3-CD56+ NK cells, along with concomitant increase in degranulation and cytotoxic activity of these cells. Optimal activation required accessory cells like CD14+ myeloid and CD14- cells and triggered increased secretion of numerous inflammatory cytokines. VP40-induced IFN-γ and TNF-α secretion by NK cells was dependent on IL-12 and IL-18 and suppressed by IL-10. In contrast, their increased degranulation was dependent on IL-12 with little influence of IL-18 or IL-10. These results demonstrate that Ebola VP40 stimulates NK cell functions in an IL-12 and IL-18 dependent manner that involves CD14+ and CD14- accessory cells. These novel findings may help in designing improved intervention strategies required to control viral transmission during Ebola outbreaks.

Authors

Hung Le, Paul Spearman, Stephen N. Waggoner, Karnail Singh

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Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a psoriasis susceptibility locus that is negatively related to IL36G
Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a post-translational regulator of the low-density lipoprotein receptor (LDLR). Recent studies have proposed a role for PCSK9 in regulating...
Published July 21, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.141193.
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Research In-Press Preview Dermatology

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a psoriasis susceptibility locus that is negatively related to IL36G

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Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a post-translational regulator of the low-density lipoprotein receptor (LDLR). Recent studies have proposed a role for PCSK9 in regulating immune responses. Using RNA-sequencing-based variant discovery, we identified a novel psoriasis susceptibility locus at 1p32.3, located within PCSK9 (rs662145 C>T). This finding was verified in independently acquired genomic and RNA-sequencing datasets. Single-cell RNA-sequencing (scRNA-seq) identified keratinocytes as the primary source of PCSK9 in human skin. PCSK9 expression, however, was not uniform across keratinocyte subpopulations. scRNA-seq and immunohistochemistry demonstrated an epidermal gradient of PCSK9, with expression being highest in basal and early spinous layer keratinocytes and lowest in granular layer keratinocytes. IL-36G expression followed the opposite pattern, with expression highest in granular layer keratinocytes. PCSK9 siRNA knockdown experiments confirmed this inverse relationship between PCSK9 and IL36G expression. Other immune genes were also linked to PCSK9 expression including, IL27RA, IL1RL1, ISG20, and STX3. In both cultured keratinocytes and nonlesional human skin, homozygosity for PCSK9 SNP rs662145 C>T was associated with lower PCSK9 expression and higher IL36G expression, when compared to heterozygous skin or cell lines. Together these results support PCSK9 as a novel psoriasis susceptibility locus and establish a putative link between PCSK9 and inflammatory cytokine expression.

Authors

Alexander Merleev, Antonio Ji-Xu, Atrin Toussi, Lam C. Tsoi, Stephanie T. Le, Guillaume Luxardi, Xianying Xing, Rachael Wasikowski, William Liakos, Marie-Charlotte Brüggen, James T. Elder, Iannis E. Adamopoulos, Yoshihiro Izumiya, Annie Riera-Leal, Qinyuan Li, Nikolay Yu Kuzminykh, Amanda Kirane, Alina I. Marusina, Johann E. Gudjonsson, Emanual Maverakis

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Spatiotemporal regulation of human IFNε and innate immunity in the female reproductive tract
Although published studies have demonstrated that interferon epsilon (IFNε) has a crucial role in regulating protective immunity in the mouse female reproductive tract (mFRT), expression and...
Published July 21, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.135407.
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Research In-Press Preview Immunology Reproductive biology

Spatiotemporal regulation of human IFNε and innate immunity in the female reproductive tract

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Although published studies have demonstrated that interferon epsilon (IFNε) has a crucial role in regulating protective immunity in the mouse female reproductive tract (mFRT), expression and regulation of IFNε in the human female reproductive tract (hFRT) have not been characterised. To characterise human IFNε, we obtained hFRT samples from a well- characterized cohort of women, enabling us to comprehensively assess ex vivo IFNε expression in the hFRT at various stages of the menstrual cycle. We found that among the various types of IFNs, IFNε is uniquely selectively and constitutively expressed in the hFRT epithelium. It has distinct expression patterns in the surface and glandular epithelia of the upper hFRT compared with basal layers of the stratified squamous epithelia of the lower hFRT. There is cyclical variation of IFNε expression in the endometrial epithelium of the upper hFRT and not in the distal FRT, consistent with selective endometrial expression of the progesterone receptor and regulation of the IFNE promoter by progesterone. Since we show IFNε stimulates important protective IFN-regulated genes (IRGs) in FRT epithelium, this characterisation is a key element in understanding the mechanisms of hormonal control of mucosal immunity.

