Men who have sex with men (MSM) with HIV are at high risk for squamous intraepithelial lesion (SIL) and anal cancer. Identifying local immunological mechanisms involved in the development of anal dysplasia could aid treatment and diagnostics. Here we studied 111 anal biopsies obtained from 101 MSM with HIV, who participated in an anal screening program. We first assessed multiple immune subsets by flow cytometry, in addition to histological examination, in a discovery cohort (n = 54). Selected molecules were further evaluated by immunohistochemistry in a validation cohort (n = 47). Pathological samples were characterized by the presence of Resident Memory T cells with low expression of CD103 and by changes in Natural Killer cell subsets, affecting residency and activation. Furthermore, potentially immune suppressive subsets, including CD15+CD16+ mature neutrophils, gradually increased as the anal lesion progressed. Immunohistochemistry confirmed the association between the presence of CD15 in the epithelium and SIL diagnosis, with a sensitivity of 80% and specificity of 71% (AUC 0.762) for the correlation with high-grade SIL. A complex immunological environment with imbalanced proportions of resident effectors and immune suppressive subsets characterizes pathological samples. Neutrophil infiltration, determined by CD15 staining, may represent a valuable pathological marker associated with the grade of dysplasia.
Joaquín Burgos, Aleix Benítez-Martínez, Cristina Mancebo-Pérez, Nuria Massana, Antonio Astorga-Gamaza, Josep Castellvi, Stefania Landolfi, Adrià Curran, Jorge N. Garcia-Perez, Vicenç Falcó, María J. Buzón, Meritxell Genescà
Diabetes mellitus (DM) is an independent risk factor for atrial fibrillation (AF). The mechanisms underlying DM-associated AF are unclear. AF and DM are both related to inflammation. We investigated whether DM-associated inflammation contributed to AF risk. Mice were fed with high fat diet to induce type II DM and were subjected to IL-1β antibodies, macrophage depletion by Clodronate liposomes, a mitochondrial antioxidant (mitoTEMPO), or a cardiac ryanodine receptor (RyR2) stabilizer (S107). All tests were performed at 36-38 weeks of age. DM mice presented with increased AF inducibility, enhanced mitochondrial reactive oxygen species (mitoROS) generation, and activated innate immunity in the atria as evidenced by enhanced monocyte chemoattractant protein-1 (MCP-1) expression, macrophage infiltration, and IL-1β levels. Signs of aberrant RyR2 Ca2+ leak were observed in the atria of DM mice. IL-1β neutralization, macrophage depletion, mitoTEMPO, and S107 significantly ameliorated the AF vulnerability in DM mice. Atrial overexpression of MCP-1 increased AF occurrence in normal mice through the same mechanistic signaling cascade as observed in DM mice. In conclusion, macrophage-mediated IL-1β contributed to DM-associated AF risk through mitoROS modulation of RyR2 Ca2+ leak.
Xiaoxu Zhou, Hong Liu, Feng Feng, Gyeoung-Jin Kang, Man Liu, Yugene Guo, Samuel C. Dudley Jr.
Loss-of-function mutations of the gene encoding the trafficking protein particle complex subunit 9 (trappc9) cause autosomal recessive intellectual disability and obesity by unknown mechanisms. Genome-wide analysis links trappc9 to non-alcoholic fatty liver disease (NAFLD). Trappc9-deficient mice have been shown to appear overweight shortly after weaning. Here, we analyzed serum biochemistry and histology of adipose and liver tissues to determine the incidence of obesity and NAFLD in trappc9-deficient mice and combined transcriptomic and proteomic analyses, pharmacological studies, and biochemical and histological examinations of postmortem mouse brains to unveil mechanisms involved. We found that trappc9-deficient mice presented with systemic glucose homeostatic disturbance, obesity and NAFLD, which were relieved upon chronic treatment combining dopamine receptor D2 (DRD2) agonist quinpirole and DRD1 antagonist SCH23390. Blood glucose homeostasis in trappc9-deficient mice was restored upon administrating quinpirole alone. RNA-sequencing analysis of DRD2-containing neurons and proteomic study of brain synaptosomes revealed signs of impaired neurotransmitter secretion in trappc9-deficient mice. Biochemical and histological studies of mouse brains showed that trappc9-deficient mice synthesized dopamine normally, but their dopamine-secreting neurons had a lower abundance of structures for releasing dopamine in the striatum. Our study suggests that trappc9 loss-of-function causes obesity and NAFLD by constraining dopamine synapse formation.
