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Abstract
Overexpression and long terminal repeat (LTR) polymorphism of the HRES-1/Rab4 human endogenous retrovirus locus have been associated with T-cell activation and disease manifestations in systemic lupus erythematosus (SLE). Although genomic DNA methylation is overall diminished in SLE, its role in HRES-1/Rab4 expression is unknown. Therefore, we determined how lupus-associated polymorphic rs451401 alleles of the LTR regulate transcription from the HRES-1/Rab4 promoter and thus impact T-cell activation. The results show that cytosine-119 is hypermethylated while cytosine-51 of the promoter and the LTR-enhancer are hypomethylated in SLE. Pharmacological or genetic inactivation of DNA-methyltransferase-1 augmented the expression of HRES-1/Rab4. The minimal promoter was selectively recognized by metabolic stress sensor NRF1 when cytosine-119 but not cytosine-51 was methylated, and NRF1 stimulated HRES-1/Rab4 expression in human T cells. In turn, IRF2 and PSIP1 bound to the LTR-enhancer and exerted control over HRES-1/Rab4 expression in rs451401-genotype and methylation-dependent manners. The LTR-enhancer conferred markedly greater expression of HRES-1/Rab4 in subjects with rs451401CC over rs451401GG alleles that in turn promoted mechanistic target of rapamycin (mTOR) activation upon T-cell receptor stimulation. HRES-1/Rab4 alone robustly activated mTOR in human T cells. These findings identify HRES-1/Rab4 as a methylation and rs451401 allele-dependent transducer of environmental stress and controller of T-cell activation.
Authors
Aparna Godavarthy, Ryan Kelly, John Jimah, Miguel Beckford, Tiffany Caza, David Fernandez, Nick Huang, Manuel Duarte, Joshua Lewis, Hind J. Fadel, Eric M. Poeschla, Katalin Banki, Andras Perl
Abstract
Biallelic mutations of the gene encoding the transcription factor NEUROG3 are associated with a rare disorder that presents in neonates as generalized malabsorption – due to a complete absence of enteroendocrine cells – followed, in early childhood or beyond, by insulin-dependent diabetes mellitus (IDDM). The commonly delayed onset of IDDM suggests a differential requirement for NEUROG3 in endocrine cell generation in the human pancreas versus the intestine. However, previously identified human mutations were hypomorphic, and hence may have had residual function in pancreas. We report two patients with biallelic functionally null variants of the NEUROG3 gene who nonetheless did not present with IDDM during infancy, but instead developed permanent IDDM during middle childhood ages. The variants show no evidence of function in traditional promoter-based assays of NEUROG3 function and also fail to exhibit function in a variety of novel in vitro and in vivo molecular assays designed to discern residual NEUROG3 function. These findings imply that unlike in mice, pancreatic endocrine cell generation in humans is not entirely dependent on NEUROG3 expression, and hence suggests the presence of unidentified redundant in vivo pathways in human pancreas capable of yielding beta-cell mass sufficient to maintain euglycemia until early childhood.
Authors
R. Sergio Solorzano-Vargas, Matthew Bjerknes, Jiafang Wang, S. Vincent Wu, Manuel G. Garcia-Careaga, Duke Pisit, Hazel Cheng, Michael S. German, Senta Georgia, Martin G. Martín
Abstract
Influenza is a highly contagious viral pathogen with more than 200,000 cases reported in the U.S. during the 2017-2018 season. Annual vaccination is recommended by the World Health Organization with the goal to reduce influenza severity and transmission. Currently available vaccines are ~60% effective and vaccine effectiveness varies from season to season, as well as between different influenza subtypes within a single season. Immunological imprinting from early life influenza infection can prominently shape the immune response to subsequent infections. Here, the impact of pre-existing B cell memory in the response to quadrivalent influenza vaccine was assessed using blood samples collected from healthy subjects (18 to 85 years old) prior to and 21-28 days following influenza vaccination. Influenza vaccination increased both HA-specific antibodies and memory B cells frequency. Despite no apparent differences in antigenicity between vaccine components, most individuals were biased towards one of the vaccine strains. Specifically, responses to H3N2 were reduced in magnitude relative to the other vaccine components. Overall, this study unveils a new mechanism underlying differential vaccine effectiveness against distinct influenza subtypes.
