Neonatal CD71+ Erythroid Cells Do Not Modify Murine Sepsis Mortality James L. Wynn, Philip O. Scumpia, Blair T. Stocks, Joann Romano-Keeler, Mhd Wael Alrifai, Jin-Hua Liu, Annette S. This information is current as Kim, Catherine E. Alford, Pranathi Matta, Jörn-Hendrik of September 24, 2021. Weitkamp and Daniel J. Moore J Immunol published online 22 June 2015 http://www.jimmunol.org/content/early/2015/06/20/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 22, 2015, doi:10.4049/jimmunol.1500771 The Journal of Immunology

Neonatal CD71+ Erythroid Cells Do Not Modify Murine Sepsis Mortality

James L. Wynn,* Philip O. Scumpia,† Blair T. Stocks,‡ Joann Romano-Keeler,* Mhd Wael Alrifai,* Jin-Hua Liu,* Annette S. Kim,‡ Catherine E. Alford,x Pranathi Matta,* Jo¨rn-Hendrik Weitkamp,* and Daniel J. Moore‡,{

Sepsis is a major cause of neonatal mortality and morbidity worldwide. A recent report suggested that murine neonatal host defense against infection could be compromised by immunosuppressive CD71+ erythroid splenocytes. We examined the impact of CD71+ erythroid splenocytes on murine neonatal mortality to endotoxin challenge or polymicrobial sepsis and characterized circulating CD71+ erythroid (CD235a+) cells in human neonates. Adoptive transfer or an Ab-mediated reduction in neonatal CD71+ erythroid splenocytes did not alter murine neonatal survival to endotoxin challenge or polymicrobial sepsis challenge. Ex vivo immuno- suppression of stimulated adult CD11b+ cells was not limited to neonatal splenocytes; it also occurred with adult and neonatal Downloaded from bone marrow. Animals treated with anti-CD71 Ab showed reduced splenic bacterial load following bacterial challenge compared with isotype-treated mice. However, adoptive transfer of enriched CD71+ erythroid splenocytes to CD71+-reduced animals did not reduce bacterial clearance. Human CD71+CD235a+ cells were common among cord blood mononuclear cells and were shown to be . In summary, a lack of effect on murine survival to polymicrobial sepsis following adoptive transfer or diminution of CD71+ erythroid splenocytes under these experimental conditions suggests that the impact of these cells on neonatal infection risk and progression may be limited. An unanticipated immune priming effect of anti-CD71 Ab treatment, rather than a reduction in http://www.jimmunol.org/ immunosuppressive CD71+ erythroid splenocytes, was likely responsible for the reported enhanced bacterial clearance. In humans, the well-described rapid decrease in circulating reticulocytes after birth suggests that they may have a limited role in reducing inflammation secondary to microbial colonization. The Journal of Immunology, 2015, 195: 000–000.

eonatal sepsis remains a significant global threat and immune response to sepsis (5, 6). In particular, neonatal mice and kills .1 million newborns each year (1). In developed humans exhibit greater mortality and an attenuated inflammatory countries, 60% of the most prematurely born neonates response to sepsis compared with adults (7). However, neonatal-

N by guest on September 24, 2021 develop sepsis (2). Death or major disability occurs in 4 of every specific mechanistic investigations into the pathophysiology of 10 septic neonates, even with antimicrobial treatment (3). Despite sepsis have lagged behind those of adults. multiple attempts over the last three decades to improve survival, Recently, murine neonates were reported to harbor an immu- neonatal sepsis management remains limited to antimicrobial nosuppressive CD71+ (also known as transferrin receptor), ery- treatment and supportive care (4). Developmental age strongly throid (Ter119+) population of splenocytes, which were absent in influences innate and adaptive immune function and the host adult (8). Evidence was presented that suggested these cells provided the beneficial effect of reducing the inflammatory *Division of Neonatology, Department of Pediatrics, Vanderbilt University, Nash- response associated with early life microbial colonization via ville, TN 37232; †Department of Dermatology, University of California, Los arginase-2 but at the untoward expense of an increased risk for Angeles, Los Angeles, CA 90095; ‡Department of Pathology, Microbiology and x infection. CD71 is expressed on rapidly dividing cell populations, Immunology, Vanderbilt University, Nashville, TN 37232; Department of Pathology, Tennessee Valley Healthcare System, Veterans Affairs, Nashville, TN 37232; and including normal and malignant cells, and is markedly induced { Division of Endocrinology, Department of Pediatrics, Vanderbilt University, Nash- following Ag and mitogen stimulation (9). Sites of extramedullary ville, TN 37232 hematopoiesis, including the neonatal liver and , are nec- Received for publication April 1, 2015. Accepted for publication June 2, 2015. essary to support the rapid fetal and postnatal growth in the setting This work was supported by a Vanderbilt Clinical and Translational Science (CTSA) of significantly reduced erythroid reservoirs compared with adults Award (UL1 TR000445) from the National Center for Advancing Translational Scien- + ces, Vanderbilt CTSA Grant UL1 RR024975-01 from the National Center for Research (10). As such, the CD71 erythroid population represents a large Resources (National Institutes of Health), the Eunice Kennedy Shriver National Institute portion of murine fetal liver, neonatal spleen, and adult bone of Child Health and Human Development (T32HD068256 to J.R.-K. and HD061607 to marrow (9–11). Predictably, adult and neonatal spleens in both J.-H.W.), National Institutes of Health Grants DK090146 (to D.J.M.) and GM106143 (to J.L.W.), National Institutes of Health Medical Scientist Training Program rodents and humans are different in cellular composition and (5T32GM007347-33 to B.T.S.), and funds from the Department of Pediatrics, Vander- function (12–14), which is an important consideration for exper- bilt University. imental comparisons of the spleen between animals at different Address correspondence and reprint requests to Dr. James L. Wynn at the current developmental ages. address: Division of Neonatal-Perinatal Medicine, Department of Pediatrics, Univer- sity of Florida, 1600 SW Archer Road, P.O. Box 100296, Gainesville, FL 32610-0296. In this article we show that neonatal survival to endotoxin E-mail address: [email protected]fl.edu challenge and neonatal polymicrobial sepsis survival were not The online version of this article contains supplemental material. affected by provision of neonatal CD71+ erythroid cells; an Ab- + Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; CBMC, cord mediated reduction in CD71 cells did not modify neonatal sepsis blood mononuclear cell; HKLM, heat-killed Listeria monocytogenes; LD, lethal survival; the ex vivo immunomodulatory effects mediated by dose; MFI, mean fluorescence intensity; nRBC, nucleated RBC. murine neonatal splenocytes also occurred with hematopoietic Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 tissue from neonatal and adult bone marrow; enhanced bacterial

