We describe a new mechanism responsible for Systemic Lupus Erythematosus (SLE). In humans with SLE and in two SLE murine models, there is marked enrichment of isolevuglandin (isoLG)-adducted proteins in monocytes and dendritic cells (DCs). We found that antibodies form against isoLG adducts in both SLE-prone mice and humans with SLE. In addition, isoLG ligation of the transcription factor PU.1 at a critical DNA binding site markedly reduces transcription of all C1q subunits. Treatment of SLE prone mice with the specific isoLG scavenger 2-hydroxybenzlyamine (2HOBA) ameliorates parameters of autoimmunity including plasma cell expansion, circulating IgG levels, and anti-dsDNA antibody titers. 2-HOBA also lowers blood pressure, attenuates renal injury, and reduces inflammatory gene expression uniquely in C1q expressing dendritic cells. Thus, isoLG adducts play an essential role in the genesis and maintenance of systemic autoimmunity and hypertension in SLE.
David M. Patrick, Néstor de la Visitación, Jaya Krishnan, Wei Chen, Michelle J. Ormseth, C. Michael Stein, Sean S. Davies, Venkataraman Amarnath, Leslie J. Crofford, Jonathan M. Williams, Shilin Zhao, Charles D. Smart, Sergey Dikalov, Anna Dikalova, Liang Xiao, Justin P. Van Beusecum, Mingfang Ao, Agnes B. Fogo, Annet Kirabo, David G. Harrison
Nephrolithiasis is a common and recurrent disease affecting 9% of the US population. Hyperoxaluria is major risk factor for calcium oxalate kidney stones, which constitutes two-thirds of all kidney stones. Gastrointestinal epithelia play an important role in oxalate handling due to the presence of SLC26A family anion exchangers that facilitate oxalate transport. SLC26A3 (originally named DRA, down-regulated in adenoma) is an anion exchanger of chloride, bicarbonate and oxalate thought to facilitate intestinal oxalate absorption, as evidenced by ~70% reduced urine oxalate excretion in knock-out mice. We previously identified, by high-throughput screening and medicinal chemistry, a small molecule SLC26A3 inhibitor (DRAinh-A270) that selectively inhibited SLC26A3-mediated chloride/bicarbonate exchange (IC50 ~ 35 nM), and, as found here, oxalate/chloride exchange (IC50 ~ 60 nM). In colonic closed loops in mice, luminal DRAinh-A270 inhibited oxalate absorption by 70%. In a model of acute hyperoxaluria produced by oral sodium oxalate loading, DRAinh-A270 largely prevented the ~2.5-fold increase in urine oxalate/creatinine ratio. In a mouse model of oxalate nephropathy produced by a high-oxalate low-calcium diet, vehicle-treated mice by day 14 developed marked hyperoxaluria with elevated serum creatinine, renal calcium oxalate crystal deposition and renal injury, which were largely prevented by DRAinh-A270 (10 mg/kg twice daily). DRAinh-A270 administered over 7 days to healthy mice did not show significant toxicity as assessed by CBC, serum chemistries and tissue histology. Our findings support a major role of SLC26A3 in intestinal oxalate absorption and suggest the therapeutic utility of SLC26A3 inhibition for treatment of hyperoxaluria and prevention of calcium oxalate nephrolithiasis.
Onur Cil, Qi Tifany Chu, Sujin Lee, Peter M. Haggie, Alan S. Verkman
Chronic type 2 (T2) inflammatory diseases of the respiratory tract are characterized by mucus overproduction and disordered mucociliary function, which are largely attributed to the effects of IL-13 on common epithelial cell types (mucus secretory and ciliated cells). The role of rare cells in airway T2 inflammation is less clear, though tuft cells have been shown to be critical in the initiation of T2 immunity in the intestine. Using bulk and single cell RNA sequencing of airway epithelium and mouse modeling, we find that IL-13 expands and programs airway tuft cells towards eicosanoid metabolism, and that tuft cell deficiency leads to a reduction in airway prostaglandin E2 (PGE2)concentration. Allergic airway epithelia bear a signature of prostaglandin E2 activation, and PGE2 activation leads to CFTR-dependent ion and fluid secretion and accelerated mucociliary transport. Together these data reveal a role for tuft cells in regulating epithelial mucociliary function in the allergic airway.