Authors
Rodrigo B. Abreu, Greg A. Kirchenbaum, Emily F. Clutter, Giuseppe A. Sautto, Ted M. Ross
Abstract
Background: Serological tools for the accurate detection of recent malaria exposure are needed to guide and monitor malaria control efforts. IgG responses against P. vivax and P. falciparum merozoite surface protein-10 (MSP10) were measured as a potential way to identify recent malaria exposure in the Peruvian Amazon. Methods: A field-based study included 470 participants in a longitudinal cohort who completed a comprehensive evaluation: light microscopy and PCR on enrollment; at least one monthly follow-up by light microscopy; a second PCR; and serum and dried blood spots for serological analysis at the end of the follow-up. IgG titers against novel mammalian cell-produced recombinant PvMSP10 and PfMSP10 were determined by ELISA. Results: During the follow-up period, 205 participants were infected, including 171 with P. vivax, 26 with P. falciparum, 6 with infections by both species but at different times, and 2 with mixed infections. Exposure to P. vivax was more accurately identified when serological responses to PvMSP10 were obtained from serum (sensitivity, 58.1%; specificity, 81.8%; AUC: 0.76) than from dried blood spots (sensitivity, 35.2; specificity, 83.5%; AUC: 0.64) (PAUC < 0.001). Sensitivity was highest (serum, 82.9%; dried blood spot, 45.7%) with confirmed P. vivax infections occurring 7-30 days before sample collection; sensitivity decreased significantly in relation to time since last documented infection. PvMSP10 serological data did not show evidence of inter-species cross-reactivity. Anti-PfMSP10 responses poorly discriminated between P. falciparum exposed- and non-exposed individuals (AUC = 0.59, P > 0.05). Conclusions: Anti-PvMSP10 IgG indicates recent exposure to P. vivax at the population level in the Amazon region. Serum, not dried blood spots, should be used for such serological tests.
Authors
Angel Rosas-Aguirre, Kailash P. Patra, Maritza Calderón, Katherine Torres, Dionicia Gamboa, Edith Arocutipa, Edith Málaga, Katherine Garro, Carlos Fernández, Grace Trompeter, Yossef Alnasser, Alejandro Llanos-Cuentas, Robert H. Gilman, Joseph M. Vinetz
Abstract
Gigaxonin (also known as KLHL16) is an E3 ligase adaptor protein that promotes the ubiquitination and degradation of intermediate filament (IF) proteins. Mutations in human gigaxonin cause the fatal neurodegenerative disease giant axonal neuropathy (GAN), in which IF proteins accumulate and aggregate in axons throughout the nervous system, impairing neuronal function and viability. Despite this pathophysiological significance, the upstream regulation and downstream effects of normal and aberrant gigaxonin function remain incompletely understood. Here, we report that gigaxonin is modified by O-linked β-N-acetylglucosamine (O-GlcNAc), a prevalent form of intracellular glycosylation, in a nutrient- and growth factor-dependent manner. Mass spectrometry analyses of human gigaxonin revealed nine candidate sites of O-GlcNAcylation, two of which – serine 272 and threonine 277 – are required for its ability to mediate IF turnover in novel gigaxonin-deficient human cell models that we created. Taken together, these results suggest that nutrient-responsive gigaxonin O-GlcNAcylation forms a regulatory link between metabolism and IF proteostasis. Our work may have significant implications for understanding the non-genetic modifiers of GAN phenotypes and for the optimization of gene therapy for this disease.
Authors
Po-Han Chen, Jimin Hu, Jianli Wu, Duc T. Huynh, Timothy J. Smith, Samuel Pan, Brittany J. Bisnett, Alexander B. Smith, Annie Lu, Brett M. Condon, Jen-Tsan Chi, Michael Boyce
Abstract
Background: Hepatic encephalopathy (HE) is associated with poor outcomes. A prior randomized, pilot trial demonstrated safety after oral capsular FMT in HE with favorable changes in microbial composition and cognition. However, microbial functional changes are unclear. Aim: Determine impact of FMT on gut-brain axis compared to placebo using microbial function based on bile acids (BA), inflammation (serum IL-6, lipopolysaccharide-binding protein,LBP), and EncephalApp. Methods: 20 cirrhotic patients were randomized 1:1 into receiving one-time FMT capsules from a donor enriched in Lachnospiraceae and Ruminococcaceae, or placebo capsules with 5-month follow-up for safety outcomes. Stool microbiota and BA, serum IL-6, BA and LBP, and EncephalApp were analyzed at baseline and 4-weeks post-FMT/placebo. Correlation networks between microbiota, BAs, EncephalApp, IL-6 and LBP were performed pre/post-FMT. Results: FMT-assigned participants had one HE recurrence and 2 unrelated infections. Six placebo-assigned participants developed negative outcomes. FMT, but not placebo, was associated with reduced serum IL-6 and LBP and improved EncephalApp. FMT-assigned participants demonstrated higher deconjugation and secondary BA formation in feces and serum compared to baseline. No change was seen in placebo. Correlation networks showed greater complexity post-FMT compared to baseline. Beneficial taxa such as Ruminococcaceae, Verrucomicrobiaceae and Lachnospiraceae were correlated with cognitive improvement and decrease in inflammation post-FMT. Fecal/serum secondary/primary ratios and PiCRUST secondary BA pathways did not increase in participants who developed poor outcomes. Conclusions: Gut microbial function in cirrhosis is beneficially affected by capsular FMT with improved inflammation and cognition. Lower secondary BAs in FMT recipients could select for participants who develop negative outcomes.
