Esophageal Adenocarcinoma (EAC) develops from Barrett’s Esophagus (BE), a chronic inflammatory state that can progress through a series of transformative dysplastic states before tumor development. While molecular and genetic changes of EAC tumors have been studied, immune microenvironment changes during Barrett’s progression to EAC remain poorly understood. In this study, we identify potential immunologic changes which can occur during BE to EAC progression. RNA Sequencing (RNA-Seq) analysis on tissue samples from EAC patients undergoing surgical resection demonstrated that a subset of chemokines and cytokines, most notably IL-6 and IL-8, increased during BE progression to EAC. xCell deconvolution analysis investigating immune cell population changes demonstrated that the largest changes in expression during BE progression occurred in M2 macrophages, pro B-cells, and eosinophils. Multiplex immunohistochemical staining of tissue microarrays showed increased immune cell populations during Barrett’s progression to high grade dysplasia. In contrast, EAC tumor sections were relatively immune poor, with a rise in PD-L1 expression and loss of CD8+ T-cells. These data demonstrate the EAC microenvironment is characterized by poor cytotoxic effector cell infiltration and increased immune inhibitory signaling. These findings suggest an immune suppressive microenvironment, highlighting the need for further studies to explore immune modulatory therapy in EAC.
Kiran H. Lagisetty, Dyke P. McEwen, Derek J. Nancarrow, Johnathon G. Schiebel, Daysha Ferrer-Torres, Dipankar Ray, Timothy L. Frankel, Jules Lin, Andrew C. Chang, Laura A. Kresty, David G. Beer
Extra-pulmonary manifestations of COVID-19 are associated with a much higher mortality rate. Yet, little is known about the pathogenesis of systemic complications of COVID-19. Here, we create a murine model of SARS-CoV-2 induced severe systemic toxicity and multi-organ involvement by expressing the human ACE2 transgene in multiple tissues via viral delivery followed by systemic administration of SARS-CoV-2. The animals develop a profound phenotype within 7 days with severe weight loss, morbidity and failure to thrive. We demonstrate there is metabolic suppression of oxidative phosphorylation and the tri-carboxylic acid (TCA) cycle in multiple organs with neutrophilia, lymphopenia and splenic atrophy mirroring human COVID-19 phenotypes. Animals had a significantly lower heart rate and electron microscopy demonstrated myofibrillar disarray and myocardial edema, a common pathogenic cardiac phenotype in human COVID-19. We perform metabolomic profiling of peripheral blood and identify a panel of TCA cycle metabolites that serve as biomarkers of depressed oxidative phosphorylation. Finally, we observed that SARS-CoV-2 induces epigenetic changes of DNA methylation, that affects expression of immune response genes and could in part contribute to COVID-19 pathogenesis. Our model suggests that SARS-CoV-2 induced metabolic reprogramming and epigenetic changes in internal organs could contribute to systemic toxicity and lethality in COVID-19.
Shen Li, Feiyang Ma, Tomohiro Yokota, Gustavo Garcia Jr., Amelia Palermo, Yijie Wang, Colin Farrell, Yu-Chen Wang, Rimao Wu, Zhiqiang Zhou, Calvin Pan, Marco Morselli, Michael A. Teitell, Sergey Ryazantsev, Gregory A. Fishbein, Johanna ten Hoeve, Valerie A. Arboleda, Joshua Bloom, Barbara J. Dillon, Matteo Pellegrini, Aldons J. Lusis, Thomas G. Graeber, Vaithilingaraja Arumugaswami, Arjun Deb
Those under the age of 6 months are at significant risk from influenza virus infection; however, there is currently no vaccine approved for this age group. Influenza virus neuraminidase has emerged as a potential additional target for vaccine strategies. In this study, we sought to understand the ability of newborns to mount an antibody response to neuraminidase. Here we employed a nonhuman primate model given the similarities to humans in the immune system and development. We measured antibody to neuraminidase following infection with an H1N1 virus or following vaccination and challenge. Administration of an inactivated virus vaccine was not capable of eliciting detectable NA-specific antibody, even in the presence of adjuvants previously shown to increase total virus-specific IgG. However, both naïve and vaccinated newborns generated a neuraminidase-specific antibody response following virus infection. Interestingly, the presence of the vaccine-induced response did not prevent generation of systemic antibody to neuraminidase following challenge, although the respiratory response was reduced in a significant portion of newborns. These findings are the first, to our knowledge, to evaluate the newborn response to the influenza neuraminidase protein as well as the impact of previous vaccination on generation of these antibodies following virus infection.