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500771 2 CD71+ CELLS DO NOT MODIFY NEONATAL INFLAMMATORY MORTALITY clearance following anti-CD71 treatment was the result of im- blocks for sectioning were made on dry ice in embedding medium (Tissue- mune priming rather than the result of a reduction in immuno- Tek; Sakura, Torrance, CA). Murine tissue sections (8 mm) were stained with suppressive cells; and human neonatal CD71+CD235a+ cells are DAPI-gold (Molecular Probes), anti-CD71 Ab (Abcam), and appropriate secondary Ab (Invitrogen). Tissue was examined using an Olympus IX81 exquisitely sensitive to hypotonic lysis and are predominantly enu- microscope with a 12-bit charge-coupled device (Orca ERII; Hamamatsu) cleated reticulocytes. We conclude that murine neonatal CD71+ ery- camera, and images were acquired using SlideBook digital microscopy soft- throcytes have no effect on neonatal survival with endotoxemia or ware. Mean fluorescence intensity (MFI) was measured using Adobe Photo- sepsis and that there is no clinical role for targeting the subset of shop CS6. Cytospins were performed on sorted human cells with subsequent microscopic examination following Wright’s stain or methylene blue. erythroid CD71+ cells to attenuate neonatal sepsis. Reticulocytes have been extensively characterized in human neonates and are not present Experimental sepsis and endotoxemia in all newborns. However, when present, they dramatically decline Mice were made septic using polymicrobial peritonitis, as previously de- within hours after birth, at the same time as microbial colonization scribed (7). Briefly, a 6–8-wk-old nonpregnant female wild-type (C57BL/6) dramatically increases, suggesting that they may have a limited role mouse was euthanized within 2 wk of arrival from the vendor, and the cecum in reducing inflammation secondary to microbial colonization. was isolated. Cecal contents were expressed, weighed, suspended in 5% dextrose at a concentration of 80 mg/ml, and administered via i.p. injection at the desired lethal dose (LD), as indicated in each figure legend. Where Materials and Methods indicated, sepsis was generated by cecal slurry administration 24 h after Mice the second dose of anti-CD71 or isotype Ab (described below). Mice were monitored after injection, as previously described (7, 14, 15). Ultrapure LPS All studies were approved by the Institutional Animal Care and Use (Escherichia coli 0111:B4; InvivoGen), given via an i.p. injection, was used Committee at Vanderbilt University. Specific pathogen–free, male and to generate endotoxemia. The LPS dose used (20 mg/g body weight) was female C57BL/6 mice were purchased from The Jackson Laboratory (Bar determined empirically and reproducibly generated 35–40% mortality in Downloaded from Harbor, ME) between 6 and 8 wk of age and allowed a minimum of 7 d to neonatal pups. For LPS-priming experiments, mice received an i.p. injection equilibrate to their environment before any breeding or experimental use. of 1 mg LPS/g body weight 24 h prior to infectious challenge (14). Mice were maintained on breeder chow and water ad libitum. Pups aged + 5–7 d were considered neonates (7, 14, 15). Adoptive cell transfer and Ab-mediated depletion of CD71 erythroid cells Humans Neonatal CD71+ erythroid splenocytes were enriched as described above. http://www.jimmunol.org/ All studies were approved by the Vanderbilt Institutional Review Board. Neonatal mice received 3 3 106 FACS-enriched CD71+ erythroid neonatal Peripheral blood from healthy adult volunteers or cord blood from term splenocytes via an i.p. injection 30 min prior to sepsis generated by cecal healthy infants was collected fresh into blood-collection tubes containing slurry injection. Adoptive transfer of immunomodulating cells is commonly lithium heparin (BD Biosciences). Cord blood mononuclear cells (CBMCs) performed at the same time as the insult (17–19). We delayed endotoxin or and adult PBMCs were immediately isolated by Ficoll gradient separation cecal slurry injection slightly to avoid excessive volume into the limited (Ficoll-Paque Plus; GE Healthcare), as previously described (16). peritoneal space of the murine neonate. In our experiments, the peritoneum Isolation of murine bone marrow and splenocytes was the primary site of the insult (sepsis or LPS), so we administered the adoptive cells into the peritoneum. Control mice received an identical Both femurs and tibias from individual animals were collected and flushed number of enriched B220+ cells or saline via i.p. injection. For endotoxemia, with cold PBS (15). Spleens were harvested, and single-cell suspensions 3 3 106 FACS-enriched CD71+ erythroid neonatal splenocytes were given + were created by passing the cells through a 70-mm pore size cell strainer i.p. 30 min prior to LPS injection. Resident CD71 erythroid splenocytes by guest on September 24, 2021 (Falcon-type). Splenocyte and bone marrow suspensions were then sub- were diminished via i.p. administration of anti-CD71 mAb (150 mg/mouse; jected to RBC lysis using ammonium chloride solution (KD Medical, Bio X Cell, clone R17 217.1.3/TIB-219) daily for 2 d. This dose was chosen Columbia, MD). Lysed fractions were washed twice with cold PBS and based on a previous report and our empiric results (8). suspended in DMEM (Life Technologies) with 10% FBS + penicillin/ streptomycin. Cell viability before plating was determined manually us- Cell culture supernatant inflammatory mediator measurements ing a hemocytometer with trypan blue exclusion. Prior to FACS, spleens Cytokine/chemokine concentrations were determined on supernatants using were disrupted in PBS containing 0.2 M EDTA (Sigma) and were not a multiplex assay for 32 analytes [IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL- exposed to ammonium chloride. 