Authors
Jasmohan S. Bajaj, Nita Salzman, Chathur Acharya, Hajime Takei, Genta Kakiyama, Andrew Fagan, Melanie B. White, Edith A. Gavis, Mary L. Holtz, Michael Hayward, Hiroshi Nittono, Philip B. Hylemon, I. Jane Cox, Roger Williams, Simon D. Taylor-Robinson, Richard K. Sterling, Scott C. Matherly, Michael Fuchs, Hannah Lee, Puneet Puri, R. Todd Stravitz, Arun J. Sanyal, Lola Ajayi, Adrien Le Guennec, R. Andrew Atkinson, Mohammad S. Siddiqui, Velimir A. Luketic, William M. Pandak, Masoumeh Sikaroodi, Patrick M. Gillevet
Abstract
As sufficient extracellular arginine is crucial for T cell function, depletion of extracellular arginine by elevated Arginase 1 (Arg1) activity has emerged as a hallmark immunosuppressive mechanism. However, the potential cell-autonomous roles of arginases in T cells have remained unexplored. Here we show that the arginase isoform expressed by T cells, the mitochondrial Arginase 2 (Arg2), is a cell-intrinsic regulator of CD8+ T cell activity. Both germ-line Arg2 deletion and adoptive transfer of Arg2-/- CD8+ T cells significantly reduced tumor growth in preclinical cancer models by enhancing CD8+ T cell activation, effector function and persistence. Transcriptomic, proteomic and high-dimensional flow cytometry characterization revealed a CD8+ T cell-intrinsic role of Arg2 in modulating T cell activation, anti-tumor cytoxicity and memory formation, independently of extracellular arginine availability. Furthermore, specific deletion of Arg2 in CD8+ T cells strongly synergized with PD-1 blockade for the control of tumor growth and animal survival. These observations coupled with the finding that pharmacologic arginase inhibition accelerates activation of ex vivo human T cells unveil Arg2 as a new therapeutic target for T cell-based cancer therapies.
Authors
Adrià-Arnau Martí i Líndez, Isabelle Dunand-Sauthier, Mark Conti, Florian Gobet, Nicolás Núñez, J. Thomas Hannich, Howard Riezman, Geiger Roger, Alessandra Piersigilli, Kerstin Hahn, Sylvain Lemeille, Burkhard Becher, Thibaut De Smedt, Stéphanie Hugues, Walter Reith
Abstract
Proteinuric chronic kidney disease (CKD) remains a major health problem worldwide. While the progression of primary glomerular disease to induce tubulointerstitial lesions is well established, the effect of tubular injury to trigger glomerular damage is poorly understood. We hypothesized that injured tubules secrete mediators that adversely affect glomerular health. To test this, we utilized conditional knockout mice with tubule-specific ablation of β-catenin (Ksp-β-cat-/-), and subjected them to chronic angiotensin II (Ang II) infusion or adriamycin. Compared to control mice, Ksp-β-cat-/- mice were dramatically protected from proteinuria and glomerular damage. Matrix metalloproteinase-7 (MMP-7), a downstream target of β-catenin, was upregulated in treated control mice, but this induction was blunted in the Ksp-β-cat-/- littermates. Incubation of isolated glomeruli with MMP-7 ex vivo led to nephrin depletion and impaired glomerular permeability. Furthermore, MMP-7 specifically and directly degraded nephrin in cultured glomeruli or cell-free systems, and this effect was dependent on its proteolytic activity. In vivo, expression or infusion of exogenous MMP-7 caused proteinuria, and genetic ablation of MMP-7 protected mice from Ang II-induced proteinuria and glomerular injury. Collectively, these results demonstrate that beta-catenin-driven MMP-7 release from renal tubules promotes glomerular injury via direct degradation of the key slit diaphragm protein nephrin.
