Human Macrophage and Dendritic Cell-Specific Silencing of High-Mobility

Human macrophage and dendritic cell-specific silencing of high-mobility group protein B1 ameliorates sepsis in a humanized mouse model Chunting Ye, Jang-Gi Choi, Sojan Abraham, Haoquan Wu, Dolores Diaz, Daniel Terreros, Premlata Shankar, and N. Manjunath1 Center of Excellence in Infectious Disease, Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX 79905 Edited by Kevin J. Tracey, Feinstein Institute for Medical Research, Manhasset, NY, and accepted by the Editorial Board October 24, 2012 (received for review September 21, 2012) Hypersecretion of cytokines by innate immune cells is thought to sepsis pathogenesis or the impact of its neutralization on human initiate multiple organ failure in murine models of sepsis. Whether cells remain unclear. human cytokine storm also plays a similar role is not clear. Here, we RNA interference can be used to silence virtually any gene, show that human hematopoietic cells are required to induce sepsis- including multiple genes, as long as a way can be found to in- induced mortality following cecal ligation and puncture (CLP) in the troduce small interfering (si)RNAs into relevant cell types in vivo − − severely immunodeficient nonobese diabetic (NOD)/SCID/IL2Rγ / without toxicity. Several advances have been made in developing mice, and siRNA treatment to inhibit HMGB1 release by human mac- methods to deliver siRNA in vivo to different cell types, most – rophages and dendritic cells dramatically reduces sepsis-induced successfully to the liver cells (reviewed in refs. 15 17). A lipid-like mortality. Following CLP, compared with immunocompetent WT nanoparticle called C12-200, which had been developed for liver- − − fi mice, NOD/SCID/IL2Rγ / mice did not show high levels of serum speci c delivery of siRNA, was recently also shown to deliver HMGB1 or murine proinflammatory cytokines and were relatively siRNA to murine monocytes, and silencing C-C chemokine re- resistant to sepsis-induced mortality. In contrast, NOD/SCID/IL2Rγ−/− ceptor type 2 (CCR2) in monocytes using this reagent was ef- fective in reducing atherosclerosis, islet transplantation and mice transplanted with human hematopoietic stem cells [human- MEDICAL SCIENCES tumors (18). Whether this reagent also targets human DCs and ized bone marrow liver thymic mice (BLT) mice] showed high serum monocytes/macrophages is unclear. We have reported previously levels of HMGB1, as well as multiple human but not murine proin- that a short 29-aa peptide derived from the rabies virus glyco- flammatory cytokines, and died uniformly, suggesting human fi protein (RVG), fused to 9R residues (RVG-9R), can deliver cytokines are suf cient to induce organ failure in this model. siRNA to murine macrophages and brain cells by specific binding Moreover, targeted delivery of HMGB1 siRNA to human macro- to its ligand acetylcholine receptor (AchR) (19, 20). Because phages and dendritic cells using a short acetylcholine receptor AchR is also expressed on human macrophages and DCs (21) and (AchR)-binding peptide [rabies virus glycoprotein (RVG)-9R] effec- also because the acetylcholine-binding site on the α7 subunit is tively suppressed secretion of HMGB1, reduced the human cyto- highly conserved, we reasoned that RVG-9R might also be used kine storm, human lymphocyte apoptosis, and rescued humanized to target human macrophages and DCs. In this study, we validate mice from CLP-induced mortality. siRNA treatment was also effec- this hypothesis in vitro, as well as in vivo, using human hema- tive when started after the appearance of sepsis symptoms. These topoietic stem cell–engrafted nonobese diabetic NOD/SCID/ − − results show that CLP in humanized mice provides a model to study IL2Rγ / mice that lack mouse innate and adaptive immune human sepsis, HMGB1 siRNA might provide a treatment strategy systems (22). More importantly, we also show that silencing hu- for human sepsis, and RVG-9R provides a tool to deliver siRNA to man HMGB1 using this delivery reagent in this mouse model human macrophages and dendritic cells that could potentially be substantially reduces human lymphocyte apoptosis and cytokine used to suppress a variety of human inflammatory diseases. storm and protects mice from sepsis-induced mortality. epsis is an important cause of mortality in intensive-care Results Human Cytokines Are Necessary to Induce Sepsis Following CLP in Sunits, with more than 750,000 individuals developing severe − − / sepsis in North America annually and a mortality rate varying Humanized Mice. NOD/SCID/IL2Rγ mice lack a functional between 35 and 50% (1, 2). The pathogenesis of sepsis includes immune system and are also defective in multiple cytokine sig- countless disturbances of the host immune system starting with naling (22). However, whether the residual defective monocytes/ a harmful, infection-triggered exaggerated inflammatory cascade macrophages secrete murine cytokines that could cause tissue that