Fibroblast-derived extracellular vesicles contain SFRP1 and mediate pulmonary fibrosis

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Abstract

Idiopathic pulmonary fibrosis (IPF) is a lethal chronic lung disease characterized by aberrant intercellular communication, extracellular matrix deposition, and destruction of functional lung tissue. While extracellular vesicles (EVs) accumulate in the IPF lung, their cargo and biological effects remain unclear. We interrogated the proteome of EV and non-EV fractions during pulmonary fibrosis and characterized their contribution to fibrosis. EVs accumulated 14 days after bleomycin challenge, correlating with decreased lung function and initiated fibrogenesis in healthy precision-cut lung slices. Label-free proteomics of bronchoalveolar lavage fluid EVs (BALF-EVs) collected from mice challenged with bleomycin or control identified 107 proteins enriched in fibrotic vesicles. Multiomic analysis revealed fibroblasts as a major cellular source of BALF-EV cargo, which was enriched in secreted frizzled related protein 1 (SFRP1). Sfrp1 deficiency inhibited the activity of fibroblast-derived EVs to potentiate lung fibrosis in vivo. SFRP1 led to increased transitional cell markers, such as keratin 8, and WNT/β-catenin signaling in primary alveolar type 2 cells. SFRP1 was expressed within the IPF lung and localized at the surface of EVs from patient-derived fibroblasts and BALF. Our work reveals altered EV protein cargo in fibrotic EVs promoting fibrogenesis and identifies fibroblast-derived vesicular SFRP1 as a fibrotic mediator and potential therapeutic target for IPF.

Authors

Olivier Burgy, Christoph H. Mayr, Déborah Schenesse, Efthymios Fousekis Papakonstantinou, Beatriz Ballester, Arunima Sengupta, Yixin She, Qianjiang Hu, Maria Camila Melo-Narvaéz, Eshita Jain, Jeanine C. Pestoni, Molly Mozurak, Adriana Estrada-Bernal, Ugochi Onwuka, Christina Coughlan, Tanyalak Parimon, Peter Chen, Thomas Heimerl, Gert Bange, Bernd T. Schmeck, Michael Lindner, Anne Hilgendorff, Clemens Ruppert, Andreas Güenther, Matthias Mann, Ali Önder Yildirim, Oliver Eickelberg, Anna Lena Jung, Herbert B. Schiller, Mareike Lehmann, Gerald Burgstaller, Melanie Königshoff

Respiratory neuropathology in spinocerebellar ataxia type 7

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Abstract

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurological disorder caused by deleterious CAG repeat expansion in the coding region of the ataxin 7 gene (polyQ-ataxin-7). Infantile-onset SCA7 leads to severe clinical manifestation of respiratory distress, but the exact cause of respiratory impairment remains unclear. Using the infantile-SCA7 mouse model, the SCA7266Q/5Q mouse, we examined the impact of pathological polyQ-ataxin-7 on hypoglossal (XII) and phrenic motor units. We identified the transcript profile of the medulla and cervical spinal cord and investigated the XII and phrenic nerves and the neuromuscular junctions in the diaphragm and tongue. SCA7266Q/5Q astrocytes showed significant intranuclear inclusions of ataxin-7 in the XII and putative phrenic motor nuclei. Transcriptomic analysis revealed dysregulation of genes involved in amino acid and neurotransmitter transport and myelination. Additionally, SCA7266Q/5Q mice demonstrated blunted efferent output of the XII nerve and demyelination in both XII and phrenic nerves. Finally, there was an increased number of neuromuscular junction clusters with higher expression of synaptic markers in SCA7266Q/5Q mice compared with WT controls. These preclinical findings elucidate the underlying pathophysiology responsible for impaired glial cell function and death leading to dysphagia, aspiration, and respiratory failure in infantile SCA7.

Authors

Debolina D. Biswas, Yihan Shi, Léa El Haddad, Ronit Sethi, Meredith Huston, Sean Kehoe, Evelyn R. Scarrow, Laura M. Strickland, Logan A. Pucci, Justin S. Dhindsa, Ani Hunanyan, Albert R. La Spada, Mai K. ElMallah

T cell responses and clinical symptoms among infants with congenital cytomegalovirus infection

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Abstract

BACKGROUND Congenital cytomegalovirus (cCMV) infection can cause developmental impairment and sensorineural hearing loss (SNHL). To determine the relationship between immune responses to cCMV infection and neurologic sequelae, T cell responses were compared for their connection to clinical symptoms at birth and neurodevelopmental outcomes.METHODS Thirty cCMV-infected and 15 uninfected infants were enrolled in a single-center prospective observational case-control study. T cell pp65-specific cytokine responses; CD57, CD28, and PD-1 expression; and memory subsets were compared.RESULTS Infected neonates (73% symptomatic at birth) lacked pp65-specific cytokine-secreting T cells, with elevated frequencies of CD57+, CD28–, and PD-1+CD8+ T cells and effector memory subsets. Though frequencies overlapped between cCMV symptom groups, asymptomatic infants had higher frequencies of CD57+PD-1+CD8+ T cells. Neonates with subsequent developmental delay lacked detectable CMV-specific T cell responses, with patterns resembling those of uninfected infants. Two children with progressive SNHL had high frequencies of PD-1+CD8+ T cells over the first year compared with children without progressive SNHL.CONCLUSION Similar to published reports, neonatal viral antigen–specific cytokine-secreting T cell responses were not detected, but overall patterns indicate that globally differentiated memory CD8+ T cell populations were induced by cCMV infection, with higher frequencies of terminally differentiated PD-1+CD8+ T cells potentially associated with asymptomatic infection. In this cohort, a lack of in utero T cell differentiation was associated with developmental delay, and high frequencies of PD-1+CD8+ T cells persisted only in children with progressive SNHL. Further work is needed to define the specificity of these T cells and their mechanistic connection to these outcomes.FUNDING This study was funded through an intramural research award at Nationwide Children’s Hospital, the Pediatric Infectious Disease Society Fellowship Award funded by Stanley and Susan Plotkin and Sanofi Pasteur, the Abigail Wexner Research Institute at Nationwide Children’s Hospital, and the Pichichero Family Foundation Vaccines for Children Initiative Research Award from the Pediatric Infectious Diseases Society Foundation.