Authors
Roderick J. Tan, Yingjian Li, Brittney M. Rush, Débora Malta Cerqueira, Dong Zhou, Haiyan Fu, Jacqueline Ho, Donna Beer Stolz, Youhua Liu
Abstract
Background: Bacille Calmette-Guérin (BCG) vaccine is protective in children but its efficacy wanes with age. Consequently, determining if BCG revaccination augments anti-TB immunity in young adults in TB endemic regions is vital. Methods: 200 healthy adults, BCG vaccinated at birth were tested for their IGRA status. Of these, 28 IGRA+ and 30 IGRA- were BCG revaccinated and 24 IGRA+ and 23 IGRA- subjects served as unvaccinated controls. T and innate cell responses to mycobacterial antigens were analyzed by 14-colour flow cytometry over 34 weeks. Results: IFN-γ and/or IL-2 Ag85A and BCG-specific CD4+ and CD8+ T-cell responses were boosted by revacciantion at 4 and 34 weeks respectively and were >2-fold higher in IGRA+ compared to IGRA- vaccinees. Polyfunctional Ag85A, BCG and Mtb latency Ag (LTAg)-specific CD4+ T-cells expressing up to 8 cytokines were also significantly enhanced in both IGRA+ and IGRA- vaccinees relative to unvaccinated controls, most markedly in IGRA+ vaccinees. A focussed analysis of Th17 responses revealed expansion of Ag85A, BCG and LTAg-specific total IL-17A+IL-17F+IL-22+ and IL-10+ CD4+ T-cell effectors in both IGRA+ and IGRA- subjects. Also, innate IFN-γ+ NK/γδ/NKT responses were higher in both IGRA+ and IGRA- vaccinees compared to controls. This is the first evidence that BCG revaccination significantly boosts anti-mycobacterial Th1/Th17 responses in IGRA+ and IGRA- subjects. Summary: These data show that BCG revaccination is immunogenic in IGRA- and IGRA+ subjects implying that Mtb pre-infection in IGRA+ subjects does not impact immunogenicity. This has implications for public health and vaccine development strategies. Funding: This work was funded principally by DBT-NIH (BT/MB/Indo-US/HIPC/2013).
Authors
Srabanti Rakshit, Asma Ahmed, Vasista Adiga, Bharath K. Sundararaj, Pravat Nalini Sahoo, John Kenneth, George D'Souza, Wesley Bonam, Christina Johnson, Kees L.M.C. Franken, Tom H.M. Ottenhoff, Greg Finak, Raphael Gottardo, Kenneth D. Stuart, Stephen C. De Rosa, M. Juliana McElrath, Annapurna Vyakarnam
Abstract
Introduction: The airways of obese asthmatics have been shown to be nitric oxide (NO) deficient, which contributes to airway dysfunction and reduced response to inhaled corticosteroids (ICS). In cultured airway epithelial cells, L-citrulline, a precursor of L-arginine recycling and NO formation, has been shown to prevent asymmetric di-methyl arginine (ADMA)-mediated NO synthase (NOS2) uncoupling, restoring NO and reducing oxidative stress. Methods: In a proof of concept, pre – post open label pilot study, we hypothesized that 15g/day of L-citrulline for two weeks would: a) increase the fractional excretion of NO (FeNO); b) improve asthma control and c) improve lung function. To do this, we recruited obese (body mass index [BMI] >30) asthmatics on controller therapy with a baseline fractional exhaled nitric oxide (FeNO) ≤ 30 ppb from the University of Colorado Medical Center and Duke University Health System. Results: A total of 41 subjects with an average FeNO of 17 ppb (95% 19 - 20) and poorly controlled asthma (average asthma control questionnaire [ACQ] 1.5 [95% 1.2 – 1.8) completed the study. Compared to baseline, L-citrulline increased (values represent the mean delta and 95%CI): plasma L-citrulline (190uM, 84 – 297), plasma L-arginine (67uM, 38 – 95), plasma L-arginine/ADMA (117, 67 - 167), but not ADMA or arginase concentration. FeNO increased by 4.2ppb (1.7 – 6.7); ACQ decreased by -0.46 (-0.67 – -0.27); the forced vital capacity (FVC) and forced exhalation volume in one second (FEV1) respectively changed by 86 ml (10 – 161), and 52 ml (-11 – 132). In a secondary analysis, the greatest FEV1 increments occurred in those subjects with late onset asthma (>12 years) (63 ml [95%CI 1 – 137]), in females (80 ml [95%CI 5 – 154]), with a greater change seen in late onset females (100ml, [95%CI 2 – 177]). The changes in lung function or asthma control were not significantly associated with the pre-post changes in L-arginine/ADMA or FeNO. Conclusion: Short-term L-citrulline treatment improved asthma control and FeNO levels in obese asthmatics with low or normal FeNO. Larger FEV1 increments were observed in those with late onset asthma and in females.
