specialized pro -resolving mediators (spm) rescue...

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Specialized Pro-resolving Mediators (SPM) Rescue Infant Mice from Lethal Citrobacter 1

rodentium Infection and Promote Immunity against Re-infection 2

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Luis Alonso Diaza, Norman H. Altmanb, Wasif Khana, Charles N. Serhanc, and Becky 5

Adkinsa# 6

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Department of Microbiology and Immunology, University of Miami Miller School of 9

Medicine, Miami, FL, USAa; Department of Pathology and Laboratory Medicine, 10

University of Miami Miller School of Medicine, Miami, FL, USAb; Center for Experimental 11

Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and 12

Pain Medicine, Brigham and Women’s Hospital, Transformative Medicine Blg and 13

Harvard Medical School, Boston, MA, USAc 14

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Running Head: Pro-resolving mediators in infant intestinal infection 17

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#Address correspondence to Becky Adkins, [email protected] 20

IAI Accepted Manuscript Posted Online 10 July 2017Infect. Immun. doi:10.1128/IAI.00464-17Copyright © 2017 American Society for Microbiology. All Rights Reserved.

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ABSTRACT 21

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Infants are generally highly susceptible to oral pathogens. Intestinal infection and the 23

associated diarrhea are significant global causes of morbidity and mortality in infants. 24

Among the enteric pathogens, enteropathogenic E. coli (EPEC) stands out as showing 25

the highest risk for infection-induced death in infants ≤ 12 months old. We have 26

developed an experimental model of infant infection with EPEC, using the murine 27

specific pathogen Citrobacter rodentium. Our murine infant model is similar to EPEC 28

infection in human infants since infant mice are much more susceptible to C. rodentium 29

infection than adult mice; infants infected with fifty fold fewer bacteria than the standard 30

adult dose uniformly succumbed to the infection. Infant infection is characterized by 31

high early and sustained bacterial titers and profound intestinal inflammation associated 32

with extensive necrosis and systemic dissemination of the bacteria. Therefore, it seems 33

likely that infant deaths result from sepsis secondary to intestinal damage. Recently, 34

specialized pro-resolving mediators (SPM) have been found to exert profound beneficial 35

effects in adult models of infection. Thus, we investigated the actions of two pro-36

resolving lipid mediators, RvD1 and RvD5, on the course of infection in infants. 37

Strikingly, post-infection treatment with RvD1 and RvD5 reduced bacterial loads, 38

mitigated inflammation, and rescued the infants from death. Furthermore, post-infection 39

treatment with RvD1 and RvD5 led to protection from re-infection associated with C. 40

rodentium-specific IgG responses comparable to those in adults. These results indicate 41

that SPM may provide novel therapeutic tools for the treatment of pathological intestinal 42

infections in infants. 43

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INTRODUCTION 45

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Intestinal disease in infants infected with enteropathogens constitutes a substantial 47

global health burden. Infants infected with oral pathogens can develop pathological 48

diarrhea and experience significant morbidity and mortality. Why infants are so 49

susceptible to oral pathogens is poorly understood but the reasons may be 50

multifactorial. For example, poor colonization resistance (1, 2) is likely to be an 51

important contributing factor but developmental disparities in the immune system almost 52

certainly play a role. In particular, it is known that many of these pathogens, including 53

the bacteria Enteropathogenic E. coli (EPEC) (3-5) and Shigella (5-7), rotavirus (8), and 54

the parasite Cryptosporidium (9, 10), induce profound innate intestinal inflammation in 55

infants. The relative non-specificity of innate function may lead to damage of the 56

intestinal barrier and contribute importantly to disease pathology. Additionally, 57

excessive innate inflammation may fail to adequately promote the development of 58

adaptive immunity (11, 12) and, thereby, further contribute to susceptibility. At present, 59

however, the relationship between inflammation and immune-mediated susceptibility to 60

pathological disease in infants is not known. 61

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In adult animals, active resolution of innate inflammation is critical for healing and the 63

return to homeostasis (11-16). A number of SPM have been identified and shown to 64

regulate the phagocytosis and killing of infecting microbes, the curtailment of neutrophil 65

recruitment, apoptosis of neutrophils, phagocytosis of apoptotic neutrophils by 66

macrophages (efferocytosis), and a shift in the overall cytokine and chemokine milieu 67


