RAGE-mediated suppression of IL-10 results in enhanced mortality in a murine model of 1 Acinetobacter baumannii sepsis. 2 Michael J. Noto1, Kyle W. Becker2, Kelli L. Boyd2, Ann Marie Schmidt3, and Eric P. Skaar4,2 3 4 1 Department of Medicine, Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt 5 University Medical Center, Nashville, TN, USA. 6 2 Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical 7 Center, Nashville, TN, USA. 8 3 New York University Langone Medical Center, New York, NY, USA. 9 4 Tennessee Valley Healthcare System, US Department of Veterans Affairs, Nashville, TN, 10 USA. 11 12 Corresponding author: 13 Eric P. Skaar 14 Vanderbilt University School of Medicine 15 Department of Pathology, Microbiology and Immunology 16 MCN A-5301 17 Nashville, TN 37232-2363 18 Phone: 615-343-0002 19 Fax: 615-343-7492 20 E-mail: eric.skaar@vanderbilt.edu 21 22 Condensed title: RAGE enhances mortality from A. baumannii sepsis 23 24 Conflict of interest statement: The authors have declared that no conflicts of interest exists. 25 26

IAI Accepted Manuscript Posted Online 4 January 2017Infect. Immun. doi:10.1128/IAI.00954-16Copyright © 2017 American Society for Microbiology. All Rights Reserved.

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Abstract 27 The receptor for advanced glycation end products (RAGE) is a pattern recognition 28 receptor capable of recognizing multiple pathogen-associated and danger-associated molecular 29 patterns that contributes to the initiation and potentiation of inflammation in many disease 30 processes. During infection, RAGE functions to either exacerbate disease severity or enhance 31 pathogen clearance depending on the pathogen studied. Acinetobacter baumannii is an 32 opportunistic human pathogen capable of causing severe infections, including pneumonia and 33 sepsis, in impaired hosts. The role of RAGE signaling in response to opportunistic bacterial 34 infections is largely unknown. In murine models of A. baumannii pneumonia, RAGE signaling 35 alters neither inflammation nor bacterial clearance. In contrast, RAGE-/- mice systemically 36 infected with A. baumannii exhibit increased survival and reduced bacterial burdens in the liver 37 and spleen. The increased survival of RAGE-/- mice is associated with increased circulating 38 levels of the anti-inflammatory cytokine, IL-10. Neutralization of IL-10 in RAGE-/- mice results in 39 decreased survival during systemic A. baumannii infection that mirrors that of WT mice, and 40 exogenous IL-10 administration to WT mice enhances survival in this model. These findings 41 demonstrate the role for RAGE-dependent IL-10 suppression as a key modulator of mortality 42 from Gram-negative sepsis. 43 44 45 46 47 48 49 50 51 52

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Introduction 53 An effective immune response to an invading pathogen depends upon host recognition 54

of the infecting organism and resultant initiation of an immune response. This process is 55 accomplished through the recognition of pathogen-associated molecular patterns (PAMPs) by 56 host pattern recognition receptors (PRRs) (1). Signaling downstream of PRRs, including toll-like 57 receptors (TLRs) activates transcription factors such as nuclear factor kappa B (NFκB), inducing 58 expression of pro-inflammatory genes, thereby altering cytokine and chemokine production, 59 resulting in inflammation and effector cell recruitment to the site of infection (1, 2). In addition to 60 PAMPs, many PRRs have also evolved to detect altered host proteins, referred to as danger-61 associated molecular patterns (DAMPs) (3). The signaling mechanisms required to initiate an 62 appropriate response to an invading pathogen may also potentiate inflammation during the 63 course of infection and this inflammatory response may enhance tissue damage and exacerbate 64 disease (4). The maladaptive potentiation of inflammation in response to infection is 65 responsible for many of the severe manifestations of infectious diseases, including septic shock, 66 the most extreme manifestation of invasive bacterial infection (5). In this manner, the 67 consequences of PRR signaling in response to infectious insults may either benefit or harm the 68 host. 69