Authors

Alexandra K. Medoro, Ravi Dhital, Pablo J. Sánchez, Kaitlyn Flint, Brianna Graber, Traci Pifer, Rachelle Crisan, William C. Ray, Christopher C. Phelps, Jonathan R. Honegger, Jing Peng, Ursula Findlen, Prashant Malhotra, Oliver Adunka, Masako Shimamura

Endothelial cell–specific LAT1 ablation normalizes tumor vasculature

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Abstract

Some endothelial cells in the tumor vasculature express a system L amino acid transporter, LAT1. To elucidate the role of LAT1 in tumor-related endothelial cells, tumor cells were injected into endothelial cell–specific LAT1 conditional knockout mice (Slc7a5flox/flox; Cdh5-Cre-ERT2), and we found that the shape of the tumor vasculature was normalized and the size and numbers of lung metastasis was reduced. TNF-α–induced expression of VCAM1 and E-selectin at the surface of HUVEC, both of which are responsible for enhanced monocyte attachment and premetastatic niche formation, was reduced in the presence of LAT1 inhibitor, nanvuranlat. Deprivation of tryptophan, a LAT1 substrate, mimicked LAT1 inhibition, which led to activation of MEK1/2-ERK1/2 pathway and subsequent cystathionine γ lyase (CTH) induction. Increased production of hydrogen sulfide (H2S) by CTH was at least partially responsible for tumor vascular normalization, leading to decreased leakiness and enhanced delivery of chemotherapeutic agents to the tumor.

Authors

Jun-ichi Suehiro, Toru Kimura, Toshiyuki Fukutomi, Hisamichi Naito, Yasuharu Kanki, Youichiro Wada, Yoshiaki Kubota, Nobuyuki Takakura, Hiroyuki Sakurai

Engineered cytokine/antibody fusion proteins improve IL-2 delivery to pro-inflammatory cells and promote antitumor activity

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Abstract

Progress in cytokine engineering is driving therapeutic translation by overcoming these proteins’ limitations as drugs. The IL-2 cytokine is a promising immune stimulant for cancer treatment but is limited by its concurrent activation of both pro-inflammatory immune effector cells and antiinflammatory regulatory T cells, toxicity at high doses, and short serum half-life. One approach to improve the selectivity, safety, and longevity of IL-2 is complexing with anti–IL-2 antibodies that bias the cytokine toward immune effector cell activation. Although this strategy shows potential in preclinical models, clinical translation of a cytokine/antibody complex is complicated by challenges in formulating a multiprotein drug and concerns regarding complex stability. Here, we introduced a versatile approach to designing intramolecularly assembled single-agent fusion proteins (immunocytokines, ICs) comprising IL-2 and a biasing anti–IL-2 antibody that directs the cytokine toward immune effector cells. We optimized IC construction and engineered the cytokine/antibody affinity to improve immune bias. We demonstrated that our IC preferentially activates and expands immune effector cells, leading to superior antitumor activity compared with natural IL-2, both alone and combined with immune checkpoint inhibitors. Moreover, therapeutic efficacy was observed without inducing toxicity. This work presents a roadmap for the design and translation of cytokine/antibody fusion proteins.

Authors

Elissa K. Leonard, Jakub Tomala, Joseph R. Gould, Michael I. Leff, Jian-Xin Lin, Peng Li, Mitchell J. Porter, Eric R. Johansen, Ladaisha Thompson, Shanelle D. Cao, Shenda Hou, Tereza Henclova, Maros Huliciak, Paul R. Sargunas, Charina S. Fabilane, Ondřej Vaněk, Marek Kovar, Bohdan Schneider, Giorgio Raimondi, Warren J. Leonard, Jamie B. Spangler

Combination of locoregional radiotherapy with a TIM-3 aptamer improves survival in diffuse midline glioma models

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Abstract

Pediatric diffuse midline gliomas (DMG) with altered H3-K27M are aggressive brain tumors that arise during childhood. Despite advances in genomic knowledge and the significant number of clinical trials testing new targeted therapies, patient outcomes are still poor. Immune checkpoint blockades with small molecules, such as aptamers, are opening new therapeutic options that represent hope for this orphan disease. Here, we demonstrated that a TIM-3 aptamer (TIM-3 Apt) as monotherapy increased the immune infiltration and elicited a strong specific immune response with a tendency to improve the overall survival of treated DMG-bearing mice. Importantly, combining TIM-3 Apt with radiotherapy increased the overall median survival and led to long-term survivor mice in 2 pediatric DMG orthotopic murine models. Interestingly, TIM-3 Apt administration increased the number of myeloid populations and the proinflammatory CD8-to-Tregs ratios in the tumor microenvironment as compared with nontreated groups after radiotherapy. Importantly, the depletion of T cells led to a major loss of the therapeutic effect achieved by the combination. This work uncovers TIM-3 targeting as an immunotherapy approach to improve the radiotherapy outcome in DMGs and offers a strong foundation for propelling a phase I clinical trial using radiotherapy and TIM-3 blockade combination as a treatment for these tumors.