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from an inflammatory to a resolving phenotype (11, 12). In various adult models of 68

bacterial infection, SPM have been found to promote bacterial clearance and diminish 69

the duration of the inflammatory response (15, 17). However, whether and how SPM 70

may impact infection in early life is largely unknown. 71

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We have recently established in our laboratory an infant mouse model of infection with 73

the EPEC-related Citrobacter rodentium. EPEC is a particularly important pediatric 74

enteropathogen since it shows the highest risk for infection-induced death in infants ≤ 75

12 months old (18). Our murine model resembles human infant EPEC infection 76

because C. rodentium infected infant mice are also highly susceptible to infection. 77

Moreover, infected murine infants demonstrate profound intestinal inflammation and 78

damage and are severely compromised in the development of specific antibody 79

responses. Here, we present compelling evidence that the SPM resolvin D1 (RvD1) 80

and resolvin D5 (RvD5) exert substantial protective effects in infant C. rodentium 81

infection. RvD1 and RvD5, administered 2 days post infection, diminished bacterial 82

loads, ameliorated inflammation, led to the development of protective memory 83

responses associated with mature or nearly mature B cell adaptive responses, and 84

rescued infants from death. These findings raise the intriguing possibility that SPM may 85

represent powerful new tools for not only the mitigation of acute disease in early life but 86

also the prevention of recurrent childhood infection. 87

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RESULTS 90

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Murine infants are highly susceptible to C. rodentium infection. EPEC is an 92

attaching and effacing (A/E) bacterial enteropathogen that causes severe disease 93

burden in human infants (19-22). Thus, it would be valuable to have a pediatric animal 94

model that mimics the human disease condition. It has recently been reported (23) that 95

murine infants are susceptible to EPEC infection. However, normal adult mice do not 96

support EPEC infection, making comparative developmental studies impossible. Thus, 97

we chose to study 15 day old mouse infants, roughly corresponding to human infants 98

during the first year of life, and infect them with the EPEC-related C. rodentium, an 99

excellent and well-accepted experimental model for EPEC infection in adult mice (24-100

26). Others have reported(27, 28) that 14 day old infant mice are more susceptible than 101

adults to a single, relatively high infectious dose of C. rodentium. We extended these 102

studies to determine the sensitivity of infant mice to a broad range of doses (Fig. 1A). 103

As previously reported by many other laboratories, adult C57BL/6 mice all survived a 104

dose of 5 x 108 CFU. In striking contrast, it was necessary to reduce the inoculum to 105

103 CFU before most infant mice survived (Fig. 1A). It is noteworthy that mice of this 106

age weigh approximately 5 grams, or ~4 fold less than adults but the reduction in dose 107

necessary for survival was over 5 logs! These results indicate that mouse infants, like 108

human infants, are highly susceptible to A/E enterobacterial infections. The 109

susceptibility of infant mice to the high lethal dose (107 CFU) was associated with early 110

high and sustained bacterial burdens in the colons of infants (Fig. 1B). 111

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Lethal infection of infants is associated with severe intestinal pathology and 113

systemic dissemination. Our earlier studies (29, 30) showed that 7 day old neonatal 114

mice infected with the oral pathogen Yersinia enterocolitica mounted profound innate 115

inflammatory responses in the mesenteric lymph node (MLN) that exceeded those of 116

adults. We reasoned that the high bacterial load in infants infected with C. rodentium 117

may similarly elicit strong inflammation in the intestines. Indeed, by 10 days post 118

infection (p.i.), infants displayed marked inflammation in the colon, dominated by 119

neutrophils (Fig. 2A). This inflammatory response was associated with extensive 120

necrosis (Fig. 2B) and dissemination to systemic tissues (Fig. 2C). 121

These results indicate that infants manifest profound late inflammation that may 122

contribute importantly to damage of the intestinal barrier, systemic dissemination, and 123

death by sepsis. 124

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SPM reduce bacterial loads and inflammation, rescue infants from death, and lead 126

to adaptive responses comparable to adults. RvD1 and RvD5 have been previously 127

found to decrease bacteremia and increase survival in an adult murine peritonitis model 128

