Eighty-six infants born without a thymus have been treated with allogeneic cultured thymus tissue implantation (CTTI). These infants, who lack T cells and are profoundly immunodeficient at birth, after CTTI from an unmatched donor develop T cells similar to those of recipient that are tolerant to both their own major histocompatibility antigens and those of the donor. We tested use of CTTI with the goal of inducing tolerance to unmatched heart transplants in immunocompetent rats. We thymectomized and T cell–depleted Lewis rats. The rats were then given cultured thymus tissue from F1 (Lewis × Dark Agouti ) under the kidney capsule and vascularized Dark Agouti (DA) heart transplants in the abdomen. Cyclosporine was administered for 4 months. The control group did not receive CTTI. Recipients with CTTI showed repopulation of naive and recent thymic emigrant CD4 T cells; controls had none. Recipients of CTTI did not reject DA cardiac allografts. Control animals did not reject DA grafts, due to lack of functional T cells. To confirm donor-specific unresponsiveness, MHC-mismatched Brown Norway (BN) hearts were transplanted 6 months after the initial DA heart transplant. LW rats with LWxDA CTTI rejected the third-party BN hearts (mean survival time 10 days); controls did not. CTTI recipients produced antibody against third-party BN donor but not against the DA thymus donor, demonstrating humoral donor-specific tolerance. Taken together, F1(LWxDA) CTTI given to Lewis rats resulted in specific tolerance to the allogeneic DA MHC expressed in the donor thymus, with resulting long-term survival of DA heart transplants after withdrawal of all immunosuppression.
Jean Kwun, Jie Li, Clay Rouse, Jae Berm Park, Alton B. Farris, Maragatha Kuchibhatla, Joseph W. Turek, Stuart J. Knechtle, Allan D. Kirk, M. Louise Markert
Mitochondrial quality control is mediated by the PTEN-induced kinase 1 (PINK1), a cytoprotective protein that is dysregulated in inflammatory lung injury and neurodegenerative diseases. Here, we show that a ubiquitin E3 ligase receptor component, FBXO7, targets PINK1 for its cellular disposal. FBXO7, by mediating PINK1 ubiquitylation and degradation, was sufficient to induce mitochondrial injury and inflammation in experimental pneumonia. A computational simulation–based screen led to the identification of a small molecule, BC1464, which abrogated FBXO7 and PINK1 association, leading to increased cellular PINK1 concentrations and activities, and limiting mitochondrial damage. BC1464 exerted antiinflammatory activity in human tissue explants and murine lung inflammation models. Furthermore, BC1464 conferred neuroprotection in primary cortical neurons, human neuroblastoma cells, and patient-derived cells in several culture models of Parkinson’s disease. The data highlight a unique opportunity to use small molecule antagonists that disrupt PINK1 interaction with the ubiquitin apparatus to enhance mitochondrial quality, limit inflammatory injury, and maintain neuronal viability.
Yuan Liu, Travis B. Lear, Manish Verma, Kent Z.Q. Wang, P. Anthony Otero, Alison C. McKelvey, Sarah R. Dunn, Erin Steer, Nicholas W. Bateman, Christine Wu, Yu Jiang, Nathaniel M. Weathington, Mauricio Rojas, Charleen T. Chu, Bill B. Chen, Rama K. Mallampalli
HIV infection is associated with an increase in the proportion of activated CD8+ memory T cells (Tmem) that express CX3CR1, but how these cells are generated and maintained in vivo is unclear. We demonstrate that increased CX3CR1 expression on CD8+ Tmem in people living with HIV (PLWH) is dependent on coinfection with human CMV, and CX3CR1+CD8+ Tmem are enriched for a putatively immunosenescent CD57+CD28– phenotype. The cytokine IL-15 promotes the phenotype, survival, and proliferation of CX3CR1+CD57+CD8+ Tmem in vitro, whereas T cell receptor stimulation leads to their death. IL-15–driven survival is dependent on STAT5 and Bcl-2 activity, and IL-15–induced proliferation requires STAT5 and mTORC1. Thus, we identify mechanistic pathways that could explain how “inflammescent” CX3CR1+CD57+ CD8+ Tmem dominate the overall memory T cell pool in CMV-seropositive PLWH and that support reevaluation of immune senescence as a nonproliferative dead end.