Patrick K. Shultz, Kali F. Crofts, Beth C. Holbrook, Martha A. Alexander-Miller
In this work, we have explored natural unmodified low- and high-density lipoproteins (LDL and HDL) as selective delivery vectors in colorectal cancer therapy. We show in vitro in cultured cells and in vivo (NanoSPECT/CT) in the CT-26 mice colorectal cancer model that LDLs are mainly taken up by cancer cells, while HDLs are preferentially taken up by macrophages. We loaded LDLs with cisplatin and HDLs with the heat shock protein-70 inhibitor AC1LINNC, turning them into a pair of “Trojan horses” delivering drugs selectively to their target cells as demonstrated in vitro in human colorectal cancer cells and macrophages, and in vivo. Coupling of the drugs to lipoproteins and stability was assessed by mass and raman spectrometry analysis. Cisplatin vectorized in LDLs led to better tumor growth suppression with strongly reduced adverse effects such as a renal or liver toxicity. AC1LINNC vectorized into HDLs induced a strong oxidative burst in macrophages and innate anti-cancer immune response. Cumulative anti-tumor effect was observed for both drug-loaded lipoproteins. Altogether, our data show that lipoproteins from patient’s blood can be used as natural nanocarriers allowing cell specific targeting, paving the way toward more efficient, safer and personalized use of chemo-and immunotherapeutic drugs in cancer.
Tarik Hadi, Christophe Ramseyer, Thomas Gautier, Pierre-Simon Bellaye, Tatiana Lopez, Antonin Schmitt, Foley Sarah, Semen Yesylevskyy, Thibault Minervini, Romain Douhard, Lucile Dondaine, Lil Proukhnitzky, Samir Messaoudi, Maeva Wendremaire, Mathieu Moreau, Fabrice Neiers, Bertrand Collin, Franck Denat, Laurent Lagrost, Carmen Garrido, Frederic Lirussi
Inflammatory damage contributes to β-cell failure in type 1 and 2 diabetes (T1D and T2D). Mitochondria are damaged by inflammatory signaling in β-cells, resulting in impaired bioenergetics and initiation of pro-apoptotic machinery. Hence, the identification of protective responses to inflammation could lead to new therapeutic targets. Here we report that mitophagy serves as a protective response to inflammatory stress in both human and rodent β-cells. Utilizing in vivo mitophagy reporters, we observed that diabetogenic pro-inflammatory cytokines induced mitophagy in response to nitrosative/oxidative mitochondrial damage. Mitophagy-deficient β-cells were sensitized to inflammatory stress, leading to the accumulation of fragmented dysfunctional mitochondria, increased β-cell death, and hyperglycemia. Overexpression of CLEC16A, a T1D gene and mitophagy regulator whose expression in islets is protective against T1D, ameliorated cytokine-induced human β-cell apoptosis. Thus, mitophagy promotes β-cell survival and prevents diabetes by countering inflammatory injury. Targeting this pathway has the potential to prevent β-cell failure in diabetes and may be beneficial in other inflammatory conditions.
