Research
Abstract
Despite advances in identifying the key immunoregulatory roles of many of the human leukocyte immunoglobulin (Ig)-like receptor (LILR) family members, the function of the inhibitory molecule LILRB3 (ILT5, CD85a, LIR3) remains unclear. Studies indicate a predominant myeloid expression; however, high homology within the LILR family and a relative paucity of reagents have hindered progress for this receptor. To investigate its function and potential immunomodulatory capacity, a panel of LILRB3-specific monoclonal antibodies (mAb) was generated. LILBR3-specific mAb bound to discrete epitopes in either Ig-like domain two or four. LILRB3 ligation on primary human monocytes by an agonistic mAb resulted in phenotypic and functional changes, leading to potent inhibition of immune responses in vitro, including significant reduction in T cell proliferation. Importantly, agonizing LILRB3 in humanized mice induced tolerance and permitted efficient engraftment of allogeneic cells. Our findings reveal powerful immunosuppressive functions of LILRB3 and identify it as an important myeloid checkpoint receptor.
Authors
Muchaala J. Yeboah, Charys Papagregoriou, Des C. Jones, H. T. Claude Chan, Guangan Hu, Justine S. McPartlan, Torbjörn Schiött, Ulrika T. Mattson, C. Ian Mockridge, Ulla-Carin Tornberg, Björn Hambe, Anne Ljungars, Mikael Mattsson, Ivo Tews, Martin J. Glennie, Stephen M. Thirdborough, John Trowsdale, Björn Frendéus, Jianzhu Chen, Mark S. Cragg, Ali Roghanian
Abstract
Hidradenitis Suppurativa (HS) is a debilitating chronic inflammatory skin disease characterized by chronic abscess formation and development of multiple draining sinus tracts in the groin, axillae, and perineum. Utilizing proteomic and transcriptomic approaches, we characterized the inflammatory responses in HS in depth, revealing immune responses centered around IFN-γ, IL-36, and TNF, with lesser contribution from IL-17A. We further identified B cells and plasma cells, with associated increases in immunoglobulin production and complement activation, as pivotal players in HS pathogenesis, with BTK and SYK pathway activation as a central signal transduction networks in HS. These data provide preclinical evidence to accelerate the path towards clinical trials targeting BTK and SYK signaling in moderate to severe HS.
Authors
Johann E. Gudjonsson, Lam C. Tsoi, Feiyang Ma, Allison C. Billi, Kelsey R. van Straalen, Allard R.J.V. Vossen, H.H. Zee, Paul W. Harms, Rachael Wasikowski, Christine M. Yee, Syed Monem Rizvi, Xianying Xing, Enze Xing, Olesya Plazyo, Chang Zeng, Matthew T. Patrick, Margaret M. Lowe, Richard E. Burney, Jeffrey H. Kozlow, Jill R. Cherry-Bukowiec, Yanyun Jiang, Joseph Kirma, Stephan Weidinger, Kelly C. Cushing, Michael D. Rosenblum, Celine C. Berthier, Amanda S. MacLeod, John J. Voorhees, Fei Wen, J. Michelle Kahlenberg, Emanual Maverakis, Robert L. Modlin, Errol P. Prens
Abstract
Based on its clinical benefits, Trikafta, the combination of folding correctors VX-661 (tezacaftor), VX-445 (elexacaftor), and the gating potentiator VX-770 (ivacaftor) was FDA-approved for treatment of cystic fibrosis (CF) patients carrying deletion of phenylalanine 508 (F508del) of the CF Transmembrane Conductance Regulator (CFTR) on at least one allele. Neither the mechanism of action of VX-445, nor the susceptibility of rare CF folding mutants to Trikafta are known. Here we show that in human bronchial epithelial cells, VX-445 synergistically restores F508del-CFTR processing in combination with type I or II correctors that target the nucleotide binding domain 1 (NBD1)-membrane spanning domains (MSDs) interface and NBD2, respectively, consistent with a type III corrector mechanism. This inference was supported by the VX-445 binding to and unfolding suppression of the isolated F508del-NBD1 of CFTR. The VX-661+VX-445 treatment restored F508del-CFTR chloride channel function in the presence of VX-770 to ~62% of wild-type CFTR in homozygous nasal epithelia. Substantial rescue of rare misprocessing mutations (S13F, R31C, G85E, E92K, V520F, M1101K and N1303K), confined to MSD1, MSD2, NBD1 and NBD2 of CFTR, was also observed in airway epithelia, suggesting an allosteric correction mechanism and the possible application of Trikafta for patients with rare misfolding mutants of CFTR.