Stephen R. Morris, Bonnie Chen, Joseph C. Mudd, Soumya Panigrahi, Carey L. Shive, Scott F. Sieg, Cheryl M. Cameron, David A. Zidar, Nicholas T. Funderburg, Souheil-Antoine Younes, Benigno Rodriguez, Sara Gianella, Michael M. Lederman, Michael L. Freeman
BACKGROUND Epidemiologic studies suggest that metformin has antitumor effects. Laboratory studies indicate metformin impacts cancer stem-like cells (CSCs). As part of a phase II trial, we evaluated the impact of metformin on CSC number and on carcinoma-associated mesenchymal stem cells (CA-MSCs) and clinical outcomes in nondiabetic patients with advanced-stage epithelial ovarian cancer (EOC).METHODS Thirty-eight patients with stage IIC (n = 1)/III (n = 25)/IV (n = 12) EOC were treated with either (a) neoadjuvant metformin, debulking surgery, and adjuvant chemotherapy plus metformin or (b) neoadjuvant chemotherapy and metformin, interval debulking surgery, and adjuvant chemotherapy plus metformin. Metformin-treated tumors, compared with historical controls, were evaluated for CSC number and chemotherapy response. Primary endpoints were (a) a 2-fold or greater reduction in aldehyde dehydrogenase–positive (ALDH+) CD133+ CSCs and (b) a relapse-free survival at 18 months of more than 50%.RESULTS Metformin was well tolerated. Median progression-free survival was 18.0 months (95% CI 14.0–21.6) with relapse-free survival at 18 months of 59.3% (95% CI 38.6–70.5). Median overall survival was 57.9 months (95% CI 28.0–not estimable). Tumors treated with metformin had a 2.4-fold decrease in ALDH+CD133+ CSCs and increased sensitivity to cisplatin ex vivo. Furthermore, metformin altered the methylation signature in CA-MSCs, which prevented CA-MSC–driven chemoresistance in vitro.CONCLUSION Translational studies confirm an impact of metformin on EOC CSCs and suggest epigenetic change in the tumor stroma may drive the platinum sensitivity ex vivo. Consistent with this, metformin therapy was associated with better-than-expected overall survival, supporting the use of metformin in phase III studies.TRIAL REGISTRATION ClinicalTrials.gov NCT01579812.
Jason R. Brown, Daniel K. Chan, Jessica J. Shank, Kent A. Griffith, Huihui Fan, Robert Szulawski, Kun Yang, R. Kevin Reynolds, Carolyn Johnston, Karen McLean, Shitanshu Uppal, J. Rebecca Liu, Lourdes Cabrera, Sarah E. Taylor, Brian C. Orr, Francesmary Modugno, Pooja Mehta, Michael Bregenzer, Geeta Mehta, Hui Shen, Lan G. Coffman, Ronald J. Buckanovich
The initiation of puberty is driven by an upsurge in hypothalamic gonadotropin-releasing hormone (GnRH) secretion. In turn, GnRH secretion upsurge depends on the development of a complex GnRH neuroendocrine network during embryonic life. Although delayed puberty (DP) affects up to 2% of the population, is highly heritable, and is associated with adverse health outcomes, the genes underlying DP remain largely unknown. We aimed to discover regulators by whole-exome sequencing of 160 individuals of 67 multigenerational families in our large, accurately phenotyped DP cohort. LGR4 was the only gene remaining after analysis that was significantly enriched for potentially pathogenic, rare variants in 6 probands. Expression analysis identified specific Lgr4 expression at the site of GnRH neuron development. LGR4 mutant proteins showed impaired Wnt/β-catenin signaling, owing to defective protein expression, trafficking, and degradation. Mice deficient in Lgr4 had significantly delayed onset of puberty and fewer GnRH neurons compared with WT, whereas lgr4 knockdown in zebrafish embryos prevented formation and migration of GnRH neurons. Further, genetic lineage tracing showed strong Lgr4-mediated Wnt/β-catenin signaling pathway activation during GnRH neuron development. In conclusion, our results show that LGR4 deficiency impairs Wnt/β-catenin signaling with observed defects in GnRH neuron development, resulting in a DP phenotype.