6, IL-7, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17a, G-CSF, Cell purification, immunophenotyping, and stimulation M-CSF, GM-CSF, IFN-g, CXCL1 (KC), CXCL10 (IP-10), CXCL5 (LIX), LIF, CCL2 (MCP-1), CCL3 (MIP-1a), CCL4 (MIP-1b), CXCL2 (MIP2), CD11b+ effector cells were enriched from unlysed adult bone marrow CXCL9 (MIG), CCL11 (eotaxin), CCL5 (RANTES), TNF-a,VEGF] using anti-CD11b magnetic beads, according to the manufacturer’s pro- (Millipore) on a Luminex 100, according to the manufacturer’s protocol. tocol (Miltenyi Biotec). One million enriched CD11b+ effector cells were used for coculture with and without stimulation via 5 3 106 heat-killed Statistics + Listeria monocytogenes (HKLM; InvivoGen). Murine neonatal CD71 Endotoxemia or sepsis survival was compared between groups using the erythroid splenocytes were targeted and enriched using FACS on a BD Fisher exact test. Values were considered significant only if the two-tailed FACSAria III. All FACS Abs were obtained from either eBioscience or BD confidence level was p , 0.05. Cytokine concentrations and leukocyte Biosciences. Isolated or enriched murine splenic leukocytes were pheno- phenotypes were compared using the Student t test. If the descriptive typed by cell surface staining with B220, CD71, Ter119, and 7-amino- analyses passed normality and equal variance, then a post hoc Tukey actinomycin D (7-AAD) in FACS buffer (PBS with 3% FBS with no azide) multiple range test was used to compare all groups to determine whether on a BD Fortessa. Human PBMCs were processed for same-day flow there was a difference between groups. If the descriptive analyses failed cytometry by washing with FACS buffer containing 20% heat-inactivated normality or tests of equal variance, a comparison using Kruskal–Wallis FBS, followed by staining with 7-AAD as viability dye (Molecular Probes), ANOVA on ranks and Dunn’s method was performed versus sham-treated anti-CD235(GlyA)–FITC (Invitrogen), and anti-CD71–PE or anti-CD71– controls. A Student t test or a Wilcoxon signed-rank test was used to allophycocyanin (BD Biosciences). For compensation, we used Ab-capture compare results from two groups. Values were considered significant if beads (CompBeads; BD Biosciences). Stained cells were washed and p , 0.05. Analyses were performed using Prism 6. resuspended in 100 ml FACS buffer prior to acquisition on the cytometer (FACSCanto II; Becton Dickinson). To remove erythrocytes after initial data collection, samples were treated with Pharm Lyse buffer (BD Biosciences) Results + and washed. FACS samples were analyzed using FlowJo software. A min- Provision of enriched neonatal CD71 erythroid cells does not 2 imum of 3 3 104 nondebris, live (7-AAD ) cells was used for analysis. reduce mortality to endotoxin challenge Immunofluorescence and cytospin staining We hypothesized that immunosuppressive neonatal CD71+ ery- Neonatal small intestine was collected, and tissues were placed in 10% throid cells would reduce mortality to systemic inflammation. To formalin (Fisher Scientific) at 4˚C for 1 h, 15% sucrose (Research Products address this hypothesis, we performed adoptive transfer of 3 3 International, Mount Prospect, IL) overnight, and 30% sucrose for 6 h; 106 highly enriched (.90% purity) CD71+ erythroid neonatal The Journal of Immunology 3 splenocytes (Supplemental Fig. 1A) and measured survival to challenge (Fig. 1A), we found no change in sepsis survival among + endotoxin challenge (LD40) as a noninfectious model of systemic neonates that received enriched neonatal CD71 erythroid inflammation. Three million CD71+ erythroid neonatal splenocytes splenocytes compared with PBS or neonatal B220+ cells (Fig. represents an increase of 60% over the endogenous CD71+ ery- 1B). To answer the question of whether adoptive transfer modifies throid neonatal splenocytes that mediate the reported immunosup- cytokine production but not mortality, we examined early plasma pressive effect. Survival was not modified in two separate assessments cytokine production in septic neonates with or without antecedent of neonates that received enriched neonatal CD71+ erythroid adoptive transfer of CD71+ erythroid cells. We did not find a sig- splenocytes (65%) compared with neonates that received PBS nificant difference in the median concentration of plasma TNF-a (69%) (Fig. 1A). Endotoxin challenge is a useful immunologic tool when it was measured at 2 h after sepsis (the timing of peak plasma to generate systemic inflammation, yet it lacks clinical relevance TNF-a using our model) in mice (n = 5/group) that received either 5 and the multiorgan system complexity associated with sepsis. 3 106 CD71 erythroid cells or PBS (67 versus 72 pg/ml, p =0.82). Therefore, we next examined the impact of adoptive transfer of These results demonstrate that, despite the reported ex vivo im- neonatal CD71+ erythroid splenocytes on sepsis survival using munomodulatory properties associated with neonatal CD71+ ery- a well-characterized model of polymicrobial sepsis (7). throid cells, provision of additional immunosuppressive cells does not modify in vivo plasma concentration of TNF-a or mortality in Provision of enriched CD71+ erythroid splenocytes does not our murine model of polymicrobial sepsis. modify polymicrobial sepsis survival + CD71+ erythroid splenocytes reportedly reduce the inflammatory Depletion of CD71 erythroid cells does not alter neonatal polymicrobial sepsis survival response associated with microbial colonization but with the un- Downloaded from toward consequence of increased risk for infection (8). To directly Single pathogen challenge following Ab-mediated depletion of assess the clinically relevant possibility of infection risk, we next CD71+ erythroid cells in neonatal mice was associated with a de- tested the hypothesis that provision of immunosuppressive CD71+ creased splenic bacterial load compared with mice that did not erythroid cells would reduce survival to sepsis by compromising receive depleting Ab (8). However, mortality to this challenge the host response to infection using a well-characterized model of following CD71+ erythroid reduction was not reported. Study of 3 6 polymicrobial sepsis (7). We adoptively transferred 5 10 highly CD71-null mice is not possible because loss of CD71 is embry- http://www.jimmunol.org/ enriched (.90% purity) neonatal CD71+ erythroid splenocytes via onically lethal (20). Therefore, we examined the impact of Ab- i.p. injection prior to generating polymicrobial sepsis using the mediated CD71+ erythroid depletion on polymicrobial sepsis cecal slurry model (7). The number of injected CD71+ erythroid survival. Ab-mediated depletion resulted in a 58% reduction in cells represented a doubling of the number of endogenous neo- CD71+ erythroid splenocyte representation (from 60 to 25% of the natal CD71+ splenocytes and was greater that what we used in live splenocytes, an average absolute reduction of 3 3 106 CD71+ mice given LPS to account for the increased severity of the septic erythroid cells), resulting in a gross change in splenic appearance insult. Control animals received an identical number of highly (Fig. 1C). Remarkably, sepsis survival for mice with diminished enriched neonatal B220+ splenocytes (Supplemental Fig. 1B) or CD71+ erythroid splenocytes was not altered compared with

