Issue published January 9, 2025
- Volume 10, Issue 1
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Khedr et al. report findings supporting that the cGAS/STING pathway represents a therapeutic target to ameliorate the progression of diabetic kidney disease to end-stage kidney disease. The cover image shows a type 2 diabetic human kidney with extensive infiltration of inflammatory cells stained for the STING molecule.
On the cover: Role of cGAS/STING pathway in aging and sexual dimorphism in diabetic kidney disease
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Research Articles
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Abstract
Diabetic kidney disease (DKD) is the leading cause of chronic renal pathology. Understanding the molecular underpinnings of DKD is critical to designing tailored therapeutic approaches. Here, we focused on sex differences and the contribution of aging toward the progression of DKD. To explore these questions, we utilized young (12 weeks old) and aged (approximately 50 weeks old) type 2 diabetic nephropathy (T2DN) rats. We revealed that the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway was upregulated in T2DN rats compared with nondiabetic Wistar rats and in type 2 diabetic human kidneys. The activation of the cGAS/STING signaling pathway exhibited distinct protein expression profiles between male and female T2DN rats, with these differences becoming more pronounced with aging. RNA-Seq analysis of the kidney cortex in both male and female T2DN rats, at both younger and older ages, revealed several key molecules, highlighting crucial genes within the cGAS/STING pathway. Thus, our study delved deep into understanding the intricate sexual differences in the development and progression of DKD and we propose the cGAS/STING pathway as an essential contributor to disease development.
Authors
Sherif Khedr, Lashodya V. Dissanayake, Ammar J. Alsheikh, Adrian Zietara, Denisha R. Spires, Romica Kerketta, Angela J. Mathison, Raul Urrutia, Oleg Palygin, Alexander Staruschenko
×Abstract
The role played by anionic channels in diabetic kidney disease (DKD) is not known. Chloride channel accessory 1 (CLCA1) facilitates the activity of TMEM16A (Anoctamin-1), a Ca2+-dependent Cl– channel. We examined if CLCA1/TMEM16A had a role in DKD. In mice with type 2 diabetes, renal cortical CLCA1 and TMEM16A content was increased. CLCA1 and TMEM16A content was associated with hydrogen sulfide (H2S) deficiency, mTOR complex 1 (mTORC1) activation, albuminuria, and matrix increase. Administering sodium hydrosulfide (NaHS), a source of H2S, mitigated these changes. In proximal tubular epithelial (MCT) cells, high glucose rapidly increased CLCA1 by recruiting the IL-6/STAT3 axis and augmented TMEM16A expression by stimulating its mRNA translation; these changes were abolished by NaHS. Patch clamp experiments showed that high glucose increased Cl– current in MCT cells that was ameliorated by NaHS and a TMEM16A chemical inhibitor. siRNA against CLCA1 or TMEM16A and TMEM16A inhibitor abolished high glucose–induced mTORC1 activation and matrix protein increase. Tubular expression of TMEM16A correlated with albuminuria in kidney biopsies from people with type 2 diabetes. We report a pathway for DKD in which H2S deficiency results in kidney injury by the recruitment of the CLCA1/TMEM16A/Cl– current system.
Authors
Hak Joo Lee, Yuyang Sun, Falguni Das, Wenjun Ju, Viji Nair, Christopher G. Kevil, Shankara Varadarajan, Guanshi Zhang, Goutam Ghosh Choudhury, Brij B. Singh, Matthias Kretzler, Robert G. Nelson, Kumar Sharma, Balakuntalam S. Kasinath
×Abstract
Macrophages play a crucial role in promoting perfusion recovery and revascularization after ischemia through antiinflammatory polarization, a process essential for the treatment of peripheral artery disease (PAD). Mitochondrial dynamics, particularly regulated by the fission protein DRP1, are closely linked to macrophage metabolism and inflammation. However, the role of DRP1 in reparative neovascularization remains unexplored. Here, we show that DRP1 expression was increased in F4/80+ macrophages within ischemic muscle on day 3 after hind limb ischemia (HLI), an animal model of PAD. Mice lacking Drp1 in myeloid cells exhibited impaired limb perfusion recovery, angiogenesis, and muscle regeneration after HLI. These effects were associated with increased proinflammatory M1-like macrophages, p-NF-κB, and TNF-α, and reduced antiinflammatory M2-like macrophages and p-AMPK in ischemic muscle of myeloid Drp1–/– mice. In vitro, Drp1-deficient macrophages under hypoxia serum starvation (HSS), an in vitro PAD model, demonstrated enhanced glycolysis via reducing p-AMPK as well as mitochondrial dysfunction, and excessive mitochondrial ROS production, resulting in increased proinflammatory M1-gene and reduced antiinflammatory M2-gene expression. Conditioned media from HSS-treated Drp1–/– macrophages exhibited increased proinflammatory cytokine secretion, leading to suppressed angiogenesis in endothelial cells. Thus, macrophage DRP1 deficiency under ischemia drives proinflammatory metabolic reprogramming and macrophage polarization, limiting revascularization in experimental PAD.