Alessandra Mancini, Sasha R. Howard, Federica Marelli, Claudia P. Cabrera, Michael R. Barnes, Michael J.E. Sternberg, Morgane Leprovots, Irene Hadjidemetriou, Elena Monti, Alessia David, Karoliina Wehkalampi, Roberto Oleari, Antonella Lettieri, Valeria Vezzoli, Gilbert Vassart, Anna Cariboni, Marco Bonomi, Marie Isabelle Garcia, Leonardo Guasti, Leo Dunkel
Loss of melanocytes is the pathological hallmark of vitiligo, a chronic inflammatory skin depigmenting disorder induced by exaggerated immune response, including autoreactive CD8 T cells producing high levels of type 1 cytokines. However, the interplay between this inflammatory response and melanocyte disappearance remains to be fully characterized. Here, we demonstrate that vitiligo skin contains a significant proportion of suprabasal melanocytes, associated with disruption of E-cadherin expression, a major protein involved in melanocyte adhesion. This phenomenon is also observed in lesional psoriatic skin. Importantly, apoptotic melanocytes were mainly observed once cells were detached from the basal layer of the epidermis, suggesting that additional mechanism(s) could be involved in melanocyte loss. The type 1 cytokines IFN-γ and TNF-α induce melanocyte detachment through E-cadherin disruption and the release of its soluble form, partly due to MMP-9. The levels of MMP-9 are increased in the skin and sera of patients with vitiligo, and MMP-9 is produced by keratinocytes in response to IFN-γ and TNF-α. Inhibition of MMP-9 or the JAK/STAT signaling pathway prevents melanocyte detachment in vitro and in vivo. Therefore, stabilization of melanocytes in the basal layer of the epidermis by preventing E-cadherin disruption appears promising for the prevention of depigmentation occurring in vitiligo and during chronic skin inflammation.
Nesrine Boukhedouni, Christina Martins, Anne-Sophie Darrigade, Claire Drullion, Jérôme Rambert, Christine Barrault, Julien Garnier, Clément Jacquemin, Denis Thiolat, Fabienne Lucchese, Franck Morel, Khaled Ezzedine, Alain Taieb, François-Xavier Bernard, Julien Seneschal, Katia Boniface
Tumor-associated macrophages (TAMs) contribute to the maintenance of a strong immunosuppressive environment, supporting tumor progression and resistance to treatment. To date, the mechanisms that drive acquisition of these immunosuppressive features are still poorly defined. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme that catabolizes free heme. It displays important cytoprotective, antiinflammatory, and antioxidant properties. A growing body of evidence suggests that HO-1 may also promote tumor development. Herein, we show that HO-1 is highly expressed in monocytic cells in the tumor microenvironment (TME) once they differentiate into TAMs. Deletion of HO-1 in the myeloid compartment enhances the beneficial effects of a therapeutic antitumor vaccine by restoring CD8+ T cell proliferation and cytotoxicity. We further show that induction of HO-1 plays a major role in monocyte education by tumor cells by modulating their transcriptional and epigenetic programs. These results identify HO-1 as a valuable therapeutic target to reprogram the TME and synergize with current cancer therapies to facilitate antitumor response.
Emmanuelle Alaluf, Benoît Vokaer, Aurélie Detavernier, Abdulkader Azouz, Marion Splittgerber, Alice Carrette, Louis Boon, Frédérick Libert, Miguel Soares, Alain Le Moine, Stanislas Goriely
Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy. In the present study, when human induced pluripotent stem cells (hiPSCs) were differentiated into myoblasts, the myoblasts derived from DMD patient hiPSCs (DMD hiPSC–derived myoblasts) exhibited an identifiable DMD-relevant phenotype: myogenic fusion deficiency. Based on this model, we developed a DMD hiPSC–derived myoblast screening platform employing a high-content imaging (BD Pathway 855) approach to generate parameters describing morphological as well as myogenic marker protein expression. Following treatment of the cells with 1524 compounds from the Johns Hopkins Clinical Compound Library, compounds that enhanced myogenic fusion of DMD hiPSC–derived myoblasts were identified. The final hits were ginsenoside Rd and fenofibrate. Transcriptional profiling revealed that ginsenoside Rd is functionally related to FLT3 signaling, while fenofibrate is linked to TGF-β signaling. Preclinical tests in mdx mice showed that treatment with these 2 hit compounds can significantly ameliorate some of the skeletal muscle phenotypes caused by dystrophin deficiency, supporting their therapeutic potential. Further study revealed that fenofibrate could inhibit mitochondrion-induced apoptosis in DMD hiPSC–derived cardiomyocytes. We have developed a platform based on DMD hiPSC–derived myoblasts for drug screening and identified 2 promising small molecules with in vivo efficacy.