PBS prior to sepsis. Similar to our negative results with endotoxin neonatal mice that received isotype-control Ab (Fig. 1D). by guest on September 24, 2021

FIGURE 1. Impact of CD71+ erythroid cells on inflammatory-mediated mortality in neonates. (A) Survival after adoptive transfer with endotoxin ex- posure. Neonatal mice received 3 3 106 highly enriched neonatal CD71+ erythroid (Ter119+) cells via i.p. injection 30 min prior to endotoxin challenge 6 + (LD40). (B) Survival after adoptive transfer with polymicrobial sepsis. Neonatal mice received 5 3 10 highly enriched neonatal splenic CD71 erythroid + + cells, B220 cells, or PBS via i.p. injection 30 min prior to sepsis (LD60). (C) Gross spleen appearance and splenic CD71 erythroid cell representation by FACS in previously healthy neonatal mice following anti-CD71 or isotype Ab treatment. Data are mean + SD. (D) Impact of anti-CD71 treatment or isotype-control Ab on sepsis survival following low (top;LD20) and high (bottom;LD60) mortality challenges. *p , 0.05, t test. 4 CD71+ CELLS DO NOT MODIFY NEONATAL INFLAMMATORY MORTALITY

Immunomodulation occurs with coculture of adult or neonatal tissues examined (Fig. 2B). Taken together, these results support hematopoietic tissues that broad immunomodulatory effects on CD11b+ effector cells Because we did not observe an effect on mortality after inflam- occur with coculture of actively hematopoietic tissues from matory or infectious challenge with either adoptive transfer or neonates and adults, rather than as a unique property of neonatal reduction of CD71+ erythroid cells and no difference in plasma splenocytes. TNF-a production in vivo by septic neonatal mice following CD71+ cells are prominent in neonatal ileum, and anti-CD71 adoptive transfer of CD71+ erythroid cells, we determined whether treatment diminishes intestinal barrier function there was immunosuppression ex vivo. Importantly, CD71+ ery- Enhanced bacterial clearance was demonstrated following Ab- throid cells represent a large portion of hematopoietic tissues, mediated reduction of CD71+ erythroid cells (8). This effect including murine fetal liver, neonatal spleen and bone marrow, might be the result of reducing CD71+ immunosuppressive cells and adult bone marrow (9–11) (Supplemental Fig. 1D). Because or an unrelated effect of exposure to the Ab used in the reduction the neonatal spleen is largely hematopoietic, we hypothesized procedure. It is noteworthy that anti-CD71 treatment only acti- that the immunosuppressive effects previously observed with co- vated intestinal immune cells, and their activation was depen- culture of neonatal splenocytes would also be present in other dent upon intestinal microbiota. These results suggest that the hematopoietic tissues from adults and neonates. To address this observed effect could result from a breach of intestinal barrier hypothesis, we highly enriched CD11b+ effector cells via mag- function after anti-CD71 treatment. Because CD71 is robustly netic bead–based positive selection from adult whole bone marrow + expressed on gut epithelium (21), anti-CD71 treatment prior to (Supplemental Fig. 1E). CD11b effector cells were cocultured + pathogenic challenge would not uniquely target splenic CD71 Downloaded from with neonatal splenocytes and stimulated with HKLM. We con- erythroid cells and could alter intestinal barrier function. There- firmed that increasing the ratio of neonatal splenocytes progres- fore, we hypothesized that enhanced bacterial clearance after anti- sively suppressed adult CD11b+ effector cell TNF-a production CD71 treatment was the result of immune priming by leaked with HKLM stimulation (p , 0.05, Student t test, compared with microbiota rather than the absence of CD71+ erythroid cells (14, CD11b+ effector cells alone, Fig. 2A). In addition to TNF-a,we 22). To differentiate between immune priming and the absence uncovered cell ratio–dependent suppression of G-CSF, CXCL5,