Authors
Guido Veit, Ariel Roldan, Mark A. Hancock, Dillon F. Da Fonte, Haijin Xu, Maytham Hussein, Saul Frenkiel, Elias Matouk, Tony Velkov, Gergely L. Lukacs
Abstract
African green monkeys (AGMs) are natural hosts of Simian immunodeficiency virus (SIV) that post-thymically down-regulate CD4 to maintain a large population of CD4-CD8aa+ virus-resistant cells with T-helper functionality, which can result in AGMs becoming apparently cured of SIVagm infection. To understand the mechanisms of this process we performed genome-wide transcriptional analysis on T cells induced to down-regulate CD4 in vitro from AGMs and closely-related Patas monkeys, and T cells that maintain CD4 expression from rhesus macaques. In T cells that down-regulated CD4, pathway analysis revealed an atypical regulation ofthe DNA methylation machinery, which was reversible when pharmacologically targeted with 5-aza-2deoxycytidine. This signature was driven largely by the dioxygenase TET3 that became down-regulated with loss of CD4 expression. CpG motifs within the AGM CD4 promoter region became methylated during CD4 downregulation in vitro and were stably imprinted in AGM CD4-CD8aa+ T cells sorted directly ex vivo. These results suggest AGMs employ epigenetic mechanisms to durably silence the CD4 gene. Manipulation of these mechanisms could provide avenues for modulating SIV and human immunodeficiency virus (HIV)-1 entry receptor expression in hosts that become progressively SIV-infected, which could lead to novel therapeutic interventions aimed to reduce HIV viremia in vivo.
Authors
Joseph C. Mudd, Stephen Lai, Sanjana Shah, Andrew R. Rahmberg, Jacob K. Flynn, Carly E. Starke, Molly R. Perkins, Amy Ransier, Samuel Darko, Daniel Douek, Vanessa Hirsch, Mark J. Cameron, Jason M. Brenchley
Abstract
Lynch syndrome is the most common colorectal cancer (CRC) hereditary form and it is characterized by DNA mismatch repair (MMR) deficiency. The term Lynch-like syndrome (LLS) is used for patients with MMR-deficient tumors and neither germline mutation in MLH1, MSH2, MSH6, PMS2, or EPCAM, nor MLH1 somatic methylation. Biallelic somatic inactivation or cryptic germline MMR variants undetected during genetic testing have been proposed to be involved. Sixteen patients with early-onset LLS CRC were selected for germline and tumor whole-exome sequencing. Two potentially pathogenic germline MCM8 variants were detected in a LLS male patient with fertility problems. A knockout cellular model for MCM8 was generated by CRISPR-Cas9 and detected genetic variants were produced by mutagenesis. DNA damage, microsatellite instability and mutational signatures were monitored. DNA damage was evident for MCM8KO cells and the analyzed genetic variants. Microsatellite instability and mutational signatures in MCM8KO cells were compatible with the involvement of MCM8 in MMR. Replication in an independent familial cancer cohort detected additional carriers. Unexplained MMR-deficient CRC cases, even showing somatic biallelic MMR inactivation, may be caused by underlying germline defects in genes different than the MMR genes. We suggest MCM8 as a new gene involved in CRC germline predisposition with a recessive pattern of inheritance.