Vaibhav Sidarala, Gemma L. Pearson, Vishal S. Parekh, Benjamin Thompson, Lisa Christen, Morgan A. Gingerich, Jie Zhu, Tracy Stromer, Jianhua Ren, Emma C. Reck, Biaoxin Chai, John A. Corbett, Thomas Mandrup-Poulsen, Leslie S. Satin, Scott A. Soleimanpour
Cardiopulmonary bypass (CPB) is required during most cardiac surgeries. CBP drives systemic inflammation and multi-organ dysfunction that is more severe in neonatal patients. Limited understanding of molecular mechanisms underlying CPB-associated inflammation presents a significant barrier to improving clinical outcomes. To better understand these clinical issues, we performed the first mRNA-sequencing on total circulating leukocytes from neonatal patients undergoing CPB. These data identified myeloid cells, particularly monocytes, as the major cell type driving transcriptional responses to CPB. Furthermore, Interleukin-8 (IL8) and Tumor Necrosis Factor-α (TNFα) were inflammatory cytokines robustly upregulated in leukocytes from both patients and piglets exposed to CPB. To delineate a molecular mechanism, we exposed THP-1 human monocytic cells to CPB-like conditions including artificial surfaces, high shear stress, and cooling/rewarming. Here, shear stress was found to drive cytokine upregulation via calcium-dependent signaling pathways. We also observed a subpopulation of THP-1 cells died via TNFα-mediated necroptosis in our model, which we hypothesize contributes to post-CPB inflammation. Together, our study identifies a shear-stress modulated molecular mechanism that drives systemic inflammation in pediatric CPB patients. These are also the first data to demonstrate that shear-stress causes necroptosis. Finally, we observe that calcium and TNFα signaling are novel targets to ameliorate post-CPB inflammation.
Lan N. Tu, Lance Hsieh, Masaki Kajimoto, Kevin Charette, Nataliya Kibiryeva, Adriana Forero, Sarah E. Hampson, Jennifer Marshall, James O’Brien, Marta Scatena, Michael A. Portman, Ram Savan, Christopher Benner, Alberto Aliseda, Muhammad Nuri, Douglas Bittel, Peter Pastuszko, Vishal Nigam
Background. Clostridioides difficile is a major cause of healthcare-associated diarrhea. Severity ranges from mild to life-threatening, but this variability remains poorly understood. Microbiological diagnosis of C. difficile infection (CDI) is straightforward, but offers little insight into the patient's prognosis, nor into pathophysiological determinants of clinical trajectory. The aim of this study was to discover host-derived, CDI-specific, fecal biomarkers involved in disease severity. Methods. Subjects without and with diarrhea were recruited. CDI was established by commercial, diagnostic real-time PCR assay of tcdB. CDI severity was based on IDSA/SHEA criteria. We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) approach to identify host-derived protein biomarkers from stool and applied it to diagnostic samples for cohort-wise comparison (CDI-negative vs. non-severe CDI vs. severe CDI). Selected biomarkers were orthogonally confirmed and subsequently verified in a CDI mouse model. Results. We identified a protein signature from stool, consisting of alpha-2-macroglobulin (A2M), matrix metalloproteinase-7 (MMP7) and alpha-1-antitrypsin (A1AT), that not only discriminates CDI-positive samples from non-CDI ones, but is potentially associated with disease severity. In the mouse model, this signature with the murine homologs of the corresponding proteins was also identified. Conclusions. A2M, MMP7 and A1AT serve as biomarkers in patients with CDI and define novel components of the host response that may determine disease severity.
Makan Golizeh, Kaitlin Winter, Lucie Roussel, Marija Landekic, Melanie Langelier, Vivian G. Loo, Momar Ndao, Donald C. Vinh
Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short life spans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of ~ 40 days and died of gastrointestinal disease, but therapeutic regimens aimed at restoring gastrointestinal transit extended median survival to ~ 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression pattern in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF-like abnormalities in the bioelectric properties of the upper and lower airways. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1 year-old CF rabbits as compared to WT littermates, which were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.