Authors

Nollaig M. Bourke, Sharon L. Achilles, Stephanie U-Shane Huang, Helen E. Cumming, San S. Lim, Irene Papageorgiou, Linden J. Gearing, Ross Chapman, Suruchi Thakore, Niamh E. Mangan, Sam Mesiano, Paul J. Hertzog

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ARMC5-CUL3 E3 ligase targets full-length SREBF in adrenocortical tumor
Inactivating mutations of ARMC5 are responsible for the development of bilateral macronodular adrenal hyperplasia (BMAH). Although ARMC5 inhibits adrenocortical tumor growth and is considered as...
Published July 21, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.151390.
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Research In-Press Preview Endocrinology

ARMC5-CUL3 E3 ligase targets full-length SREBF in adrenocortical tumor

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Inactivating mutations of ARMC5 are responsible for the development of bilateral macronodular adrenal hyperplasia (BMAH). Although ARMC5 inhibits adrenocortical tumor growth and is considered as tumor-suppressor gene, its molecular function is poorly understood. In this study, through biochemical purification using SREBF (SREBP) as bait, we identified the interaction between SREBF and ARMC5 through its Armadillo repeat. We also found that ARMC5 interacted with CUL3 through its BTB domain and underwent self-ubiquitination. ARMC5 colocalized with SREBF1 in the cytosol and induced proteasome-dependent degradation of full-length SREBF through ubiquitination. Introduction of missense mutations in Armadillo repeat of ARMC5 attenuated the interaction between SREBF, and introduction of mutations found in BMAH completely abolished its ability to degrade full-length SREBF. In H295R adrenocortical cells, silencing of ARMC5 increased full-length SREBFs and upregulated SREBF2 target genes. siARMC5-mediated cell growth was abrogated by simultaneous knockdown of SREBF2 in H295R cells. Our results demonstrated that ARMC5 was a substrate adaptor protein between full-length SREBF and CUL3-based E3 ligase, and suggested the involvement of SREBF pathway in the development of BMAH.

Authors

Yosuke Okuno, Atsunori Fukuhara, Michio Otsuki, Iichiro Shimomura

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Human T-bet+ B cell development is associated with BTK activity and suppressed by evobrutinib
Recent clinical trials show promising results for the next-generation Bruton’s tyrosine kinase (BTK) inhibitor evobrutinib in the treatment of multiple sclerosis (MS). BTK has a central role in...
Published July 19, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.160909.
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Research In-Press Preview Immunology

Human T-bet+ B cell development is associated with BTK activity and suppressed by evobrutinib

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Recent clinical trials show promising results for the next-generation Bruton’s tyrosine kinase (BTK) inhibitor evobrutinib in the treatment of multiple sclerosis (MS). BTK has a central role in signaling pathways that govern the development of B cells. Whether and how BTK activity shapes B cells as key drivers of MS is currently unclear. In contrast to BTK protein, we found higher levels of phospho-BTK in ex vivo blood memory B cells of relapsing and secondary progressive MS patients versus controls. In these MS groups, BTK activity was induced to a lesser extent after anti-IgM stimulation. BTK positively correlated with CXCR3 expression, both of which were increased in blood B cells of clinical responders to natalizumab (anti-VLA-4 antibody) treatment. Under in vitro TFH-like conditions, BTK phosphorylation was enhanced by T-bet-inducing stimuli IFN-γ and CpG-ODN, whilst the expression of T-bet and T-bet-associated molecules CXCR3, CD21 and CD11c were affected by evobrutinib. Furthermore, evobrutinib interfered with in vitro class switching as well as memory recall responses, and disturbed CXCL10-mediated migration of CXCR3+ switched B cells through human brain endothelial monolayers. These findings demonstrate a functional link between BTK activity and disease-relevant B cells and offer valuable insights into how next-generation BTK inhibitors could modulate the clinical course in MS patients.

