Broad-spectrum antibiotics are widely used in patients on intensive care units (ICU), many of which develop hospital-acquired infections with Pseudomonas aeruginosa. Although preceding antimicrobial therapy is known as a major risk factor for P. aeruginosa-induced pneumonia, the underlying mechanisms remain incompletely understood. Here we demonstrate that depletion of the resident microbiota by broad-spectrum antibiotic treatment inhibited TLR-dependent production of a proliferation inducing ligand (APRIL), resulting in a secondary IgA deficiency in the lung in mice and human ICU patients. Microbiota-dependent local IgA contributed to early antibacterial defense against P. aeruginosa. Consequently, Pseudomonas-binding IgA purified from lamina propria culture or IgA hybridomas enhanced resistance of antibiotic-treated mice to P. aeruginosa infection after transnasal substitution. Our study provides a mechanistic explanation for the well-documented risk of P. aeruginosa infection following antimicrobial therapy, and we propose local administration of IgA as a novel prophylactic strategy.
Oliver H. Robak, Markus M. Heimesaat, Andrey A. Kruglov, Sandra Prepens, Justus Ninnemann, Birgitt Gutbier, Katrin Reppe, Hubertus Hochrein, Mark Suter, Carsten J. Kirschning, Veena Marathe, Jan Buer, Mathias W. Hornef, Markus Schnare, Pascal Schneider, Martin Witzenrath, Stefan Bereswill, Ulrich Steinhoff, Norbert Suttorp, Leif E. Sander, Catherine Chaput, Bastian Opitz
Enterotoxigenic Escherichia coli (ETEC) infections are highly prevalent in developing countries where clinical presentations range from asymptomatic colonization to severe cholera-like illness. The molecular basis for these varied presentations, that may involve strain-specific virulence features as well as host factors, have not been elucidated. We demonstrate that when challenged with ETEC strain H10407, originally isolated from a case of cholera-like illness, blood group A human volunteers developed severe diarrhea more frequently than individuals from other blood groups. Interestingly, a diverse population of ETEC strains, including H10407, secrete a novel adhesin molecule, EtpA. As many bacterial adhesins also agglutinate red blood cells, we combined the use of glycan arrays, biolayer inferometry, and non-canonical amino acid labeling with hemagglutination studies to demonstrate that EtpA is a dominant ETEC blood group A specific lectin/hemagglutinin. Importantly, we also show that EtpA interacts specifically with glycans expressed on intestinal epithelial cells from blood group A individuals, and that EtpA-mediated bacterial-host interactions accelerate bacterial adhesion and the effective delivery both heat-labile and heat-stable toxins of ETEC. Collectively, these data provide additional insight into the complex molecular basis of severe ETEC diarrheal illness that may inform rational design of vaccines to protect those at highest risk.
Pardeep Kumar, F. Matthew Kuhlmann, Subhra Chakroborty, A. Louis Bourgeois, Jennifer Foulke-Abel, Brunda Tumala, Tim J. Vickers, David A. Sack, Barbara DeNearing, Clayton D. Harro, W. Shea Wright, Jeffrey C. Gildersleeve, Matthew A. Ciorba, Srikanth Santhanam, Chad K. Porter, Ramiro L. Gutierrez, Michael G. Prouty, Mark S. Riddle, Alexander Polino, Alaullah Sheikh, Mark Donowitz, James M. Fleckenstein
Cancer progression is associated with alterations of intra- and extramedullary hematopoiesis to support a systemic tumor-promoting myeloid response. However, the functional specialty, mechanism, and clinical relevance of extramedullary hematopoiesis (EMH) remain unclear. Here we showed that the heightened splenic myelopoiesis in tumor-bearing hosts was not only characterized by the accumulation of myeloid precursors, but also associated with profound functional alterations of splenic early hematopoietic stem/progenitor cells (HSPCs). With the distinct capability to produce and respond to granulocyte-macrophage colony-stimulating factor (GM-CSF), these splenic HSPCs were “primed” and committed to generating immunosuppressive myeloid cells. Mechanistically, the CCL2-CCR2 axis-dependent recruitment and the subsequent local education by the splenic stroma were critical for eliciting this splenic HSPC response. Selective abrogation of this splenic EMH was sufficient to synergistically enhance the therapeutic efficacy of immune checkpoint blockade. Clinically, patients with different types of solid tumors exhibited increased splenic HSPC levels associated with poor survival. These findings reveal a unique and important role of splenic hematopoiesis in the tumor-associated myelopoiesis.