Authors
Shikha Yadav, Vijay C. Ganta, Sudhahar Varadarajan, Vy Ong, Yang Shi, Archita Das, Dipankar Ash, Sheela Nagarkoti, Malgorzata McMenamin, Stephanie Kelley, Tohru Fukai, Masuko Ushio-Fukai
×Abstract
Tumor-associated macrophages (TAMs) are one of the key immunosuppressive components in the tumor microenvironment (TME) and contribute to tumor development, progression, and resistance to cancer immunotherapy. Several reagents targeting TAMs have been tested in preclinical and clinical studies, but they have had limited success. Here, we show that a unique reagent, FF-10101, exhibited a sustained inhibitory effect against colony-stimulating factor 1 receptor by forming a covalent bond and reduced immunosuppressive TAMs in the TME, which led to strong antitumor immunity. In preclinical animal models, FF-10101 treatment significantly reduced immunosuppressive TAMs and increased antitumor TAMs in the TME. In addition, tumor antigen-specific CD8+ T cells were increased; consequently, tumor growth was significantly inhibited. Moreover, combination treatment with an anti–programmed cell death 1 (anti–PD-1) antibody and FF-10101 exhibited a far stronger antitumor effect than either treatment alone. In human cancer specimens, FF-10101 treatment reduced programmed cell death 1 ligand 1 (PD-L1) expression on TAMs, as observed in animal models. Thus, FF-10101 acts as an immunomodulatory agent that can reduce immunosuppressive TAMs and augment tumor antigen-specific T cell responses, thereby generating an immunostimulatory TME. We propose that FF-10101 is a potential candidate for successful combination cancer immunotherapy with immune checkpoint inhibitors, such as PD-1/PD-L1 blockade.
Authors
Takahiko Sato, Daisuke Sugiyama, Jun Koseki, Yasuhiro Kojima, Satomi Hattori, Kazuki Sone, Hitomi Nishinakamura, Tomohiro Ishikawa, Yuichi Ishikawa, Takuma Kato, Hitoshi Kiyoi, Hiroyoshi Nishikawa
×Abstract
Soft tissue trauma can cause immune system disturbance and neuropathological invasion, resulting in heterotopic ossification (HO) due to aberrant chondrogenic differentiation of mesenchymal stem cells (MSCs). However, the molecular mechanisms behind the interaction between the immune and nervous systems in promoting HO pathogenesis are unclear. In this study, we found that mast cell–specific deletion attenuated localized tissue inflammation, with marked inhibition of HO endochondral osteogenesis. Likewise, blockage of nerve growth factor (NGF) receptor, known as tropomyosin receptor kinase A (TrkA), led to similar attenuations in tissue inflammation and HO. Moreover, while NGF/TrkA signaling did not directly affect MSCs chondrogenic differentiation, it modulated mast cell activation in traumatic soft tissue. Mechanistically, lipid A in LPS binding to TrkA enhanced NGF-induced TrkA phosphorylation, synergistically stimulating mast cells to release neurotrophin-3 (NT3), thereby promoting MSC chondrogenic differentiation in situ. Finally, analysis of single-cell datasets and human pathological specimens confirmed the important role of mast cell–mediated neuroinflammation in HO pathogenesis. In conclusion, NGF regulates mast cells in soft tissue trauma and drives HO progression via paracrine NT3. Targeted early inhibition of mast cells holds substantial promise for treating traumatic HO.