Authors

Liza Rijvers, Jamie van Langelaar, Laurens Bogers, Marie-José Melief, Steven C. Koetzier, Katelijn M. Blok, Annet F. Wierenga-Wolf, Helga E. De Vries, Jasper Rip, Odilia B.J. Corneth, Rudi W. Hendriks, Roland Grenningloh, Ursula Boschert, Joost Smolders, Marvin M. van Luijn

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KLF4 is a therapeutically tractable brake on fibroblast activation which promotes resolution of pulmonary fibrosis
There is a paucity of information about potential molecular brakes on the activation of fibroblasts that drive tissue fibrosis. The transcription factor Kruppel-like factor 4 (KLF4) is best known...
Published July 19, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.160688.
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Research In-Press Preview Pulmonology

KLF4 is a therapeutically tractable brake on fibroblast activation which promotes resolution of pulmonary fibrosis

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There is a paucity of information about potential molecular brakes on the activation of fibroblasts that drive tissue fibrosis. The transcription factor Kruppel-like factor 4 (KLF4) is best known as a determinant of cell stemness and a tumor suppressor. We found that its expression was diminished in fibroblasts from fibrotic lung. Gain- and loss-of-function studies showed that KLF4 inhibits fibroblast proliferation, collagen synthesis, and differentiation to myofibroblasts, while restoring their sensitivity to apoptosis. Conditional deletion of KLF4 from fibroblasts potentiated the peak degree of pulmonary fibrosis and abrogated the subsequent spontaneous resolution that follows in a model of transient fibrosis. A small molecule inducer of KLF4 was able to restore its expression in fibrotic fibroblasts and elicit resolution in an experimental model characterized by more clinically relevant persistent pulmonary fibrosis. These data identify KLF4 as a pivotal brake on fibroblast activation whose induction represents a new therapeutic approach in fibrosis of the lung, and perhaps other organs.

Authors

Loka Raghu Kumar Penke, Jennifer M. Speth, Steven K. Huang, Sean M. Fortier, Jared Baas, Marc Peters-Golden

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An in vivo model of glioblastoma radiation resistance identifies long non-coding RNAs and targetable kinases
Key molecular regulators of acquired radiation resistance in recurrent glioblastoma (GBM) are largely unknown with a dearth of accurate pre-clinical models. To address this, we generated 8 GBM...
Published July 19, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.148717.
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Research In-Press Preview Oncology

An in vivo model of glioblastoma radiation resistance identifies long non-coding RNAs and targetable kinases

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Key molecular regulators of acquired radiation resistance in recurrent glioblastoma (GBM) are largely unknown with a dearth of accurate pre-clinical models. To address this, we generated 8 GBM patient-derived xenograft (PDX) models of acquired radiation therapy-selected (RTS) resistance compared with same-patient, treatment naïve (RTU) PDX. These unique models mimic the longitudinal evolution of patient recurrent tumors following serial radiation therapy. Indeed, while whole exome sequencing confirmed retention of major genomic alterations in the RTS lines, we did detect a chromosome 12q14 amplification that is associated with clinical GBM recurrence in two RTS models. A novel bioinformatics pipeline was applied to analyze phenotypic, transcriptomic and kinomic alterations, which identified long non-coding RNAs (lncRNAs) and targetable, PDX-specific kinases. We observed differential transcriptional enrichment of DNA damage repair (DDR) pathways in our RTS models which correlated with several lncRNAs. Global kinomic profiling separated RTU and RTS models, but pairwise analyses indicated that there are multiple molecular routes to acquired radiation-resistance. RTS model-specific kinases were identified and targeted with clinically relevant small molecule inhibitors (SMIs). This unique cohort of in vivo radiation therapy-selected patient-derived models will enable future preclinical therapeutic testing to help overcome the treatment resistance seen in GBM patients.

Authors

Christian T. Stackhouse, Joshua C. Anderson, Zongliang Yue, Thanh Nguyen, Nicholas J. Eustace, Catherine P. Langford, Jelai Wang, James R. Rowland IV, Chuan Xing, Fady M. Mikhail, Xiangqin Cui, Hasan Alrefai, Ryan E. Bash, Kevin J. Lee, Eddy S. Yang, Anita B. Hjelmeland, C. Ryan Miller, Jake Y. Chen, G. Yancey Gillespie, Christopher D. Willey

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ARID1A-deficient bladder cancer is dependent on PI3K signaling and sensitive to EZH2 and PI3K inhibitors
Metastatic urothelial carcinoma is generally incurable with current systemic therapies. Chromatin modifiers are frequently mutated in bladder cancer, with ARID1A-inactivating mutations present in...
Published July 19, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.155899.
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Research In-Press Preview Oncology