The receptor for advanced glycation end-products (RAGE) functions as a PRR, belongs 70 to the immunoglobulin superfamily, exists in soluble or transmembrane isoforms, and is notable 71 for its ability to potentiate inflammation. Full length RAGE consists of three extracellular 72 immunoglobulin-like domains, a transmembrane domain, and an intracellular domain, which is 73 responsible for intracellular signaling (6, 7). RAGE interacts with three dimensional structures 74 rather than specific amino acid sequences and therefore functions as a multi-ligand receptor (8). 75 RAGE can be ligated by PAMPs, such as bacterial lipopolysaccharide, and a number of host 76 DAMPs, including advanced glycation end-products, S100 proteins, and high mobility group box 77 1 (HMGB1) (6, 8). Engagement of RAGE results in the rapid activation and nuclear translocation 78

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of NFκB, which increases transcription of pro-inflammatory cytokines, vasoactive agents, 79 prothrombotic factors, RAGE itself, and NFκB p65 mRNA (9-14). The latter is thought to be 80 partially responsible for the prolonged activation of NFκB induced by RAGE, as de novo NFκB 81 synthesis overwhelms counter-regulatory machinery thereby potentiating the inflammatory 82 response (15). RAGE has been shown to either contribute to the control of infection or enhance 83 disease severity depending on the pathogen and infection model studied. RAGE contributes to 84 host defense in a murine model of Klebsiella pneumoniae pneumonia but loss of RAGE results 85 in decreased mortality in a murine model of Streptococcus pneumoniae pneumonia (16-18). 86 Similarly, RAGE-/- mice have reduced mortality in a cecal ligation and puncture model of sepsis, 87 but the loss of RAGE does not alter disease severity in a S. pneumoniae model of sepsis (19-88 21). Therefore, RAGE signaling impacts disease severity in a pathogen-specific manner. 89

Acinetobacter baumannii is a Gram-negative bacterium and a frequent cause of severe 90 infections in persons with impaired immunity. A. baumannii is among the most prominent 91 nosocomial pathogens where it is a frequent cause of nosocomial pneumonia, including 92 ventilator-associated pneumonia, burn and wound infections, and sepsis (22-25). The 93 pathogenicity of A. baumannii is propelled by the organism’s ability to develop multi- and pan-94 resistance to antimicrobial agents, which complicates treatment of infections and allows its 95 persistence in the healthcare setting (23, 26). As an opportunistic pathogen, A. baumannii 96 capitalizes on impaired immune function and rarely causes disease in healthy persons, 97 suggesting intact innate immune functions are sufficient to prevent infection. Studies examining 98 the role of RAGE in infectious diseases have focused on animal models employing professional 99 pathogens or overwhelming polymicrobial sepsis, and studies of opportunistic pathogens are 100 lacking. To address this, we investigated the role of RAGE in murine models of A. baumannii 101 pneumonia and systemic infection and uncover RAGE-dependent suppression of IL-10 102 production as a key contributor to mortality from A. baumannii sepsis. 103

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Methods 104 Bacterial strains and growth conditions. 105 All experiments were performed using A. baumannii strain ATCC 17978 (Ab17978) grown in 106 lysogeny broth or agar at 37°C with shaking. 107 A. baumannii infections. 108 Wildtype C57BL/6J mice were purchased from Jackson Laboratories. RAGE-/- mice were 109 provided by Ann Marie Schmidt at New York University Langone Medical Center (27, 28). All 110 animal experiments were approved by the Vanderbilt University Institutional Animal Care and 111 Use Committee. The murine model of A. baumannii pneumonia was performed as previously 112 described (29-34). Briefly, Ab17978 were back-diluted 1:1000 from overnight culture and grown 113 for 3.5 hours at 37°C with shaking, washed twice with cold PBS, and resuspended in PBS at an 114 appropriate cell density for infection. Eight-week old, female mice were infected intranasally 115 with 3×108 colony forming units Ab17978 in 30 µl PBS. At 36 hours post infection mice were 116 euthanized and organs and blood were harvested. For systemic infection, Ab17978 was 117 prepared in a similar manner and mice were infected retro-orbitally with 9×108 colony forming 118 units of Ab17978 in 100 μl PBS and survival was monitored (35). To minimize discomfort, mice 119 that exhibited signs of severe disease, defined as a combination of; abnormal posture, impaired 120 activity, absence of eye opening, and an inability to right themselves within 10 seconds when 121 positioned on their backs, were humanely euthanized and considered to meet the mortality end 122 point. At 24 or 48 hours post infection, mice were euthanized and organs and blood were 123 harvested. Lungs, livers, and spleens were homogenized and plated onto lysogeny agar for 124 bacterial enumeration. Blood was harvested by cardiac puncture immediately after the mice 125 were humanely euthanized. For histological analyses, lungs were inflated with 1 ml of 10 126 percent formalin, fixed, embedded and stained as described previously (29). Rat anti-mouse IL-127 10 neutralizing and IgG2a isotype control antibody were purchased from BioLegend. RAGE-/- 128 mice were dosed intraperitoneally with 75 μg in deionized water immediately prior to systemic 129