Authors
Fernando Holguin, Hartmut Grasemann, Sunita Sharma, Daniel Winnica, Karen Wasil, Vong Smithphone, Margaret H. Cruse, Nancy Perez, Erika Coleman, Timothy J. Scialla, Loretta Que
Abstract
Patients with Duchene Muscular Dystrophy (DMD) commonly present severe ventricular arrhythmias that contribute to heart failure. Arrhythmias and lethality are also consistently observed in adult Dmdmdx mice, a mouse model of DMD, after acute β-adrenergic stimulation. These pathological features were previously linked to aberrant expression and remodeling of the cardiac gap junction protein connexin 43 (Cx43). Here, we report that remodeled Cx43 protein forms Cx43 hemichannels in the lateral membrane of Dmdmdx cardiomyocytes and that the β -adrenergic agonist isoproterenol (Iso) aberrantly activates these hemichannels. Block of Cx43 hemichannels or a reduction in Cx43 levels (using Dmdmdx:Cx43+/- mice) prevents the abnormal increase in membrane permeability, plasma membrane depolarization and Iso-evoked electrical activity in these cells. Additionally, Iso treatment promotes nitric oxide (NO) production and S-nitrosylation of Cx43 hemichannels in Dmdmdx heart. Importantly, inhibition of NO production prevents arrhythmias evoked by Iso. We found that NO directly activates Cx43 hemichannels by S-nitrosylation of cysteine at the position 271. Our results demonstrate that opening of remodeled and S-nitrosylated Cx43 hemichannels play a key role in the development of arrhythmias in DMD mice and may serve as therapeutic targets to prevent fatal arrhythmias in DMD patients.
Authors
Mauricio A. Lillo, Eric Himelman, Natalia Shirokova, Lai-Hua Xie, Diego Fraidenraich, Jorge E. Contreras
Abstract
Immune activation is associated with increased risk of tuberculosis (TB) disease in infants. We performed a case control analysis to identify drivers of immune activation and disease risk. Among 49 infants who developed TB disease over the first two years of life, and 129 matched controls who remained healthy, we found the cytomegalovirus (CMV) stimulated IFNγ response at age 4-6 months to be associated with CD8+ T cell activation (Spearmans rho, P = 6 x 10-8). A CMV specific IFNγ response was also associated with increased risk of developing TB disease (Conditional Logistic Regression, P = 0.043, OR 2.2, 95% CI 1.02-4.83), and shorter time to TB diagnosis (Log Rank Mantel-Cox P = 0.037). CMV positive infants who developed TB disease had lower expression of natural killer cell associated gene signatures and a lower frequency of CD3−CD4−CD8− lymphocytes. We identified transcriptional signatures predictive of risk of TB disease among CMV ELISpot positive (AUROC 0.98, accuracy 92.57%) and negative (AUROC 0.9, accuracy 79.3%) infants; the CMV negative signature validated in an independent infant study (AUROC 0.71, accuracy 63.9%). Understanding and controlling the microbial drivers of T cell activation, such as CMV, could guide new strategies for prevention of TB disease in infants.
Authors
Julius Müller, Rachel Tanner, Magali Matsumiya, Margaret A. Snowden, Bernard Landry, Iman Satti, Stephanie A. Harris, Matthew K. O'Shea, Lisa Stockdale, Leanne Marsay, Agnieszka Chomka, Rachel Harrington-Kandt, Zita-Rose Manjaly Thomas, Elena Stylianou, Vivek Naranbhai, Stanley Kimbung Mbandi, Mark Hatherill, Gregory Hussey, Hassan Mahomed, Michele Tameris, J. Bruce McClain, Willem A. Hanekom, Thomas G. Evans, Thomas J. Scriba, Helen McShane, Helen A. Fletcher
Abstract
WHIM syndrome immunodeficiency is caused by autosomal dominant gain-of-function mutations in chemokine receptor CXCR4. Patient WHIM-09 was spontaneously cured by chromothriptic deletion of one copy of 164 genes, including the CXCR4WHIM allele, presumably in a single hematopoietic stem cell (HSC) that repopulated HSCs and the myeloid lineage. Testing the specific contribution of CXCR4 hemizygosity to her cure, we previously demonstrated enhanced engraftment of Cxcr4+/o HSCs after transplantation in WHIM (Cxcr4+/w) model mice, but the potency was not quantitated. We now report graded-dose competitive transplantation experiments using lethally irradiated Cxcr4+/+ recipients in which mixed BM cells containing ~5 Cxcr4+/o HSCs and a 100-fold excess of Cxcr4+/w HSCs achieved durable 50% Cxcr4+/o myeloid and B cell chimerism in blood and ~20% Cxcr4+/o HSC chimerism in BM. In Cxcr4+/o/Cxcr4+/w parabiotic mice, we observed 80-100% Cxcr4+/o myeloid and lymphoid chimerism in the blood and 15% Cxcr4+/o HSC chimerism in BM from the Cxcr4+/w parabiont, which was durable after separation from the Cxcr4+/o parabiont. Thus, CXCR4 haploinsufficiency likely significantly contributed to the selective repopulation of HSCs and the myeloid lineage from a single chromothriptic HSC in WHIM-09. Moreover, the results suggest that WHIM allele silencing of patient HSCs is a viable gene therapy strategy.