Chong Wu, Huiheng Ning, Mingyu Liu, Jie Lin, Shufeng Luo, Wenjie Zhu, Jing Xu, Wen-Chao Wu, Jing Liang, Chun-Kui Shao, Jiaqi Ren, Bin Wei, Jun Cui, Min-Shan Chen, Limin Zheng
Tumor angiogenesis occurs through regulation of genes that orchestrate endothelial sprouting and vessel maturation, including deposition of a vessel-associated extracellular matrix. CD93 is a transmembrane receptor that is up-regulated in tumor vessels in many cancers, including high-grade glioma. Here, we demonstrate that CD93 regulates integrin-β1-signaling and organization of fibronectin fibrillogenesis during tumor vascularization. In endothelial cells and mouse retina, CD93 was found to be expressed in endothelial filopodia and to promote filopodia formation. The CD93 localization to endothelial filopodia was stabilized by interaction with multimerin-2 (MMRN2), which inhibited its proteolytical cleavage. The CD93-MMRN2 complex was required for activation of integrin-β1, phosphorylation of focal adhesion kinase (FAK) and fibronectin fibrillogenesis in endothelial cells. Consequently, tumor vessels in gliomas implanted orthotopically in CD93-deficient mice showed diminished activation of integrin-β1 and lacked organization of fibronectin into fibrillar structures. These findings demonstrate a key role of CD93 in vascular maturation and organization of the extracellular matrix in tumors, identifying it as a potential target for therapy.
Roberta Lugano, Kalyani Vemuri, Di Yu, Michael Bergqvist, Anja Smits, Magnus Essand, Staffan Johansson, Elisabetta Dejana, Anna Dimberg
Tumor Necrosis Factor (TNF) is an important mediator in numerous inflammatory diseases, e.g., in inflammatory bowel diseases (IBD). In IBD, acute increases in TNF production can lead to disease flares. Glucocorticoids (GCs), which are steroids that bind and activate the glucocorticoid receptor (GR), are able to protect animals and humans against acute TNF-induced inflammatory symptoms. Mice with a poor transcriptional response of GR-dimer-dependent target genes were studied in a model of TNF-induced lethal inflammation. In contrast to the GRwt/wt mice, these GRdim/dim mice displayed a significant increase in TNF sensitivity and a lack of protection by the GC dexamethasone (DEX). Unchallenged GRdim/dim mice had a strong interferon-stimulated gene (ISG) signature, along with STAT1 upregulation and phosphorylation. This ISG signature was gut specific and, based on our studies with antibiotics, depended on the gut microbiota. GR dimers directly bound to short DNA sequences in the STAT1 promoter known as inverted repeat negative GRE (IR-nGRE) elements. Poor control of STAT1 in GRdim/dim mice led to failure to repress ISG genes resulting in excessive necroptosis induction by TNF. Our findings support a critical interplay between gut microbiota, interferons, necroptosis and GR in both the basal response to acute inflammatory challenges and in the pharmacological intervention by GCs.