(31). Thus, we investigated whether RvD1 and RvD5 could affect the course of C. 129

rodentium infection in infants. As a first test of this idea, infants were pre-treated with 130

RvD1 and RvD5 two days prior to infection and then infected with a threshold lethal 131

dose (104 CFU) or a high lethal dose (107 CFU). C. rodentium titers in the colons were 132

evaluated 2 days p.i. (Fig. 3A) Remarkably, we found no colonies whatsoever in the 133

colons of infants infected with 104 CFU and significantly reduced titers in those infected 134

with 107 CFU (Fig. 3B). 135


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To test the therapeutic potential of these resolvins, we infected infants with the 136

lethal dose (107 CFU) and treated with vehicle (PBS) or RvD1 and RvD5 two days after 137

infection. Colonic C. rodentium loads were measured at 4 and 10 days p.i. (Fig. 4A). 138

Small but statistically significant decreases in titers were observed as early as 2 days 139

post RvD1 and RvD5 treatment (4 days p.i.) and substantial and significant decreases 140

were seen 10 days p.i. (Fig. 4B). Strikingly, dramatically reduced neutrophilic 141

inflammation was detected 10 days p.i. following this single RvD1/5 treatment at 2 days 142

p.i. (Fig. 4C&D). Moreover, and remarkably, this treatment rescued approximately 143

33.3% of the infected infants from death (Fig. 4E). These results indicate that SPM 144

improve survival of infants infected with a lethal dose of C. rodentium. This may result, 145

in part, from reduced bacterial burdens in the colons, similar to related earlier reports in 146

infected adult mice (31-33), but also from a dramatic reduction in pathological 147

inflammation. 148

We next tested the impact of RvD1 and RvD5 on infection with a threshold lethal 149

dose (104 CFU) of C. rodentium. Infants infected with 104 CFU and treated with RvD1 150

and RvD5 two days p.i. showed undetectable bacterial loads 10 days p.i. (Fig. 6B). 151

Remarkably, 100% of infected infants treated with RvD1 and RvD5 survived, in contrast 152

to only ~50-60% survival in the untreated or vehicle treated group (Fig. 6C). Thus, 153

RvD1 and RvD5 treatment of infants infected with a threshold lethal dose of C. 154

rodentium abolished bacterial burdens and promoted survival of all infants. 155

Strikingly, early administration of RvD1 and RvD5 in infected infants also 156

appeared to lead to the development of immunological memory, as assessed by (a) the 157

development of vigorous adaptive responses and (b) resistance to colonization on re-158


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infection. First, adults were infected with 5 x 108 CFU and infants were infected with the 159

high lethal dose of 107 CFU. The infected mice were treated 2 days p.i. with vehicle or 160

100ng (infants) or 400ng (adults; to account for weight differences) each of RvD1 and 161

RvD5. Thirty days later, all surviving animals were challenged with 5 x 108 CFU; 10 162

days later, sera were collected and anti-C. rodentium IgG responses were tested in 163

ELISA. Strikingly, surviving infants infected with a lethal dose (107 CFU) and treated 164

with RvDs developed serum IgG responses comparable with adults (Fig. 6B). Adult IgG 165

titers in the vehicle and RvD1 + RvD5 groups were completely overlapping; for the sake 166

of clarity, only the adult + RvD titers are shown. Second, parallel groups of treated 167

infant mice showed significantly reduced colon bacterial loads, relative to naïve (i.e., 168

never previously infected or treated) adult mice upon challenge 30 days following initial 169

infection (Fig. 6c). Note that we cannot obtain data from vehicle treated infants infected 170

with 107 CFU at this late time point because they have all died by 20 days p.i. (see Fig. 171

4E). However, this comparison can be obtained with infants infected with a threshold 172

lethal dose (104 CFU), at which some vehicle treated animals survive. Under these 173

conditions, significant protection against reinfection developed in the infants treated with 174

RvD1 + RvD5 (Fig. 6C). Moreover, memory IgG titers in the infants infected with 104 175

CFU and treated with RvD1 + RvD5 were markedly increased over vehicle treated 176

infants, largely achieving the levels seen in adults (Fig. 6D). It is well established that B 177

cells and IgG contribute importantly to immunity against C. rodentium in adult mice (34-178