Yan Li, Usman Muhammad, Ellen Sapp, Yuting Ke, Zejian Wang, Adel Boudi, Marian DiFiglia, Xueyi Li
Mucus plugs occlude airways to obstruct airflow in asthma. Studies in patients and in mouse models show that mucus plugs occur in the context of type 2 inflammation, and studies in human airway epithelial cells (HAECs) show that interleukin 13 (IL-13) activated cells generate pathologic mucus independently of immune cells. To determine how HAECs autonomously generate pathologic mucus, we used a magnetic microwire rheometer to characterize the viscoelastic properties of mucus secreted under varying conditions. We found that normal HAEC mucus exhibits viscoelastic liquid behavior and that mucus secreted by IL-13 activated HAECs exhibits solid-like behavior caused by mucin cross-linking. In addition, IL-13 activated HAECs show increased peroxidase activity in apical secretions, and an overlaid thiolated polymer (thiomer) solution shows an increase in solid behavior that is prevented by peroxidase inhibition. Furthermore, gene expression for thyroid peroxidase (TPO), but not lactoperoxidase (LPO), is increased in IL-13 activated HAECs and both TPO and LPO catalyze the formation of oxidant acids that cross-link thiomer solutions. Finally, gene expression for TPO in airway epithelial brushings is increased in asthma patients with high airway mucus plug scores. Together, our results show that IL-13 activated HAECs autonomously generate pathologic mucus via peroxidase-mediated cross-linking of mucin polymers.
Maude A. Liegeois, Margaret Braunreuther, Annabelle R. Charbit, Wilfred W. Raymond, Monica Tang, Prescott G. Woodruff, Stephanie A. Christenson, Mario Castro, Serpil C. Erzurum, Elliot Israel, Nizar N. Jarjour, Bruce D. Levy, Wendy C. Moore, Sally E. Wenzel, Gerald G. Fuller, John V. Fahy
A defining feature of systemic lupus erythematosus (SLE) is loss of tolerance to self-DNA, and DNASE1L3 deficiency, the main enzyme responsible for chromatin degradation in blood, is also associated with SLE. This association includes an ultra-rare pediatric population with DNASE1L3 deficiency who develop SLE, adult patients with loss of function variants of DNASE1L3 who are at a higher risk for SLE, and patients with sporadic SLE who have neutralizing autoantibodies to DNASE1L3. To mitigate the pathogenic effects of inherited and acquired DNASE1L3 deficiencies, we engineered a long-acting enzyme biologic with dual DNASE1/DNASE1L3 activity that is resistant to DNASE1 and DNASE1L3 inhibitors. Notably, we found that the biologic prevented the development of lupus in Dnase1–/–/Dnase1L3–/– double knockout mice and rescued animals from death in pristane-induced lupus. Finally, we confirmed that the human isoform of the enzyme biologic was not recognized by autoantibodies in SLE and efficiently degrades genomic and mitochondrial cell free DNA, as well as microparticle DNA, in SLE plasma. Our findings suggest that autoimmune diseases characterized by aberrant DNA accumulation, such as SLE, can be effectively treated with a replacement DNASE tailored to bypass pathogenic mechanisms, both genetic and acquired, that restrict DNASE1L3 activity.