of immunosuppression, we examined the splenic bacterial load http://www.jimmunol.org/ CXCL1, CCL3, and CCL4 (Fig. 2B). To directly address our in two groups of anti-CD71–treated neonates following a nonlethal hypothesis regarding an immunosuppressive effect of other he- septic challenge: neonates completely replenished with CD71+ matopoietic tissues, we cocultured enriched HKLM-stimulated erythroid cells by adoptive transfer and neonates that did not adult CD11b+ effector cells with neonatal or adult bone marrow receive cells by adoptive transfer. We found no difference in and measured supernatant inflammatory mediators. We found splenic bacterial load among nonlethal sepsis–challenged similar cell ratio–based alterations in TNF-a production for CD71-depleted neonates that received CD71+ erythroid cells by neonatal and adult bone marrow (Fig. 2A). Interestingly, we also adoptive transfer versus those that received no cells (Fig. 3A), found cell ratio–dependent enhancement of CXCL10 and VEGF indicating that CD71+ cells are not immunosuppressive in this production associated with decreasing coculture ratios of all setting. To determine whether Ab-mediated disruption of in- by guest on September 24, 2021 testinal epithelium occurred, we first examined healthy neonatal ileum for the expression of CD71 and found that CD71 was prominent along the villi epithelium (Fig. 3B). Next, we ex- amined CD71 staining following anti-CD71 Ab exposure or isotype-control Ab at 3200 magnification (Fig. 3C, 3D) and 3600 magnification (Supplemental Fig. 1C). We found a re- duction in the MFI for CD71 in the intestine of anti-CD71– treated animals and the presence of skip lesions along the villi compared with animals treated with isotype-control Ab. Next, we examined peritoneal washes of healthy neonates that re- ceived anti-CD71 or isotype Ab treatment alone (with no in- fectious challenge) and found peritoneal colonization only among the anti-CD71–treated mice (Fig. 3E). To determine whether enhanced bacterial clearance occurred with bacterial challenge following anti-CD71 treatment, we examined micro- bial colonization of the spleen in nonlethal sepsis–challenged mice following LPS priming [used as a positive control (14)], CD71 depletion, or isotype Ab treatment (Fig. 3F). Consistent with immune priming, we found enhanced bacterial clearance with both LPS and anti-CD71 treatment compared with isotype- control treatment. Therefore, anti-CD71 treatment results in decreased bacterial burden in the spleen; this decrease is not reversed by the restoration of CD71+ cells by adoptive transfer, FIGURE 2. Dose-dependent supernatant cytokine inhibition and pro- indicating that these cells are not responsible for the observed motion ex vivo. (A) Supernatant TNF-a measurements from coculture of effect of the Ab treatment on bacterial burden. enriched and HKLM-stimulated adult CD11b+ effector cells with the in- dicated hematopoietic tissues. Data are mean + SD. (B) Fold change in Human CD71+ erythroid cells in cord blood are exquisitely enhanced and suppressed supernatant inflammatory mediators following sensitive to hypotonic lysis 1:1 coculture of the indicated tissues with enriched CD11b+ effector cells stimulated with HKLM. *p , 0.05, t test, versus CD11b+ effector cells Investigations in mice may provide key mechanistic insights, but alone. translation to clinical benefit requires overlap of murine findings The Journal of Immunology 5 Downloaded from

FIGURE 3. Anti-CD71 treatment is associated with enhanced bacterial clearance due to immune priming. (A) Splenic bacterial load 24 h after nonlethal sepsis challenge (0.6 mg of cecal slurry/g body weight given 24 h after the last Ab dose) in CD71-depleted neonates that received either 5 3 106 highly http://www.jimmunol.org/ enriched neonatal splenic CD71+ erythroid cells by adoptive transfer or no cells. (B) CD71 expression in healthy neonatal ileum (original magnification 3200). (C) Isotype Ab–treated neonatal ileum and anti-CD71–treated ileum 24 h after the last dose of Ab (original magnification 3200). (D) Reduction in CD71 MFI in ileum of anti-CD71–treated and isotype control–treated neonates 24 h after the last dose of Ab. (E) CFU recovered from peritoneal washes of healthy neonates 24 h after last dose of isotype Ab or anti-CD71 treatment alone. (F) Splenic bacterial load 24 h after nonlethal sepsis challenge (0.6 mg of cecal slurry/g body weight given 24 h after the last Ab dose) among neonates that received treatment with anti-CD71 Ab or isotype Ab or priming with a single dose of LPS (1 mg/g, 24 h prior to challenge). *p , 0.05, ANOVA. in humans. The mononuclear fraction from healthy human cord CD71+CD235a+ cells (85% reduction, Fig. 4B). These results are by guest on September 24, 2021 blood contains a large population of nucleated RBCs (nRBCs) and in stark contrast to the stability of murine neonatal CD71+ ery- reticulocytes (both express CD71 and CD235a) (8, 23, 24). Similar throid splenocytes to hypotonic lysis (19% reduction, data not to the immunosuppressive function by neonatal murine CD71+ shown) and suggest that these cells are predominantly reticulo- erythroid splenocytes, human cord blood CD71+ erythroid cells cytes (23, 24). FACS enrichment of the neonatal human CD71+ can inhibit TNF-a production ex vivo (8). To determine whether CD235a+ cells with cytospin examination after Wright’s stain or the human cells were nRBCs or reticulocytes, we first examined methylene blue confirmed that these cells were predominantly PBMCs isolated from the peripheral blood of healthy adult donors enucleated reticulocytes (Fig. 4A). (n = 5) and CBMCs from term newborns (n = 6). As expected, we confirmed the robust representation of CD71+CD235a+ cells Discussion among CBMCs (mean 50%, range 31–69%) and the minimal Developmental age significantly impacts the host immune response representation of CD71+CD235a+ cells among PBMCs (Fig. 4A). to sepsis (5). Specifically, the structure, cellular composition, and Hypotonic lysis resulted in a substantial reduction in neonatal function of the murine spleen are heavily influenced by chrono-