results in tissue injury and rapidly leads to massive apoptosis injury during sepsis is not known. If they are incapable of se- creting murine cytokines, then the only source of cytokines in of immune cells (2, 3). This is followed by a secondary immune − − NOD/SCID/IL2Rγ / mice transplanted with human hemato- paralysis phase accompanied by uncontrolled growth of bacteria poietic stem cells (HSCs) would be human cells and this would and tissue damage. Although therapy to suppress the immediate β allow us to test the impact of human cytokines in sepsis. There- cytokine response, such as treatment with TNF and IL-1 anti- fore, we compared immunocompetent WT BALB/c mice, im- – − − bodies, have failed in clinical trials (4 6), it has now come to be munodeficient NOD/SCID/IL2Rγ / mice, and NOD/SCID/ recognized that, at least in animal models, high-mobility group protein 1 (HMGB1), which is secreted from macrophages and dendritic cells (DCs) but not lymphocytes late in the disease, acts Author contributions: H.W., P.S., and N.M. designed research; C.Y., J.-G.C., D.D., and S.A. as a master regulator of late and sustained cytokine storm, up- performed research; C.Y., D.T., and N.M. analyzed data; and C.Y. and N.M. wrote the paper. α β regulating many cytokines including TNF- , IL-6, IL-1 , and IL-8 The authors declare no conflict of interest. – (reviewed in refs. 7 11). In fact, injection of mice with HMGB1 is This article is a PNAS Direct Submission.K.J.T.isaguesteditorinvitedbythe enough to induce the lethal organ damage seen in sepsis (12), Editorial Board. whereas treatment with neutralizing HMGB1 antibody can rescue 1To whom correspondence should be addressed. E-mail: [email protected]. mice and rats from experimental sepsis (13, 14). However, al- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. though HMGB1 is also secreted in human sepsis (12), its role in 1073/pnas.1216195109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1216195109 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 − − − − IL2Rγ / mice transplanted with human HSCs [humanized bone NOD/SCID/IL2Rγ / mice (Fig. 1E). We also performed TUNEL marrow liver thymic mice (BLT) mice] for sepsis induction fol- staining of liver sections to detect apoptotic cells. Whereas high + lowing CLP. Twenty hours after CLP, blood was tested for the levels of TUNEL cells were detected in humanized mice, + − − key late inflammatory mediator HMGB1, as well as other TUNEL cells were substantially reduced in NOD/SCID/IL2Rγ / downstream mouse and human proinflammatory cytokines, and mice (Fig. 1F). Taken together, these results suggest that NOD/ − − the mice were followed for survival. Whereas high levels of SCID/IL2Rγ / mice are resistant to CLP-induced sepsis and that HMGB1 (Fig. 1A) and murine cytokines (Fig. 1B) could be human cytokines are required to induce organ damage and mor- − − detected in the sera of WT mice, NOD/SCID/IL2Rγ / mice tality in humanized mice. These results are also consistent with an showed minimal levels of these cytokines. In contrast, humanized earlier report that showed that elevated levels of human cytokines BLT mice showed elevated levels of human HMGB1 and mul- are seen in humanized mice after CLP (23). tiple human, but not murine cytokines (Fig. 1 A–C). Corre- It was surprising that immunodeficient mice, compared with WT spondingly, both WT and humanized mice died quickly, with all and humanized mice, were resistant to CLP-induced sepsis. Thus, − − mice dying within 3 d after CLP, whereas ∼70% of NOD/SCID/ we investigated the mechanism by which NOD/SCID/IL2Rγ / − − IL2Rγ / mice survived symptom-free for up to a 12-d period of mice resist CLP-induced mortality. First, we compared the blood − − observation, and in the few mice that died, the death was delayed bacterial load in WT, NOD/SCID/IL2Rγ / , and humanized mice. by 2–4 d (Fig. 1D). Histological examination of liver and lungs Although all three groups of mice had equivalent bacterial titres − − showed evidence of disseminated intravascular coagulation with 1 d after CLP, in NOD/SCID/IL2Rγ / mice, bacterial load marked congestion and fibrin thrombi in the blood vessels, focal steadily declined and was undetectable between days 7–9 (Fig. vacuolar changes, chromatin disruption, and spotty apoptosis 1G). WT and humanized mice could not be serially followed be- − − following CLP in BALB/c and humanized BLT mice but not in cause they all died by day 3. Although NOD/SCID/IL2Rγ / mice − − Fig. 1. Human hematopoietic cells are required to induce sepsis in NOD/SCID/IL2Rγ / mice. Mice underwent sham or CLP surgery and 24 h post-CLP, sera were tested for HMGB1 by ELISA (A), murine proinflammatory cytokines were tested by cytometric bead array (B), and human proinflammatory cytokines were tested by cytometric bead array (C). Mean ± SD from seven mice per group is shown. (D) Kaplan–Meier survival curves for mice following CLP are shown (n = 14 per group).

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