Authors
Tao Jiang, Xiang Ao, Xin Xiang, Jie Zhang, Jieyi Cai, Jiaming Fu, Wensheng Zhang, Zhenyu Zheng, Jun Chu, Minjun Huang, Zhongmin Zhang, Liang Wang
×Abstract
The oral mucosa undergoes daily insults, and stem cells in the epithelial basal cell layer regenerate gingiva tissue to maintain oral health. The Iroquois Homeobox 1 (IRX1) protein is expressed in the stem cell niches in human/mouse oral epithelium and mesenchyme under homeostasis. We found that Irx1+/– heterozygous (Het) mice have delayed wound closure, delayed morphological changes of regenerated epithelium, and defective keratinocyte proliferation and differentiation during wound healing. RNA-Seq analyses between WT and Irx1+/– mice at 3 days postinjury (dpi) found impaired epithelial migration and decreased keratinocyte-related genes upon injury. IRX1-expressing cells are found in the gingival epithelial basal cell layer, a stem cell niche for gingival maintenance. IRX1-expressing cells are also found in cell niches in the underlying stroma. IRX1 activates SOX9 in the transient amplifying layer to increase cell proliferation, and EGF signaling is activated to induce cell migration. Krt14CreERT lineage tracing experiments reveal defects in the stratification of the Irx1+/– HET mouse oral epithelium. IRX1 is primed at the base of the gingiva in the basal cell layer of the oral epithelium, facilitating rapid and scarless wound healing through activating SOX9 and the EGF signaling pathway.
Authors
Dan Su, Tadkamol Krongbaramee, Samuel Swearson, Yan Sweat, Mason Sweat, Fan Shao, Steven Eliason, Brad A. Amendt
×Abstract
Urinary neutrophils are a hallmark of urinary tract infection (UTI), yet the mechanisms governing their activation, function, and efficacy in controlling infection remain incompletely understood. Tamm-Horsfall glycoprotein (THP), the most abundant protein in urine, uses terminal sialic acids to bind an inhibitory receptor and dampen neutrophil inflammatory responses. We hypothesized that neutrophil modulation is an integral part of THP-mediated host protection. In a UTI model, THP-deficient mice showed elevated urinary tract bacterial burdens, increased neutrophil recruitment, and more severe tissue histopathological changes compared with WT mice. Furthermore, THP-deficient mice displayed impaired urinary NETosis during UTI. To investigate the effect of THP on NETosis, we coupled in vitro fluorescence-based NET assays, proteomic analyses, and standard and imaging flow cytometry with peripheral human neutrophils. We found that THP increases proteins involved in respiratory chain, neutrophil granules, and chromatin remodeling pathways; enhances NETosis in an ROS-dependent manner; and drives NET-associated morphologic features including nuclear decondensation. These effects were observed only in the presence of a NETosis stimulus and could not be solely replicated with equivalent levels of sialic acid alone. We conclude that THP is a critical regulator of NETosis in the urinary tract, playing a key role in host defense against UTI.
Authors
Vicki Mercado-Evans, Holly Branthoover, Claude Chew, Camille Serchejian, Alexander B. Saltzman, Marlyd E. Mejia, Jacob J. Zulk, Ingrid Cornax, Victor Nizet, Kathryn A. Patras
×Abstract
CD4+ T helper 1 (Th1) cells coordinate adaptive immune responses to intracellular pathogens, including viruses. Key to this function is the ability of Th1 cells to migrate within secondary lymphoid tissues, as well as to sites of inflammation, which relies on signals received through the chemokine receptor CXCR3. CXCR3 expression is driven by the Th1 lineage-defining transcription factor T-bet and the cytokine-responsive STAT family members STAT1 and STAT4. Here, we identify the Ikaros zinc finger (IkZF) transcription factor Aiolos (Ikzf3) as an additional positive regulator of CXCR3 both in vitro and in vivo using a murine model of influenza virus infection. Mechanistically, we found that Aiolos-deficient CD4+ T cells exhibited decreased expression of key components of the IFN-γ/STAT1 signaling pathway, including JAK2 and STAT1. Consequently, Aiolos deficiency resulted in decreased levels of STAT1 tyrosine phosphorylation and reduced STAT1 enrichment at the Cxcr3 promoter. We further found that Aiolos and STAT1 formed a positive feedback loop via reciprocal regulation of each other downstream of IFN-γ signaling. Collectively, our study demonstrates that Aiolos promotes CXCR3 expression on Th1 cells by propagating the IFN-γ/STAT1 cytokine signaling pathway.
Authors
Melissa R. Leonard, Devin M. Jones, Kaitlin A. Read, Srijana Pokhrel, Jasmine A. Tuazon, Robert T. Warren, Jacob S. Yount, Kenneth J. Oestreich
×Abstract
Bone homeostasis primarily stems from the balance between osteoblasts and osteoclasts, wherein an augmented number or heightened activity of osteoclasts is a prevalent etiological factor in the development of bone loss. Nuclear Dbf2-related kinase (NDR2), also known as STK38L, is a member of the Hippo family with serine/threonine kinase activity. We unveiled an upregulation of NDR2 expression during osteoclast differentiation. Manipulation of NDR2 levels through knockdown or overexpression facilitated or hindered osteoclast differentiation, respectively, indicating a negative feedback role for NDR2 in the osteoclastogenesis. Myeloid NDR2-dificient mice (Lysm+NDR2fl/fl) showed lower bone mass and further exacerbated ovariectomy-induced or aging-related bone loss. Mechanically, NDR2 enhanced autophagy and mitophagy through mediating ULK1 instability. In addition, ULK1 inhibitor (ULK1-IN2) ameliorated NDR2 conditional KO–induced bone loss. Finally, we clarified a significant inverse association between NDR2 expression and the occurrence of osteoporosis in patients. The NDR2/ULK1/mitophagy axis is a potential innovative therapeutic target for the prevention and management of bone loss.