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infection with A. baumannii. Recombinant murine IL-10 (rIL-10) was purchased from 130 BioLegend. Twenty μg of rIL-10 or vehicle control (deionized water) were administered 131 intraperitoneally to WT mice immediately prior to systemic infection with A. baumannii. 132 Histology. 133 Paraffin-embedded mouse tissue sections were stained with hematoxylin and eosin by the 134 Vanderbilt University Medical Center Translational Pathology Shared Resource (TPSR). Lung 135 inflammation was scored by K. L. B. who was blinded to the treatment assignment according to 136 the following definitions; 0, no pathology; 1, minimal infiltrates of neutrophils in alveolar spaces; 137 2, mild numbers of neutrophils in alveoli; 3, moderate numbers of neutrophils and hemorrhage 138 in alveoli with occasional lobar involvement, focal necrosis of alveolar walls neutrophils in 139 bronchioles; 4, marked numbers of neutrophils, consolidation and widespread alveolar necrosis. 140 Complete blood count and serum chemistry analyses. 141 Complete blood counts were performed on EDTA-treated whole blood by the Vanderbilt TPSR. 142 Measurements of serum chemistries (BUN, AST, and ALT) were performed on serum using the 143 Alfa Wasserman ACE Alera® by the Vanderbilt TPSR. 144 Cytokine quantification. 145 Determination of cytokine levels was performed on serum from infected animals harvested 24 146 hours post-infection. Cytokines were quantified using the MAP Mouse Cytokine/Chemokine 147 Magnetic Bead Panel (Millipore) on a Luminex Flexmap 3D platform (Luminex) according to the 148 manufacturer’s recommended protocol. 149 Statistics. 150 Statistical analyses were performed using GraphPad Prism version 6. Mean comparisons were 151 performed using unpaired Welch’s t-test. Median survival times were compared using a Kaplan-152 Meier analysis and log-rank test. No adjustments were made for multiple comparisons. P 153 values less than 0.05 were considered statistically significant. 154 155

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Results 156 RAGE signaling does not alter disease severity induced by A. baumannii pneumonia. 157

RAGE is highly expressed in the lung (36) and has been shown to contribute to host 158 defense against Klebsiella pneumoniae pneumonia but exacerbates disease in mouse models 159 of Streptococcus pneumoniae pneumonia (16, 17), suggesting that RAGE signaling impacts 160 disease severity in a pathogen-specific manner. To advance the understanding of RAGE 161 signaling in host defense against bacterial pneumonia, the impact of RAGE on outcomes of A. 162 baumannii was investigated. WT and RAGE-/- mice were challenged intranasally with A. 163 baumannii in a non-lethal pneumonia model and bacterial burdens in the lung were determined 164 at 24 hours post-infection. WT and RAGE-/- mice exhibited no significant difference in bacterial 165 burdens recovered from the lung and no difference in extra-pulmonary dissemination to the liver 166 (Fig. 1a), indicating that RAGE signaling does not alter bacterial clearance. RAGE is involved in 167 initiating and sustaining the inflammatory response to pathogens (19) and may potentiate lung 168 inflammation and injury without altering bacterial burdens. To address this possibility, WT and 169 RAGE-/- mice were challenged with A. baumannii and lungs were subjected to histopathologic 170 examination at 48 hours post-infection. Both groups of mice developed interstitial pneumonia, 171 characterized by infiltrates of neutrophils in alveolar spaces and proliferation of alveolar 172 macrophages involving 25-35% of the parenchyma without necrosis of the alveolar walls (Fig. 173 1b). Additionally, when compared with WT mice, RAGE-/- mice had no difference in lung 174 inflammation or injury induced by A. baumannii (Fig. 1c). Taken together, these findings indicate 175 that RAGE does not contribute to bacterial clearance or pulmonary inflammation induced by A. 176 baumannii pneumonia. 177 178 RAGE increases mortality caused by A. baumannii sepsis. 179