Jie Xu, Alessandra Livraghi-Butrico, Xia Hou, Carthic Rajagopalan, Jifeng Zhang, Jun Song, Hong Jiang, Hong-guang Wei, Hui Wang, Mohamad Bouhamdan, Jinxue Ruan, Dongshan Yang, Yining Qiu, Xie Youming, Ronald P. Barrett, Sharon A. McClellan, Hongmei Mou, Qingtian Wu, Xuequn Chen, Troy D. Rogers, Kristen J. Wilkinson, Rodney C. Gilmore, Charles R. Esther Jr., Khalequz Zaman, Xiubin Liang, Michael Sobolic, Linda Hazlett, Kezhong Zhang, Raymond A. Frizzell, Martina Gentzsch, Wanda K. O'Neal, Barbara R. Grubb, Y Eugene Chen, Richard C. Boucher, Fei Sun
Immune and inflammatory responses to SARS-CoV-2 contribute to disease severity of COVID-19. However, the utility of specific immune-based biomarkers to predict clinical outcome remains elusive. Here, we analyzed levels of 66 soluble biomarkers in 175 Italian patients with COVID-19 ranging from mild/moderate to critical severity, and assessed type-I IFN-, type-II IFN-, and NF-κB-dependent whole blood transcriptional signatures. A broad inflammatory signature was observed, implicating activation of various immune and non-hematopoietic cell subsets. Discordance between IFN-α2a protein and IFNA2 transcript levels in blood suggests that type-I IFNs during COVID-19 may be primarily produced by tissue-resident cells. Multivariable analysis of patients’ first samples revealed 12 biomarkers (CCL2, IL-15, sST2, NGAL, sTNFRSF1A, ferritin, IL-6, S100A9, MMP-9, IL-2, sVEGFR1, IL-10) that when increased were independently associated with mortality. Multivariate analyses of longitudinal biomarker trajectories identified 8 of the aforementioned biomarkers (IL-15, IL-2, NGAL, CCL2, MMP-9, sTNFRSF1A, sST2, IL-10) and two additional biomarkers (lactoferrin, CXCL9) that were significantly associated with mortality when increased, while IL-1α was associated with mortality when decreased. Among these, sST2, sTNFRSF1A, IL-10, and IL-15 were consistently higher throughout the hospitalization in patients who died versus those who recovered, suggesting that these biomarkers may provide an early warning of eventual disease outcome.
Michael S. Abers, Ottavia M. Delmonte, Emily E. Ricotta, Jonathan Fintzi, Danielle Fink, Adriana A. de Jesus, Kol A. Zarember, Sara Alehashemi, Vasileios Oikonomou, Jigar V. Desai, Scott W. Canna, Bita Shakoory, Kerry Dobbs, Luisa Imberti, Alessandra Sottini, Eugenia Quiros-Roldan, Francesco Castelli, Camillo Rossi, Duilio Brugnoni, Andrea Biondi, Laura R. Bettini, Mariella D’Angio’, Paolo Bonfanti, Riccardo Castagnoli, Daniela Montagna, Amelia Licari, Gian Luigi Marseglia, Emily Gliniewicz, Elana R. Shaw, Dana Kahle, Andre T. Rastegar, Michael A Stack, Katherine Myint-Hpu, Susan L. Levinson, Mark J. DiNubile, Daniel W. Chertow, Peter Burbelo, Jeffrey I. Cohen, Katherine R. Calvo, John S. Tsang, Helen C. Su, John I. Gallin, Douglas B. Kuhns, Raphaela Goldbach-Mansky, Michail S Lionakis, Luigi D Notarangelo
Glioblastoma multiforme (GBM) is a fatal human cancer in part because GBM stem cells are resistant to therapy and recurrence is inevitable. Previously, we demonstrated Zika virus (ZIKV) targets GBM stem cells and prevents death of mice with gliomas. Here, we evaluated the immunological basis of ZIKV-mediated protection against GBM. Introduction of ZIKV into the brain tumor increases recruitment of CD8+ T and myeloid cells to the tumor microenvironment. CD8+ T cells are required for ZIKV-dependent tumor clearance, as survival benefits are lost with CD8+ T cell depletion. Moreover, while anti-PD1 antibody therapy alone moderately improves tumor survival, when co-administered with ZIKV, survival increases. ZIKV-mediated tumor clearance also results in durable protection against syngeneic tumor re-challenge, which also depends on CD8+ T cells. To address safety concerns, we generated an immune-sensitized ZIKV strain, which is effective alone or in combination with immunotherapy. Thus, oncolytic ZIKV treatment can be leveraged by immunotherapies, which may prompt combination treatment paradigms for adult GBM patients.
Sharmila Nair, Luciano Mazzoccoli, Arijita Jash, Jennifer Govero, Sachendra S. Bais, Tong Hu, Camila R. Fontes-Garfias, Chao Shan, Hideho Okada, Sujan Shresta, Jeremy N. Rich, Pei-Yong Shi, Michael S. Diamond, Milan G. Chheda
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