Authors
Ji-Liang Gao, Albert Owusu-Ansah, Andrea Paun, Kimberly Beacht, Erin Yim, Marie Siwicki, Alexander Yang, Qian Liu, David H. McDermott, Philip M. Murphy
Abstract
Accumulation of senescent cells is associated with the progression of pulmonary fibrosis but mechanisms accounting for this linkage are not well understood. To explore this issue, we investigated whether a class of biologically active profibrotic lipids, the leukotrienes (LT), is part of the senescence-associated secretory phenotype. The analysis of conditioned medium (CM) lipid extracts and gene expression of LT biosynthesis enzymes revealed that senescent cells secreted LT regardless of the origin of the cells or the modality of senescence induction. The synthesis of LT was biphasic and followed by anti-fibrotic prostaglandin (PG) secretion. The LT-rich CM of senescent lung fibroblasts (IMR90) induced pro-fibrotic signaling in naïve fibroblasts, which were abrogated by inhibitors of ALOX5, the principal enzyme in LT biosynthesis. The bleomycin-induced expression of genes encoding LT and PG synthases, level of cysteinyl leukotriene in the bronchoalveolar lavage, and overall fibrosis were reduced upon senescent cells removal either in a genetic mouse model or after senolytic treatment. Quantification of ALOX5+cells in lung explants obtained from idiopathic pulmonary fibrosis (IPF) patients indicated that half of these cells were also senescent (p16Ink4a+). Unlike human fibroblasts from unused donor lungs made senescent by irradiation, senescent IPF fibroblasts secreted LTs but failed to synthesize PGs. This study demonstrates for the first time that senescent cells secrete functional LTs, significantly contributing to the LTs pool known to cause or exacerbate IPF.
Authors
Christopher D. Wiley, Alexis N. Brumwell, Sonnet S. Davis, Julia R. Jackson, Alexis Valdovinos, Cheresa Calhoun, Fatouma Alimirah, Carlos A. Castellanos, Richard Ruan, Ying Wei, Harold A. Chapman, Arvind Ramanathan, Judith Campisi, Claude Jourdan Le Saux
Abstract
The HER2-specific monoclonal antibody (mAb), Trastuzumab, has been the mainstay of therapy for HER2+ breast cancers (BC) for ~20 years. However, its therapeutic mechanism of action (MOA) remains unclear, with antitumor responses to Trastuzumab remaining heterologous and metastatic HER2+ BC remaining incurable. Consequently, understanding its MOA could enable rational strategies to enhance its efficacy. Using both novel murine and human versions of Trastuzumab, we found its antitumor activity dependent on Fcγ-Receptor stimulation of tumor-associated-macrophages (TAM) and Antibody-Dependent-Cellular-Phagocytosis (ADCP), but not cytotoxicity (ADCC). Trastuzumab also stimulated TAM activation and expansion, but did not require adaptive immunity, natural killer cells, and/or neutrophils. Moreover, inhibition of the innate immune ADCP checkpoint, CD47, significantly enhanced Trastuzumab-mediated ADCP, TAM expansion and activation, resulting in the emergence of a unique hyper-phagocytic macrophage population, improved antitumor responses and prolonged survival. In addition, we found tumor-associated CD47 expression was inversely associated with survival in HER2+ BC patients and that human HER2+ BC xenografts treated with Trastuzumab+CD47 inhibition underwent complete tumor regression. Collectively, our study identifies Trastuzumab-mediated ADCP as a significant antitumor MOA that may be clinically enabled by CD47 blockade to augment therapeutic efficacy.