Authors
Xiangxi Kong, Zhi Shan, Yihao Zhao, Siyue Tao, Jingyun Chen, Zhongyin Ji, Jiayan Jin, Junhui Liu, Wenlong Lin, Xiao-jian Wang, Jian Wang, Fengdong Zhao, Bao Huang, Jian Chen
×Abstract
Although biologics have been revolutionizing the treatment of inflammatory bowel diseases (IBD) over the past decade, a significant number of patients still fail to benefit from these drugs. Overcoming the nonresponse to biologics is one of the top challenges in IBD treatment. In this study, we revealed that hyaluronan (HA), an extracellular matrix (ECM) component in the gut, is associated with nonresponsiveness to infliximab and vedolizumab therapy in patients with IBD. In murine colitis models, inhibition of HA synthase 2–mediated (HAS2-mediated) HA synthesis sensitized the therapeutic response to infliximab. Mechanistically, HA induced the expression of MMP3 in colonic fibroblasts by activating STAT3 signaling, thereby mediating the proteolytic cleavage of multiple IgG1 biologics. Finally, we found that macrophage-derived factors upregulated HAS2 expression in fibroblasts, thereby contributing to infliximab nonresponse. In summary, we identified a pathogenic connection between abnormal ECM remodeling and biologics nonresponse and provided insights for the precise therapy for IBD.
Authors
Peng Xiao, Zhehang Chen, Xuechun Cai, Wenhao Xia, Xia Liu, Zhangfa Song, Huijuan Wang, Yuening Zhao, Youling Huang, Yu Zhang, Ke Guo, Haotian Chen, Rongbei Liu, Changcheng Meng, Yanfei Fang, Yunkun Lu, Qian Cao
×Abstract
CD4+ T cells contribute to antitumor immunity and are implicated in the efficacy of cancer immunotherapies. In particular, CD4+ T helper 2 (Th2) cells were recently found to block spontaneous breast carcinogenesis. However, the antitumor potential of Th2 cells in targeting established breast cancer remains uncertain. Herein, we demonstrate that Th2 cells induced by the topical calcipotriol/thymic stromal lymphopoietin cytokine axis suppressed the growth of established mammary tumors in mice. Interleukin-24 (IL-24), an anticancer cytokine, was highly upregulated in macrophages infiltrating calcipotriol-treated mammary tumors. Macrophages expressed IL-24 in response to IL-4 signaling in combination with Toll-like receptor 4 (TLR4) agonists (e.g., HMGB1) in vitro. Calcipotriol treatment significantly increased HMGB1 release by tumor cells in vivo. CD4+ T cell depletion reduced HMGB1 and IL-24 expression, reversing calcipotriol’s therapeutic efficacy. Macrophage depletion and TLR4 inhibition also reduced the therapeutic efficacy of calcipotriol. Importantly, calcipotriol treatment failed to control mammary tumors lacking the IL-24 receptor on tumor cells. Collectively, our findings reveal that Th2 cell–macrophage crosstalk leads to IL-24–mediated tumor cell death, highlighting a promising therapeutic strategy to tackle breast cancer.
Authors
Bo Wang, Yun Xia, Can Zhou, Yuhan Zeng, Heehwa G. Son, Shadmehr Demehri
×Abstract
The impact of diet-induced maternal obesity on offspring airway hyperresponsiveness was studied in a diversity outbred mouse model that mirrors human genetic diversity. Female mice were started on high-fat or regular diet 8 weeks before breeding and throughout pregnancy and lactation. After weaning, all offspring were fed a regular diet. By 12 weeks, body weight and fat were increased in offspring of high-fat diet–fed dams, which was accompanied by metabolic dysfunction and hyperinsulinemia. This was followed by increased epithelial sensory innervation and increased bronchoconstriction to inhaled 5-hydroxytryptamine at 16 weeks. Bronchoconstriction was nerve mediated and blocked by vagotomy or atropine. A high-fat diet before pregnancy exerted the most influence on offspring airway physiology. Maternal obesity induced metabolic dysfunction and hyperinsulinemia, resulting in hyperinnervation and subsequent increased reflex-mediated hyperresponsiveness in their offspring. This is relevant to our understanding of asthma inheritance, considering the genetic diversity of humans.