Authors

Iker Ausejo-Mauleon, Naiara Martinez-Velez, Andrea Lacalle, Daniel de la Nava, Javier Cebollero, Helena Villanueva, Noelia Casares, Javier Marco-Sanz, Virginia Laspidea, Oren Becher, Ana Patiño-García, Sara Labiano, Fernando Pastor, Marta M. Alonso

The aberrant tonsillar microbiota modulates autoimmune responses in rheumatoid arthritis

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Abstract

Palatine tonsils are the only air-contacted lymphoid organs that constantly engage in crosstalk with commensal microorganisms and serve as the first handling sites against microbial antigens. While tonsil inflammations have been implicated in various autoimmune diseases, including rheumatoid arthritis (RA), the precise role of tonsillar microbiota in autoimmune pathogenesis remains inadequately characterized. In this study, we profiled the tonsillar microbiota and identified a notable dysbiosis in patients with RA, particularly within the Streptococcus genus. Specifically, patients with RA exhibited an enrichment of pathogenic Streptococcus species, including S. pyogenes, S. dysgalactiae, and S. agalactiae. Colonization with these bacteria significantly exacerbated arthritis severity and increased autoimmune responses in collagen-induced arthritis (CIA). Furthermore, immunization with peptides derived from these pathogenic Streptococcus species directly induced experimental arthritis. Conversely, patients with RA demonstrated a marked deficiency in commensal Streptococcus members, notably S. salivarius. Treatment of CIA mice with S. salivarius attenuated the progression of arthritis and downregulated autoimmune responses. These findings highlight a pathogenic link of tonsillar microbiota with RA, shedding light on their contribution to autoimmunity.

Authors

Jing Li, Shenghui Li, Jiayang Jin, Ruochun Guo, Yuebo Jin, Lulu Cao, Xuanlin Cai, Peishi Rao, Yan Zhong, Xiaohong Xiang, Xiaolin Sun, Jianping Guo, Fanlei Hu, Hua Ye, Yuan Jia, Wenjing Xiao, Yuan An, Xuan Zhang, BinBin Xia, Rentao Yang, Yuanjie Zhou, Lijun Wu, Junjie Qin, Jing He, Jun Wang, Zhanguo Li

Pyruvate metabolism dictates fibroblast sensitivity to GLS1 inhibition during fibrogenesis

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Abstract

Fibrosis is a chronic disease characterized by excessive extracellular matrix production, which leads to disruption of organ function. Fibroblasts are key effector cells of this process, responding chiefly to the pleiotropic cytokine transforming growth factor–β1 (TGF-β1), which promotes fibroblast to myofibroblast differentiation. We found that extracellular nutrient availability profoundly influenced the TGF-β1 transcriptome of primary human lung fibroblasts and that biosynthesis of amino acids emerged as a top enriched TGF-β1 transcriptional module. We subsequently uncovered a key role for pyruvate in influencing glutaminase (GLS1) inhibition during TGF-β1–induced fibrogenesis. In pyruvate-replete conditions, GLS1 inhibition was ineffective in blocking TGF-β1–induced fibrogenesis, as pyruvate can be used as the substrate for glutamate and alanine production via glutamate dehydrogenase (GDH) and glutamic-pyruvic transaminase 2 (GPT2), respectively. We further show that dual targeting of either GPT2 or GDH in combination with GLS1 inhibition was required to fully block TGF-β1–induced collagen synthesis. These findings embolden a therapeutic strategy aimed at additional targeting of mitochondrial pyruvate metabolism in the presence of a glutaminolysis inhibitor to interfere with the pathological deposition of collagen in the setting of pulmonary fibrosis and potentially other fibrotic conditions.

Authors

Greg Contento, Jo-Anne A.M. Wilson, Brintha Selvarajah, Manuela Platé, Delphine Guillotin, Valle Morales, Marcello Trevisani, Vanessa Pitozzi, Katiuscia Bianchi, Rachel C. Chambers

dldh^cri3 zebrafish exhibit altered mitochondrial ultrastructure, morphology, and dysfunction partially rescued by probucol or thiamine

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Abstract

Dihydrolipoamide dehydrogenase (DLD) deficiency is a recessive mitochondrial disease caused by variants in DLD, the E3 subunit of mitochondrial α-keto (or 2-oxo) acid dehydrogenase complexes. DLD disease symptoms are multisystemic, variably manifesting as Leigh syndrome, neurodevelopmental disability, seizures, cardiomyopathy, liver disease, fatigue, and lactic acidemia. While most DLD disease symptoms are attributed to dysfunction of the pyruvate dehydrogenase complex, the effects of other α-keto acid dehydrogenase deficiencies remain unclear. Current therapies for DLD deficiency are ineffective, with no vertebrate animal model available for preclinical study. We created a viable Danio rerio (zebrafish) KO model of DLD deficiency, dldhcri3. Detailed phenotypic characterization revealed shortened larval survival, uninflated swim bladder, hepatomegaly and fatty liver, and reduced swim activity. These animals displayed increased pyruvate and lactate levels, with severe disruption of branched-chain amino acid catabolism manifest as increased valine, leucine, isoleucine, α-ketoisovalerate, and α-ketoglutarate levels. Evaluation of mitochondrial ultrastructure revealed gross enlargement, severe cristae disruption, and reduction in matrix electron density in liver, intestines, and muscle. Therapeutic modeling of candidate therapies demonstrated that probucol or thiamine improved larval swim activity. Overall, this vertebrate model demonstrated characteristic phenotypic and metabolic alterations of DLD disease, offering a robust platform to screen and characterize candidate therapies.