Authors
Li-Chung Tsao, Erika J. Crosby, Timothy N. Trotter, Pankaj Agarwal, Bin-Jin Hwang, Chaitanya Acharya, Casey W. Shuptrine, Tao Wang, Junping Wei, Xiao Yang, Gangjun Lei, Cong-Xiao Liu, Christopher A. Rabiola, Lewis A. Chodosh, William J. Muller, Herbert Kim Lyerly, Zachary C. Hartman
Abstract
Conventional treatments for inflammatory bowel disease (IBD) have multiple potential side effects. Therefore, alternative treatments are desperately needed. This work demonstrated that systemic administration of exosomes from human bone marrow-derived mesenchymal stromal cells (MSC-Exos) significantly mitigated colitis in various models of IBD. MSC-Exos treatment downregulated inflammatory responses, maintained intestinal barrier integrity and polarized M2b macrophages, but did not favor intestinal fibrosis. Mechanistically, infused MSC-Exos mainly acted on colonic macrophages and macrophages from colitic colons acquired obvious resistance to inflammatory re-stimulation when prepared from mice treated with MSC-Exos versus untreated mice. The beneficial effect of MSC-Exos was blocked by macrophage depletion. Besides, the induction of IL-10 production from macrophages was partially involved in the beneficial effect of MSC-Exos. MSC-Exos were enriched in proteins involved in regulating multiple biological processes associated with the anti-colitic benefit of MSC-Exos. Particularly, metallothionein-2 in MSC-Exos was required for the suppression of inflammatory responses. Taken together, MSC-Exos are critical regulators of inflammatory responses and may be promising candidates for IBD treatment.
Authors
Huashan Liu, Zhenxing Liang, Fengwei wang, Chi Zhou, Xiaobin Zheng, Tuo Hu, Xiaowen He, Xianrui Wu, Ping Lan
Abstract
γδ T cells account for a large fraction of human intestinal intraepithelial lymphocytes (IELs) endowed with potent anti-tumor activities. However, little is known about their origin, phenotype and clinical relevance in colorectal cancer (CRC). To determine γδ IEL gut-specificity, homing and functions, γδ T cells were purified from human healthy blood, lymph nodes, liver, skin, intestine either disease-free or affected by CRC or generated from thymic precursors. The constitutive expression of NKp46 specifically identifies a new subset of cytotoxic Vδ1 T cells representing the largest fraction of gut-resident IELs. The ontogeny and gut-tropism of NKp46pos/Vδ1 IELs depends both on distinctive features of Vδ1 thymic precursors and gut-environmental factors. Either the constitutive presence of NKp46 on tissue-resident Vδ1 intestinal IELs or its induced-expression on IL-2/IL-15 activated Vδ1 thymocytes are associated with anti-tumor functions. Higher frequencies of NKp46pos/Vδ1 IELs in tumor-free specimens from CRC patients correlate with a lower risk of developing metastatic III/IV disease stages. Additionally, our in vitro settings reproducing CRC tumor-microenvironment inhibited the expansion of NKp46pos/Vδ1 cells from activated thymic precursors. These results parallel the very low frequencies of NKp46pos/Vδ1 IELs able to infiltrate CRC, thus providing new insights to either follow-up cancer progression or develop novel adoptive cellular therapies.