Paul R. Stabach, Dominique Sims, Eduardo Gomez-Bañuelos, Sandra Zehentmeier, Kris Dammen-Brower, Andrew Bernhisel, Sophia E. Kujawski, Sam G. Lopez, Michelle Petri, Daniel W. Goldman, Ethan R. Lester, Quan Le, Tayyaba Ishaq, Hana Kim, Shivani Srivastava, Deepika Kumar, Joao P. Pereira, Kevin J. Yarema, Fotios Koumpouras, Felipe Andrade, Demetrios T. Braddock
The excessive formation and release of neutrophil extracellular traps (NETs) in sepsis may represent a substantial mechanism contributing to multi-organ damage, which is associated with a poorer prognosis. However, the precise role of NETs in mediating the transition from innate immunity to adaptive immunity during the progression of inflammation and sepsis remains incompletely elucidated. In this study, we provide evidence that, despite a reduction in the number of CD4+ T-cells in the late stage of sepsis, there is a notable upregulation in the proportion of regulatory T-cells (Tregs). Mechanistically, we have identified that NETs can induce metabolic reprogramming of naïve CD4+ T-cells through the Akt-mTOR-SREBP2 pathway, resulting in enhanced cholesterol metabolism, thereby promoting their conversion into Tregs and augmenting their functional capacity. Collectively, our findings highlight the potential therapeutic strategy of targeting intracellular cholesterol normalization for the management of immunosuppressed patients with sepsis.
Yuxin Shi, Dan Wu, Yanghanzhao Wang, Yuwen Shao, Fu Zeng, Di Zhou, Hao Zhang, Changhong Miao
Cystic fibrosis (CF) is a genetic disorder that disrupts CF transmembrane conductance regulator (CFTR) anion channels and impairs airway host defenses. Airway inflammation is ubiquitous in CF and suppressing it has generally been considered to improve outcomes. However, the role of inflammation in people taking CFTR modulators, small-molecule drugs that restore CFTR function, is not well-understood. We previously showed that inflammation enhances the efficacy of CFTR modulators. To further elucidate this relationship, we treated human ∆F508-CF epithelia with TNFα and IL-17, two inflammatory cytokines that are elevated in CF airways. TNFα+IL-17 enhanced CFTR modulator-evoked anion secretion through mechanisms that raise intracellular Cl– (Na+/K+/2Cl– co-transport) and HCO3– (carbonic anhydrases and Na+/HCO3– co-transport). This enhancement required p38 MAPK signaling. Importantly, CFTR modulators did not affect CF airway surface liquid viscosity under control conditions, but prevented the rise in viscosity in epithelia treated with TNFα+IL-17. Lastly, anti-inflammatory drugs limited CFTR modulator responses in TNFα+IL-17-treated epithelia. These results provide critical insights into mechanisms by which inflammation increases responses to CFTR modulators. They also suggest an equipoise between potential benefits versus limitations of suppressing inflammation in people taking modulators, call into question current treatment approaches, and highlight a need for additional studies.
Tayyab Rehman, Alejandro A. Pezzulo, Andrew L. Thurman, Rachel L. Zemans, Michael J. Welsh
Genetic defects affecting steroid biosynthesis cause cortisol deficiency and differences of sex development; among them recessive mutations in the steroidogenic enzymes CYP11A1 and CYP11B, whose function is supported by reducing equivalents donated by ferredoxin reductase (FDXR) and ferredoxin. So far, mutations in the mitochondrial flavoprotein FDXR have been associated with a progressive neuropathic mitochondriopathy named FDXR-Related Mitochondriopathy (FRM), but cortisol insufficiency has not been documented. However, FRM patients often experience worsening or demise following stress associated with infections. We investigated two female FRM patients carrying the novel homozygous FDXR mutation p.G437R with ambiguous genitalia at birth and sudden death in the first year of life; they presented with cortisol deficiency and androgen excess compatible with 11-hydroxylase deficiency. In addition, steroidogenic FDXR-variant cell lines reprogrammed from three FRM patients’ fibroblasts displayed deficient mineralocorticoid and glucocorticoid production. Finally, Fdxr-mutant mice allelic to the severe p.R386W human variant, showed reduced progesterone and corticosterone production. Therefore, our comprehensive studies show that human FDXR variants may cause compensated, but possibly life-threatening adrenocortical insufficiency in stress by affecting adrenal glucocorticoid and mineralocorticoid synthesis through direct enzyme inhibition, most likely in combination with disturbed mitochondrial redox balance.