37). Thus, a synthesis of these observations indicates that the promotion of mature IgG 179

levels by SPM in infected infant mice may markedly enhance protective immunity 180

against re-exposure. 181

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DISCUSSION 183

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We have described a murine infant model of infection with the intestinal pathogen 185

EPEC, using the related murine-specific bacterium C. rodentium. As with human infants 186

and EPEC infection, murine infants were highly susceptible to C. rodentium infection 187

and developed severe inflammation dominated by neutrophils. At late time points post 188

infection, infant intestines showed extensive necrosis and this was associated with 189

systemic dissemination of the bacteria. It seems likely that the infected infants 190

ultimately succumbed to sepsis due to damage to the intestinal barrier and bacterial 191

spread throughout the body. Using this system, we investigated the effects of 192

exogenous treatment with two SPM, RvD1 and RvD5, on the course of infection in 193

infants. Strikingly, post-infection treatment with RvD1 and RvD5 reduced the bacterial 194

burdens, alleviated inflammation, and rescued the infants from death. In addition, 195

infants treated early post infection with RvD1 and RvD5 were protected against re-196

infection and this was linked to the development of C. rodentium specific IgG responses 197

comparable to those in adults. These results identify SPM as outstanding candidates 198

for treating infectious intestinal diseases in human infants. 199

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SPM have been shown to accelerate the clearance of pathogens and increase survival 201

in numerous infection models in adult mice (13, 15). Bacterial-mediated diseases 202

include pneumonia (38-40), peritonitis (31, 32, 41, 42), skin infections (43), and cecal 203

ligation and puncture-induced sepsis (33, 44, 45). However, this is the first 204

demonstration that these SPM have potent activity in a setting of intestinal infection with 205

a bacterial enteropathogen. Furthermore, this is the first description that resolvins can 206


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relieve disease burden and dramatically improve survival in infant intestinal disease. 207

Importantly, this approach has strong potential applicability to human infants, as 208

supported by observations that infants treated with topical SPM (46) or their oral 209

precursors (47) show, respectively, reduced severity of skin disease or reduced 210

incidence of respiratory or diarrheal illnesses. 211

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The dramatic effects of RvD1 and RvD5 on survival in infant infection may arise through 213

multiple processes. First, as in infected adult animals (31, 33, 38, 41-43), SPM 214

treatment post infection reduced the bacterial load in infected infants. This is unlikely to 215

be due to a direct anti-bacterial effect since our earlier studies demonstrated that SPM 216

lack microbicidal activity (45). Thus, it is possible that the resolvins may act in infants 217

as they do in adults - to increase phagocytosis of bacteria by innate immune cells. 218

Interestingly, the majority of findings support the idea that, under physiological 219

conditions, infant and adult phagocytes have comparable capacities for phagocytosing 220

and killing various species of bacteria (48-53). Thus, infant phagocytes appear to be 221

able to respond to resolvins and increase their phagocytic capacity, in a manner similar 222

to that of adults. Together, these findings indicate that the receptors for RvD1 and 223

RvD5 and their downstream signaling pathways may be fully mature by mid-infancy. Of 224

interest, human breast milk contains RvD1 and RvD5 (54, 55) which may function in the 225

newborn. Second, the major reduction in intestinal inflammation induced by RvD1 and 226

RvD5 treatment almost certainly contributes to the enhanced survival of infants. 227

Indeed, resolvin treatment appears to allow maintenance of the intestinal barrier as 228

systemic dissemination of the bacteria was completely abolished in treated infants 229

(Supplementary Fig. 1). How inflammation is so substantially reduced is currently 230


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unclear. Although there are a number of potential mechanisms, the possibility that SPM 231

mediators enhance suboptimal efferocytosis in infants is compelling. This idea is 232

supported by observations that infants with protracted bacterial bronchitis show 233

dysfunction in alveolar macrophage efferocytosis (56). This idea is currently under 234

investigation. 235

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Antibody responses in early life are generally reduced in both quality and quantity, 237

compared with adults (reviewed in (57-62)). Achieving robust IgG response is the major 238

goal of many pediatric vaccines and the importance of B cells and IgG responses in 239

protection against extracellular microbial pathogens, such as C. rodentium (34-37), is 240

well established. Thus, our observations that RvD1 and RvD5 support the development 241

of resistance against re-infection associated with nearly mature IgG responses have 242

major implications for infant vaccine responses. How these mature responses are 243

elicited is not currently known but it is tempting to speculate that resolvins may support 244

the development of mature germinal center (GC) reactions in the intestinal lymphoid 245

tissues. This possibility is based on observations (63, 64) from C.A. Siegrist and 246

colleagues that the poor IgG responses of neonatal mice to systemic vaccination with 247

inert antigens are linked with poor GC development and that T follicular helper cell and 248