Authors
Joanna Mikulak, Ferdinando Oriolo, Elena Bruni, Alessandra Roberto, Federico S. Colombo, Anna Villa, Marita Bosticardo, Ileana Bortolomai, Elena Lo Presti, Serena Meraviglia, Francesco Dieli, Stefania Vetrano, Silvio Danese, Silvia Della Bella, Michele M. Carvello, Matteo Sacchi, Giovanni Cugini, Giovanni Colombo, Marco Klinger, Paola Spaggiari, Massimo Roncalli, Immo Prinz, Sarina Ravens, Biagio di Lorenzo, Emanuela Marcenaro, Bruno Silva-Santos, Antonino Spinelli, Domenico Mavilio
Abstract
Rationale: Reflex-mediated sympathoexcitation is central to the pathogenesis of arrhythmias and heart disease; neuraxial modulation can favorably attenuate these cardiac reflexes leading to cardioprotection. Objective: The purpose of this study was to define the mechanism by which cardiac neural decentralization and spinal cord stimulation (SCS) reduces ischemia-induced ventricular fibrillation (VF) and sudden cardiac death (SCD) by utilizing direct neurotransmitter measurements in the heart. Methods and Results: Direct measurement of norepinephrine (NE) levels in the left ventricular (LV) interstitial fluid (ISF) by microdialysis in response to transient left anterior descending coronary artery occlusion (CAO: 15 min) in anesthetized canines. Responses were studied with: (i) intact neuraxis and were compared to those in which the (ii) intrathoracic component of the cardiac neuraxis (stellate ganglia),(iii) the intrinsic cardiac neuronal (ICN) system were surgically delinked from the central nervous system versus (iv) subjects with intact neuraxis subjected to pre-emptive SCS (T1-T3 spinal level). With an intact neuraxis, animals with exaggerated NE-ISF levels in response to CAO were at increased risk for VF and SCD. During CAO there was a 152% increase in NE level when the entire neuraxis was intact compared to 114% following intrathoracic neuraxial decentralization (removal of the stellates) and 16% increase following ICN decentralization, when the entire heart and ICN was delinked from the other levels of the neuraxis. During SCS, CAO increased NE levels by 59%. Risk for CAO-induced VF was 38% in controls, 8% following total decentralization and 11% following SCS. Conclusions: These data indicate that ischemia related afferent neuronal transmission engages central and intrathoracic sympathetic reflexes which amplifies sympathoexcitation and results in an increase in regional ventricular NE release that causes VF and SCD. Surgical decentralization or SCS prevents this amplification of sympathoexcitation, attenuating the resultant NE release, and reduces VF and SCD.
Authors
Jeffrey L. Ardell, Robert D. Foreman, J. Andrew Armour, Kalyanam Shivkumar
Abstract
At diagnosis, most people with type 1 diabetes (T1D) produce measurable levels of endogenous insulin, but the rate at which insulin secretion declines is heterogeneous. To explain this heterogeneity, we sought to identify a composite signature predictive of insulin secretion, using a collaborative assay evaluation and analysis pipeline that incorporated multiple cellular and serum measures reflecting beta cell health and immune system activity. The ability to predict decline in insulin secretion would be useful for patient stratification for clinical trial enrollment or therapeutic selection. Analytes from 12 qualified assays were measured in shared samples from subjects newly diagnosed with T1D. We developed a computational tool to identify a composite panel associated with decline in insulin secretion over 2 years after diagnosis. The tool employs multiple filtering steps to reduce data dimensionality, incorporates error-estimation techniques including cross-validation and sensitivity analysis, and is flexible to assay type, clinical outcome and disease setting. Using this novel analytical tool, we identified a panel of immune markers that, in combination, are highly associated with loss of insulin secretion. The methods used here represent a novel process for identifying combined immune signatures that predict outcomes relevant for complex and heterogeneous diseases like T1D.
Authors
Cate Speake, Samuel O. Skinner, Dror Berel, Elizabeth Whalen, Matthew J. Dufort, William Chad Young, Jared M. Odegard, Anne M. Pesenacker, Frans K. Gorus, Eddie A. James, Megan K. Levings, Peter S. Linsley, Eitan M. Akirav, Alberto Pugliese, Martin J. Hessner, Gerald T. Nepom, Raphael Gottardo, S. Alice Long
Abstract
Pulmonary drug delivery presents a unique opportunity to target lower airway inflammation, which is often characterized by the massive recruitment of neutrophils from blood. However specific therapies are lacking that can modulate airway neutrophil function, and difficult challenges must be overcome to achieve therapeutic efficacy against pulmonary inflammation, notably drug hydrophobicity, mucociliary and macrophage-dependent clearance, and high extracellular protease burden. Here, we present a multi-stage, aerodynamically favorable delivery platform that uses extracellular proteolysis to its advantage in order to deliver nanoparticle-embedded hydrophobic drugs to neutrophils within the lower airways. Our design consists in a self-regulated nanoparticle-in-microgel system, in which microgel activation is triggered by extracellular elastase (degranulated by inflammatory neutrophils), and nanoparticles are loaded with Nexinhib20, a potent neutrophil degranulation inhibitor. Successful in vivo delivery of Nexinhib20 to the airways and into neutrophils promoted resolution of the inflammatory response by dampening neutrophil recruitment and degranulation, pro-inflammatory cytokine production in both airway and systemic compartments, as well as the presence of neutrophil-derived pathological extracellular vesicles in the lung fluid. Our findings showcase a new platform that overcomes challenges in pulmonary drug delivery and allows customization to match the proteolytic footprint of given diseases.
Authors
Joscelyn C Mejías, Osric A Forrest, Camilla Margaroli, David A. Frey Rubio, Liliana Viera, Jindong Li, Xin Xu, Amit Gaggar, Rabindra Tirouvanziam, Krishnendu Roy
