Introduction: PD-1 and PD-L1 have been studied interchangeably in the clinic as checkpoints to reinvigorate T cells in diverse tumor types. Data for biologic effects of checkpoint blockade in human premalignancy are limited. Methods: We analyzed the immunologic effects of PD-L1 blockade in a clinical trial of atezolizumab in patients with asymptomatic multiple myeloma (AMM), a precursor to clinical malignancy. Genomic signatures of PD-L1 blockade in purified monocytes and T cells in vivo were also compared to those following PD-1 blockade in lung cancer patients. Effects of PD-L1 blockade on monocyte-derived dendritic cells were analyzed to better understand its effects on myeloid antigen-presenting cells. Results: In contrast to anti-PD-1 therapy, anti-PD-L1 therapy led to a distinct inflammatory signature in CD14+ monocytes and increase in myeloid-derived cytokines (e.g. IL-18) in vivo. Treatment of AMM patients with atezolizumab led to rapid activation and expansion of circulating myeloid cells which persisted in the bone marrow. Blockade of PD-L1 on purified monocyte-derived dendritic cells (DCs) led to rapid inflammasome activation and synergized with CD40L-driven DC maturation, leading to greater antigen-specific T cell expansion. Conclusions: These data show that PD-L1 blockade leads to distinct systemic immunologic effects compared to PD-1 blockade in vivo in humans, particularly manifest as rapid myeloid activation. These findings also suggest an additional role for PD-L1 as a checkpoint for regulating inflammatory phenotype of myeloid cells and antigen-presentation in DCs, which may be harnessed to improve PD-L1-based combination therapies. Trial Registration: NCT02784483 Funding: NCI CA197603, CA238471, CA208328
Noffar Bar, Federica Costa, Rituparna Das, Alyssa Duffy, Mehmet K. Samur, Samuel S. McCachren, Scott Gettinger, Natalia Neparidze, Terri L. Parker, Jithendra Kini Bailur, Katherine E. Pendleton, Richa Bajpai, Lin Zhang, Mina L. Xu, Tara Anderson, Nicola Giuliani, Ajay K. Nooka, Hearn J. Cho, Aparna Raval, Mala Shanmugam, Kavita M. Dhodapkar, Madhav Dhodapkar
BACKGROUND. Severe acute respiratory coronavirus 2 (SARS-CoV-2) caused coronavirus disease 2019 (COVID-19) has become a pandemic. This study addressed the clinical and immunopathological characteristics of severe COVID-19. METHODS. Sixty-nine COVID-19 patients were classified into as severe and non-severe groups to analyze their clinical and laboratory characteristics. A panel of blood cytokines was quantified over time. Biopsy specimens from two deceased cases were obtained for immunopathological, ultrastructural, and in situ hybridization examinations. RESULTS. Circulating cytokines, including IL8, IL6, TNFα, IP10, MCP1, and RANTES, were significantly elevated in severe COVID-19 patients. Dynamic IL6 and IL8 were associated with disease progression. SARS-CoV-2 was demonstrated to infect type II, type I pneumocytes and endothelial cells, leading to severe lung damage through cell pyroptosis and apoptosis. In severe cases, lymphopenia, neutrophilia, depletion of CD4+ and CD8+ T lymphocytes, and massive macrophage and neutrophil infiltrates were observed in both blood and lung tissues. CONCLUSIONS. A panel of circulating cytokines could be used to predict disease deterioration and inform clinical interventions. Severe pulmonary damage was predominantly attributed to both SARS-CoV-2 caused cytopathy and immunopathologic damage. Strategies that encourage pulmonary recruitment and overactivation of inflammatory cells by suppressing cytokine storm might improve the outcomes of severe COVID-19 patients.
Shaohua Li, Lina Jiang, Xi Li, Fang Lin, Yijin Wang, Boan Li, Tianjun Jiang, Weimin An, Shuhong Liu, Hongyang Liu, Pengfei Xu, Lihua Zhao, Lixin Zhang, Jinsong Mu, Hongwei Wang, Jiarui Kang, Yan Li, Lei Huang, Caizhong Zhu, Shousong Zhao, Jiangyang Lu, Junsheng Ji, Jingmin Zhao
Refractory neonatal seizures do not respond to first-line anti-seizure medications (ASMs) like phenobarbital (PB), a positive allosteric modulator for GABAA receptors. GABAA receptor-mediated inhibition is dependent upon electroneutral cation-chloride transporter KCC2 which mediates neuronal chloride extrusion and its age-dependent increase, postnatally shifts GABAergic signaling from depolarizing to hyperpolarizing. BDNF-TrkB activation following excitotoxic injury recruits downstream targets like PLCγ1, leading to KCC2 hypofunction. Here, the anti-seizure efficacy of TrkB agonists LM22A-4, HIOC, and Deoxygedunin (DG), on PB-refractory seizures, and post-ischemic TrkB-pathway activation was investigated in a mouse model (CD-1, P7) of refractory neonatal seizures. LM, a BDNF loop II mimetic, rescued PB-refractory seizures in a sexually dimorphic manner. Efficacy was associated with a significant reduction in the post-ischemic phosphorylation of TrkB at Y816, a site known to mediate post-ischemic KCC2 hypofunction via PLCγ1 activation. LM rescued ischemia-induced pKCC2-S940 dephosphorylation, preserving its membrane stability. Full TrkB agonists HIOC and DG similarly rescued PB-refractoriness. Chemogenetic inactivation of TrkB significantly reduced post-ischemic neonatal seizure burdens at P7. Sex differences identified in developmental expression profiles of TrkB and KCC2 may underlie the sexually dimorphic efficacy of LM. These results support a novel role for the TrkB receptor in the emergence of age-dependent refractory neonatal seizures.