The finding that RAGE does not alter the course of A. baumannii pneumonia suggests 180 that RAGE may not play a role in the host response to A. baumannii infection. In addition to 181

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pneumonia, A. baumannii is a frequent cause of sepsis in humans (37) and RAGE has been 182 implicated in increasing mortality in several murine sepsis (cecal ligation and puncture, Listeria 183 monocytogenes) models (19, 20) but does not alter S. pneumoniae systemic infection (21). To 184 determine the impact of RAGE signaling in an A. baumannii model of systemic infection, WT 185 and RAGE-/- mice were challenged retro-orbitally with A. baumannii and survival was assessed 186 over time. WT mice rapidly succumbed to infection with less than 20% of mice alive at 50 hours 187 post-infection. RAGE-/- mice also experienced significant mortality in this model; however, 188 survival was significantly improved compared to WT with over 40% of mice alive at 50 hours 189 post-infection (Fig. 2a). To determine if the enhanced mortality of WT mice correlates with 190 increased bacterial dissemination, bacterial burdens in the lungs, livers, and spleens were 191 quantified at 24 hours following systemic challenge. This early time point was chosen to 192 determine bacterial burdens prior to the onset of death. Consistent with their enhanced survival, 193 RAGE-/- mice have significant reductions in bacterial burdens in the liver and spleen without a 194 significant difference in lung burdens compared with WT mice (Fig. 2b). Therefore, RAGE 195 signaling results in impaired bacterial clearance and increased mortality from A. baumannii 196 sepsis. 197 198 RAGE does not contribute to liver or kidney dysfunction resulting from A. baumannii sepsis. 199 To determine the mechanism by which RAGE signaling leads to increased death from A. 200 baumannii sepsis, organ dysfunction was assessed. Sepsis commonly results in renal and 201 hepatic dysfunction marked by increased blood levels of urea, or hepatic transaminases, 202 respectively. Kidney and liver dysfunction was assessed in WT and RAGE-/- mice 24 hours after 203 systemic infection with A. baumannii by measuring serum levels of blood urea nitrogen (BUN), 204 aspartate aminotransferase (AST), and alanine aminotransferase (ALT). RAGE-/- mice do not 205 exhibit differential kidney and liver dysfunction as compared with WT mice, as no significant 206 differences in BUN, AST, or ALT were present between groups (Fig. 3a). Therefore, these data 207

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do not support that differential organ dysfunction is responsible for enhanced mortality of WT 208 mice systemically infected by A. baumannii. 209 210 Blood leukocytes do not differ between systemically-infected WT and RAGE-/- mice. 211 Consistent with its role in inflammation, RAGE signaling may alter leukocyte mobilization 212 in response to infection. To test this hypothesis, complete blood counts were performed on 213 blood of WT and RAGE-/- mice 24 hours following systemic infection with A. baumannii. No 214 significant differences in total white blood cells, neutrophils, lymphocytes, or monocytes exist 215 between WT and RAGE-/- mice post-infection (Fig. 3b). Similarly, there were no significant 216 differences between groups in hemoglobin and hematocrit levels, or in total number of platelets 217 (data not shown). These data indicate that RAGE does not alter leukocyte mobilization, 218 erythrocyte, or platelet levels in response to A. baumannii infection. 219 220 RAGE suppresses serum levels of IL-10 in response to A. baumannii sepsis. 221 Sepsis is associated with the marked production of proinflammatory cytokines, which 222 contribute to the enhanced vascular permeability and organ dysfunction characteristic of 223 advanced disease (5). To determine if RAGE signaling alters the production of inflammatory 224 cytokines, serum cytokine levels were quantified from WT and RAGE-/- mice 24 hours after 225 systemic A. baumannii infection. WT mice produce significantly increased levels of IFN-γ 226 without increases in TNF-α or IL-1β when compared with RAGE-/- mice (Fig. 4). WT mice also 227 have a significant reduction in the anti-inflammatory cytokine IL-10 in response to A. baumannii 228 infection compared with RAGE-/- mice. Therefore, RAGE signaling results in increased IFN-γ and 229 a marked reduction in IL-10 production following A. baumannii infection. 230 231 RAGE-mediated IL-10 suppression results in increased mortality from A. baumannii sepsis. 232