Authors
Mariano Golubicki, Laia Bonjoch, José G. Acuña-Ochoa, Marcos Díaz-Gay, Jenifer Muñoz, Miriam Cuatrecasas, Teresa Ocaña, Soledad Iseas, Guillermo Mendez, Daniel Cisterna, Stephanie A. Schubert, Maartje Nielsen, Tom van Wezel, Yael Goldberg, Eli Pikarsky, Juan Robbio, Enrique Roca, Antoni Castells, Francesc Balaguer, Marina Antelo, Sergi Castellví-Bel
Abstract
Myeloid cells orchestrate the anti-tumor immune response and influence the efficacy of immune checkpoint blockade (ICB) therapies. We and others have previously shown that interleukin 32 (IL-32) mediates dendritic cell (DC) differentiation and macrophage activation. Here, we demonstrate that IL-32 expression in human melanoma positively correlates with overall survival, response to ICB, and an immune inflamed tumor microenvironment (TME) enriched in mature DC, M1 macrophages and CD8+ T cells. Treatment of B16F10 murine melanomas with IL-32 increased the frequencies of activated, tumor-specific CD8+ T cells, leading to the induction of systemic tumor immunity. Our mechanistic in vivo studies revealed a novel role of IL-32 in activating intra-tumoral DC and macrophages to act in concert to prime CD8+ T cells and recruit them into the TME through CCL5. Thereby, IL-32 treatment reduced tumor growth and rendered ICB resistant B16F10 tumors responsive to anti-PD-1 therapy without toxicity. Furthermore, increased baseline IL-32 gene expression was associated with response to nivolumab and pembrolizumab in two independent human melanoma patient cohorts, implying IL-32 as a predictive biomarker for anti-PD-1 therapy. Collectively, this study suggests IL-32 as a potent adjuvant in immunotherapy to enhance the efficacy of ICB to patients with non-T cell inflamed TME.
Authors
Thomas Gruber, Mirela Kremenovic, Hassan Sadozai, Nives Rombini, Lukas Baeriswyl, Fabienne Maibach, Robert L. Modlin, Michel Gilliet, Diego Von Werdt, Robert E. Hunger, Giulia Parisi, Gabriel Abril-Rodriguez, Antoni Ribas, Mirjam Schenk
Abstract
Arrhythmogenic cardiomyopathy (AC) is a heart disease often caused by mutations in genes coding for desmosomal proteins including desmoglein-2 (DSG2), plakoglobin (PG), and desmoplakin (DP). Therapy is symptomatic to limit arrhythmia since the mechanisms by which desmosomal components control cardiomyocyte function are largely unknown. A new paradigm would be to stabilize desmosomal cardiomyocyte adhesion and hyper-adhesion, which renders desmosomal adhesion independent from Ca2+. Here, we further characterized the mechanisms behind enhanced cardiomyocyte adhesion and hyper-adhesion. Dissociation assays performed in HL-1 cells and murine ventricular cardiac slice cultures allowed us to define a set of signaling pathways regulating cardiomyocyte adhesion under basal and hyper-adhesive conditions. Adrenergic signaling, activation of PKC and inhibition of p38MAPK enhanced cardiomyocyte adhesion, referred to as positive adhesiotropy, and induced hyper-adhesion. Activation of ERK1/2 paralleled positive adhesiotropy, whereas adrenergic signaling induced Pg phosphorylation at S665 under both basal and hyper-adhesive conditions. Adrenergic signaling and p38MAPK inhibition recruited DSG2 to cell junctions. In PG-deficient mice with an AC phenotype, only PKC activation and p38MAPK inhibition enhanced cardiomyocyte adhesion. Our results demonstrate that cardiomyocyte adhesion can be stabilized by different signaling mechanisms, which are in part off-set in PG-deficient AC.
Authors
Maria Shoykhet, Sebastian Trenz, Ellen Kempf, Tatjana Williams, Brenda Gerull, Camilla Schinner, Sunil Yeruva, Jens Waschke
Abstract
ETV6 is an ETS family transcription factor which plays a key role in hematopoiesis and megakaryocyte development. Our group and others have identified germline mutations in ETV6 resulting in autosomal dominant thrombocytopenia and predisposition to malignancy; however, molecular mechanisms defining the role of ETV6 in megakaryocyte development have not been well established. Using a combination of molecular, biochemical, and sequencing approaches in patient-derived PBMCs, we demonstrate abnormal cytoplasmic localization of ETV6 and the HDAC3/NCOR2 repressor complex that leads to overexpression of HDAC3-regulated interferon response genes. This transcriptional dysregulation is also reflected in patient-derived platelet transcripts, and drives aberrant proplatelet formation in megakaryocytes. Our results suggest that aberrant transcription may predispose patients with ETV6 mutations to bone marrow inflammation, dysplasia, and megakaryocyte dysfunction.