ARID1A-deficient bladder cancer is dependent on PI3K signaling and sensitive to EZH2 and PI3K inhibitors

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Metastatic urothelial carcinoma is generally incurable with current systemic therapies. Chromatin modifiers are frequently mutated in bladder cancer, with ARID1A-inactivating mutations present in about 20% of tumors. EZH2, a histone methyltransferase, acts as an oncogene that functionally opposes ARID1A. In addition, PI3K signaling is activated in more than 20% of bladder cancers. Using a combination of in vitro and in vivo data, including patient derived xenografts, we show that ARID1A-mutant tumors are more sensitive to EZH2 inhibition than ARID1A-wild type tumors. Mechanistic studies reveal that: 1) ARID1A deficiency results in a dependency on PI3K/AKT/mTOR signaling via novel upregulation of a non-canonical PI3K regulatory subunit, PIK3R3, and downregulation of MAPK signaling, and: 2) EZH2 inhibitor sensitivity is due to upregulation of PIK3IP1, a protein inhibitor of PI3K signaling. We show for the first time that PIK3IP1 inhibits PI3K signaling by inducing proteasomal degradation of PIK3R3. Further, ARID1A deficient bladder cancer is sensitive to combination therapies with EZH2 and PI3K inhibitors, in a synergistic manner. Thus, our studies suggest that bladder cancers with ARID1A mutations can be treated with inhibitors of EZH2 and/or PI3K, and reveal mechanistic insights into the role of non-canonical PI3K constituents in bladder cancer biology.

Authors

Hasibur Rehman, Darshan S. Chandrashekar, Chakravarthi Balabhadrapatruni, Saroj Nepal, Sai Akshaya Hodigere Balasubramanya, Abigail K. Shelton, Kasey R. Skinner, Ai-Hong Ma, Ting Rao, Marie-Lisa Eich, Alyncia D. Robinson, Gurudatta Naik, Upender Manne, George J. Netto, C. Ryan Miller, Chong-xian Pan, Guru Sonpavde, Sooryanarayana Varambally, James E. Ferguson 3rd

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Integrated single-cell transcriptomics and proteomics reveal cellular-specific response and microenvironment remodeling in aristolochic acid nephropathy
Aristolochic acid nephropathy (AAN) is characterized by acute proximal tubule necrosis and immune cell infiltration, contributing to the global burden of chronic kidney disease and urothelial...
Published July 19, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.157360.
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Research In-Press Preview Cell biology Nephrology

Integrated single-cell transcriptomics and proteomics reveal cellular-specific response and microenvironment remodeling in aristolochic acid nephropathy

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Aristolochic acid nephropathy (AAN) is characterized by acute proximal tubule necrosis and immune cell infiltration, contributing to the global burden of chronic kidney disease and urothelial cancer. Although the proximal tubule has been defined as the primary target of aristolochic acids I (AAI), the mechanistic underpinning of gross renal deterioration caused by AAI has not been explicitly explained, prohibiting effective therapeutic intervention. To this point, we employed integrated single-cell RNA-sequencing, bulk RNA-sequencing, and mass spectrometry-based proteomics to analyze mouse kidney post-acute AAI exposure. Our results revealed a dramatic reduction of proximal tubule epithelial cells, associated with apoptotic and inflammatory pathways, indicating permanent damage beyond repair. We found the enriched development pathways in other nephron segments, suggesting activation of reparative programs triggered by AAI. The divergent response may be attributed to the segment-specific distribution of organic anions channels along the nephron, including OAT1 and OAT3. Moreover, we observed dramatic activation and recruitment of cytotoxic T and macrophage M1 cells, highlighting inflammation as a principal contributor to permanent renal injury. Ligand-receptor pairing revealed critical intercellular crosstalk underpins damage-induced activation of immune cells. These results provide novel insight into AAI-induced kidney injury and point out potential pathways for future therapeutic intervention.