Authors
Kayla R. Williams, Hoyt A.T.K. Bright, Allison D. Fryer, David B. Jacoby, Zhenying Nie
×Abstract
The degeneration of retinal ganglion cells (RGC) due to mitochondrial dysfunctions manifests optic neuropathy. However, the molecular components of RGC linked to optic neuropathy manifestations remain largely unknown. Here, we identified a potentially novel optic atrophy-causative CRYAB gene encoding a highly conserved major lens protein acting as mitochondrial chaperone and possessing antiapoptotic activities. The heterozygous CRYAB mutation (c.313G>A, p. Glu105Lys) was cosegregated with autosomal dominant inheritance of optic atrophy in 3 Chinese families. The p.E105K mutation altered the structure and function of CRYAB, including decreased stability, reduced formation of oligomers, and decreased chaperone activity. Coimmunoprecipitation indicated that the p.E105K mutation reduced the interaction of CRYAB with apoptosis-associated cytochrome c and voltage-dependent anion channel protein. The cell lines carrying the p.E105K mutation displayed promotion of apoptosis and defective assembly, stability, and activities of oxidative phosphorylation system as well as imbalance of mitochondrial dynamics. Involvement of CRYAB in optic atrophy was confirmed by phenotypic evaluations of Cryabp.E105K-knockin mice. These mutant mice exhibited ocular lesions that included alteration of intraretinal layers, degeneration of RGCs, photoreceptor deficits, and abnormal retinal vasculature. Furthermore, Cryab-deficient mice displayed elevated apoptosis and mitochondrial dysfunctions. Our findings provide insight of pathophysiology of optic atrophy arising from RGC degeneration caused by CRYAB deficiency–induced elevated apoptosis and mitochondrial dysfunctions.
Authors
Chenghui Wang, Liyao Zhang, Zhipeng Nie, Min Liang, Hanqing Liu, Qiuzi Yi, Chunyan Wang, Cheng Ai, Juanjuan Zhang, Yinglong Gao, Yanchun Ji, Min-Xin Guan
×Abstract
Lung cancer is the leading cause of cancer deaths in the United States. New targeted therapies against the once-deemed undruggable oncogenic KRAS are changing current therapeutic paradigms. However, resistance to targeted KRAS inhibitors almost inevitably occurs; resistance can be driven by tumor cell–intrinsic changes or by changes in the microenvironment. Here, we utilized a genetically engineered mouse model of KRASG12D-driven lung cancer that allows for inducible and reversible expression of the oncogene: activation of oncogenic KRASG12D induces tumor growth; conversely, inactivation of KRASG12D causes tumor regression. We showed that in addition to regulating cancer cell growth and survival, oncogenic KRAS regulated the transcriptional status of cancer-associated fibroblasts and macrophages in this model. Utilizing ex vivo approaches, we showed that secreted factors from cancer cells induced the expression of multiple cytokines in lung fibroblasts, and in turn drove expression of immunosuppressive factors, such as arginase 1, in macrophages. In summary, fibroblasts emerged as a key source of immune regulatory signals, and a potential therapeutic target for improving the efficacy of KRAS inhibitors in lung cancer.
Authors
Emily L. Lasse-Opsahl, Ivana Barravecchia, Elyse McLintock, Jennifer M. Lee, Sarah F. Ferris, Carlos E. Espinoza, Rachael Hinshaw, Sophia Cavanaugh, Marzia Robotti, Lily Rober, Kristee Brown, Kristena Y. Abdelmalak, Craig J. Galban, Timothy L. Frankel, Yaqing Zhang, Marina Pasca di Magliano, Stefanie Galban
×Abstract
High apolipoprotein B–containing (apoB-containing) low-density lipoproteins (LDLs) and low apoA1–containing high-density lipoproteins (HDLs) are associated with atherosclerotic cardiovascular diseases. In search of a molecular regulator that could simultaneously and reciprocally control both LDL and HDL levels, we screened a microRNA (miR) library using human hepatoma Huh-7 cells. We identified miR-541-3p that both significantly decreases apoB and increases apoA1 expression by inducing mRNA degradation of 2 different transcription factors, Znf101 and Casz1. We found that Znf101 enhances apoB expression, while Casz1 represses apoA1 expression. The hepatic knockdown of Casz1 in mice increased plasma apoA1, HDL, and cholesterol efflux capacity. The hepatic knockdown of Zfp961, an ortholog of Znf101, reduced lipogenesis and production of triglyceride-rich lipoproteins and atherosclerosis, without causing hepatic lipid accumulation. This study identifies hepatic Znf101/Zfp961 and Casz1 as potential therapeutic targets to alter plasma lipoproteins and reduce atherosclerosis without causing liver steatosis.