GC responses can be substantially boosted with specific adjuvant. Thus, it will be of 249

considerable interest to examine the effects of SPM on GC formation during infant 250

intestinal bacterial infection. 251

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It is well appreciated that bacterial and viral infections of infants often lead to profound 253

inflammation (65-67). Yet, most in vitro tests of innate immune function in infancy show 254


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depressed or deficient proinflammatory function (68, 69). Of course, in vitro tests 255

largely read out the initiation of inflammation and are not designed to measure the 256

accumulation of inflammation. Perhaps our results provide insights into a possible 257

reconciliation of these observations. That is, that the severe inflammation often seen in 258

infant infections in vivo is largely independent of early events in inflammation and, 259

instead, occurs due to immaturity in the later phases of inflammation. 260

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MATERIALS AND METHODS 262

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Mice. Adult C57BL/6 were purchased from Harlan Laboratories. All mice were bred 264

and housed under barrier conditions in the Division of Veterinary Resources of the 265

University of Miami Miller School of Medicine. Mice were regularly screened for specific 266

common pathogens. Adult mice (6-10 weeks of age) and infant mice (15 days of age) 267

were used in experiments. Infant mice were weaned at 3 ½ weeks of age. All mice 268

were handled in compliance with the Institutional Animal Care and Use Committee 269

(IACUC) of the University of Miami Miller School of Medicine, Miami, Florida. 270

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Bacteria infections and SPM treatments. GPM1831a (gift from Dr. G. P. Munson, 272

University of Miami) is a kanamycin-resistant derivative of C. rodentium DBS100 (ATCC 273

51459). It was constructed by lambda RED mediated recombination of a kanamycin 274

cassette into the non-coding region ca. 400 bp upstream of an unnamed gene encoding 275

a putative membrane protein (NCBI Accession number KIQ50635). The bacteria were 276

grown at 37oC in Luria-Bertani broth (LB) medium or MacConkey Agar (Sigma Aldrich, 277

United States) plates containing 25µg/ml kanamycin. Adults were inoculated 278


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orogastrically with 5 x 108 CFU using a 22-gauge, round-tipped feeding needle (Fine 279

Science Tools, Foster City, CA) attached to a 1-ml syringe (Becton Dickinson, Franklin 280

Lakes, NJ). Infants were inoculated orogastrically with the indicated doses using PE-10 281

tubing (polyethylene tubing with an outside diameter of 0.61 mm) [Clay Adams, Sparks, 282

MD] attached to a 30-gauge needle and Hamilton syringe (70). The actual administered 283

dose was determined by plating serial dilutions of the suspensions on Luria Broth (LB) 284

plates and incubating for 24 hr at 37°C. 285

RvD1 and RvD5 were obtained from Cayman Chemical Company (Ann Arbor, 286

MI). The RvDs were stored in undiluted aliquots in the dark at -80oC. Upon thawing, 287

the RvDs were diluted in cold sterile PBS to 5mg/ml and used immediately. Infant mice 288

were injected i.p. with 20µl containing 100ng each of RvD1 and RvD5 using a Hamilton 289

25µl gas tight syringe with a 30 gauge attachable needle; adult mice were injected i.p. 290

with 100µl containing 400ng each of RvD1 and RvD5 using a 1.0ml syringe and a 25 291

gauge needle . The times of injection relative to the day of infection are indicated in the 292

text and figure legends. 293

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Bacterial enumeration from organs of infected mice. To measure C. rodentium 295

titers, colons, livers, or spleens were weighed and homogenized in HBSS using a 296

Seward Biomaster 80 Stomacher (Brinkman, Westbury, NY) for 4 min at high speed. 297

Individual mesenteric lymph nodes were homogenized in 400ul (neonates) or 500ul 298