Maya E Kotas, Camille M. Moore, Jose G. Gurrola II, Steven D. Pletcher, Andrew N. Goldberg, Raquel Alvarez, Sheyla Yamato, Preston E. Bratcher, Ciaran A. Shaughnessy, Pamela L. Zeitlin, Irene H Zhang, Yingchun Li, Michael T. Montgomery, Keehoon Lee, Emily K. Cope, Richard M. Locksley, Max A. Seibold, Erin D. Gordon
The transcription factor Signal transducer and activator of transcription 1 (STAT1) plays a critical role in modulating the differentiation of CD4+ T cells producing IL-17 and GM-CSF, which promote the development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). The protective role of STAT1 in MS and EAE has been largely attributed to its ability to limit pathogenic T helper (Th) cells and promote regulatory T (Treg) cells. Using mice with selective deletion of STAT1 in T cells (STAT1CD4-Cre), we identify a novel mechanism by which STAT1 regulates neuroinflammation independently of Foxp3+ Treg cells. STAT1-deficient effector T cells become the target of NK cell-mediated killing, limiting their capacity to induce EAE. STAT1-deficient T cells promoted their own killing by producing more IL-2 that in return activated NK cells. Elimination of NK cells restored EAE susceptibility in STAT1CD4-Cre mice. Therefore, our study suggests that the STAT1 pathway can be manipulated to limit autoreactive T cells during autoimmunity directed against the central nervous system.
Carlos A. Arbelaez, Pushpalatha Palle, Jonathan Charaix, Estelle Bettelli
The Aedes aegypti mosquito transmits both dengue (DENV) and Zika (ZIKV) viruses. Individuals in endemic areas are at risk for infection with both viruses as well as repeated DENV infection. In the presence of anti-DENV antibodies, outcomes of secondary DENV infection range from mild to life-threatening. Further, the role of cross-reactive antibodies on the course of ZIKV infection remains unclear. We assessed the ability of cross-reactive DENV monoclonal antibodies or polyclonal immunoglobulin isolated after DENV vaccination to upregulate type I interferon (IFN) production by plasmacytoid dendritic cells (pDCs) in response to both heterotypic DENV- and ZIKV- infected cells. We found a range in the ability of antibodies to increase pDC IFN production and a positive correlation between IFN production and the ability of an antibody to bind to the infected cell surface. Engagement of Fc receptors on the pDC and Fab binding of an epitope on infected cells was required to mediate increased IFN production by providing specificity to and promoting pDC sensing of DENV or ZIKV. This represents a mechanism independent of neutralization by which pre-existing cross-reactive DENV antibodies could protect a subset of individuals from severe outcomes during secondary heterotypic DENV or ZIKV infection.
Laura K. Aisenberg, Kimberly E. Rousseau, Katherine Cascino, Guido Massaccesi, William H. Aisenberg, Wensheng Luo, Kar Muthumani, David B. Weiner, Stephen S. Whitehead, Michael A. Chattergoon, Anna P. Durbin, Andrea L. Cox
Macrophages play a crucial role in the inflammatory response to the human stomach pathogen Helicobacter pylori, which infects half of the world’s population and causes gastric cancer. Recent studies have highlighted the importance of macrophage immunometabolism in their activation state and function. We have demonstrated that the cysteine-producing enzyme, cystathionine g-lyase (CTH), is upregulated in humans and mice with H. pylori infection. Here we show that induction of CTH in macrophages by H. pylori promotes persistent inflammation. Cth–/– mice have reduced macrophage and T-cell activation in H. pylori-infected tissues, an altered metabolome, and decreased enrichment of immune-associated gene networks, culminating in decreased H. pylori-induced-gastritis. CTH is downstream of the proposed anti-inflammatory molecule, S-adenosylmethionine (SAM). While Cth–/– mice exhibit gastric SAM accumulation, WT mice treated with SAM did not display protection against H. pylori-induced inflammation. Instead, we demonstrate that Cth-deficient macrophages exhibit alterations in the proteome, decreased NF-kB activation, diminished expression of macrophage activation markers, and impaired oxidative phosphorylation and glycolysis. Thus, through altering cellular respiration, CTH is a key enhancer of macrophage activation contributing to a pathogenic inflammatory response that is the universal precursor for the development of H. pylori-induced gastric disease.
