Deficiency of inhibitory TLR4 homolog RP105 exacerbates fibrosis

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Abstract

Activation of Toll-like receptor 4 (TLR4) by its cognate damage-associated endogenous ligands (DAMPs) elicits potent profibrotic effects and myofibroblasts activation in systemic sclerosis (SSc), while genetic targeting of TLR4 or its DAMPs in mice accelerates fibrosis resolution. To prevent aberrant DAMP-TLR4 activity, a variety of negative regulators evolved to dampen the magnitude and duration of the signaling. These include radioprotective 105 KDa (RP105), a transmembrane TLR4 homolog that competitively inhibits DAMP recognition of TLR4, blocking TLR4 signaling in immune cells. The role of RP105 in TLR4-dependent fibrotic responses in SSc is unknown. Using unbiased transcriptome analysis of skin biopsies, we found that both TLR4 and its adaptor protein MD2 were elevated in SSc skin and significantly correlated with each other (r=0.54, p=0.0062). Expression of RP105 was negatively associated with myofibroblast differentiation in SSc (r=-0.53). Importantly, RP105-TLR4 association was reduced while TLR4- TLR4 showed strong association in SSc fibroblasts as shown by PLA assays. Moreover, RP105 adaptor MD1 expression was significantly reduced in SSc skin biopsies and explanted SSc skin fibroblasts. Exogenous RP105-MD1 abrogated, while loss of RP105 exaggerated, fibrotic cellular responses. Importantly, ablation of RP105 in mice was associated with augmented TLR4 signaling and aggravated skin fibrosis in complementary disease models. Thus, we identify RP105-MD1 as a novel cell-intrinsic negative regulator of TLR4-MD2-driven sustained fibroblast activation, representing a critical regulatory network governing the fibrotic process. Impaired RP105 function in SSc might contribute to persistence of progression of the disease.

Authors

Wenxia Wang, Swarna Bale, Bharath Yalavarthi, Priyanka Verma, Pei-Suen Tsou, Ken M. Calderone, Dibyendu Bhattacharyya, Gary J. Fisher, John Varga, Swati Bhattacharyya

Unraveling unique features of plasma cell clones in POEMS syndrome by single-cell analysis

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Abstract

POEMS syndrome is a rare monoclonal plasma cell disorder with unique symptoms distinct from other plasma cell neoplasms, including high serum VEGF levels. Since the prospective isolation of POEMS clones has not yet been successful, their real nature remains unclear. We herein performed the single-cell RNA sequencing of bone marrow plasma cells from patients with POEMS syndrome and identified POEMS clones that had immunoglobulin λ light chain (IGL) sequences (IGLV1-36, 40, 44, and 47) with amino acid changes specific to POEMS syndrome. The proportions of POEMS clones in plasma cells were markedly smaller (median: 12.9%) than in multiple myeloma (MM) (96–100%) and monoclonal gammopathy of undetermined significance (MGUS) patients (57–81%). Single-cell transcriptomes revealed that POEMS clones were CD19-negative, CD138-positive, and MHC class II-low, which allowed for their prospective isolation. POEMS clones expressed significantly lower levels of c-MYC and CCND1 than MM, accounting for their small size. VEGF mRNA was not up-regulated in POEMS clones, directly indicating that VEGF is not produced by POEMS clones. These results reveal unique features of POEMS clones and enhance our understanding of the pathogenesis of POEMS syndrome.

Authors

Yusuke Isshiki, Motohiko Oshima, Naoya Mimura, Kensuke Kayamori, Yurie Miyamoto-Nagai, Masahide Seki, Yaeko Nakajima-Takagi, Takashi Kanamori, Eisuke Iwamoto, Tomoya Muto, Shokichi Tsukamoto, Yusuke Takeda, Chikako Ohwada, Sonoko Misawa, Jun-ichiro Ikeda, Masashi Sanada, Satoshi Kuwabara, Yutaka Suzuki, Emiko Sakaida, Chiaki Nakaseko, Atsushi Iwama

Cell-free DNA topology is unique to its sub-cellular and cellular origins in cancer