(adults) of HBSS using a VWR disposable pellet mixer with cordless motor (VWR 299

International). C. rodentium titers were enumerated by plating dilutions of homogenates 300

on MacConkey agar containing 25µg/ml kanamycin. 301

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Histology and inflammation. Sections of colon were fixed in 10% neutral buffered 303

formalin, sectioned, and stained with hematoxylin and eosin. The slides were examined 304

for histological changes by a board certified veterinary pathologist. Slides were 305

assessed blindly; inflammation scores were determined on a scale of 0-3 (0, none; 306

1,mild; 2, moderate, 3, severe). 307

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ELISA. Overnight cultures of C. rodentium were homogenized in bicarbonate buffer, pH 309

9.5, using the VWR disposable pellet mixer. ELISA plates were coated o.n. at room 310

temperature with 100µl of 20µg/ml bacterial lysate in bicarbonate buffer. The wells 311

were blocked with PBS containing 2% BSA for 1 hr at room temperature, washed, and 312

serum dilutions in PBS were incubated o.n. at room temperature. Rabbit anti-mouse 313

IgG-peroxidase (Sigma A9044) was added for 2 hr at room temperature and the wells 314

were developed with TMB solution (Life Technologies) for 30 min. 315

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Statistical Analyses. All experiments were performed at least two times. Statistical 317

tests were performed using GraphPad Prism software, as follows: Mann Whitney test 318

for the bacterial colonization experiments; Log-rank (Mantel-Cox) test for the survival 319

experiments. The significance threshold was P ≤0.05. 320

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ACKNOWLEDGEMENTS 323

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This research project received no specific grant from any funding agency in the public, 325

commercial, or not-for-profit sectors. There are no conflicting interests relevant to the 326

study. CNS is supported by NIH grant GM38765. 327


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527

528

FIGURE LEGENDS 529

530

Figure 1. Infants are highly sensitive to primary infection with C. rodentium; 531

association with high early and sustained bacterial titers. (A) Survival of 2 wk old 532

infant and adult C57BL/6 mice orally infected with the indicated doses of C. rodentium. 533

≥ 11 mice per group in 2 independent experiments; *, p<0.01 by Log-rank (Mantel-Cox) 534

test, compared to adult survival curves. (B) CFU in the colons at the indicated days after 535

oral infection of adults with 5 x 108 or infants with 1 x 10

7 CFU. Each symbol represents 536

an individual animal. *, p≤0.01 by Mann Whitney test. 537

538

Figure 2. Infant susceptibility to C. rodentium infection is associated with late 539

severe inflammation in the colon and systemic dissemination. Infants and adults 540

were orally infected with 107 and 5 x 10

8 CFU, respectively, of C. rodentium and 541

examined 10 days p.i. (A) colon sections were stained with hematoxylin & eosin and 542

examined for inflammation; a total of 4 mice in each group was examined. Scores were 543

determined on a scale of 0-3 (0, none; 1,mild; 2, moderate, 3, severe). Of note, the 544

pathologist was only given numbers for each sample and was unaware of any specifics 545

of the experiments. (B) Representative sections of colons. Adult (AD), mild 546

inflammation limited to the mucosal surface; Infant (INF), severe inflammation with 547

necrosis extending from the mucosa to the submucosa and muscularis (25X). (C) 548

Organs were dissected and homogenized for CFU counts; Colon (COL), Mesenteric 549


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Lymph node (MLN); Spleen (SP); Liver (LIV). Each symbol represents an individual 550

mouse; *, p≤ 0.01 by Mann Whitney analyses. 551

552

553

Figure 3. Pretreatment with RvD1 and RvD5 diminishes early bacterial loads in C. 554

rodentium infected infants. Infant mice were left untreated or treated with vehicle 555

(PBS) or 100ng each of RvD1 and RvD5 i.p. Two days later, the mice were infected 556

with the indicated doses of C. rodentium and CFU in the colons were assessed 2 days 557

after that. (A) Experimental scheme. (B) Colon CFU. Symbols represent individual 558

animals. The dashed line indicates the limit of detection of the assay. *, p<0.01 by 559

Mann Whitney analysis. . 560

561

Figure 4. Treatment with RvD1 and RvD5 two days after infection with a lethal 562

dose diminishes bacterial loads and inflammation and improves survival in C. 563

rodentium infected infants. Infant mice were left untreated or treated with vehicle 564