Authors

Jiayun Chen, Piao Luo, Chen Wang, Chuanbin Yang, Yunmeng Bai, Xueling He, Qian Zhang, Junzhe Zhang, Jing Yang, Shuang Wang, Jigang Wang

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Multicenter analysis of neutrophil extracellular trap dysregulation in adult and pediatric COVID-19
Dysregulation in neutrophil extracellular trap (NET) formation and degradation may play a role in the pathogenesis and severity of COVID-19; however, its role in the pediatric manifestations of...
Published July 19, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.160332.
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Research In-Press Preview Infectious disease Inflammation

Multicenter analysis of neutrophil extracellular trap dysregulation in adult and pediatric COVID-19

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Abstract

Dysregulation in neutrophil extracellular trap (NET) formation and degradation may play a role in the pathogenesis and severity of COVID-19; however, its role in the pediatric manifestations of this disease including multisystem inflammatory syndrome in children (MIS-C) and chilblain-like lesions (CLL), otherwise known as “COVID toes”, remains unclear. Studying multinational cohorts, we found that, in CLL, NETs were significantly increased in serum and skin. There was geographic variability in the prevalence of increased NETs in MIS-C, in association with disease severity. MIS-C and CLL serum samples displayed decreased NET degradation ability, in association with C1q and G-actin or anti-NET antibodies, respectively, but not with genetic variants of DNases. In adult COVID-19, persistent elevations in NETs post-disease diagnosis were detected but did not occur in asymptomatic infection. COVID-19-affected adults displayed significant prevalence of impaired NET degradation, in association with anti-DNase1L3, G-actin, and specific disease manifestations, but not with genetic variants of DNases. NETs were detected in many organs of adult patients who died from COVID-19 complications. Infection with the Omicron variant was associated with decreased levels of NETs when compared to other SARS-CoV-2 strains. These data support a role for NETs in the pathogenesis and severity of COVID-19 in pediatric and adult patients.

Authors

Carmelo Carmona-Rivera, Yu Zhang, Kerry Dobbs, Tovah E. Markowitz, Clifton L. Dalgard, Andrew J. Oler, Dillon R. Claybaugh, Deborah Draper, Meng Truong, Ottavia M. Delmonte, Francesco Licciardi, Ugo Ramenghi, Nicoletta Crescenzio, Luisa Imberti, Alessandra Sottini, Virginia Quaresima, Chiara Fiorini, Valentina Discepolo, Andrea Lo Vecchio, Alfredo Guarino, Luca Pierri, Andrea Catzola, Andrea Biondi, Paolo Bonfanti, Maria Cecilia Poli Harlowe, Yazmin Espinosa, Camila A. Astudillo, Emma Rey-Jurado, Cecilia Vial, Javiera De la Cruz, Ricardo Gonzalez, Cecilia Pinera, Jacqueline W. Mays, Ashley Ng, Andrew Platt, Beth A. Drolet, John Moon, Edward W. Cowen, Heather Kenney, Sarah E. Weber, Riccardo Castagnoli, Mary K. Magliocco, Michael Austin Stack, Gina A. Montealegre Sanchez, Karyl Barron, Danielle L. Fink, Douglas B. Kuhns, Stephen M. Hewitt, Lisa M. Arkin, Daniel S. Chertow, Helen C. Su, Luigi D. Notarangelo, Mariana J. Kaplan

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The UIP/IPF fibroblastic focus is a collagen biosynthesis factory embedded in a distinct extracellular matrix
Usual Interstitial Pneumonia (UIP) is a histological pattern characteristic of Idiopathic Pulmonary Fibrosis (IPF). The UIP pattern is patchy with histologically normal lung adjacent to dense...
Published July 19, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.156115.
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Research In-Press Preview Pulmonology

The UIP/IPF fibroblastic focus is a collagen biosynthesis factory embedded in a distinct extracellular matrix

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Abstract

Usual Interstitial Pneumonia (UIP) is a histological pattern characteristic of Idiopathic Pulmonary Fibrosis (IPF). The UIP pattern is patchy with histologically normal lung adjacent to dense fibrotic tissue. At this interface, fibroblastic foci (FF) are present and are sites where myofibroblasts and extracellular matrix (ECM) accumulate. Utilizing laser capture microdissection coupled mass spectrometry (LCM-MS), we interrogated the FF, adjacent mature scar, and adjacent alveoli in 6 fibrotic (UIP/IPF) specimens plus 6 non-fibrotic alveolar specimens as controls. The data were subject to qualitative and quantitative analysis, and histologically validated. We found that the fibrotic alveoli protein signature is defined by immune deregulation as the strongest category. The fibrotic mature scar classified as end-stage fibrosis whereas the FF contained an overabundance of a distinctive ECM compared to non-fibrotic control. Furthermore, the FF is positive for both TGFB1 and TGFB3, whereas the aberrant basaloid cell lining of the FF is predominantly positive for TGFB2. In conclusion, spatial proteomics demonstrated distinct protein compositions in the histologically defined regions of UIP/IPF tissue. These data revealed that the FF is the main site of collagen biosynthesis and that the adjacent alveoli are abnormal. This new and essential information will inform future mechanistic studies on fibrosis progression.