Authors

Manuela Lavorato, Donna Iadarola, Cristina Remes, Prabhjot Kaur, Chynna Broxton, Neal D. Mathew, Rui Xiao, Christoph Seiler, Eiko Nakamaru-Ogiso, Vernon E. Anderson, Marni J. Falk

Dysregulated immunologic landscape of the early host response in melioidosis

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Abstract

Melioidosis, a neglected tropical infection caused by Burkholderia pseudomallei, commonly presents as pneumonia or sepsis with mortality rates up to 50% despite appropriate treatment. A better understanding of the early host immune response to melioidosis may lead to new therapeutic interventions and prognostication strategies to reduce disease burden. Whole blood transcriptomic signatures in 164 patients with melioidosis and in 70 patients with other infections hospitalized in northeastern Thailand enrolled within 24 hours following hospital admission were studied. Key findings were validated in an independent melioidosis cohort. Melioidosis was characterized by upregulation of interferon (IFN) signaling responses compared with other infections. Mortality in melioidosis was associated with excessive inflammation, enrichment of type 2 immune responses, and a dramatic decrease in T cell–mediated immunity compared with survivors. We identified and independently confirmed a 5-gene predictive set classifying fatal melioidosis (validation cohort area under the receiver operating characteristic curve 0.83; 95% CI, 0.67–0.99). This study highlights the intricate balance between innate and adaptive immunity during fatal melioidosis and can inform future precision medicine strategies for targeted therapies and prognostication in this severe infection.

Authors

Patpong Rongkard, Lu Xia, Barbara Kronsteiner, Thatcha Yimthin, Rungnapa Phunpang, Adul Dulsuk, Viriya Hantrakun, Gumphol Wongsuvan, Parinya Chamnan, Lara Lovelace-Macon, Emanuele Marchi, Nicholas P.J. Day, Ali Shojaie, Direk Limmathurotsakul, Narisara Chantratita, Paul Klenerman, Susanna J. Dunachie, T. Eoin West, Sina A. Gharib

Phosphodiesterase-5 inhibition collaborates with vaccine-based immunotherapy to reprogram myeloid cells in pancreatic ductal adenocarcinoma

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Abstract

Pancreatic ductal adenocarcinoma (PDAC) is highly lethal and resistant to immunotherapy. Although immune recognition can be enhanced with immunomodulatory agents including checkpoint inhibitors and vaccines, few patients experience clinical efficacy because the tumor immune microenvironment (TiME) is dominated by immunosuppressive myeloid cells that impose T cell inhibition. Inhibition of phosphodiesterase-5 (PDE5) was reported to downregulate metabolic regulators arginase and inducible NOS in immunosuppressive myeloid cells and enhance immunity against immune-sensitive tumors, including head and neck cancers. We show for the first time to our knowledge that combining a PDE5 inhibitor, tadalafil, with a mesothelin-specific vaccine, anti–programmed cell death protein 1, and anti–cytotoxic T lymphocyte–associated protein 4 yields antitumor efficacy even against immune-resistant PDAC. To determine immunologic advantages conferred by tadalafil, we profiled the TiME using mass cytometry and single-cell RNA-sequencing analysis with Domino to infer intercellular signaling. Our analyses demonstrated that tadalafil reprograms myeloid cells to be less immunosuppressive. Moreover, tadalafil synergized with the vaccine, enhancing T cell activation including mesothelin-specific T cells. Tadalafil treatment was also associated with myeloid/T cell signaling axes important for antitumor responses (e.g., Cxcr3, Il12). Our study shows that PDE5 inhibition combined with vaccine-based immunotherapy promotes pro-inflammatory states of myeloid cells, activation of T cells, and enhanced myeloid/T cell crosstalk to yield antitumor efficacy against immune-resistant PDAC.

Authors

Nicole E. Gross, Zhehao Zhang, Jacob T. Mitchell, Soren Charmsaz, Alexei G. Hernandez, Erin M. Coyne, Sarah M. Shin, Diana Carolina Vargas Carvajal, Dimitrios N. Sidiropoulos, Yeonju Cho, Guanglan Mo, Xuan Yuan, Courtney Cannon, Jayalaxmi Suresh Babu, Melissa R. Lyman, Todd Armstrong, Luciane T. Kagohara, Katherine M. Bever, Dung T. Le, Elizabeth M. Jaffee, Elana J. Fertig, Won Jin Ho

Human gut microbiota–reactive DP8α Tregs prevent acute graft-versus-host disease in a CD73-dependent manner

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Abstract

Graft-versus-host disease (GvHD) is a life-threatening complication frequently occurring following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Since gut microbiota and regulatory T cells (Tregs) are believed to play roles in GvHD prevention, we investigated whether DP8α Tregs, which we have previously described to harbor a T cell receptor specificity for the gut commensal Faecalibacterium prausnitzii, could protect against GvHD, thereby linking the microbiota and its effect on GvHD. We observed a decrease in CD73+ DP8α Treg frequency in allo-HSCT patients 1 month after transplantation, which was associated with acute GvHD (aGvHD) development at 1 month after transplantation, as compared with aGvHD-free patients, without being correlated to hematological disease relapse. Importantly, CD73 activity was shown to be critical for DP8α Treg suppressive function. Moreover, the frequency of host-reactive DP8α Tregs was also lower in aGvHD patients, as compared with aGvHD-free patients, which could embody a protective mechanism responsible for the maintenance of this cell subset in GvHD-free patients. We also showed that human DP8α Tregs protected mice against xenogeneic GvHD through limiting deleterious inflammation and preserving gut integrity. Altogether, these results demonstrated that human DP8α Tregs mediate aGvHD prevention in a CD73-dependent manner, likely through host reactivity, advocating for the use of these cells for the development of innovative therapeutic strategies to preclude aGvHD-related inflammation.