Congshan Sun, In Young Choi, Yazmin I. Rovira Gonzalez, Peter Andersen, C. Conover Talbot Jr., Shama R. Iyer, Richard M. Lovering, Kathryn R. Wagner, Gabsang Lee
With the effectiveness of antimicrobials declining as antimicrobial resistance continues to threaten public health, we must look to alternative strategies for the treatment of infections. In this study, we investigated an innovative, drug-free, dual-wavelength irradiation approach that combines 2 wavelengths of light, 460 nm and 405 nm, against methicillin-resistant Staphylococcus aureus (MRSA). MRSA was initially irradiated with 460-nm light (90–360 J/cm2) and subsequently irradiated with aliquots of 405-nm light (54–324 J/cm2). For in vivo studies, mouse skin was abraded and infected with approximately 107 CFUs of MRSA and incubated for 3 hours before irradiating with 460 nm (360 J/cm2) and 405 nm (342 J/cm2). Naive mouse skin was also irradiated to investigate apoptosis. We found that staphyloxanthin, the carotenoid pigment in MRSA cells, promoted resistance to the antimicrobial effects of 405-nm light. In addition, we found that the photolytic effect of 460-nm light on staphyloxanthin attenuated resistance of MRSA to 405-nm light killing. Irradiation of 460 nm alone did not elicit any antimicrobial effect on MRSA. In a proof-of-principle mouse skin abrasion infection model, we observed significant killing of MRSA using the dual-wavelength irradiation approach. However, when either wavelength of light was administered alone, no significant decrease in bacterial viability was observed. Moreover, exposure of the dual-wavelength irradiation to naive mouse skin did not result in any visible apoptosis. In conclusion, a dual-wavelength irradiation strategy may offer an innovative, effective, and safe approach for the treatment of skin infections caused by MRSA.
Leon G. Leanse, Xueping Sharon Goh, Ji-Xin Cheng, David C. Hooper, Tianhong Dai
Next-generation sequencing (NGS) has not revealed all the mechanisms underlying resistance to genomically matched drugs. Here, we performed in 1417 tumors whole-exome tumor (somatic)/normal (germline) NGS and whole-transcriptome sequencing, the latter focusing on a clinically oriented 50-gene panel in order to examine transcriptomic silencing of putative driver alterations. In this large-scale study, approximately 13% of the somatic single nucleotide variants (SNVs) were unexpectedly not expressed as RNA; 23% of patients had ≥1 nonexpressed SNV. SNV-bearing genes consistently transcribed were TP53, PIK3CA, and KRAS; those with lower transcription rates were ALK, CSF1R, ERBB4, FLT3, GNAS, HNF1A, KDR, PDGFRA, RET, and SMO. We also determined the frequency of tumor mutations being germline, rather than somatic, in these and an additional 462 tumors with tumor/normal exomes; 33.8% of germline SNVs within the gene panel were rare (not found after filtering through variant information domains) and at risk of being falsely reported as somatic. Both the frequency of silenced variant transcription and the risk of falsely identifying germline mutations as somatic/tumor related are important phenomena. Therefore, transcriptomics is a critical adjunct to genomics when interrogating patient tumors for actionable alterations, because, without expression of the target aberrations, there will likely be therapeutic resistance.
Jacob J. Adashek, Shumei Kato, Rahul Parulkar, Christopher W. Szeto, J. Zachary Sanborn, Charles J. Vaske, Stephen C. Benz, Sandeep K. Reddy, Razelle Kurzrock
Depletion of CD4+ T cells during HIV-1 infection is mostly mediated by inflammatory cells via indirect but not clearly defined mechanisms. In this report, we used single-cell RNA-Seq (scRNA-Seq) technology to study HIV-induced transcriptomic change in innate immune cells in lymphoid organs. We performed scRNA-Seq on hCD45+hCD3–hCD19– human leukocytes isolated from spleens of humanized NOD/Rag2–/–γc–/– (NRG) mice transplanted with human CD34+ hematopoietic stem progenitor cells (NRG-hu HSC mice). We identified major populations of innate immune cells, including plasmacytoid dendritic cells (pDCs), myeloid dendritic cells (mDCs), macrophages, NK cells, and innate lymphoid cells (ILCs). HIV-1 infection significantly upregulated genes involved in type I IFN inflammatory pathways in each of the innate immune subsets. Interestingly, we found that TRAIL was upregulated in the innate immune populations, including pDCs, mDCs, macrophages, NK cells, and ILCs. We further demonstrated that blockade of the TRAIL signaling pathway in NRG-hu HSC mice prevented HIV-1–induced CD4+ T cell depletion in vivo. In summary, we characterized HIV-induced transcriptomic changes of innate immune cells in the spleen at single-cell levels, identified the TRAIL+ innate immune cells, and defined an important role of the TRAIL signaling pathway in HIV-1–induced CD4+ T cell depletion in vivo.