(PBS) or 100ng each of RvD1 and RvD5 i.p. two days after infection with 107 CFU C. 565

rodentium. Colon CFU, inflammation, and survival were scored. (A) Scheme of the 566

experiment. (B) Colon titers 4 and 10 days p.i. Symbols represent individual animals. #, 567

p<0.04; *, p<0.01 by Mann Whitney analysis. (C) Colon sections 10 days p.i. were 568

stained with hematoxylin and eosin and assessed as in the legend for Figure 2. Age-569

matched control infants (uninfected, untreated) showed no score (i.e., scores = 0) (D) 570

Representative sections of colons 10 days p.i. from the indicated groups (50X). (E) 571


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Survival of similarly treated infants. ≥12 mice per group in 2 independent experiments. 572

*, p<0.01 by Log-rank (Mantel-Cox) test. 573

574

Figure 5. Treatment with RvD1 and RvD5 two days after infection with a 575

threshold lethal dose leads vastly diminished bacterial loads 10 days p.i. and 576

complete survival. Infant mice were left untreated or treated with vehicle (PBS) or 577

100ng each of RvD1 and RvD5 i.p. two days after infection with 104 CFU of C. 578

rodentium. Colonic titers were assessed at 10 days p.i. Survival was monitored in 579

parallel groups of animals. (A) Experimental scheme. (B) Colon CFU. Symbols 580

represent individual animals. *, p<0.01 by Mann Whitney analysis. (C) Survival curves. 581

≥ 8 mice per group in 2 independent experiments; p<0.01 by Log-rank (Mantel-Cox) 582

test. 583

584

Figure 6. Treatment of infant mice with RvD1 and RvD5 after infection leads to 585

vigorous memory responses. Adult mice were infected with 5 x 108 CFU; Infant 586

mice were infected with 107 or 104 CFU. Two days p.i., mice were treated with vehicle 587

or 100ng (infants) or 400ng (adults) each of RvD1 and RvD5 i.p.. Thirty days later, all 588

mice were challenged with 5 x 108 CFU and ,10 days later, sera were collected and 589

colon CFU measured. Serum anti-C. rodentium IgG responses were measured by 590

ELISA. (A) Experimental scheme. (B) Serum IgG titers in infants infected with 107 CFU 591

and in infected adults. (C) Colon titers in the indicated mice. *, p<0.01 by Mann-592

Whitney analyses. (D) Serum IgG titers in infants infected with 104 CFU and in infected 593

adults. In B and D, IgG titers for vehicle treated adult mice are not shown due to 594


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22

complete overlap with the IgG titers in +RvD1/5 treated adult mice. Symbols represent 595

individual animals. 596


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Figure 1. Infants are highly sensitive to primary infection with C. rodentium; association with high

early and sustained bacterial titers. (A) Survival of 2 wk old infant and adult C57BL/6 mice orally

infected with the indicated doses of C. rodentium. ≥ 11 mice per group in 2 independent experiments; *, p<0.01 by Log-rank (Mantel-Cox) test, compared to adult survival curves. (B) CFU in the colons at the

indicated days after oral infection of adults with 5 x 108 or infants with 1 x 107 CFU. Each symbol

represents an individual animal. *, p≤0.01 by Mann Whitney test.

A. B.


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AD INF

Figure 2. Infant susceptibility to C. rodentium infection is associated with late

severe inflammation in the colon and systemic dissemination. Infants and adults

were orally infected with 107 and 5 x 108 CFU, respectively, of C. rodentium and

examined 10 days p.i. (A) colon sections were stained with hematoxylin & eosin and

examined for inflammation ; a total of 4 mice in each group was examined. Scores were

determined on a scale of 0-3 (0, none; 1,mild; 2, moderate, 3, severe). Of note, the

pathologist was only given numbers for each sample and was unaware of any specifics of

the experiments. (B) Representative sections of colons. Adult (AD), mild inflammation

limited to the mucosal surface; Infant (INF), severe inflammation with necrosis extending

from the mucosa to the submucosa and muscularis (25X). (C) Organs were dissected

and homogenized for CFU counts; Colon (COL), Mesenteric Lymph node (MLN); Spleen

(SP); Liver (LIV). Each symbol represents an individual mouse; *, p≤ 0.01 by Mann

Whitney analyses.