Marlies Ballegeer, Kelly Van Looveren, Steven Timmermans, Melanie Eggermont, Sofie Vandevyver, Fabien Thery, Karen Dendoncker, Jolien Souffriau, Jolien Vandewalle, Lise Van Wyngene, Riet De Rycke, Nozomi Takahashi, Peter Vandenabeele, Jan Tuckermann, Holger M. Reichardt, Francis Impens, Rudi Beyaert, Karolien De Bosscher, Roosmarijn E. Vandenbroucke, Claude Libert
BACKGROUND. Recombinant leptin (metreleptin) ameliorates hyperphagia and metabolic abnormalities in leptin-deficient humans with lipodystrophy. We aimed to determine whether metreleptin improves glucose and lipid metabolism in humans when food intake is held constant. METHODS. Patients with lipodystrophy were hospitalized for 19 days with food intake held constant by controlled diet in an inpatient metabolic ward. In a non-randomized cross-over design, previously metreleptin-treated patients (n = 8) were continued on-metreleptin for five days, and off-metreleptin for the next 14 days (withdrawal cohort). This order was reversed in metreleptin-naïve patients (n = 14), who were restudied after six months of metreleptin treatment on an ad libitum diet (initiation cohort). Outcomes included insulin sensitivity by hyperinsulinemic-euglycemic clamp, fasting glucose and triglycerides, lipolysis measured using isotopic tracers, and liver fat by magnetic resonance spectroscopy. RESULTS. With food intake constant, peripheral insulin sensitivity decreased by 41% after stopping metreleptin for 14 days (withdrawal cohort) and increased by 32% after starting metreleptin for 14 days (initiation cohort). In the initiation cohort only, metreleptin decreased fasting glucose by 11%, triglycerides by 41%, and increased hepatic insulin sensitivity. Liver fat decreased from 21.8% to 18.7%. In the initiation cohort, lipolysis did not change independent of food intake, but decreased after six months on metreleptin on an ad libitum diet by 30% (palmitate turnover) to 35% (glycerol turnover). CONCLUSION. Using lipodystrophy as a human model of leptin deficiency and replacement, we showed that metreleptin improves insulin sensitivity, and decreases hepatic and circulating triglycerides, independent of its effects on food intake. TRIAL REGISTRATION. ClinicalTrials.gov, NCT01778556. FUNDING. This research was supported by the intramural research program of the National Institute of Diabetes and Digestive and Kidney Diseases.
Rebecca J. Brown, Areli Valencia, Megan Startzell, Elaine Cochran, Peter J. Walter, H. Martin Garraffo, Hongyi Cai, Ahmed M. Gharib, Ronald Ouwerkerk, Amber B. Courville, Shanna Bernstein, Robert J. Brychta, Kong Y. Chen, Mary Walter, Sungyoung Auh, Phillip Gorden
Haemostasis requires conversion of fibrinogen to fibrin fibres that generate a characteristic network, interact with blood cells, and initiate tissue repair. The fibrin network is porous and highly permeable, but the spatial arrangement of the external clot face is unknown. Here we show that fibrin transitioned to the blood-air interface through Langmuir film formation, producing a protective film confining the clot. We demonstrated that only fibrin is required to form the film, and that it occurred in vitro and in vivo. The fibrin film connected to the underlying clot network through tethering fibres. It was digested by plasmin and formation of the film was prevented with surfactants. Functionally, the film retained blood cells and protected against penetration by bacterial pathogens in a murine model of dermal infection. Our data show a remarkable aspect of blood clotting, in which fibrin forms a protective film covering the external surface of the clot, defending the organism against microbial invasion.
Fraser L. Macrae, Cédric Duval, Praveen Papareddy, Stephen R. Baker, Nadira Yuldasheva, Katherine J. Kearney, Helen R. McPherson, Nathan Asquith, Joke Konings, Alessandro Casini, Jay L. Degen, Simon D. Connell, Helen Philippou, Alisa S. Wolberg, Heiko Herwald, Robert A.S. Ariëns
HIV-1 acquisition occurs most commonly after sexual contact. To establish infection, HIV-1 must infect cells that support high level replication, namely CD4+ T cells, which are absent from the outermost genital epithelium. Dendritic cells (DCs), present in mucosal epithelia, potentially facilitate HIV-1 acquisition. We show that vaginal epithelial DCs, termed CD1a+ VEDCs, are unlike other blood and tissue derived DCs because they express langerin but not DC-SIGN, and unlike skin-based langerin+ DC subset, Langerhans cells (LC), they do not harbor Birbeck granules. Individuals primarily acquire HIV-1 that utilize the CCR5 receptor (termed either R5 or R5X4) during heterosexual transmission, and the mechanism for the block against variants that only use the CXCR4 receptor (classified as X4) remains unclear. We show that X4 as compared to R5 HIV-1 show limited to no replication in CD1a+ VEDCs. This differential replication occurs post-fusion suggesting that receptor usage influences post-entry steps in the virus life-cycle. Furthermore, CD1a+ VEDCs isolated from HIV-1 infected virologically suppressed women harbor HIV-1 DNA. Thus, CD1a+ VEDCs are potentially both infected early during heterosexual transmission and retain virus during treatment. Understanding the interplay between HIV-1 and CD1a+ VEDCs will be important for future prevention and cure strategies.