Authors

Emmanuelle Godefroy, Patrice Chevallier, Fabienne Haspot, Caroline Vignes, Véronique Daguin, Sylvia Lambot, Margaux Verdon, Margaux De Seilhac, Valentin Letailleur, Anne Jarry, Annabelle Pédron, Thierry Guillaume, Pierre Peterlin, Alice Garnier, Marie-Anne Vibet, Maxence Mougon, Amandine Le Bourgeois, Maxime Jullien, Francine Jotereau, Frédéric Altare

The HIV latency reversing agent HODHBt inhibits the phosphatases PTPN1 and PTPN2

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Abstract

Nonreceptor tyrosine phosphatases (NTPs) play an important role in regulating protein phosphorylation and have been proposed as attractive therapeutic targets for cancer and metabolic diseases. We have previously identified that 3-Hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) enhanced STAT activation upon cytokine stimulation, leading to increased reactivation of latent HIV and effector functions of NK and CD8 T cells. Here, we demonstrate that HODHBt interacted with and inhibited the NTPs PTPN1 and PTPN2 through a mixed inhibition mechanism. We also confirm that PTPN1 and PTPN2 specifically controlled the phosphorylation of different STATs. The small molecule ABBV-CLS-484 (AC-484) is an active site inhibitor of PTPN1 and PTPN2 currently in clinical trials for advanced solid tumors. We compared AC-484 and HODHBt and found similar effects on STAT5 and immune activation, albeit with different mechanisms of action leading to varying effects on latency reversal. Our studies provide the first specific evidence to our knowledge that enhancing STAT phosphorylation via inhibition of PTPN1 and PTPN2 is an effective tool against HIV.

Authors

J. Natalie Howard, Thomas D. Zaikos, Callie Levinger, Esteban Rivera, Elyse K. McMahon, Carissa S. Holmberg, Joshua Terao, Marta Sanz, Dennis C. Copertino Jr., Weisheng Wang, Natalia Soriano-Sarabia, R. Brad Jones, Alberto Bosque

Metabolic landscape of the healthy pancreas and pancreatic tumor microenvironment

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Abstract

Pancreatic cancer, one of the deadliest human malignancies, is characterized by a fibro-inflammatory tumor microenvironment and wide array of metabolic alterations. To comprehensively map metabolism in a cell type–specific manner, we harnessed a unique single-cell RNA-sequencing dataset of normal human pancreata. This was compared with human pancreatic cancer samples using a computational pipeline optimized for this study. In the cancer cells we observed enhanced biosynthetic programs. We identified downregulation of mitochondrial programs in several immune populations, relative to their normal counterparts in healthy pancreas. Although granulocytes, B cells, and CD8+ T cells all downregulated oxidative phosphorylation, the mechanisms by which this occurred were cell type specific. In fact, the expression pattern of the electron transport chain complexes was sufficient to identify immune cell types without the use of lineage markers. We also observed changes in tumor-associated macrophage (TAM) lipid metabolism, with increased expression of enzymes mediating unsaturated fatty acid synthesis and upregulation in cholesterol export. Concurrently, cancer cells exhibited upregulation of lipid/cholesterol receptor import. We thus identified a potential crosstalk whereby TAMs provide cholesterol to cancer cells. We suggest that this may be a new mechanism boosting cancer cell growth and a therapeutic target in the future.

Authors

Monica E. Bonilla, Megan D. Radyk, Matthew D. Perricone, Ahmed M. Elhossiny, Alexis C. Harold, Paola I. Medina-Cabrera, Padma Kadiyala, Jiaqi Shi, Timothy L. Frankel, Eileen S. Carpenter, Michael D. Green, Cristina Mitrea, Costas A. Lyssiotis, Marina Pasca di Magliano

Cytomegalovirus immunity in high-risk liver transplant recipients following preemptive antiviral therapy versus prophylaxis

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Abstract

CMV-specific T cells, NK cells, and neutralizing antibodies (nAbs) were assessed in a randomized trial of CMV prevention with preemptive antiviral therapy (PET) versus prophylactic antiviral therapy (PRO) in donor-seropositive/recipient-seronegative (D+R–) liver transplant recipients (LTxR) at 100 days (end of intervention) and at 6 and 12 months after transplant. The PET group had significantly increased numbers of circulating polyfunctional T cells, NK cells, and nAbs compared with the PRO group at day 100, and several CMV immune parameters remained significantly higher by 12 months after transplant. Among PET recipients, preceding CMV viremia (vs. no preceding viremia) was associated with significantly higher levels of most CMV immune parameters at day 100. Higher numbers of CMV-specific polyfunctional T cells and NKG2C+ NK cells at day 100 were associated with a decreased incidence of CMV disease in multivariable Cox regression. The strongest associations with protection against CMV disease were with increased numbers of CMV-specific polyfunctional CD4+ T cells, CD3negCD56dimCD57negNKG2Cpos cells, and CD3negCD56dimCD57posNKG2Cpos NK cells. Our results suggest that PET is superior to PRO for CMV disease prevention by allowing low-level CMV replication and associated antigen exposure that is promptly controlled by antiviral therapy and facilitates enhanced CMV protective immunity in D+R– LTxR.