Liang Cheng, Haisheng Yu, John A. Wrobel, Guangming Li, Peng Liu, Zhiyuan Hu, Xiao-Ning Xu, Lishan Su
The sympathetic nervous system plays an important role in the occurrence of ventricular tachycardia (VT). Many patients, however, experience VT despite maximal doses of beta blocker therapy, possibly due to the effects of sympathetic cotransmitters such as neuropeptide Y (NPY). The purpose of this study was to determine, in a porcine model, whether propranolol at doses higher than clinically recommended could block ventricular electrophysiological effects of sympathoexcitation via stellate ganglia stimulation, and if any residual effects are mediated by NPY. Greater release of cardiac NPY was observed at higher sympathetic stimulation frequencies (10 and 20 vs. 4 Hz). Despite treatment with even higher doses of propranolol (1.0 mg/kg), electrophysiological effects of sympathetic stimulation remained, with residual shortening of activation recovery interval (ARI), a surrogate of action potential duration (APD). Adjuvant treatment with the NPY Y1 receptor antagonist BIBO 3304, however, reduced these electrophysiological effects while augmenting inotropy. These data demonstrate that high-dose beta blocker therapy is insufficient to block electrophysiological effects of sympathoexcitation, and a portion of these electrical effects in vivo are mediated by NPY. Y1 receptor blockade may represent a promising adjuvant therapy to beta-adrenergic receptor blockade.
Jonathan D. Hoang, Siamak Salavatian, Naoko Yamaguchi, Mohammed Amer Swid, David Hamon, Marmar Vaseghi
The severity of Duchenne muscular dystrophy (DMD), an incurable disease caused by the lack of dystrophin, might be modulated by different factors, including miRNAs. Among them, miR-378 is considered of high importance for muscle biology, but intriguingly, its role in DMD and its murine model (mdx mice) has not been thoroughly addressed so far. Here, we demonstrate that dystrophic mice additionally globally lacking miR-378 (double-KO [dKO] animals) exhibited better physical performance and improved absolute muscle force compared with mdx mice. Accordingly, markers of muscle damage in serum were significantly decreased in dKO mice, accompanied by diminished inflammation, fibrosis, and reduced abundance of regenerating fibers within muscles. The lack of miR-378 also normalized the aggravated fusion of dystrophin-deficient muscle satellite cells (mSCs). RNA sequencing of gastrocnemius muscle transcriptome revealed fibroblast growth factor 1 (Fgf1) as one of the most significantly downregulated genes in mice devoid of miR-378, indicating FGF1 as one of the mediators of changes driven by the lack of miR-378. In conclusion, we suggest that targeting miR-378 has the potential to ameliorate DMD pathology.
Paulina Podkalicka, Olga Mucha, Iwona Bronisz-Budzyńska, Magdalena Kozakowska, Katarzyna Pietraszek-Gremplewicz, Anna Cetnarowska, Urszula Głowniak-Kwitek, Karolina Bukowska-Strakova, Maciej Cieśla, Maria Kulecka, Jerzy Ostrowski, Michał Mikuła, Anna Potulska-Chromik, Anna Kostera-Pruszczyk, Alicja Józkowicz, Agnieszka Łoboda, Józef Dulak
Extramedullary hematopoietic cells are present in the liver of normal neonates in the first few days of life and persist in infants with biliary atresia. Based on a previous report that liver genes are enriched by erythroid pathways, we examined the liver gene expression pattern at diagnosis and found the top 5 enriched pathways are related to erythrocyte pathobiology in children who survived with the native liver beyond 2 years of age. Using immunostaining, anti-CD71 antibodies identified CD71+ erythroid cells among extramedullary hematopoietic cells in the livers at the time of diagnosis. In mechanistic experiments, the preemptive antibody depletion of hepatic CD71+ erythroid cells in neonatal mice rendered them resistant to rhesus rotavirus–induced (RRV-induced) biliary atresia. The depletion of CD71+ erythroid cells increased the number of effector lymphocytes and delayed the RRV infection of livers and extrahepatic bile ducts. In coculture experiments, CD71+ erythroid cells suppressed the activation of hepatic mononuclear cells. These data uncover an immunoregulatory role for CD71+ erythroid cells in the neonatal liver.