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Abstract

Cancer cells release large quantities of cell-free DNA (cfDNA) into the surrounding tissue and circulation. As cfDNA is a common source of biomarkers for liquid biopsy and has been implicated as a functional mediator for intercellular communication, fundamental characterization of cfDNA topology has widespread biological and clinical ramifications. Whether the topology of cfDNA is such that it exists predominantly in membrane-bound extracellular vesicles (EVs) or in non-vesicular DNA-protein complexes remains poorly understood. Here, we employed a DNA-targeted approach to comprehensively assess total cfDNA topology in cancer. Using preclinical models and patient samples, we demonstrate that nuclear cfDNA is predominantly associated with nucleosomal particles and not EVs, while a substantial subset of mitochondrial cfDNA is membrane-protected and disproportionately derived from non-tumour cells. In addition, discrimination between membrane-protected and accessible mitochondrial cfDNA added diagnostic and prognostic value in a cohort of head and neck cancer patients. Our results support a revised model for cfDNA topology in cancer. Due to its abundance, nuclear cfDNA within nucleosomal particles is the most compelling liquid biopsy substrate, while EV-bound and accessible mitochondrial cfDNA represent distinct reservoirs of potential cancer biomarkers whose structural conformations may also influence their extracellular stability and propensity for uptake by recipient cells.

Authors

Ethan Z. Malkin, Steven De Michino, Meghan Lambie, Rita G. Gill, Zhen Zhao, Ariana Rostami, Andrea Arruda, Mark D. Minden, Scott V. Bratman

Hyperglycemia exacerbates dengue virus infection by facilitating poly(A)-binding protein-mediated viral translation

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Abstract

Diabetes mellitus (DM) is highly comorbid with severe dengue diseases; however, the underlying mechanisms are unclear. DM patients display a 1.61-fold increased risk of developing dengue hemorrhagic fever. In search of host factors involved in DENV infection, this study utilizes high glucose (HG) treatment and shows that HG increases viral protein expression and virion release but has no effects on the early stages of viral infection. Following HG stimulation, DEN-Luc-transfected assay and cellular replicon-based assay display increased viral translation, while using the glucose uptake inhibitor phloretin blocks this effect. HG treatment increases the translational factor poly(A)-binding protein (PABP) in a glucose transporter-associated PI3K/AKT-regulated manner. Silencing PABP significantly decreases HG-prompted virion production. HG enhances the formation of the PABP-eIF4G complex, which is regulated by protein-disulfide isomerase. Hyperglycemia increases PABP expression, mortality, viral protein expression, and viral loads in streptozotocin-induced DM mice. Overall, hyperglycemic stress facilitates DENV infection by strengthening PABP-mediated viral translation.

Authors

Ting-Jing Shen, Chia-Ling Chen, Tsung-Ting Tsai, Ming-Kai Jhan, Chyi-Huey Bai, Yu-Chun Yen, Ching-Wen Tsai, Cheng-Yi Lee, Po-Chun Tseng, Chia-Yi Yu, Chiou-Feng Lin

Blockade of TGF-β signaling reactivates HIV-1/SIV reservoirs and immune responses in vivo

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Abstract

TGF-β plays a critical role in maintaining immune cells in a resting state by inhibiting cell activation and proliferation. Resting HIV-1 target cells represent the main cellular reservoir after long-term ART. We hypothesized that releasing cells from TGF-β-driven signaling would promote latency reversal. To test our hypothesis, we compared HIV-1 latency models with and without TGF-β and a TGF-β-Type-1 receptor (TGFBR1) inhibitor, galunisertib. We tested the effect of galunisertib in SIV-infected, ART-treated macaques by monitoring SIV-env expression via PET/CT using the Cu64-anti-gp120 Fab(7D3) probe, along with plasma and tissue viral loads (VL). Exogenous TGF-β reduced HIV-1 reactivation in U1 and ACH2 models. Galunisertib increased HIV-1 latency reversal ex vivo and in PBMC from HIV-1 infected, ART-treated aviremic donors. In vivo, oral galunisertib promoted increased total standardized uptake values (SUVtot) in PET/CT images in gut and lymph nodes of 5 out of 7 aviremic, long-term ART-treated, SIV-infected, macaques. This increase correlated with an increase in SIV-RNA in the gut. Two of the 7 animals also exhibited increases in pVL. Higher anti-SIV T cell responses and antibody titers were detected after galunisertib treatment. In summary, our data suggest that blocking TGF-β signaling simultaneously increases retroviral reactivation events and enhances anti-SIV immune responses.