Authors

Danniel Zamora, Sayan Dasgupta, Terry Stevens-Ayers, Bradley Edmison, Drew J. Winston, Raymund R. Razonable, Aneesh K. Mehta, G. Marshall Lyon, Michael Boeckh, Nina Singh, David M. Koelle, Ajit P. Limaye

Potent neutralizing human monoclonal antibodies protect from Rift Valley fever encephalitis

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Abstract

Rift Valley fever (RVF) is an emerging arboviral disease affecting both humans and livestock. In humans, RVF displays a spectrum of clinical manifestations, including encephalitis. To date, there are no FDA-approved vaccines or therapeutics for human use, although several are in preclinical development. Few small-animal models of RVF encephalitis exist, further complicating countermeasure assessment. Human mAbs RVFV-140, RVFV-268, and RVFV-379 are recombinant potently neutralizing antibodies that prevent infection by binding the RVFV surface glycoproteins. Previous studies showed that both RVFV-268 and RVFV-140 improve survival in a lethal mouse model of disease, and RVFV-268 has prevented vertical transmission in a pregnant rat model of infection. Despite these successes, evaluation of mAbs in the context of brain disease has been limited. This is the first study to our knowledge to assess neutralizing antibodies for prevention of RVF neurologic disease using a rat model. Administration of RVFV-140, RVFV-268, or RVFV-379 24 hours prior to aerosol exposure to the virulent ZH501 strain of RVFV resulted in substantially enhanced survival and lack of neurological signs of disease. These results using a stringent and highly lethal aerosol infection model support the potential use of human mAbs to prevent the development of RVF encephalitis.

Authors

Kaleigh A. Connors, Nathaniel S. Chapman, Cynthia M. McMillen, Ryan M. Hoehl, Jackson J. McGaughey, Zachary D. Frey, Morgan Midgett, Connor Williams, Douglas S. Reed, James E. Crowe Jr., Amy L. Hartman

An intranasally administered adenovirus-vectored SARS-CoV-2 vaccine induces robust mucosal secretory IgA

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Abstract

BACKGROUND The level of nasal spike-specific secretory IgA (sIgA) is inversely correlated with the risk of SARS-CoV-2 Omicron infection. This study aimed to evaluate the safety and immunogenicity of intranasal vaccination using Ad5-S-Omicron (NB2155), a replication-incompetent human type 5 adenovirus carrying Omicron BA.1 spike.METHODS An open-label, single-center, investigator-initiated trial was carried out on 128 health care workers who had never been infected with SARS-CoV-2 and had previously received 2 or 3 injections of inactivated whole-virus vaccines, with the last dose given 3–19 months previously (median 387 days, IQR 333–404 days). Participants received 2 intranasal sprays of NB2155 at 28-day intervals between November 30 and December 30, 2022. Safety was evaluated by solicited adverse events and laboratory tests. The elevation of nasal mucosal spike-specific sIgA and serum neutralizing activities were assessed. All participants were monitored for infection by antigen tests, disease symptoms, and the elevation of nucleocapsid-specific sIgA in the nasal passage.RESULTS The vaccine-related solicited adverse events were mild. Nasal spike-specific sIgA against 10 strains had a mean geometric mean fold increase of 4.5 after the first dose, but it increased much higher to 51.5 after the second dose. Serum neutralizing titers also increased modestly to 128.1 (95% CI 74.4–220.4) against authentic BA.1 and 76.9 (95% CI 45.4–130.2) against BA.5 at 14 days after the second dose. Due to the lifting of the zero-COVID policy in China on December 7, 2022, 57.3% of participants were infected with BA.5 between days 15 and 28 after the first dose, whereas no participants reported having any symptomatic infections between day 3 and day 90 after the second dose. The elevation of nasal nucleocapsid-specific sIgA on days 0, 14, 42, and 118 after the first dose was assessed to verify that these 2-dose participants had no asymptomatic infections.CONCLUSION A 2-dose intranasal vaccination regimen using NB2155 was safe, was well tolerated, and could dramatically induce broad-spectrum spike-specific sIgA in the nasal passage. Preliminary data suggested that the intranasal vaccination may establish an effective mucosal immune barrier against infection and warranted further clinical studies.TRIAL REGISTRATION Chinese Clinical Trial Registry (ChiCTR2300070346).FUNDING Natural Science Foundation of China, Guangzhou Laboratory, The First Affiliated Hospital of Guangzhou Medical University.

Authors

Baoqing Sun, Qian Wang, Peiyan Zheng, Xuefeng Niu, Ying Feng, Weijie Guan, Si Chen, Jin Li, Tingting Cui, Yijun Deng, Zhangkai J. Cheng, Yongmei Li, Xinke Zhou, Yi Fang, Wei Wang, Zhongfang Wang, Ling Chen, Nanshan Zhong

Focusing HIV-1 Gag T cell responses to highly conserved regions by DNA vaccination in HVTN 119

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Abstract

BACKGROUND An HIV-1 DNA vaccine composed of 7 highly conserved, structurally important elements (conserved elements, CE) of p24Gag was tested in a phase I randomized, double-blind clinical trial (HVTN 119, NCT03181789) in people without HIV. DNA vaccination of CE prime/CE+p55Gag boost was compared with p55Gag.METHODS Two groups (n = 25) received 4 DNA vaccinations (CE/CE+p55Gag or p55Gag) by intramuscular injection/electroporation, including IL-12 DNA adjuvant. The placebo group (n = 6) received saline. Participants were followed for safety and tolerability. Immunogenicity was assessed for T cell and antibody responses.RESULTS Both regimens were safe and generally well tolerated. The p24CE vaccine was immunogenic and significantly boosted by CE+p55Gag (64% CD4+, P = 0.037; 42% CD8+, P = 0.004). CE+p55Gag induced responses to 5 of 7 CE, compared with only 2 CE by p55Gag DNA, with a higher response to CE5 in 30% of individuals (P = 0.006). CE+p55Gag induced significantly higher CD4+ CE T cell breadth (0.68 vs. 0.22 CE; P = 0.029) and a strong trend for overall T cell breadth (1.14 vs. 0.52 CE; P = 0.051). Both groups developed high cellular and humoral responses. p24CE vaccine–induced CD4+ CE T cell responses correlated (P = 0.007) with p24Gag antibody responses.CONCLUSION The CE/CE+p55Gag DNA vaccine induced T cell responses to conserved regions in p24Gag, increasing breadth and epitope recognition throughout p55Gag compared with p55Gag DNA. Vaccines focusing immune responses by priming responses to highly conserved regions could be part of a comprehensive HIV vaccine strategy.TRIAL REGISTRATION Clinical Trials.gov NCT03181789FUNDING HVTN, NIAID/NIH