Pavel A. Kipnis, Brennan J. Sullivan, Brandon M. Carter, Shilpa Kadam
Colitis is associated with the development of colorectal cancer (CRC) by largely undefined mechanisms that are critical for understanding the link between inflammation and cancer. Intestinal stem cells (ISCs) marked by LGR5 expression are of importance in both the inflammatory response to colitis and progression to colitis-associated colon cancer (CACC). Here, we report in human MUC1-transgenic mouse models of CACC that targeting the MUC1-C oncogenic protein, which is upregulated in inflammation, suppresses the (i) Lgr5+ ISC population, (ii) induction of Myc and core pluripotency stem cell factors, and (iii) severity and progression of colitis to dysplasia and cancer. By extension to human colon cancer cells, we demonstrate that MUC1-C drives MYC, forms a complex with MYC on the LGR5 promoter and activates LGR5 expression. We also show in CRC cells that MUC1-C induces the cancer stem cell (CSC) markers (BMI1, ALDH1, FOXA1, LIN28B) and the OCT4, SOX2 and NANOG pluripotency factors. Consistent with conferring the CSC state, targeting MUC1-C suppresses the capacity of CRC cells to promote wound healing, invasion, self-renewal and tumorigenicity. In analysis of human tissues, MUC1 expression associates with activation of inflammatory pathways, development of colitis and aggressiveness of CRCs. These results collectively indicate that MUC1-C is of importance for integrating stemness and pluripotency in colitis and CRC. Of clinical relevance, the findings further indicate that MUC1-C represents a previously unrecognized target that is druggable for treating progression of colitis and CRC.
Wei Li, Ning Zhang, Caining Jin, Mark D. Long, Hasan Rajabi, Yota Yasumizu, Atsushi Fushimi, Nami Yamashita, Masayuki Hagiwara, Rongbin Zheng, Jin Wang, Ling Kui, Harpal Singh, Surender Kharbanda, Qiang Hu, Song Liu, Donald W. Kufe
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 five 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 rotavirus (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 co-culture 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 M. Kinder, Sing Sing Way, Bryan Donnelly, Reena Mourya, Zhenhua Luo, Jorge A. Bezerra
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 employed the single-cell RNA sequencing (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 (pDC), myeloid dendritic cells (mDC), macrophage, nature killer (NK) cells and innate lymphoid cells (ILC). HIV-1 infection significantly upregulated genes involved in type-I interferon inflammatory pathways in each of the innate immune subsets. Interestingly, we found that the TNF-related apoptosis-inducing ligand (TRAIL) was upregulated in the innate immune populations including pDC, mDC, macrophage, NK and ILC. We further demonstrated that blockade of 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 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
Renal fibrosis features exaggerated inflammation, extracellular matrix (ECM) deposition, and peritubular capillary loss. We previously showed that IL-10 stimulates high molecular weight hyaluronan (HMW-HA) expression by fibroblasts, and we hypothesize that HMW-HA attenuates renal fibrosis by reducing inflammation and ECM remodeling. We studied the effects of IL-10 overexpression on HA production and scarring in mouse models of unilateral ureteral obstruction (UUO) and ischemia/reperfusion (I/R) to investigate whether IL-10 anti-fibrotic effects are HA-dependent. C57BL/6J mice were fed with the HA synthesis inhibitor, 4-methylumbelliferone (4-MU), prior to UUO. We observed that in vivo injury increased intratubular spaces, ECM deposition, and HA expression at day 7 and onwards. IL-10 overexpression reduced renal fibrosis in both models, promoted HMW-HA synthesis and stability in UUO, and regulated cell proliferation in I/R. 4-MU inhibited IL-10-driven anti-fibrotic effects, indicating that HMW-HA is necessary for cytokine-mediated reduction of fibrosis. We also found that IL-10 induces in vitro HMW-HA production by renal fibroblasts via STAT3-dependent upregulation of HA synthase 2. We propose that IL-10-induced HMW-HA synthesis plays cytoprotective and anti-fibrotic roles in kidney injury, thereby revealing an effective strategy to attenuate renal fibrosis in obstructive and ischemic pathologies.