Yvonne L. Latour, Johanna C. Sierra, Jordan L. Finley, Mohammad Asim, Daniel P. Barry, Margaret M. Allaman, Thaddeus M. Smith, Kara M. McNamara, Paula B. Luis, Claus Schneider, Justin Jacobse, Jeremy A. Goettel, M. Wade Calcutt, Kristie L. Rose, Kevin L Schey, Ginger L. Milne, Alberto G. Delgado, M. Blanca Piazuelo, Bindu D. Paul, Solomon Snyder, Alain P. Gobert, Keith T. Wilson
Cyclophosphamide (CPA) and doxorubicin (DOX) are key components of chemotherapy for triple-negative breast cancer (TNBC) although suboptimal outcomes are commonly associated with drug resistance and/or intolerable side-effects. Through an approach combining high-throughput screening and chemical modification, we developed CN06 as a dual activator of the constitutive androstane receptor (CAR) and nuclear factor erythroid 2-related factor 2 (Nrf2). CN06 enhances CAR-induced bioactivation of CPA (a prodrug) by provoking hepatic expression of CYP2B6, while repressing DOX-induced cytotoxicity in cardiomyocytes in vitro via stimulating Nrf2-antioxidant signaling. Utilizing a multicellular co-culture model incorporating human primary hepatocytes, TNBC cells, and cardiomyocytes, we show that CN06 increased CPA/DOX-mediated TNBC cell death via CAR-dependent CYP2B6 induction and subsequent conversion of CPA to its active metabolite 4-hydroxy-CPA, while protecting against DOX-induced cardiotoxicity by selectively activating Nrf2-antioxidant signaling in cardiomyocytes but not in TNBC cells. Further, CN06 preserves the viability and function of human iPSC-derived cardiomyocytes by modulating antioxidant defenses, decreasing apoptosis, and enhancing the kinetics of contraction and relaxation. Collectively, our findings identify CAR and Nrf2 as novel combined therapeutic targets whereby CN06 holds the potential to improve the efficacy:toxicity ratio of CPA/DOX-containing chemotherapy.
Sydney Stern, Dongdong Liang, Linhao Li, Ritika Kurian, Caitlin Lynch, Srilatha Sakamuru, Scott Heyward, Junran Zhang, Kafayat Ajoke Kareem, Young Wook Chun, Ruili Huang, Menghang Xia, Charles C. Hong, Fengtian Xue, Hongbing Wang
Nemaline Myopathy (NM) is the most common congenital myopathy, characterized by extreme weakness of the respiratory, limb, and facial muscles. Pathogenic variants in Tropomyosin 2 (TPM2), which encodes a skeletal muscle specific actin binding protein essential for sarcomere function, cause a spectrum of musculoskeletal disorders that include NM as well as Cap Myopathy, congenital fiber type disproportion, and distal arthrogryposis (DA). The in vivo pathomechanisms underlying TPM2-related disorders are unknown, so we expressed a series of dominant, pathogenic TPM2 variants in Drosophila embryos and found four variants significantly affected muscle development and muscle function. Transient overexpression of the four variants also disrupted the morphogenesis of mouse myotubes in vitro, and negatively affected zebrafish muscle development in vivo. We used transient overexpression assays in zebrafish to characterize two novel TPM2 variants and one recurring variant that we identified in DA patients (V129A, E139K, A155T), and found these variants caused musculoskeletal defects similar to those of known pathogenic variants. The consistency of musculoskeletal phenotypes in our assays correlated with the severity of clinical phenotypes observed in our DA patients, suggesting disrupted myogenesis is a novel pathomechanism of TPM2 disorders, and that our myogenic assays can predict the clinical severity of TPM2 variants.