Authors

Spyros A. Kalams, Barbara K. Felber, James I. Mullins, Hyman M. Scott, Mary A. Allen, Stephen C. De Rosa, Jack Heptinstall, Georgia D. Tomaras, Jiani Hu, Allan C. DeCamp, Margherita Rosati, Jenifer Bear, Michael N. Pensiero, John Eldridge, Michael A. Egan, Drew Hannaman, M. Juliana McElrath, George N. Pavlakis, HIV Vaccine Trials Network 119(HVTN 119) Study Team

Speed of lung inflation at birth influences the initiation of lung injury in preterm lambs

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Abstract

Gas flow is fundamental for driving tidal ventilation and, thus, the speed of lung motion, but current bias flow settings to support the preterm lung after birth do not have an evidence base. We aimed to determine the role of gas bias flow rates to generate positive pressure ventilation in initiating early lung injury pathways in the preterm lamb. Using slower speeds to inflate the lung during tidal ventilation (gas flow rates 4–6 L/min) did not affect lung mechanics, mechanical power, or gas exchange compared with those currently used in clinical practice (8–10 L/min). Speed of pressure and volume change during inflation were faster with higher flow rates. Lower flow rates resulted in less bronchoalveolar fluid protein, better lung morphology, and fewer detached epithelial cells. Overall, relative to unventilated fetal controls, there was greater protein change using 8-10 L/min, which was associated with enrichment of acute inflammatory and innate responses. Slowing the speed of lung motion by supporting the preterm lung from birth with lower flow rates than in current clinical use resulted in less lung injury without compromising tidal ventilation or gas exchange.

Authors

David G. Tingay, Monique Fatmous, Kelly Kenna, Jack Chapman, Ellen Douglas, Arun Sett, Qi Hui Poh, Sophia I. Dahm, Tuyen Kim Quach, Magdy Sourial, Haoyun Fang, David W. Greening, Prue M. Pereira-Fantini

IL-17B alleviates the pathogenesis of systemic lupus erythematosus by inhibiting FASN-mediated differentiation of B cells

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Abstract

The interleukin 17 (IL-17) family of cytokines has emerged as a critical player in autoimmune disease, including systemic lupus erythematosus (SLE). However, the role of IL-17B, a poorly understood cytokine, in the pathogenesis of SLE is still not known. In this study, we investigated the role of IL-17B in the activation and differentiation of B cells, and the pathogenesis of SLE. Intriguingly, IL-17B deficiency aggravated disease in lupus-prone mice and promoted the activation of B cells and the differentiation of germinal center B cells and plasma cells, while recombinant mouse IL-17B (rmIL-17B) significantly alleviated disease in lupus-prone mice. Mechanistically, rmIL-17B inhibited the activation of the Toll-like receptor and interferon pathways in B cells by downregulating fatty acid synthase–mediated (FASN-mediated) lipid metabolism. Loss of FASN significantly alleviated the disease in lupus-prone mice and inhibited the activation and differentiation of B cells. In addition, B cells had greater FASN expression and lower IL-17RB levels in patients with SLE than in healthy controls. Our study describes the role of IL-17B in regulating B cell activation and differentiation, and alleviating the onset of SLE. These findings will lay a theoretical foundation for further understanding of the pathogenesis of SLE.

Authors

Yucai Xiao, Yuxin Hu, Yangzhe Gao, Lin Wang, Lili Zhang, Qun Ma, Zhaochen Ning, Lu Yu, Haochen Li, Jiakun Liu, Junyu Wang, Yonghong Yang, Huabao Xiong, Guanjun Dong

Effect of metabolic status on response to SIV infection and antiretroviral therapy in nonhuman primates

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Abstract

Current antiretroviral therapy (ART) regimens efficiently limit HIV replication, thereby improving the life expectancy of people living with HIV; however, they also cause metabolic side effects. The ongoing obesity epidemic has resulted in more people with metabolic comorbidities at the time of HIV infection, yet the effect of preexisting metabolic dysregulation on infection sequelae and response to ART is unclear. Here, to investigate the impact of preexisting obesity and insulin resistance on acute infection and subsequent long-term ART, we infected a cohort of lean and obese adult male macaques with SIV and administered ART. The responses of lean and obese macaques to SIV and ART were similar with respect to plasma and cell-associated viral loads, ART drug levels in plasma and tissues, SIV-specific immune responses, adipose tissue and islet morphology, and colon inflammation, with baseline differences between lean and obese groups largely maintained. Both groups exhibited a striking depletion of CD4+ T cells from adipose tissue that did not recover with ART. However, differential responses to SIV and ART were observed for body weight, omental adipocyte size, and the adiponectin/leptin ratio, a marker of cardiometabolic risk. Thus, obesity and insulin resistance had limited effects on multiple responses to acute SIV infection and ART, while several factors that underlie long-term metabolic comorbidities were influenced by prior obesity and insulin resistance. These studies provide the foundation for future investigations into the efficacy of adjunct therapies such as metformin and glucagon-like peptide-1 receptor agonists in the prevention of metabolic comorbidities in people living with HIV.