Authors

Sadia Samer, Yanique Thomas, Mariluz Araínga, Crystal Carter, Lisa M. Shirreff, Muhammad S. Arif, Juan M. Avita, Ines Frank, Michael D. McRaven, Christopher T. Thuruthiyil, Veli B. Heybeli, Meegan R. Anderson, Benjamin Owen, Arsen Gaisin, Deepanwita Bose, Lacy M. Simons, Judd F. Hultquist, James Arthos, Claudia Cicala, Irini Sereti, Philip J. Santangelo, Ramon Lorenzo-Redondo, Thomas J. Hope, Francois Villinger, Elena Martinelli

Dicarbonyl-modified lipoproteins contribute to proteinuric kidney injury

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Abstract

Lipoprotein modification by reactive dicarbonyls, including isolevuglandin (IsoLG), produces dysfunctional particles. Kidneys participate in lipoprotein metabolism, including tubular uptake. However, the process beyond the proximal tubule is unclear, as is the effect of kidney injury on this pathway. We found that patients and animals with proteinuric injury have increased urinary apolipoprotein AI (apoAI), IsoLG, and IsoLG adduct enrichment of the urinary apoAI fraction compared with other proteins. Proteinuric mice, induced by podocyte specific injury, showed more tubular absorption of IsoLG-apoAI and increased expression of lipoprotein transporters in proximal tubular cells compared with uninjured animals. Renal lymph reflects composition of the interstitial compartment, and showed increased apoAI and IsoLG in proteinuric animals, supporting a tubular cell-interstitium-lymph pathway for renal handling of lipoproteins. IsoLG-modified apoAI was not only a marker of renal injury, but also directly damaged renal cells. IsoLG-apoAI increased inflammatory cytokines in cultured tubular epithelial cells, activated lymphatic endothelial cells and caused greater contractility of renal lymphatic vessels than unmodified apoAI. In vivo, inhibition of IsoLG by a dicarbonyl scavenger reduced both albuminuria and urinary apoAI and decreased tubular epithelial cell and lymphatic endothelial cell injury, lymphangiogenesis, and interstitial fibrosis. Our results indicate that IsoLG-modified apolipoprotein AI is a novel pathogenic mediator and therapeutic target in kidney disease.

Authors

Jianyong Zhong, Hai-Chun Yang, Elaine L. Shelton, Taiji Matsusaka, Amanda J. Clark, Valery Yermalitsky, Zahra Mashhadi, Linda S. May-Zhang, MacRae F. Linton, Agnes B. Fogo, Annet Kirabo, Sean S. Davies, Valentina Kon

Keratinocyte-derived cytokine TSLP promotes growth and metastasis of melanoma by regulating the tumor-associated immune microenvironment

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Abstract

Malignant melanoma is a major public health issue displaying frequent resistance to targeted therapy and immunotherapy. A major challenge is to better understand how melanoma cells evade immune elimination and how tumor growth and metastasis is facilitated by tumor microenvironment. Here, we show that expression of the cytokine TSLP by epidermal keratinocytes is induced by cutaneous melanoma in both mice and humans. Using genetically engineered models of melanoma and tumor cell grafting combined with TSLP knockout or overexpression, we defined a crosstalk between melanoma cells, keratinocytes and immune cells in establishing a tumor promoting microenvironment. Keratinocyte-derived TSLP is induced by signal(s) derived from melanoma cells and subsequently acts via immune cells to promote melanoma progression and metastasis. Furthermore, we show that TSLP signals through TSLPR-expressing dendritic cells to play an unrecognized role in promoting GATA3+ Tregs expressing a gene signature including ST2, CCR8, ICOS, PD-1, CTLA-4 and OX40 and exhibiting a potent suppressive activity on CD8+ T cell proliferation and IFNγ production. An analogous population of GATA3-expressing Tregs was also identified in human melanoma tumors. Together, our study provides novel insights into the role of TSLP in programming a pro-tumoral immune microenvironment in cutaneous melanoma.