A. B. C.


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Figure 3. Pretreatment with RvD1 and RvD5

diminishes early bacterial loads in C. rodentium

infected infants. Infant mice were left untreated or

treated with vehicle (PBS) or 100ng each of RvD1 and

RvD5 i.p. Two days later, the mice were infected with the

indicated doses of C. rodentium and CFU in the colons

were assessed 2 days after that. (A) Experimental

scheme, (B) Colon CFU. Symbols represent individual

animals. The dashed line indicates the limit of detection of

the assay. *, p<0.01 by Mann Whitney analysis.

-2 0 2

Infect 107

or 104 CFURvD1/5

Colon

CFUDays p.i.

A.

B.


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0 2 4 10 days p.i.

Infect

107 CFU RvD1/5

Colon

CFU

Colon CFU

Histology

Survival

A.

Figure 4. Treatment with RvD1 and RvD5 two days after infection with a lethal dose diminishes bacterial loads and

inflammation and improves survival in C. rodentium infected infants. Infant mice were left untreated or treated with vehicle

(PBS) or 100ng each of RvD1 and RvD5 i.p. two days after infection with 107 CFU C. rodentium. Colon CFU, inflammation, and

survival were scored. (A) Scheme of the experiment. (B) Colon titers 4 and 10 days p.i. Symbols represent individual animals. #,

p<0.04; *, p<0.01 by Mann Whitney analysis. (C) Colon sections 10 days p.i. were stained with hematoxylin & eosin and assessed as

in the legend for Figure 2. Age-matched control infants (uninfected, untreated) showed no score (i.e., scores = 0) (D) Representative

sections of colons 10 days p.i. from the indicated groups (50X). (E) Survival of similarly treated infants. ≥12 mice per group in 2 independent experiments. *, p<0.01 by Log-rank (Mantel-Cox) test.

Normal

control

Infected

+ RvD1/5

Infected

+ vehicle

B.

C. D.

E.


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Figure 5. Treatment with RvD1 and RvD5 two days after infection

of infants with a threshold lethal dose leads to vastly diminished

bacterial loads 10 days p.i. and complete survival. Infant mice were

left untreated or treated with vehicle (PBS) or 100ng each of RvD1 and

RvD5 i.p. two days after infection with 104 CFU of C. rodentium. Colonic

titers were assessed at 10 days p.i. Survival was monitored in parallel

groups of animals. (A) Experimental scheme. (B) Colon CFU.

Symbols represent individual animals. *, p<0.01 by Mann Whitney

analysis. (C) Survival curves. ≥ 8 mice per group in 2 independent

experiments; p<0.01, compared to either untreated or vehicle, by Log-

rank (Mantel-Cox) test.

0 2 10 days p.i.

Infect

104 CFU RvD1/5

Colon

CFU

Survival

A.

untreated vehicle +RvD1/RvD5

B. C.


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0 2 30 days p.i.

Infect infants

107, 104 CFU RvD1/5

Challenge

5 x 108 CFU

Serum IgG;

Colon CFU

A. B.

Figure 6. Treatment of infant mice with RvD1 and RvD5 after infection leads to vigorous memory responses. Adult mice were infected with 5 x 108 CFU;

Infant mice were infected with 107 or 104 CFU. Two days p.i., mice were treated with vehicle (PBS) or 100ng (infants) or 400ng (adults) each of RvD1 and RvD5 i.p.

Thirty days later, all mice were challenged with 5 x 108 CFU and 10 days later sera were collected and colon CFU measured. Serum anti-C. rodentium IgG responses

were measured by ELISA. (A) Experimental scheme. (B) Serum IgG titers in infants infected with 107 CFU and in infected adults. (C) Colon titers in the indicated

mice. *, p<0.01 by Mann-Whitney analyses. (D) Serum IgG titers in infants infected with 104 CFU and in infected adults. In B and D, IgG titers for vehicle treated adult

mice are not shown due to complete overlap with the IgG titers in the +RvD1/5 treated adult mice. Symbols represent individual animals.

C.

D.


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specialized pro -resolving mediators (spm) rescue...

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