Authors

Gabriela M. Webb, Kristin A. Sauter, Diana Takahashi, Melissa Kirigiti, Lindsay Bader, Sarah R. Lindsley, Hannah Blomenkamp, Cicely Zaro, Molly Shallman, Casey McGuire, Heather Hofmeister, Uriel Avila, Cleiton Pessoa, Joseph M. Hwang, Allyson McCullen, Matthew Humkey, Jason Reed, Lina Gao, Lee Winchester, Courtney V. Fletcher, Oleg Varlamov, Todd T. Brown, Jonah B. Sacha, Paul Kievit, Charles T. Roberts

HIF1 activation safeguards cortical bone formation against impaired oxidative phosphorylation

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Abstract

Energy metabolism, through pathways such as oxidative phosphorylation (OxPhos) and glycolysis, plays a pivotal role in cellular differentiation and function. Our study investigates the impact of OxPhos disruption in cortical bone development by deleting mitochondrial transcription factor A (TFAM). TFAM controls OxPhos by regulating the transcription of mitochondrial genes. The cortical bone, constituting the long bones’ rigid shell, is sheathed by the periosteum, a connective tissue layer populated with skeletal progenitors that spawn osteoblasts, the bone-forming cells. TFAM-deficient mice presented with thinner cortical bone, spontaneous midshaft fractures, and compromised periosteal cell bioenergetics, characterized by reduced ATP levels. Additionally, they exhibited an enlarged periosteal progenitor cell pool with impaired osteoblast differentiation. Increasing hypoxia-inducible factor 1a (HIF1) activity within periosteal cells substantially mitigated the detrimental effects induced by TFAM deletion. HIF1 is known to promote glycolysis in all cell types. Our findings underscore the indispensability of OxPhos for the proper accrual of cortical bone mass and indicate a compensatory mechanism between OxPhos and glycolysis in periosteal cells. The study opens new avenues for understanding the relationship between energy metabolism and skeletal health and suggests that modulating bioenergetic pathways may provide a therapeutic avenue for conditions characterized by bone fragility.

Authors

Mohd P. Khan, Elena Sabini, Katherine Beigel, Giulia Lanzolla, Brittany Laslow, Dian Wang, Christophe Merceron, Amato Giaccia, Fanxin Long, Deanne Taylor, Ernestina Schipani

Organelle communication maintains mitochondrial and endosomal homeostasis during podocyte lipotoxicity

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Abstract

Organelle stress exacerbates podocyte injury, contributing to perturbed lipid metabolism. Simultaneous organelle stresses can occur in the kidney in the diseased state; therefore, a thorough analysis of organelle communication is crucial for understanding the progression of kidney diseases. Although organelles closely interact with one another at membrane contact sites, limited studies have explored their involvement in kidney homeostasis. The endoplasmic reticulum (ER) protein, PDZ domain–containing 8 (PDZD8), is implicated in multiple-organelle-tethering processes and cellular lipid homeostasis. In this study, we aimed to elucidate the role of organelle communication in podocyte injury using podocyte-specific Pdzd8-knockout mice. Our findings demonstrated that Pdzd8 deletion exacerbated podocyte injury in an accelerated obesity–related kidney disease model. Proteomic analysis of isolated glomeruli revealed that Pdzd8 deletion exacerbated mitochondrial and endosomal dysfunction during podocyte lipotoxicity. Additionally, electron microscopy revealed the accumulation of abnormal, fatty endosomes in Pdzd8-deficient podocytes during obesity-related kidney diseases. Lipidomic analysis indicated that glucosylceramide accumulated in Pdzd8-deficient podocytes, owing to accelerated production and decelerated degradation. Thus, the organelle-tethering factor, PDZD8, plays a crucial role in maintaining mitochondrial and endosomal homeostasis during podocyte lipotoxicity. Collectively, our findings highlight the importance of organelle communication at the 3-way junction among the ER, mitochondria, and endosomes in preserving podocyte homeostasis.

Authors

Sho Hasegawa, Masaomi Nangaku, Yuto Takenaka, Chigusa Kitayama, Qi Li, Madina Saipidin, Yu Ah Hong, Jin Shang, Yusuke Hirabayashi, Naoto Kubota, Takashi Kadowaki, Reiko Inagi

Clinical cell-surface targets in metastatic and primary solid cancers

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Abstract

Therapies against cell-surface targets (CSTs) represent an emerging treatment class in solid malignancies. However, high-throughput investigations of CST expression across cancer types have been reliant on data sets of mostly primary tumors, despite therapeutic use most commonly in metastatic disease. We identified a total of 818 clinical trials of CST therapies with 78 CSTs. We assembled a data set spanning RNA-seq and microarrays in 7,927 benign samples, 16,866 primary tumor samples, and 6,124 metastatic tumor samples. We also utilized single-cell RNA-seq data from 36 benign tissues and 558 primary and metastatic tumor samples, and matched RNA versus protein expression in 29 benign tissue samples, 1,075 tumor samples, and 942 cell lines. High RNA expression accurately predicted high protein expression across CST therapies in benign tissues, tumor samples, and cell lines. We compared metastatic versus primary tumor expression, identified potential opportunities for repositioning, and matched cell lines to tumor types based on CST and global RNA expression. We evaluated single-cell heterogeneity across tumors, and identified rare normal cell subpopulations that may contribute to toxicity. Finally, we identified combinations of CST therapies for which bispecific approaches could improve tumor specificity. This study helps better define the landscape of CST expression in metastatic and primary cancers.

Authors

Marina N. Sharifi, Yue Shi, Matthew R. Chrostek, S. Carson Callahan, Tianfu Shang, Tracy J. Berg, Kyle T. Helzer, Matthew L. Bootsma, Martin Sjöström, Andreas Josefsson, Felix Y. Feng, Laura B. Huffman, Chris Schulte, Grace C. Blitzer, Quaovi H. Sodji, Zachary S. Morris, Vincent T. Ma, Labros Meimetis, David Kosoff, Amy K. Taylor, Aaron M. LeBeau, Joshua M. Lang, Shuang G. Zhao