Jennifer McAdow, Shuo Yang, Tiffany Ou, Gary Huang, Matthew B. Dobbs, Christina A. Gurnett, Michael J. Greenberg, Aaron N. Johnson
In situ vaccination has demonstrated the feasibility of priming local immunity for systemic antitumor responses. Although direct intratumoral delivery of adjuvant is the mainstay, tumor-draining lymph nodes (TDLNs) also play essential roles in antitumor immunity. We report that directing an adjuvant to both tumors and TDLNs during in situ vaccination can induce robust antitumor responses. Conventional intratumoral dosing leads to tumor-limited delivery of agents; however, delivery to both tumors and TDLNs can be ensured through a micellar formation. The peritumoral delivery of micellar MEDI9197 (mcMEDI), a toll-like receptor 7/8 agonist, induced significantly stronger innate and adaptive immune responses than those on conventional dosing. Optimal dosing was crucial because excessive or insufficient accumulation of the adjuvant in the TDLNs compromised therapeutic efficacy. The combination of local mcMEDI therapy significantly improved the efficacy of systemic anti-programmed death receptor-1 therapy. These data suggest that rerouting adjuvants to tumors and TDLNs can augment the therapeutic efficacy of in situ vaccination.
Moonkyoung Jeong, Heegon Kim, Junyong Yoon, Dong-Hyun Kim, Ji-Ho Park
Pregnancy confers unique immune responses to infection and vaccination across gestation. To date, there is limited data comparing vaccine versus infection-induced nAb to COVID-19 variants in mothers during pregnancy. We analyzed paired maternal and cord plasma samples from 60 pregnant individuals. Thirty women vaccinated with mRNA vaccines (from December 2020 through August 2021) were matched with 30 naturally infected women (from March 2020 through January 2021) by gestational age of exposure. Neutralization activity against the five SARS-CoV-2 Spike sequences was measured by a SARS-CoV-2 pseudotyped Spike virion assay. Effective nAbs against SARS-CoV-2 were present in maternal and cord plasma after both infection and vaccination. Compared to wild type Spike, these nAbs were less effective against the Delta and Mu Spike variants. Vaccination during the third trimester induced higher cord nAb levels at delivery than infection during the third trimester. In contrast, vaccine-induced nAb levels were lower at the time of delivery compared to infection during the first trimester. The transfer ratio (cord nAb level/maternal nAb level) was greatest in mothers vaccinated in the second trimester. SARS-CoV-2 vaccination or infection in pregnancy elicit effective nAbs with differing neutralization kinetics that is impacted by gestational time of exposure.
Yusuke Matsui, Lin Li, Mary Prahl, Arianna G. Cassidy, Nida Ozarslan, Yarden Golan, Veronica J. Gonzalez, Christine Y. Lin, Unurzul Jigmeddagva, Megan A. Chidboy, Mauricio Montano, Taha Y. Taha, Mir M. Khalid, Bharath Sreekumar, Jennifer M. Hayashi, Pei-Yi Chen, G. Renuka Kumar, Lakshmi Warrier, Alan H.B. Wu, Dongli Song, Priya Jegatheesan, Daljeet S. Rai, Balaji Govindaswami, Jordan M. Needens, Monica Rincon, Leslie Myatt, Ifeyinwa V. Asiodu, Valerie J. Flaherman, Yalda Afshar, Vanessa L. Jacoby, Amy P. Murtha, Joshua F. Robinson, Melanie Ott, Warner C. Greene, Stephanie L Gaw
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