Li Yang, Pranavkumar Shivakumar, Jeremy Kinder, Sing Sing Way, Bryan Donnelly, Reena Mourya, Zhenhua Luo, Jorge A. Bezerra
Insulin receptor signaling is crucial for white adipose tissue (WAT) function. Consequently, lack of insulin receptor (IR) in WAT results in a diabetes-like phenotype. Yet, causes for IR downregulation in WAT of patients with diabetes are not well understood. By using multiple mouse models of obesity and insulin resistance, we identify a common downregulation of IR with a reduction of mRNA expression of selenoproteins Txnrd3, Sephs2, and Gpx3 in gonadal adipose tissue. Consistently, GPX3 is also decreased in adipose tissue of insulin-resistant and obese patients. Inducing Gpx3 expression via selenite treatment enhances IR expression via activation of the transcription factor Sp1 in 3T3-L1 preadipocytes and improves adipocyte differentiation and function. Feeding mice a selenium-enriched high-fat diet alleviates diet-induced insulin resistance with increased insulin sensitivity, decreased tissue inflammation, and elevated IR expression in WAT. Again, IR expression correlated positively with Gpx3 expression, a phenotype that is also conserved in humans. Consequently, decreasing GPx3 using siRNA technique reduced IR expression and insulin sensitivity in 3T3-L1 preadipocytes. Overall, our data identify GPx3 as a potentially novel regulator of IR expression and insulin sensitivity in adipose tissue.
Robert Hauffe, Vanessa Stein, Chantal Chudoba, Tanina Flore, Michaela Rath, Katrin Ritter, Mareike Schell, Kristina Wardelmann, Stefanie Deubel, Johannes Florian Kopp, Maria Schwarz, Kai Kappert, Matthias Blüher, Tanja Schwerdtle, Anna P. Kipp, André Kleinridders
BACKGROUND The reshaping of the immune landscape by nivolumab (NIVO) and ipilimumab (IPI) and its relation to patient outcomes is not well described.METHODS We used high-parameter flow cytometry and a computational platform, CytoBrute, to define immunophenotypes of up to 15 markers to assess peripheral blood samples from metastatic melanoma patients receiving sequential NIVO > IPI or IPI > NIVO (Checkmate-064).RESULTS The 2 treatments were associated with distinct immunophenotypic changes and had differing profiles associated with response. Only 2 immunophenotypes were shared but had opposing relationships to response/survival. To understand the impact of sequential treatment on response/survival, phenotypes that changed after the initial treatment and differentiated response in the other cohort were identified. Immunophenotypic changes occurring after NIVO were predominately associated with response to IPI > NIVO, but changes occurring after IPI were predominately associated with progression after NIVO > IPI. Among these changes, CD4+CD38+CD39+CD127–GARP– T cell subsets were increased after IPI treatment and were negatively associated with response/survival for the NIVO > IPI cohort.CONCLUSION Collectively, these data suggest that the impact of IPI and NIVO on the immunophenotypic landscape of patients is distinct and that the impact of IPI may be associated with resistance to subsequent NIVO therapy, consistent with poor outcomes in the IPI > NIVO cohort of Checkmate-064.
David M. Woods, Andressa S. Laino, Aidan Winters, Jason Alexandre, Daniel Freeman, Vinay Rao, Santi S. Adavani, Jeffery S. Weber, Pratip K. Chattopadhyay
Alveolar macrophages (AM) play a central role in initiation and resolution of lung inflammation, but the integration of these opposing core functions is poorly understood. AM expression of cholesterol 25-hydroxylase (CH25H), the primary biosynthetic enzyme for 25-hydroxycholesterol (25HC), far exceeds the expression of macrophages in other tissues, but no role for CH25H has been defined in lung biology. As 25HC is an agonist for the antiinflammatory nuclear receptor, liver X receptor (LXR), we speculated that CH25H might regulate inflammatory homeostasis in the lung. Here, we show that, of natural oxysterols or sterols, 25HC is induced in the inflamed lung of mice and humans. Ch25h–/– mice fail to induce 25HC and LXR target genes in the lung after LPS inhalation and exhibit delayed resolution of airway neutrophilia, which can be rescued by systemic treatment with either 25HC or synthetic LXR agonists. LXR-null mice also display delayed resolution, suggesting that native oxysterols promote resolution. During resolution, Ch25h is induced in macrophages upon their encounter with apoptotic cells and is required for LXR-dependent prevention of AM lipid overload, induction of Mertk, efferocytic resolution of airway neutrophilia, and induction of TGF-β. CH25H/25HC/LXR is, thus, an inducible metabolic axis that programs AMs for efferocytic resolution of inflammation.