Authors

Jeremy A. Herrera, Lewis A. Dingle, M. Angeles Montero Fernandez, Rajamiyer V. Venkateswaran, John F. Blaikley, Craig Lawless, Martin A. Schwartz

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Junctional adhesion molecule-A deletion increases phagocytosis and improves survival in a murine model of sepsis
The tight junction-associated protein Junctional Adhesion Molecule-A (JAM-A) is increased in sepsis although the significance of this is unknown. Here we show that septic JAM-A-/- mice have...
Published July 12, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.156255.
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Research In-Press Preview Infectious disease

Junctional adhesion molecule-A deletion increases phagocytosis and improves survival in a murine model of sepsis

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Abstract

The tight junction-associated protein Junctional Adhesion Molecule-A (JAM-A) is increased in sepsis although the significance of this is unknown. Here we show that septic JAM-A-/- mice have increased gut permeability. Paradoxically, septic JAM-A-/- mice have decreased bacteremia and systemic TNF and IL-1β. Survival is improved in JAM-A-/- mice. However intestine-specific JAM-A-/- deletion does not alter mortality suggesting the mortality benefit conferred in mice lacking JAM-A is independent of the intestine. Septic JAM-A-/- mice have increased splenic CD44hiCD4+ T cells with decreased frequency of TNF+CD4+ cells and elevated frequency of IL-2+CD4+ cells. Septic JAM-A-/- mice have increased B cells in mesenteric lymph nodes with elevated serum IgA and intraepithelial lymphocyte IgA production. JAM-A-/- x RAG -/- mice have improved survival compared to RAG-/- mice and identical mortality as WT mice. Gut neutrophil infiltration is increased in JAM-A-/- mice and neutrophil phagocytosis is increased. Septic JAM-A-/- mice depleted of neutrophils lose their survival advantage. Therefore increased bacterial clearance via neutrophils and an altered systemic inflammatory response with increased opsonizing IgA produced through the adaptive immune system results in improved survival in septic JAM-A-/- mice. JAM-A may be a therapeutic target in sepsis via immune mechanisms not related to its role in permeability.

Authors

Nathan J. Klingensmith, Katherine T Fay, David A. Swift, Julia M.R. Bazzano, John D. Lyons, Ching-wen Chen, Mei Meng, Kimberly M. Ramonell, Zhe Liang, Eileen M. Burd, Charles A. Parkos, Mandy L. Ford, Craig M. Coopersmith

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Protective role of tissue-resident regulatory T cells in a murine model of beryllium-induced disease
CD4+ T cells drive the immunopathogenesis of chronic beryllium disease (CBD), and their recruitment to the lung heralds the onset of granulomatous inflammation. We have shown that regulatory CD4+ T...
Published July 12, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.156098.
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Research In-Press Preview Pulmonology

Protective role of tissue-resident regulatory T cells in a murine model of beryllium-induced disease

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Abstract

CD4+ T cells drive the immunopathogenesis of chronic beryllium disease (CBD), and their recruitment to the lung heralds the onset of granulomatous inflammation. We have shown that regulatory CD4+ T cells (Tregs) control granuloma formation in an HLA-DP2 transgenic (Tg) model of CBD. In these mice, Be oxide (BeO) exposure resulted in the accumulation of three distinct CD4+ T cell subsets in the lung with the majority of tissue-resident memory cells expressing FoxP3. The amount of Be regulated the number of total and antigen-specific CD4+ T cells and Tregs in the lungs of HLA-DP2 Tg mice. Depletion of Tregs increased the number of IFN-γ-producing CD4+ T cells and enhanced lung injury while mice treated with IL2/αIL-2 complexes had increased Tregs and reduced inflammation and Be-responsive T cells in the lung. BeO-experienced resident Tregs suppressed anti-CD3-induced proliferation of CD4+ T cells in a contact-dependent manner. CLTLA-4 and ICOS blockade as well as addition of LPS to BeO-exposed mice increased the Teff/Treg ratio and enhanced lung injury. Collectively, these data show that the protective role of tissue-resident Tregs is dependent on quantity of Be exposure and is overcome by blocking immune regulatory molecules or additional environmental insults.