Emanuele Pignatti, Jesse Slone, María Ángeles Gómez Cano, Teresa Margaret Campbell, Jimmy Vu, Kay-Sara Sauter, Amit V. Pandey, Francisco Martínez-Azorín, Marina Alonso-Riaño, Derek E. Neilson, Nicola Longo, Therina du Toit, Clarissa D. Voegel, Taosheng Huang, Christa E. Flück
Osteoporotic fractures are a major complication of long-term glucocorticoid therapy. Glucocorticoids transiently increase bone resorption, but they predominantly inhibit bone formation and induce osteocyte apoptosis, leading to bone loss. Current treatments of glucocorticoid-induced osteoporosis aim mainly at reducing bone resorption and are therefore inadequate. We previously showed that signaling via the NO/cGMP/protein kinase G pathway plays a key role in skeletal homeostasis. Here, we show that pharmacological PKG activation with the guanylyl cyclase-1 activator cinaciguat or expression of a constitutively-active, mutant PKG2R242Q restored proliferation, differentiation, and survival of primary mouse osteoblasts exposed to dexamethasone. Cinaciguat treatment of wild type mice or osteoblast-specific expression of PKG2R242Q in transgenic mice prevented dexamethasone-induced loss of cortical bone mass and strength. These effects of cinaciguat and PKG2R242Q expression were due to preserved bone formation parameters and osteocyte survival. The basis for PKG2’s effects appeared to be through recovery of Wnt/β-catenin signaling, which was suppressed by glucocorticoids but is critical for proliferation, differentiation, and survival of osteoblast-lineage cells. Cinaciguat reduced dexamethasone activation of osteoclasts, but this did not occur in the PKG2R242Q transgenic mice, suggesting a minor role in osteoprotection. We propose that existing PKG-targeting drugs could represent a novel therapeutic approach to prevent glucocorticoid-induced osteoporosis.
Shyamsundar Pal China, Hema Kalyanaraman, Shunhui Zhuang, Justin A. Cabriales, Robert L. Sah, Renate B. Pilz
Antibody-mediated depletion studies have demonstrated that CD8+ T cells are required for effective immune control of SIV. However, this approach is confounded by several factors, including reactive CD4+ T cell proliferation, and further provides no specificity information. We circumvented these limitations by selectively depleting CD8+ T cells specific for the Gag epitope CTPYDINQM (CM9) via the administration of immunotoxin-conjugated tetrameric complexes of CM9/Mamu-A*01. Immunotoxin administration effectively depleted circulating but not tissuelocalized CM9-specific CD8+ T cells, akin to the bulk depletion pattern observed with antibodies directed against CD8. However, we found no evidence to indicate that circulating CM9-specific CD8+ T cells suppressed viral replication in Mamu-A*01+ rhesus macaques during acute or chronic progressive infection with a pathogenic strain of SIV. This observation extended to macaques with established infection during and after continuous antiretroviral therapy. In contrast, natural controller macaques experienced dramatic increases in plasma viremia after immunotoxin administration, highlighting the importance of CD8+ T cell-mediated immunity against CM9. Collectively, these data showed that CM9-specific CD8+ T cells were necessary but not sufficient for robust immune control of SIV in a nonhuman primate model and, more generally, validated an approach that could inform the design of next-generation vaccines against HIV-1.
Jennifer Simpson, Carly E. Starke, Alexandra M. Ortiz, Amy Ransier, Sam Darko, Sian Llewellyn-Lacey, Christine M. Fennessey, Brandon F. Keele, Daniel C. Douek, David A. Price, Jason M. Brenchley
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