Authors
Abulaish Ansari, Pradeep Kumar Yadav, Liye Zhou, Binu Prakash, Laraib Ijaz, Amanda Christiano, Sameer Ahmad, Antoine Rimbert, M. Mahmood Hussain
×Abstract
Hepatic macrophages and regulatory T cells (Tregs) play an important role in the maintenance of liver immune homeostasis, but the mechanism by which hepatic macrophages regulate Tregs in acute liver injury remains largely unknown. Here, we found that the hepatic Treg proportion and β-catenin expression in hepatic macrophages were associated with acetaminophen- and d-galactosamine/LPS–induced acute liver injury. Interestingly, β-catenin was markedly upregulated only in infiltrating macrophages but not in resident Kupffer cells. Myeloid-specific β-catenin–knockout mice showed an increased inflammatory cell infiltration and hepatocyte apoptosis. Moreover, myeloid β-catenin deficiency decreased the hepatic Treg proportion in the injured liver. Mechanistically, in vitro coculture experiments revealed that macrophage β-catenin modulated its exosome composition and influenced Treg differentiation. Using mass spectrometry–based proteomics, we identified that macrophage β-catenin activation increased the level of exosomal alpha soluble NSF attachment protein (α-SNAP), which in turn promoted Treg differentiation. Overall, our findings demonstrated a molecular mechanism that macrophage β-catenin regulated the Treg proportion in the liver by enhancing the expression of exosomal α-SNAP, providing insights into the pathophysiology of acute liver injury.
Authors
Ruobin Zong, Yujie Liu, Mengya Zhang, Buwei Liu, Wei Zhang, Hankun Hu, Changyong Li
×Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has emerged as a global pandemic pathogen with high mortality. While treatments have been developed to reduce morbidity and mortality of COVID-19, more antivirals with broad-spectrum activities are still needed. Here, we identified lonafarnib (LNF), a Food and Drug Administration–approved inhibitor of cellular farnesyltransferase (FTase), as an effective anti–SARS-CoV-2 agent. LNF inhibited SARS-CoV-2 infection and acted synergistically with known anti-SARS antivirals. LNF was equally active against diverse SARS-CoV-2 variants. Mechanistic studies suggested that LNF targeted multiple steps of the viral life cycle. Using other structurally diverse FTase inhibitors and a LNF-resistant FTase mutant, we demonstrated a key role of FTase in the SARS-CoV-2 life cycle. To demonstrate in vivo efficacy, we infected SARS-CoV-2–susceptible humanized mice expressing human angiotensin-converting enzyme 2 (ACE2) and treated them with LNF. LNF at a clinically relevant dose suppressed the viral titer in the respiratory tract and improved pulmonary pathology and clinical parameters. Our study demonstrated that LNF, an approved oral drug with excellent human safety data, is a promising antiviral against SARS-CoV-2 that warrants further clinical assessment for treatment of COVID-19 and potentially other viral infections.
Authors
Mohsin Khan, Parker Irvin, Seung Bum Park, Hannah M. Ivester, Inna Ricardo-Lax, Madeleine Leek, Ailis Grieshaber, Eun Sun Jang, Sheryl Coutermarsh-Ott, Qi Zhang, Nunziata Maio, Jian-Kang Jiang, Bing Li, Wenwei Huang, Amy Q. Wang, Xin Xu, Zongyi Hu, Wei Zheng, Yihong Ye, Tracey Rouault, Charles Rice, Irving C. Allen, T. Jake Liang
×Abstract
Both CO2 retention, or hypercapnia, and skeletal muscle dysfunction predict higher mortality in critically ill patients. Mechanistically, muscle injury and reduced myogenesis contribute to critical illness myopathy, and while hypercapnia causes muscle wasting, no research has been conducted on hypercapnia-driven dysfunctional myogenesis in vivo. Autophagy flux regulates myogenesis by supporting skeletal muscle stem cell — satellite cell — activation, and previous data suggest that hypercapnia inhibits autophagy. We tested whether hypercapnia worsens satellite cell autophagy flux and myogenic potential and if autophagy induction reverses these deficits. Satellite cell transplantation and lineage-tracing experiments showed that hypercapnia undermined satellite cells’ activation, replication, and myogenic capacity. Bulk and single-cell sequencing analyses indicated that hypercapnia disrupts autophagy, senescence, and other satellite cell programs. Autophagy activation was reduced in hypercapnic cultured myoblasts, and autophagy genetic knockdown phenocopied these changes in vitro. Rapamycin stimulation led to AMPK activation and downregulation of the mTOR pathway, which are both associated with accelerated autophagy flux and cell replication. Moreover, hypercapnic mice receiving rapamycin showed improved satellite cell autophagy flux, activation, replication rate, and posttransplantation myogenic capacity. In conclusion, we have shown that hypercapnia interferes with satellite cell activation, autophagy flux, and myogenesis, and systemic rapamycin administration improves these outcomes.