Victor Pena-Cruz, Luis M. Agosto, Hisashi Akiyama, Alex Olson, Yvetane Moreau, Jean-Robert Larrieux, Andrew Henderson, Suryaram Gummuluru, Manish Sagar
For gene therapy of gain-of-function autosomal dominant diseases, either correcting or deleting the disease allele is potentially curative. To test whether there may be an advantage of one approach over the other for WHIM (warts, hypogammaglobulinemia, infections and myelokathexis) syndrome — a primary immunodeficiency disorder caused by gain-of-function autosomal dominant mutations in chemokine receptor CXCR4 — we performed competitive transplantation experiments using both lethally irradiated wild-type (Cxcr4+/+) and unconditioned WHIM (Cxcr4+/w) recipient mice. In both models, hematopoietic reconstitution was markedly superior using bone marrow (BM) cells from donors hemizygous for Cxcr4 (Cxcr4+/o) compared with BM cells from Cxcr4+/+ donors. Remarkably, only ~6% Cxcr4+/o hematopoietic stem cell (HSC) chimerism post-transplantation in unconditioned Cxcr4+/w recipient BM supported >70% long-term donor myeloid chimerism in blood and corrected myeloid cell deficiency in blood. Donor Cxcr4+/o HSCs differentiated normally and did not undergo exhaustion as late as 465 days post-transplantation. Thus, disease allele deletion resulting in Cxcr4 haploinsufficiency was superior to disease allele repair in a mouse model of gene therapy for WHIM syndrome, allowing correction of leukopenia without recipient conditioning.
Ji-Liang Gao, Erin Yim, Marie Siwicki, Alexander Yang, Qian Liu, Ari Azani, Albert Owusu-Ansah, David H. McDermott, Philip M. Murphy
Receptor tyrosine kinases (RTKs) are important drivers of cancers. In addition to genomic alterations, aberrant activation of wild type RTKs plays an important role in driving cancer progression. However, the underlying mechanisms of how RTKs drive prostate cancer remain incompletely characterized. Here we show that non-proteolytic ubiquitination of RTK regulates its kinase activity and contributes to RTK-mediated prostate cancer metastasis. TRAF4, an E3 ubiquitin ligase, is highly expressed in metastatic prostate cancer. We demonstrated here that it is a key player in regulating RTK mediated prostate cancer metastasis. We further identified TrkA, a neurotrophin RTK, as TRAF4-targeted ubiquitination substrate that promotes cancer cell invasion and inhibition of TrkA activity abolished TRAF4-dependent cell invasion. TRAF4 promoted K27 and K29-linked ubiquitination at the TrkA kinase domain and increased its kinase activity. Mutation of TRAF4-targeted ubiquitination sites abolished TrkA tyrosine auto-phosphorylation and its interaction with downstream proteins. TRAF4 knockdown also suppressed NGF-stimulated TrkA downstream p38 MAPK activation and invasion-associated gene expression. Furthermore, elevated TRAF4 levels significantly correlated with increased NGF-stimulated invasion-associated gene expression in prostate cancer patients, indicating that this signaling axis is significantly activated during oncogenesis. Our results revealed a post-translational modification mechanism contributing to aberrant non-mutated RTK activation in cancer cells.
Ramesh Singh, Dileep Karri, Hong Shen, Jiangyong Shao, Subhamoy Dasgupta, Shixia Huang, Dean P. Edwards, Michael M. Ittmann, Bert W. O’Malley, Ping Yi
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