FIGURE 4. CD71+CD235a+ cells are prominent in human neonatal cord blood and are predominantly enucleated reticulocytes. (A) Representative FACS plots for post-Ficoll mono- nuclear fraction of healthy adult peripheral blood, healthy term neonate cord blood, healthy term neonate cord blood after hypotonic lysis, enrich- ment of neonatal cord blood CD71+235a+ cells, H&E stain of enriched CD71+CD235a+ cells (top), methylene blue stain of enriched CD71+ CD235a+ cells (bottom, inset showing appearance of nucleated cell). Top, original magnification 3400; bottom, original magnification 31000. (B) Effect of hypotonic lysis on CD71+CD235a+ cells from healthy adult peripheral blood and healthy neonatal cord blood after Ficoll-based isolation of mononuclear cells. *p , 0.05 by t test. 6 CD71+ CELLS DO NOT MODIFY NEONATAL INFLAMMATORY MORTALITY logical age (12–14). In contrast to the immune function of the supporting that enhanced bacterial clearance in neonatal mice spleen in the healthy adult, the spleen is normally a major site following anti-CD71 treatment was the result of immune priming of during fetal and neonatal life. Extramedullary after exposure to microbiota rather than the result of a reduction in erythropoiesis is required in neonates to support the extremely immunosuppressive cells: repletion of CD71+ erythroid cells to rapid growth rate in the setting of significantly reduced erythroid CD71+ diminished animals did not alter bacterial clearance; anti- reservoirs compared with the adult (10). Accordingly, the murine CD71 treatment was associated with reduced intestinal barrier neonatal spleen, bone marrow, and liver are known to harbor large function, as evidenced by recovery of live bacteria in the perito- numbers of erythroid progenitors for several weeks after birth neum of unchallenged healthy animals; and anti-CD71–treated (25). Fraser et al. (26) examined the fetal liver and showed a mice demonstrated enhanced bacteria clearance similar to LPS- representation of CD71+ erythroid cells similar to that observed in primed animals following nonlethal sepsis challenge. Although the neonatal spleen and bone marrow. the potential confounders of murine modeling can be debated, the It was reported that CD71+ erythroid cells are enriched in the identification of enucleated reticulocytes as the cell type enriched murine spleen to quench inflammation secondary to microbial in human neonatal cord blood, in conjunction with the absence or colonization (8). This conclusion was supported, in part, by rapid decline in reticulocytes after birth during a time of robust microbiota-dependent immune cell activation that occurred ex- microbial colonization, does not readily support the hypothesis clusively in the intestine following Ab-mediated CD71+ erythroid that CD71+ cells are enriched to quench intestinal inflammation cell diminution. We showed that anti-CD71 treatment was asso- associated with microbial colonization. ciated with augmented pathogen clearance that was unaffected Developmental age strongly impacts the cellular composition of by replenishment of CD71+ erythroid cells by adoptive transfer, the murine spleen. Neither provision nor reduction of enriched Downloaded from which demonstrates that the beneficial effect was not due to CD71+ erythroid cells had an effect on neonatal mortality with a decrease in immunosuppression. Anti-CD71 treatment led to polymicrobial sepsis. Ex vivo immunosuppression of stimulated recoverable microbiota in the peritoneum, which would be ex- adult CD11b+ cells by neonatal splenocytes also occurred with pected to activate local intestinal immune cells. In turn, intestinal adult and neonatal bone marrow. Enhanced bacterial clearance after immune cell activation would be dependent on host microbiota. anti-CD71 treatment was the result of immune priming following