Authors

Shaikh M. Atif, Douglas G. Mack, Allison K. Martin, Andrew P. Fontenot

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Single-nucleus RNA-sequencing reveals singular gene signatures of human ductal cells during adaptation to insulin resistance
Adaptation to increased insulin demand is mediated by β-cell proliferation and neogenesis among other mechanisms. Although it is known that pancreatic β-cells can arise from ductal progenitors,...
Published July 12, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.153877.
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Research In-Press Preview Development Endocrinology

Single-nucleus RNA-sequencing reveals singular gene signatures of human ductal cells during adaptation to insulin resistance

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Abstract

Adaptation to increased insulin demand is mediated by β-cell proliferation and neogenesis among other mechanisms. Although it is known that pancreatic β-cells can arise from ductal progenitors, these observations have been limited mostly to the neonatal period. We have recently reported that the duct is a source of insulin secreting cells in adult insulin resistant states. To further explore the signaling pathways underlying the dynamic β-cell reserve during insulin resistance we undertook human islet and duct transplantations under the kidney capsule of immunodeficient NOD SCID gamma (NSG) mouse models that were either pregnant, insulin resistant or had insulin resistance superimposed upon pregnancy (pregnancy+insulin resistance), followed by single-nucleus RNA-sequencing (snRNA-seq) on snap-frozen graft samples. We observed an upregulation of proliferation markers (e.g., NEAT1), expression of islet endocrine cell markers (e.g., GCG and PPY) as well as mature β-cell markers (e.g., INS), in transplanted human duct grafts in response to high insulin demand. We also noted downregulation of ductal cell identity genes (e.g., KRT19 and ONECUT2) coupled with upregulation of β-cell development and insulin signaling pathways. These results indicate that subsets of ductal cells are able to gain β-cell identity and reflect a form of compensation during the adaptation to insulin resistance in both physiological and pathological states.

Authors

Ercument Dirice, Giorgio Basile, Sevim Kahraman, Danielle Diegisser, Jiang Hu, Rohit N. Kulkarni

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Mucosecretory lung disease: Assemblies of JAG1 and JAG2 determine tracheobronchial cell fate
Mucosecretory lung disease compromises airway epithelial function and is characterized by goblet cell hyperplasia and ciliated cell hypoplasia. These cell types are derived from tracheobronchial...
Published July 12, 2022
Citation Information: JCI Insight. 2022. https://doi.org/10.1172/jci.insight.157380.
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Research In-Press Preview Cell biology Stem cells

Mucosecretory lung disease: Assemblies of JAG1 and JAG2 determine tracheobronchial cell fate

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Abstract

Mucosecretory lung disease compromises airway epithelial function and is characterized by goblet cell hyperplasia and ciliated cell hypoplasia. These cell types are derived from tracheobronchial stem/progenitor cells via a Notch dependent mechanism. Although specific arrays of Notch receptors regulate cell fate determination, the function of the ligands Jagged1 (JAG1) and Jagged2 (JAG2) is unclear. This study examined JAG1 and JAG2 function using human air-liquid-interface cultures that were treated with γ-secretase complex inhibitors (GSC), neutralizing peptides/antibodies, or WNT/β-catenin pathway antagonists/agonists. These experiments revealed that JAG1 and JAG2 regulated cell fate determination in the tracheobronchial epithelium; however, their roles did not adhere to simple necessity and sufficiency rules. Biochemical studies indicated that JAG1 and JAG2 underwent post-translational modifications that resulted in generation of a JAG1 C-terminal peptide and regulated the abundance of full-length JAG2 on the cell surface. The GSC and glycogen synthase kinase 3 were implicated in these post-translational events but WNT agonist/antagonist studies and RNA sequencing indicated a WNT-independent mechanism. Collectively, these data suggest that post-translational modifications create distinct assemblies of JAG1 and JAG2 which regulate Notch signal strength and determine the fate of tracheobronchial stem/progenitor cells.

Authors

Susan D. Reynolds, Cynthia L. Hill, Alfahdah Alsudayri, Scott W. Lallier, Saranga Wijeratne, Zheng Hong Tan, Tendy Chiang, Estelle Cormet-Boyaka

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