Xinyi Wang, Swathi Balaji, Emily H. Steen, Alexander J. Blum, Hui Li, Christina K. Chan, Scott R. Manson, Thomas C. Lu, Meredith M. Rae, Paul F. Austin, Thomas N. Wight, Paul L. Bollyky, Jizhong Cheng, Sundeep G. Keswani
Lupus nephritis, one of the most serious manifestations of systemic lupus erythematosus (SLE), has both a heterogeneous clinical and pathological presentation. For example, proliferative nephritis identifies a more aggressive disease class that requires immunosuppression. However, the current classification system relies on the static appearance of histopathological morphology which does not capture differences in the inflammatory response. Therefore, a biomarker grounded in the disease biology is needed to understand the molecular heterogeneity of lupus nephritis and identify immunologic mechanism and pathways. Here, we analyzed the patterns of 1000 urine protein biomarkers in 30 patients with active lupus nephritis. We found that patients stratify over a chemokine gradient inducible by interferon-gamma. Higher values identified patients with proliferative lupus nephritis. After integrating the urine proteomics with the single-cell transcriptomics of kidney biopsies, it was observed that the urinary chemokines defining the gradient were predominantly produced by infiltrating CD8 T cells, along with natural killer and myeloid cells. The urine chemokine gradient significantly correlated with the number of kidney-infiltrating CD8 cells. These findings suggest that urine proteomics can capture the complex biology of the kidney in lupus nephritis. Patient-specific pathways may be noninvasively tracked in the urine in real time, enabling diagnosis and personalized treatment.
Andrea Fava, Jill P. Buyon, Chandra Mohan, Ting Zhang, H. Michael Belmont, Peter Izmirly, Robert Clancy, Jose Monroy Trujillo, Derek M. Fine, Yuji Zhang, Laurence Magder, Deepak A. Rao, Arnon Arazi, Celine C. Berthier, Anne Davidson, Betty Diamond, Nir Hacohen, David Wofsy, William Apruzzese, The Accelerating Medicines Partnership, Soumya Raychaudhuri, Michelle Petri
Autosomal co-dominant genetic disorder alpha-1 antitrypsin (AAT) deficiency (AATD) causes pulmonary and liver disease. Individuals homozygous for the mutant Z allele accumulate polymers of Z-AAT protein in hepatocytes, where AAT is primarily produced. This accumulation causes endoplasmic reticulum (ER) stress, oxidative stress, damage to mitochondria and inflammation, leading to fibrosis, cirrhosis and hepatocellular carcinoma. The magnitude of AAT reduction and duration of response from first generation intravenously-administered RNA interference (RNAi) therapeutic ARC-AAT and then with next generation subcutaneously-administered ARO-AAT were assessed by measuring AAT protein in serum of the PiZ transgenic mouse model and human volunteers. The impact of Z-AAT reduction by RNAi on liver disease phenotypes was evaluated in PiZ mice by measuring polymeric Z-AAT in the liver; expression of genes associated with fibrosis, autophagy, apoptosis and redox regulation; inflammation; Z-AAT globule parameters; and tumor formation. Ultrastructure of the ER, mitochondria and autophagosomes in hepatocytes were evaluated by electron microscopy. In mice, sustained RNAi treatment reduced hepatic Z-AAT polymer, restored ER and mitochondrial health, normalized expression of disease-associated genes, reduced inflammation and prevented tumor formation. RNAi therapy holds promise for the treatment of patients with AATD-associated liver disease. ARO-AAT is currently in Phase 2/3 clinical trials.
Christine I. Wooddell, Keith Blomenkamp, Ryan M. Peterson, Vladimir M. Subbotin, Christian Schwabe, James Hamilton, Qili Chu, Dawn R. Christianson, Julia O. Hegge, John Kolbe, Holly L. Hamilton, Maria F. Branca-Afrazi, Bruce D. Given, David L. Lewis, Edward Gane, Steven B. Kanner, Jeffrey H. Teckman
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of human coronavirus disease 2019 (COVID-19), emerged in Wuhan, China in December 2019. The virus rapidly spread globally, resulting in a public-health crisis including more than 3.1 million cases and 224,000 deaths as of May 1, 2020. Here, we describe the identification and evaluation of commercially available reagents and assays for the molecular detection of SARS-CoV-2 in infected formalin fixed paraffin embedded (FFPE) cell pellets. We identified a suitable rabbit polyclonal anti-SARS-CoV spike protein antibody and a mouse monoclonal anti-SARS-CoV nucleocapsid protein (NP) antibody for cross detection of the respective SARS-CoV-2 proteins by immunohistochemistry (IHC) and immunofluorescence assay (IFA). Next, we established RNAscope in situ hybridization (ISH) to detect SARS-CoV-2 RNA. Furthermore, we established a multiplex fluorescence ISH (mFISH) to detect positive-sense SARS-CoV-2 RNA and negative-sense SARS-CoV-2 RNA (a replicative intermediate indicating viral replication). Finally, we developed a dual staining assay using IHC and ISH to detect SARS-CoV-2 antigen and RNA in the same FFPE section. These reagents and assays will accelerate COVID-19 pathogenesis studies in humans and in COVID-19 animal models.
Jun Liu, April M. Babka, Brian J. Kearney, Sheli R. Radoshitzky, Jens H. Kuhn, Xiankun Zeng
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