Our group (14) and other investigators (27, 28) demonstrated a minor disruption in gut integrity, rather than a reduction in http://www.jimmunol.org/ similar enhancements in pathogen clearance and survival associ- immunosuppressive cells. The paucity or absence of reticulocytes ated with augmented innate immune function following minor in peripheral blood at birth does not readily support an anti- antecedent exposure to microbial products. inflammatory role in humans. Interestingly, murine intestinal epithelial inflammation associ- ated with microbial colonization is rapidly attenuated (,6 h after Disclosures exposure) via downregulation of IL-1R–associated kinase-1 (29). The authors have no financial conflicts of interest. Rapid gut colonization and early desensitization to microbiota- induced inflammation do not readily support the hypothesis that CD71+ erythroid cells persist in the murine spleen for only 2–3 wk References by guest on September 24, 2021 after birth to quench inflammation secondary to colonization. In 1. Liu, L., H. L. Johnson, S. Cousens, J. Perin, S. Scott, J. E. Lawn, I. Rudan, H. Campbell, R. Cibulskis, M. Li, et al; Child Health Epidemiology Reference fact, human neonates are colonized with microbiota during de- Group of WHO and UNICEF. 2012. Global, regional, and national causes of livery and undergo a continuous and dynamic colonization process child mortality: an updated systematic analysis for 2010 with time trends since 2000. Lancet 379: 2151–2161. for the first several years of life (30–32). 2. Stoll, B. J., N. I. Hansen, E. F. Bell, S. Shankaran, A. R. Laptook, M. C. Walsh, + + Of note, nRBCs are CD71 CD235a and may be absent or E. C. Hale, N. S. Newman, K. Schibler, W. A. Carlo, et al; Eunice Kennedy represent a very small portion of cord or peripheral blood from Shriver National Institute of Child Health and Human Development Neonatal Research Network. 2010. Neonatal outcomes of extremely preterm infants from healthy human neonates, and they are significantly increased the NICHD Neonatal Research Network. Pediatrics 126: 443–456. by intrauterine stress (hypoxia, infection [IL-6], or intrauterine 3. Brocklehurst, P., B. Farrell, A. King, E. Juszczak, B. Darlow, K. Haque, A. Salt, growth restriction) (33–38). Day-of-birth peripheral blood nRBC B. Stenson, and W. Tarnow-Mordi, INIS Collaborative Group. 2011. Treatment of neonatal sepsis with intravenous immune globulin. N. Engl. J. Med. 365: reference ranges from 14,319 term infants revealed that nRBCs 1201–1211. may be small or even absent (mean , 7% of WBCs, absolute , 4. Wynn, J. L., J. Neu, L. L. Moldawer, and O. Levy. 2009. Potential of immu- 1100/ml) and progressively decrease over the first week of life nomodulatory agents for prevention and treatment of neonatal sepsis. J. Peri- natol. 29: 79–88. (37). Similar decreases in the number of circulating reticulocytes 5. Wynn, J., T. T. Cornell, H. R. Wong, T. P. Shanley, and D. S. Wheeler. 2010. The (also CD71+CD235a+) were documented (39). The limited num- host response to sepsis and developmental impact. Pediatrics 125: 1031–1041. + + 6. Wynn, J. L., N. Z. Cvijanovich, G. L. Allen, N. J. Thomas, R. J. Freishtat, ber or absence of CD71 235a cells (nRBCs and reticulocytes) N. Anas, K. Meyer, P. A. Checchia, R. Lin, T. P. Shanley, et al. 2011. The in- from peripheral blood samples on the first day of life, coupled fluence of developmental age on the early transcriptomic response of children with a decreasing trend for these cells during the first days of life with septic shock. Mol. Med. 17: 1146–1156. 7. Wynn, J. L., P. O. Scumpia, M. J. Delano, K. A. O’Malley, R. Ungaro, (which is a period of robust microbial colonization), does not A. Abouhamze, and L. L. Moldawer. 2007. Increased mortality and altered im- preclude the potential action of quenching inflammation due to munity in neonatal sepsis produced by generalized peritonitis. Shock 28: 675–683. microbial colonization, but it strongly suggests that there are 8. Elahi, S., J. M. Ertelt, J. M. Kinder, T. T. Jiang, X. Zhang, L. Xin, V. Chaturvedi, B. S. Strong, J. E. Qualls, K. A. Steinbrecher, et al. 2013. Immunosuppressive other, more likely, mechanisms that account for the presence of CD71+ erythroid cells compromise neonatal host defence against infection. these cells. Nature 504: 158–162. We acknowledge the potential limitations surrounding a com- 9. Ned, R. M., W. Swat, and N. C. Andrews. 2003. Transferrin receptor 1 is dif- ferentially required in lymphocyte development. Blood 102: 3711–3718. parison of the host response between preclinical models using 10. Socolovsky, M., H. Nam, M. D. Fleming, V. H. Haase, C. Brugnara, and different inflammatory and pathogenic challenges. However, a lack H. F. Lodish. 2001. Ineffective erythropoiesis in Stat5a(2/2)5b(2/2) mice due to decreased survival of early erythroblasts. Blood 98: 3261–3273. of impact on murine neonatal survival to noninfectious inflam- 11. Li, H., A. C. Rybicki, S. M. Suzuka, L. von Bonsdorff, W. Breuer, C. B. Hall, matory challenge (LPS) or septic challenge following either de- Z. I. Cabantchik, E. E. Bouhassira, M. E. Fabry, and Y. Z. Ginzburg. 2010. Trans- pletion or adoptive transfer of CD71+ erythroid cells suggests that ferrin therapy ameliorates disease in beta-thalassemic mice. Nat. Med. 16: 177–182. 12. Timens, W., A. Boes, T. Rozeboom-Uiterwijk, and S. Poppema. 1989. Imma- the impact of these cells may be more limited than previously turity of the human splenic marginal zone in infancy. Possible contribution to the described. Furthermore, we presented several lines of evidence deficient infant immune response. J. Immunol. 143: 3200–3206. The Journal of Immunology 7