Jennifer H. Madenspacher, Eric D. Morrell, Kymberly M. Gowdy, Jeffrey G. McDonald, Bonne M. Thompson, Ginger Muse, Jennifer Martinez, Seddon Thomas, Carmen Mikacenic, Jerry A. Nick, Edward Abraham, Stavros Garantziotis, Renee D. Stapleton, Julie M. Meacham, Mary Jane Thomassen, William J. Janssen, Donald N. Cook, Mark M. Wurfel, Michael B. Fessler
Protease-activated receptor 2 (PAR2) has long been implicated in inflammatory and visceral pain, but the cellular basis of PAR2-evoked pain has not been delineated. Although PAR2-evoked pain has been attributed to sensory neuron expression, RNA-sequencing experiments show ambiguous F2rl1 mRNA detection. Moreover, many pharmacological tools for PAR2 are nonspecific, acting also on the Mas-related GPCR family (Mrg) that are highly enriched in sensory neurons. We sought to clarify the cellular basis of PAR2-evoked pain. We developed a PAR2–conditional knockout mouse and specifically deleted PAR2 in all sensory neurons using the PirtCre mouse line. Our behavioral findings show that PAR2 agonist–evoked mechanical hyperalgesia and facial grimacing, but not thermal hyperalgesia, are dependent on PAR2 expression in sensory neurons that project to the hind paw in male and female mice. F2rl1 mRNA is expressed in a discrete population (~4%) of mostly small-diameter sensory neurons that coexpress the Nppb and IL31ra genes. This cell population has been implicated in itch, but our work shows that PAR2 activation in these cells causes clear pain-related behaviors from the skin. Our findings show that a discrete population of DRG sensory neurons mediate PAR2-evoked pain.
Shayne N. Hassler, Moeno Kume, Juliet M. Mwirigi, Ayesha Ahmad, Stephanie Shiers, Andi Wangzhou, Pradipta R. Ray, Serge N. Belugin, Dhananjay K. Naik, Michael D. Burton, Josef Vagner, Scott Boitano, Armen N. Akopian, Gregory Dussor, Theodore J. Price
Recently, we reported that expression of endogenous retroviruses (ERVs) is associated with response to immune checkpoint blockade (ICB) in renal cell carcinoma (RCC). We show that decitabine, a DNA hypomethylating agent, activates transposable element (TE) expression (LINE1 and ERVs ERV3-2 and ERV4700) and antiviral signaling to potentially enhance response to ICB in kidney cancer cell lines and primary cells. KO of RIGI and MDA5 dsRNA sensors attenuated activation of antiviral signaling associated with DNA hypomethylation, and RIGI and MDA5 IPs showed increased ERV binding with decitabine treatment. Bioinformatic analyses showed the decitabine-induced signature could be associated with increased immune infiltration and response to ICB. Cytokine secretion induced by decitabine could modestly improve T cell activation and robustly enhanced T cell migration. In a small retrospective cohort of metastatic clear cell RCC (ccRCC) patients treated with anti-PD1/PDL1 blockade, activation of some antiviral genes was significantly higher in responders. Thus, we identified a potential strategy to induce TE expression through inhibition of DNA methylation in modulating T cell action via regulation of the innate antiviral pathway.