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IL-10 is elevated in the initial phase of bacterial sepsis and may offer protection from 233 mortality (38, 39). To test if RAGE-mediated suppression of IL-10 production is responsible for 234 the enhanced mortality of WT mice relative to RAGE-/- mice, RAGE-/- mice were treated with an 235 IL-10 neutralizing antibody or isotype control prior to systemic infection with A. baumannii. 236 Neutralization of IL-10 results in a significant increase in mortality (Fig.5a) and renders RAGE-/- 237 mice susceptible to A. baumannii sepsis with rates of death approximating that seen for WT 238 mice (Fig. 2a). The finding that RAGE-mediated suppression of IL-10 production results in 239 enhanced mortality raises the possibility that exogenous IL-10 may enhance survival of WT 240 mice, thereby recapitulating the RAGE-/- survival phenotype. To test this hypothesis, WT mice 241 were treated with recombinant IL-10 or carrier control prior to systemic A. baumannii infection. 242 Mice treated with recombinant IL-10 had a significant increase in survival when compared to the 243 vehicle treated control mice (Fig. 5b), indicating that exogenous IL-10 can compensate for 244 RAGE-mediated IL-10 suppression to enhance survival from A. baumannii sepsis. 245 246 247 248 249 250 251 252 253 254 255 256 257 258

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Discussion 259 Opportunistic bacterial pathogens capitalize on innate immune impairments to initiate 260 and sustain an infection. Given their evolution of multi- and pan-antibiotic resistant phenotypes, 261 many opportunistic bacterial pathogens, including A. baumannii, are among the greatest threats 262 to the existing antibiotic repertoire. Approaches targeting the host to enhance immunity have 263 been suggested as antibiotic-independent therapeutic strategies (40, 41). Therefore, a 264 mechanistic understanding of how intact immune functions control these pathogens to prevent 265 or combat infections is essential. RAGE is known to play a key role in inflammation, which is 266 required for the control of many bacterial infections, yet investigations into the role of this 267 receptor in antibacterial responses has centered on studies of professional pathogens and 268 pathogen-agnostic models of septic shock (16, 19, 21). Here, we investigated the role for RAGE 269 in infections caused by the important opportunistic pathogen A. baumannii, using murine models 270 of pulmonary and systemic infection. One limitation to the study of opportunistic pathogens, 271 such as A. baumannii, in non-immunosuppressed animal models is the high inoculum required 272 to induce a disease phenotype, which may not accurately reflect the initiation of infection as it 273 pertains to human disease. 274

RAGE is constitutively expressed in the lung and is known to play a role in numerous 275 lung diseases such as acute respiratory distress syndrome, asthma, and pulmonary fibrosis (42-276 44). The role of RAGE has been studied in models of murine pneumonia caused by K. 277 pneumoniae and S. pneumoniae with conflicting findings. RAGE contributes to host defense 278 against K. pneumoniae infection with a reduction in mortality, lung bacterial burdens, and extra-279 pulmonary bacterial dissemination in WT versus RAGE-/- mice (16). In contrast, RAGE 280 exacerbates S. pneumoniae pneumonia with increased mortality, bacterial lung burdens, and 281 extra-pulmonary dissemination in WT versus RAGE-/- mice (18). RAGE deficiency did not alter 282 the inflammatory response to K. pneumoniae pneumonia but resulted in reduced inflammation 283 and neutrophil recruitment to the lung in response to S. pneumoniae, suggesting the enhanced 284

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inflammatory response in WT mice facilitated S. pneumoniae pathogenesis (16, 18). RAGE 285 deficiency did not alter lung inflammation or bacterial burdens in response to A. baumannii 286 pneumonia (Fig. 1), indicating that RAGE-mediated modulation of lung inflammation does not 287 contribute to antibacterial immunity against A. baumannii. 288