13. Timens, W., T. Rozeboom, and S. Poppema. 1987. Fetal and neonatal development 26. Fraser, S. T., J. Isern, and M. H. Baron. 2007. Maturation and enucleation of of human spleen: an immunohistological study. Immunology 60: 603–609. primitive erythroblasts during mouse embryogenesis is accompanied by changes 14. Wynn, J. L., P. O. Scumpia, R. D. Winfield, M. J. Delano, K. Kelly-Scumpia, in cell-surface antigen expression. Blood 109: 343–352. T. Barker, R. Ungaro, O. Levy, and L. L. Moldawer. 2008. Defective innate 27. Ito, S., K. J. Ishii, M. Gursel, H. Shirotra, A. Ihata, and D. M. Klinman. 2005. immunity predisposes murine neonates to poor sepsis outcome but is reversed by CpG oligodeoxynucleotides enhance neonatal resistance to Listeria infection. J. TLR agonists. Blood 112: 1750–1758. Immunol. 174: 777–782. 15. Cuenca, A. G., J. L. Wynn, K. M. Kelly-Scumpia, P. O. Scumpia, L. Vila, 28. Pedras-Vasconcelos, J. A., D. Goucher, M. Puig, L. H. Tonelli, V. Wang, S. Ito, M. J. Delano, C. E. Mathews, S. M. Wallet, W. H. Reeves, K. E. Behrns, et al. and D. Verthelyi. 2006. CpG oligodeoxynucleotides protect newborn mice from 2011. Critical role for CXC ligand 10/CXC receptor 3 signaling in the murine a lethal challenge with the neurotropic Tacaribe arenavirus. J. Immunol. 176: neonatal response to sepsis. Infect. Immun. 79: 2746–2754. 4940–4949. 16. Weitkamp, J. H., and J. E. Crowe, Jr. 2001. Blood donor leukocyte reduction 29. Lotz, M., D. Gutle,€ S. Walther, S. Me´nard, C. Bogdan, and M. W. Hornef. 2006. filters as a source of human B lymphocytes. Biotechniques 31: 464, 466. Postnatal acquisition of endotoxin tolerance in intestinal epithelial cells. J. Exp. 17.Heuer,J.G.,T.Zhang,J.Zhao,C.Ding,M.Cramer,K.L.Justen, Med. 203: 973–984. S. L. Vonderfecht, and S. Na. 2005. Adoptive transfer of in vitro-stimulated 30. Dominguez-Bello, M. G., E. K. Costello, M. Contreras, M. Magris, G. Hidalgo, CD4+CD25+ regulatory T cells increases bacterial clearance and improves N. Fierer, and R. Knight. 2010. Delivery mode shapes the acquisition and survival in polymicrobial sepsis. J. Immunol. 174: 7141–7146. structure of the initial microbiota across multiple body habitats in newborns. 18. Kuhlhorn,€ F., M. Rath, K. Schmoeckel, K. Cziupka, H. H. Nguyen, Proc. Natl. Acad. Sci. USA 107: 11971–11975. P. Hildebrandt, T. Hunig,€ T. Sparwasser, J. Huehn, C. Po¨tschke, and 31. Koenig, J. E., A. Spor, N. Scalfone, A. D. Fricker, J. Stombaugh, R. Knight, B. M. Bro¨ker. 2013. Foxp3+ regulatory T cells are required for recovery from L. T. Angenent, and R. E. Ley. 2011. Succession of microbial consortia in the severe sepsis. PLoS ONE 8: e65109. developing infant gut microbiome. Proc. Natl. Acad. Sci. USA 108(Suppl. 1): 19. Nemzek, J. A., C. Fry, and B. B. Moore. 2013. Adoptive transfer of fibrocytes en- 4578–4585. hances splenic T-cell numbers and survival in septic peritonitis. Shock 40: 106–114. 32. Romano-Keeler, J., D. J. Moore, C. Wang, R. M. Brucker, C. Fonnesbeck, 20. Levy, J. E., O. Jin, Y. Fujiwara, F. Kuo, and N. C. Andrews. 1999. Transferrin J. C. Slaughter, H. Li, D. P. Curran, S. Meng, H. Correa, et al. 2014. Early life receptor is necessary for development of erythrocytes and the nervous system. establishment of site-specific microbial communities in the gut. Gut Microbes 5: Nat. Genet. 21: 396–399. 192–201. 21. Matysiak-Budnik, T., I. C. Moura, M. Arcos-Fajardo, C. Lebreton, S. Me´nard, 33. Hermansen, M. C. 2001. Nucleated red blood cells in the fetus and newborn. Downloaded from C. Candalh, K. Ben-Khalifa, C. Dugave, H. Tamouza, G. van Niel, et al. 2008. Arch. Dis. Child. Fetal Neonatal Ed. 84: F211–F215. Secretory IgA mediates retrotranscytosis of intact gliadin peptides via the 34. Ferber, A., V. K. Minior, E. Bornstein, and M. Y. Divon. 2005. Fetal “non- transferrin receptor in celiac disease. J. Exp. Med. 205: 143–154. reassuring status” is associated with elevation of nucleated counts 22. Lantier, L., F. Drouet, W. Guesdon, R. Mancassola, C. Metton, R. Lo-Man, and interleukin-6. Am. J. Obstet. Gynecol. 192: 1427–1429. C. Werts, F. Laurent, and S. Lacroix-Lamande´. 2014. Poly(I:C)-induced pro- 35. Ulich, T. R., J. del Castillo, S. M. Yin, and J. C. Egrie. 1991. The erythropoietic tection of neonatal mice against intestinal Cryptosporidium parvum infection effects of interleukin 6 and erythropoietin in vivo. Exp. Hematol. 19: 29–34. requires an additional TLR5 signal provided by the gut flora. J. Infect. Dis. 209: 36. Dulay, A. T., I. A. Buhimschi, G. Zhao, G. Luo, S. Abdel-Razeq, M. Cackovic,

457–467. V. A. Rosenberg, C. M. Pettker, S. F. Thung, M. O. Bahtiyar, et al. 2008. Nu- http://www.jimmunol.org/ 23. Wangen, J. R., L. Eidenschink Brodersen, T. T. Stolk, D. A. Wells, and cleated red blood cells are a direct response to mediators of inflammation in M. R. Loken. 2014. Assessment of normal erythropoiesis by flow cytometry: im- newborns with early-onset neonatal sepsis. Am. J. Obstet. Gynecol. 198: 426.e1–9. portant considerations for specimen preparation. Int. J. Lab. Hematol. 36: 184–196. 37. Christensen, R. D., E. Henry, R. L. Andres, and S. T. Bennett. 2011. Reference 24. Noulin, F., C. Borlon, P. van den Eede, L. Boel, C. M. Verfaillie, ranges for blood concentrations of nucleated red blood cells in neonates. Neo- U. D’Alessandro, and A. Erhart. 2012. Cryopreserved reticulocytes derived from natology 99: 289–294. hematopoietic stem cells can be invaded by cryopreserved Plasmodium vivax 38. Christensen, R. D., D. K. Lambert, and D. S. Richards. 2014. Estimating the isolates. PLoS ONE 7: e40798. ‘emergence time’ in neonates. J. Perinatol. 34: 116– 25. Wolber, F. M., E. Leonard, S. Michael, C. M. Orschell-Traycoff, M. C. Yoder, 119. and E. F. Srour. 2002. Roles of spleen and liver in development of the murine 39. Matoth, Y., R. Zaizov, and I. Varsano. 1971. Postnatal changes in some red cell hematopoietic system. Exp. Hematol. 30: 1010–1019. parameters. Acta Paediatr. Scand. 60: 317–323. by guest on September 24, 2021