Aguirre A. de Cubas, William Dunker, Andrew Zaninovich, Rachel A. Hongo, Anuj Bhatia, Anshuman Panda, Kathryn E. Beckermann, Gyan Bhanot, Shridar Ganesan, John Karijolich, W. Kimryn Rathmell
Streptococcus pyogenes (group A streptococcus; GAS) causes 600 million cases of pharyngitis annually worldwide. There is no licensed human GAS vaccine despite a century of research. Although the human oropharynx is the primary site of GAS infection, the pathogenic genes and molecular processes used to colonize, cause disease, and persist in the upper respiratory tract are poorly understood. Using dense transposon mutant libraries made with serotype M1 and M28 GAS strains and transposon-directed insertion sequencing, we performed genome-wide screens in the nonhuman primate (NHP) oropharynx. We identified many potentially novel GAS fitness genes, including a common set of 115 genes that contribute to fitness in both genetically distinct GAS strains during experimental NHP pharyngitis. Targeted deletion of 4 identified fitness genes/operons confirmed that our newly identified targets are critical for GAS virulence during experimental pharyngitis. Our screens discovered many surface-exposed or secreted proteins — substrates for vaccine research — that potentially contribute to GAS pharyngitis, including lipoprotein HitA. Pooled human immune globulin reacted with purified HitA, suggesting that humans produce antibodies against this lipoprotein. Our findings provide new information about GAS fitness in the upper respiratory tract that may assist in translational research, including developing novel vaccines.
Luchang Zhu, Randall J. Olsen, Stephen B. Beres, Matthew Ojeda Saavedra, Samantha L. Kubiak, Concepcion C. Cantu, Leslie Jenkins, Andrew S. Waller, Zhizeng Sun, Timothy Palzkill, Adeline R. Porter, Frank R. DeLeo, James M. Musser
Patients with systemic lupus erythematosus (SLE) present a high incidence of atherosclerosis, which contributes significantly to morbidity and mortality in this autoimmune disease. An impaired balance between regulatory (Treg) and follicular helper (Tfh) CD4+ T cells is shared by both diseases. However, whether there are common mechanisms of CD4+ T cell dysregulation between SLE and atherosclerosis remains unclear. Pre-B cell leukemia transcription factor 1 isoform d (Pbx1d) is a lupus susceptibility gene that regulates Tfh cell expansion and Treg cell homeostasis. Here, we investigated the role of T cells overexpressing Pbx1d in low-density lipoprotein receptor–deficient (Ldlr–/–) mice fed with a high-fat diet, an experimental model for atherosclerosis. Pbx1d-transgenic T cells exacerbated some phenotypes of atherosclerosis, which were associated with higher autoantibody production, increased Tfh cell frequency, and impaired Treg cell regulation, in Ldlr–/– mice as compared with control T cells. In addition, we showed that dyslipidemia and Pbx1d-transgenic expression independently impaired the differentiation and function of Treg cells in vitro, suggesting a gene/environment additive effect. Thus, our results suggest that the combination of Pbx1d expression in T cells and dyslipidemia exacerbates both atherosclerosis and autoimmunity, at least in part through a dysregulation of Treg cell homeostasis.
Wei Li, Ahmed S. Elshikha, Caleb Cornaby, Xiangyu Teng, Georges Abboud, Josephine Brown, Xueyang Zou, Leilani Zeumer-Spataro, Brian Robusto, Seung-Chul Choi, Kristianna Fredenburg, Amy Major, Laurence Morel
In severe cases of coronavirus disease 2019 (COVID-19), viral pneumonia progresses to respiratory failure. Neutrophil extracellular traps (NETs) are extracellular webs of chromatin, microbicidal proteins, and oxidant enzymes that are released by neutrophils to contain infections. However, when not properly regulated, NETs have the potential to propagate inflammation and microvascular thrombosis — including in the lungs of patients with acute respiratory distress syndrome. We now report that sera from patients with COVID-19 have elevated levels of cell-free DNA, myeloperoxidase-DNA (MPO-DNA), and citrullinated histone H3 (Cit-H3); the latter 2 are specific markers of NETs. Highlighting the potential clinical relevance of these findings, cell-free DNA strongly correlated with acute-phase reactants, including C-reactive protein, D-dimer, and lactate dehydrogenase, as well as absolute neutrophil count. MPO-DNA associated with both cell-free DNA and absolute neutrophil count, while Cit-H3 correlated with platelet levels. Importantly, both cell-free DNA and MPO-DNA were higher in hospitalized patients receiving mechanical ventilation as compared with hospitalized patients breathing room air. Finally, sera from individuals with COVID-19 triggered NET release from control neutrophils in vitro. Future studies should investigate the predictive power of circulating NETs in longitudinal cohorts and determine the extent to which NETs may be novel therapeutic targets in severe COVID-19.
Yu Zuo, Srilakshmi Yalavarthi, Hui Shi, Kelsey Gockman, Melanie Zuo, Jacqueline A. Madison, Christopher Blair, Andrew Weber, Betsy J. Barnes, Mikala Egeblad, Robert J. Woods, Yogendra Kanthi, Jason S. Knight