Authors
Marlie H. Fisher, Gregory D. Kirkpatrick, Brett M. Stevens, Courtney L. Jones, Michael U. Callaghan, Madhvi Rajpurkar, Joy Fulbright, Megan A. Cooper, Jesse Rowley, Christopher C. Porter, Arthur Gutierrez-Hartmann, Kenneth Jones, Craig T. Jordan, Eric M. Pietras, Jorge Di Paola
Abstract
The emergence of SARS-CoV-2 has created an international health crisis. Small animal models mirroring SARS-CoV-2 human disease are essential for medical countermeasure (MCM) development. Mice are refractory to SARS-CoV-2 infection due to low affinity binding to the murine angiotensin-converting enzyme 2 (ACE2) protein. Here we evaluated the pathogenesis of SARS-CoV-2 in male and female mice expressing the human ACE2 gene under the control of the keratin 18 promotor. In contrast to non-transgenic mice, intranasal exposure of K18-hACE2 animals to two different doses of SARS-CoV-2 resulted in acute disease including weight loss, lung injury, brain infection and lethality. Vasculitis was the most prominent finding in the lungs of infected mice. Transcriptomic analysis from lungs of infected animals revealed increases in transcripts involved in lung injury and inflammatory cytokines. In the lower dose challenge groups, there was a survival advantage in the female mice with 60% surviving infection whereas all male mice succumbed to disease. Male mice that succumbed to disease had higher levels of inflammatory transcripts compared to female mice. This is the first highly lethal murine infection model for SARS-CoV-2. The K18-hACE2 murine model will be valuable for the study of SARS-CoV-2 pathogenesis and the assessment of MCMs.
Authors
Joseph W. Golden, Curtis R. Cline, Xiankun Zeng, Aura R. Garrison, Brian D. Carey, Eric M. Mucker, Lauren E. White, Joshua D. Shamblin, Rebecca L. Brocato, Jun Liu, April M. Babka, Hypaitia B. Rauch, Jeffrey M. Smith, Bradley S. Hollidge, Collin Fitzpatrick, Catherine V. Badger, Jay W. Hooper
Abstract
Atrial fibrillation (AF) is the most common cardiac arrhythmia, yet the molecular signature of the vulnerable atrial substrate is not well understood. Here, we delineated a distinct transcriptional signature in right versus left atrial cardiomyocytes (CMs) at baseline, and identified chamber-specific gene expression changes in patients with history of AF in the setting of end-stage heart failure (AF+HF) that are not present in heart failure alone (HF). We observed that human left atrial (LA) CMs exhibit Notch pathway activation and increased ploidy in AF+HF, but not in HF alone. Transient activation of Notch signaling within adult CMs in a murine genetic model is sufficient to increase ploidy in both atrial chambers. Notch activation within LA CMs generated a transcriptomic fingerprint resembling AF, with dysregulation of transcription factor and ion channel genes including Pitx2, Tbx5, Kcnh2, Kcnq1, and Kcnip2. Notch activation also produced distinct cellular electrophysiologic responses in LA versus RA CMs, prolonging the action potential duration (APD) without altering the upstroke velocity in the LA, and reducing the maximal upstroke velocity without altering the APD in the RA. Our results support a shared human/murine model of increased Notch pathway activity predisposing to AF.
Authors
Catherine E. Lipovsky, Jesus Jimenez, Qiusha Guo, Gang Li, Tiankai Yin, Stephanie Hicks, Somya Bhatnagar, Kentaro Takahashi, David M. Zhang, Brittany D. Brumback, Uri Goldsztejn, Rangarajan D. Nadadur, Carlos Perez-Cervantes, Ivan P. Moskowitz, Shaopeng Liu, Bo Zhang, Stacey L. Rentschler
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