Authors

Wenjin Yao, Beatriz German, Dounia Chraa, Antoine Braud, Cecile Hugel, Pierre Meyer, Guillaume Davidson, Patrick Laurette, Gabrielle Mengus, Eric Flatter, Pierre Marschall, Justine Segaud, Marine Guivarch, Pierre Hener, Marie-Christine Birling, Dan Lipsker, Irwin Davidson, Mei Li

Insulin increases sensory nerve density and reflex bronchoconstriction in obese mice

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Abstract

Obesity-induced asthma responds poorly to all current pharmacological interventions, including steroids; suggesting that classic, eosinophilic inflammation is not a mechanism. As insulin resistance and hyperinsulinemia are common in obese individuals and associated with increased risk of asthma, we used diet-induced obese mice to study how insulin induces airway hyperreactivity. Inhaled 5-HT or methacholine induced dose dependent bronchoconstriction that was significantly potentiated in obese mice. Cutting the vagus nerves eliminated bronchoconstriction in both obese and non-obese animals indicating it was mediated by a neural reflex. There was significantly greater density of airway sensory nerves in obese than in non-obese mice. Deleting insulin receptors on sensory nerves prevented the increase in sensory nerve density and prevented airway hyperreactivity in obese mice with hyperinsulinemia. Our data demonstrate that high levels of insulin drives obesity-induced airway hyperreactivity by increasing sensory innervation of the lung. Therefore, pharmacological interventions to control metabolic syndrome and limit reflex-mediated bronchoconstriction may be a more effective approach to reduce asthma exacerbations in obese and asthmatic patients.

Authors

Gina N. Calco, Jessica N. Maung, David B. Jacoby, Allison D. Fryer, Zhenying Nie

Beta-cell mass expansion during puberty involves serotonin signaling and determines glucose homeostasis in adulthood

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Abstract

Puberty is associated with transient insulin resistance that normally recedes at the end of puberty; however, in overweight children insulin resistance persists leading to an increased risk of type 2 diabetes. The mechanisms whereby pancreatic β cells adapt to pubertal insulin resistance, and how they are affected by the metabolic status, have not been investigated. Here we show that puberty is associated with a transient increase in β-cell proliferation in rats and humans of both sexes. In rats, β-cell proliferation correlated with a rise in growth hormone (GH) levels. Serum from pubertal rats and humans promoted β-cell proliferation, suggesting the implication of a circulating factor. In pubertal rat islets, expression of genes of the GH/serotonin (5-HT) pathway underwent changes consistent with proliferative effect. Inhibition of the pro-proliferative 5-HT receptor isoform HTR2B blocked the increase in β-cell proliferation in pubertal islets ex vivo and in vivo. Peri-pubertal metabolic stress blunted β-cell proliferation during puberty and led to altered glucose homeostasis later in life. This study identifies a role of GH/GHR/5-HT/HTR2B signaling in the control of β-cell mass expansion during puberty and a mechanistic link between pubertal obesity and the risk of developing type 2 diabetes.

Authors

Anne-Laure Castell, Clara Goubault, Mélanie Ethier, Grace Fergusson, Caroline Tremblay, Marie Baltz, Dorothée Dal Soglio, Julien Ghislain, Vincent Poitout

Glutamine prevents acute kidney injury by modulating oxidative stress and apoptosis in tubular epithelial cells

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Abstract

Acute kidney injury (AKI) represents a common complication in critically ill patients that is associated with increased morbidity and mortality. In a murine AKI model induced by ischemia-reperfusion-injury (IRI), we show that glutamine significantly decreases kidney damage and improves kidney function. We demonstrate that glutamine causes transcriptomic and proteomic reprogramming in murine renal tubular epithelial cells (TECs), resulting in decreased epithelial apoptosis, neutrophil recruitment and improved mitochondrial functionality and respiration provoked by an ameliorated oxidative phosphorylation. We identify the proteins glutamine gamma glutamyltransferase 2 (Tgm2) and apoptosis signal-regulating kinase (Ask1) as the major targets of glutamine in apoptotic signaling. Furthermore, the direct modulation of the Tgm2-HSP70 signalosome and reduced Ask1 activation result in decreased JNK activation leading to diminished mitochondrial intrinsic apoptosis in TECs. Glutamine administration attenuated kidney damage in vivo during AKI and TEC viability in vitro under inflammatory or hypoxic conditions.

Authors

Katharina Thomas, Lisa Zondler, Nadine Ludwig, Marina Kardell, Corinna Lüneburg, Katharina Henke, Sina Mersmann, Andreas Margraf, Tilmann Spieker, Tobias Tekath, Ana Velic, Richard Holtmeier, Juliane Hermann, Vera Jankowski, Melanie Meersch, Dietmar Vestweber, Martin Westphal, Johannes Roth, Michael A. Schaefers, John A. Kellum, Clifford A. Lowell, Jan Rossaint, Alexander Zarbock