RAGE increases mortality and bacterial burdens in the liver and spleen in a systemic 289 model of A. baumannii sepsis (Fig. 2). These findings are consistent with cecal ligation and 290 puncture and LPS models of polymicrobial or endotoxic septic shock where RAGE potentiates 291 inflammation and increases mortality (19, 45). However, these findings are in contrast to the 292 limited role of RAGE in a model of systemic infection caused by S. pneumoniae where RAGE 293 did not alter multiple metrics of disease severity (21). RAGE is known to directly bind 294 lipopolysaccharide of Gram-negative bacteria (45). Therefore, these discrepancies may result 295 from RAGE-mediated amplification of maladaptive inflammation in response to systemic Gram-296 negative, but not Gram-positive, bacterial infection. 297 RAGE-/- mice have increased serum levels of IL-10 upon systemic challenge with A. 298 baumannii, suggesting RAGE signaling suppresses IL-10 production during infection (Fig. 4). 299 RAGE deficiency leads to increased IL-10 production by T cells in vitro (46) as well as increased 300 IL-10 in murine models of hemorrhagic shock (47), traumatic brain injury-induced pulmonary 301 dysfunction (48), and in mice fed a high fat diet (49). Therefore, our finding of RAGE-dependent 302 suppression of IL-10 production is consistent with the findings of others. IL-10 is an important 303 anti-inflammatory cytokine in sepsis and is known to suppress the hyper-inflammation 304 characteristic of early septic shock while contributing to the immunosuppressive features found 305 later in the course of septic shock (50). Exogenous IL-10 is known to be protective in a murine 306 model of endotoxic shock (38). Consistent with these findings, the enhanced mortality of WT 307 mice compared with RAGE-/- mice during systemic challenge with A. baumannii is reversed by 308 exogenous IL-10 administration (Fig.5) suggesting the enhanced mortality is attributable to 309 RAGE-dependent suppression of IL-10 production. Furthermore, neutralization of IL-10 in 310

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RAGE-/- mice abrogates their survival benefit from A. baumannii systemic infection and mirrors 311 the mortality experienced by WT mice. These findings emphasize the importance of RAGE-312 dependent IL-10 suppression as a mitigator of mortality from Gram-negative sepsis. 313 314 Acknowledgements 315 We thank members of the Skaar laboratory for review of the manuscript. This research was 316 supported by Department of Veterans Affairs Merit Award INFB-024-13F and 5RO1AI101171 to 317 E.P.S., Cystic Fibrosis Foundation award NOTO15D0 and National Institutes of Health 318 2T32HL087738-06 to M.J.N. E.P.S. is a Burroughs Wellcome Fellow in the Pathogenesis of 319 Infectious Diseases. All authors declare no conflicts of interest. 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336

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34. Mortensen BL, Rathi S, Chazin WJ, Skaar EP. 2014. Acinetobacter baumannii 449 response to host-mediated zinc limitation requires the transcriptional regulator Zur. 450 Journal of Bacteriology 196:2616-2626. 451 35. Noto MJ, Boyd KL, Burns WJ, Varga MG, Peek RM, Jr., Skaar EP. 2015. Toll-Like 452 Receptor 9 Contributes to Defense against Acinetobacter baumannii Infection. Infect 453 Immun 83:4134-4141. 454 36. Brett J, Schmidt AM, Yan SD, Zou YS, Weidman E, Pinsky D, Nowygrod R, 455 Neeper M, Przysiecki C, Shaw A, Migheli A, and Stern D. 1993. Survey of the 456 distribution of a newly characterized receptor for advanced glycation end products 457 in tissues. Am J Pathol 143:1699-1712. 458 37. Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond MB. 2004. 459 Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a 460 prospective nationwide surveillance study. Clin Infect Dis 39:309-317. 461 38. Howard M, Muchamuel T, Andrade S, Menon S. 1993. Interleukin 10 protects 462 mice from lethal endotoxemia. J Exp Med 177:1205-1208. 463 39. Derkx B, Marchant A, Goldman M, Bijlmer R, van Deventer S. 1995. High levels 464 of interleukin-10 during the initial phase of fulminant meningococcal septic shock. J 465 Infect Dis 171:229-232. 466 40. Spellberg B, Bartlett JG, Gilbert DN. 2013. The future of antibiotics and resistance. 467 N Engl J Med 368:299-302. 468 41. Czaplewski L, Bax R, Clokie M, Dawson M, Fairhead H, Fischetti VA, Foster S, 469 Gilmore BF, Hancock RE, Harper D, Henderson IR, Hilpert K, Jones BV, 470 Kadioglu A, Knowles D, Olafsdottir S, Payne D, Projan S, Shaunak S, Silverman 471