Authors
Joseph Balnis, Emily L. Jackson, Lisa A. Drake, Diane V. Singer, Ramon Bossardi Ramos, Harold A. Singer, Ariel Jaitovich
×Abstract
BACKGROUND Cow’s milk (CM) allergy is the most common food allergy in young children. Treatment with oral immunotherapy (OIT) has shown efficacy, but high rates of adverse reactions. The aim of this study was to determine whether baked milk OIT (BMOIT) could reduce adverse reactions while still inducing desensitization, and to identify immunological correlates of successful BMOIT.METHODS This phase II, randomized trial evaluated the safety and efficacy of BMOIT in milk-allergic children 3–18 years old. After the initial placebo-controlled first year of treatment, placebo-treated participants crossed over to active BMOIT. Double-blind, placebo-controlled oral food challenges (OFCs) were conducted with BM after year 1 and to both BM and unheated milk (UM) after year 2. IgG and IgE antibodies were measured along with CM-specific (CM+) CD4+ memory T cell populations, profiled using flow cytometry and scRNA-Seq.RESULTS Twenty-one of 30 (70%) reached the primary endpoint of tolerating 4044 mg of BM protein at month 24, and 11 of 30 tolerated 2000 mg or more of UM protein. Dosing symptoms were common, but more than 98% were mild, with no severe reactions. Immunological changes associated with desensitization included increased CM IgG4, CM+ FOXP3+ cells, and Tregs and corresponding decreases in CM IgE, CM+ Th2A cells, and CD154+ cells. T cell and antibody measurements were combined to build a model that predicted UM OFC outcomes.CONCLUSION BMOIT was well tolerated and induced desensitization to BM and UM. This desensitization corresponded to redistribution within antigen-specific antibody and T cell compartments that provided insight into the mechanistic changes that occur with OIT treatment.TRIAL REGISTRATION ClinicalTrials.gov NCT03462030.FUNDING: Myra Reinhardt Family Foundation (grant number 128388), NIH/NIAID (U19AI135731, T32AI125179, S10OD025052)
Authors
Jennifer A. Dantzer, Sloan A. Lewis, Kevin J. Psoter, Aaron Sutherland, April Frazier, Eve Richardson, Synaida Maiche, Gregory Seumois, Bjoern Peters, Robert A. Wood
×Abstract
The availability and integration of electrophysiological and molecular data from the living brain is critical in understanding and diagnosing complex human disease. Intracranial stereo electroencephalography (SEEG) electrodes used for identifying the seizure focus in patients with epilepsy could enable the integration of such multimodal data. Here, we report multimodal profiling of epileptic brain activity via explanted depth electrodes (MoPEDE), a method that recovers extensive protein-coding transcripts, including cell type markers, DNA methylation, and short variant profiles from explanted SEEG electrodes matched with electrophysiological and radiological data allowing for high-resolution reconstructions of brain structure and function. We found gene expression gradients that corresponded with the neurophysiology-assigned epileptogenicity index but also outlier molecular fingerprints in some electrodes, potentially indicating seizure generation or propagation zones not detected during electroclinical assessments. Additionally, we identified DNA methylation profiles indicative of transcriptionally permissive or restrictive chromatin states and SEEG-adherent differentially expressed and methylated genes not previously associated with epilepsy. Together, these findings validate that RNA profiles and genome-wide epigenetic data from explanted SEEG electrodes offer high-resolution surrogate molecular landscapes of brain activity. The MoPEDE approach has the potential to enhance diagnostic decisions and deepen our understanding of epileptogenic network processes in the human brain.
Authors
Anuj Kumar Dwivedi, Arun Mahesh, Albert Sanfeliu, Julian Larkin, Rebecca A. Siwicki, Kieron J. Sweeney, Donncha F. O’Brien, Peter Widdess-Walsh, Simone Picelli, David C. Henshall, Vijay K. Tiwari
×Abstract
The cytokine IL-18 has immunostimulatory effects but is negatively regulated by a secreted binding protein, IL-18BP, that limits IL-18’s anticancer efficacy. A decoy-resistant form of IL-18 (DR-18) that avoids sequestration by IL-18BP while maintaining its immunostimulatory potential has recently been developed. Here, we investigated the therapeutic potential of DR-18 in renal cell carcinoma (RCC). Using pantumor transcriptomic data, we found that clear cell RCC had among the highest expression of IL-18 receptor subunits and IL18BP of tumor types in the database. In samples from patients with RCC treated with immune checkpoint inhibitors, IL-18BP protein expression increased in the tumor microenvironment and in circulation within plasma in nonresponding patients, and it decreased in the majority of responding patients. We used immunocompetent RCC murine models to assess the efficacy of DR-18 in combination with single- and dual-agent anti–PD-1 and anti–CTLA-4. In contrast to preclinical models of other tumor types, in RCC models, DR-18 enhanced the activity of anti–CTLA-4 but not anti–PD-1 treatment. This activity correlated with intratumoral enrichment and clonal expansion of effector CD8+ T cells, decreased Treg levels, and enrichment of proinflammatory antitumor myeloid cell populations. Our findings support further clinical investigation of the combination of DR-18 and anti–CTLA-4 in RCC.
Authors
David A. Schoenfeld, Dijana Djureinovic, David G. Su, Lin Zhang, Benjamin Y. Lu, Larisa Kamga, Jacqueline E. Mann, John D. Huck, Michael Hurwitz, David A. Braun, Lucia Jilaveanu, Aaron M. Ring, Harriet M. Kluger
×Insulin mitigates acute ischemia–induced atrial fibrillation and sinoatrial node dysfunction ex vivo
Abstract
Acute atrial ischemia is a well-known cause of postoperative atrial fibrillation (POAF). However, mechanisms through which ischemia contributes to the development of POAF are not well understood. In this study, ex vivo Langendorff perfusion was used to induce acute ischemia/reperfusion in the heart to mimic POAF. Inducibility of atrial fibrillation (AF) was evaluated using programmed electrical stimulation and verified with open-atrium optical mapping. Compared with the control group without ischemia, 25 minutes of ischemia substantially increased the incidence of AF. The right atrium was more susceptible to AF than the left atrium. Administering insulin for 30 minutes before ischemia and during reperfusion with 25 minutes of ischemia greatly reduced the vulnerability to AF. However, insulin treatment during reperfusion only did not show substantial benefits against AF. Optical mapping studies showed that insulin mitigated ischemia-induced abnormal electrophysiology, including shortened action potential duration and effective refractory period, slowed conduction velocity, increased conduction heterogeneity, and altered calcium transients. In conclusion, insulin reduced the risk of acute ischemia/reperfusion–induced AF via improving the electrophysiology and calcium handling of atrial cardiomyocytes, which provides a potential therapy for POAF.
Authors
Huiliang Qiu, Fan Li, Hannah Prachyl, Alejandra Patino-Guerrero, Michael Rubart, Wuqiang Zhu
×Abstract
HIV-1 reservoir cells persist indefinitely during suppressive antiretroviral therapy (ART) in individuals who acquire infection in adulthood, but little is known about the longitudinal evolution of viral reservoir cells during long-term ART started during early infancy. We studied 2 fraternal twins who acquired HIV-1 perinatally, started ART at week 10 after birth and remained on ART for 28 years. We observed that the frequency of genome-intact proviruses, determined by single-genome near–full-length proviral sequencing, declined by approximately 4,000- to 13,000-fold during this period, indicating enhanced decay rates of intact proviruses even after adjusting for dilution effects from somatic growth. Despite analyzing more than one billion PBMC after 28 years of ART in each participant, no intact proviruses were detected in 1 participant, and 1 intact provirus was isolated in the other. The longitudinal decline of defective proviruses in the 2 participants was more similar to proviral decay kinetics reported in individuals who started ART during adulthood; moreover, clonal sequence clusters were readily detectable for defective proviruses but not for intact proviruses after 28 years of ART in the 2 twins. Together, these data suggest decreased long-term stability and increased immunological vulnerability of intact proviruses during long-term ART started in early infancy.
Authors
Liliana C. Vela, Leah Carrere, Chloe Naasz, Sruthi Kalavacherla, Toong Seng Tan, Lesley de Armas, Ce Gao, Xu G. Yu, Savita G. Pahwa, Katherine Luzuriaga, Mathias Lichterfeld