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519 Figure 1. RAGE signaling does not alter inflammation or bacterial burdens during A. 520 baumannii pneumonia. Wild type C57BL/6J or RAGE-/- mice were challenged intranasally with 521 3 x 108 A. baumannii and lungs and livers were harvested 24 hours post-infection, 522 homogenized, serially diluted, and plated onto LB agar for bacterial enumeration. (a) Bacterial 523 burdens are plotted with the columns indicating the mean and error bars show standard error of 524 the mean. Filled circles represent individual animals. (b) Hematoxylin and eosin stained lung 525 sections from infected wild type and RAGE-/- mice at 48 hours post-infection are shown at 100x 526 magnification. (c) Lung inflammation was scored and mean values are shown with error bars 527 indicating standard error of the mean. Means were compared using Welch’s t-test and no 528 statistically significant differences between groups were present. CFU/ml, colony forming units 529 per milliliter. 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544

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545 Figure 2. RAGE signaling results in increased rates of death and bacterial burdens in the 546 liver and spleen in an A. baumannii systemic infection model. (a) Wild type C57BL/6J or 547 RAGE-/- mice were challenged retro-orbitally with 9 x 108 A. baumannii and survival was 548 monitored over time (n=21 in each group, P<0.005 using a Kaplan-Meier analysis). (b) Mice 549 were infected as described for (a) and lungs, spleens, and livers were harvested 24 hours post-550 infection, homogenized, serially diluted, and plated onto LB agar for bacterial enumeration. 551 Mean bacterial burdens are depicted by columns and error bars indicate standard error of the 552 mean. Filled circles represent individual animals. Means were compared using the Welch’s t-553 test. ns, not statistically significant; *, P<0.05; **, P<0.01. CFU/g, colony forming units per gram 554 of tissue. 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570

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571 Figure 3. RAGE signaling does not result in differential serum markers of organ 572 dysfunction or differential numbers of leukocytes in the blood following systemic 573 infection with A. baumannii. (a) Wild type C57BL/6J or RAGE-/- mice were challenged retro-574 orbitally with 9 x 108 A. baumannii and serum levels of blood urea nitrogen (BUN), aspartate 575 aminotransferase (AST), and alanine aminotransferase (ALT) were determine at 24 hours post-576 infection as metrics of kidney and liver function, respectively. (b) Mice were infected as in (a) 577 and complete blood counts were performed on EDTA-treated whole blood and total leukocyte 578 (WBC) and subsets are depicted. Columns indicate means with error bars depicting standard 579 error of the mean. Filled circles indicate individual animals. Means were compared using 580 Welch’s t-test and no significant differences between groups were present. mg/dL, milligrams 581 per deciliter; μl, microliter. 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596

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597 Figure 4. RAGE signaling results in increased serum levels of IFNγ and reduced levels of 598 IL-10 during systemic A. baumannii infection. Wild type C57BL/6J or RAGE-/- mice were 599 challenged retro-orbitally with 9 x 108 A. baumannii and serum levels of the indicated cytokines 600 were determined at 24 hours post-infection. Columns depict the mean and error bars indicate 601 standard error of the mean. Means were compared using Welch’s t-test. IFNγ, interferon 602 gamma; TNFα, tumor necrosis factor alpha; IL-1β, interleukin-1 beta; IL-10, interleukin-10; 603 pg/ml, picograms per milliliter; ns, not statistically significant; *, P<0.05. 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621

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622 Figure 5. RAGE-mediated suppression of IL-10 results in enhanced rates of death from A. 623 baumannii systemic infection. (a) RAGE-/- mice were treated with intraperitoneal injection of 624 an interleukin-10 (IL-10) neutralizing or isotype control antibody prior to systemic infection with 625 A. baumannii and survival was monitored over time. Rates of death were compared using a 626 Kaplan-Meier analysis and P<0.05 (n=6 mice per group). (b) WT mice were treated with 627 recombinant IL-10 (rIL-10) or carrier control prior to systemic infection with A. baumannii and 628 survival was monitored over time. Rates of death were compared using a Kaplan-Meier analysis 629 and P<0.01 (n=6 mice per group). 630 631 632

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