Immunosuppressive CD71+ erythroid cells compromise neonatal host defence against infection

Abstract

Newborn infants are highly susceptible to infection. This defect in host defence has generally been ascribed to the immaturity of neonatal immune cells; however, the degree of hyporesponsiveness is highly variable and depends on the stimulation conditions. These discordant responses illustrate the need for a more unified explanation for why immunity is compromised in neonates. Here we show that physiologically enriched CD71(+) erythroid cells in neonatal mice and human cord blood have distinctive immunosuppressive properties. The production of innate immune protective cytokines by adult cells is diminished after transfer to neonatal mice or after co-culture with neonatal splenocytes. Neonatal CD71(+) cells express the enzyme arginase-2, and arginase activity is essential for the immunosuppressive properties of these cells because molecular inhibition of this enzyme or supplementation with L-arginine overrides immunosuppression. In addition, the ablation of CD71(+) cells in neonatal mice, or the decline in number of these cells as postnatal development progresses parallels the loss of suppression, and restored resistance to the perinatal pathogens Listeria monocytogenes and Escherichia coli. However, CD71(+) cell-mediated susceptibility to infection is counterbalanced by CD71(+) cell-mediated protection against aberrant immune cell activation in the intestine, where colonization with commensal microorganisms occurs swiftly after parturition. Conversely, circumventing such colonization by using antimicrobials or gnotobiotic germ-free mice overrides these protective benefits. Thus, CD71(+) cells quench the excessive inflammation induced by abrupt colonization with commensal microorganisms after parturition. This finding challenges the idea that the susceptibility of neonates to infection reflects immune-cell-intrinsic defects and instead highlights processes that are developmentally more essential and inadvertently mitigate innate immune protection. We anticipate that these results will spark renewed investigation into the need for immunosuppression in neonates, as well as improved strategies for augmenting host defence in this vulnerable population.

Figures

Figure 1. Infection susceptibility of neonatal mice and immunosuppressive properties of neonatal cells

a, Survival of 6-day-old (neonatal) and 8-week-old (adult) mice after L. monocytogenes (Lm) infection (200 colony-forming units (CFU)). b,Number of recoverable bacteria 48 h after infection with various doses of L. monocytogenes. c, Flow cytometric analysis showing the retained donor CD45.1+ cells from adult mice in the splenocyte population of neonatal mice. Numbers indicate the percentage of cells in the adjacent boxed areas. d, Number of recoverable bacteria after L. monocytogenes infection (200 CFU) of neonatal mice containing donor adult splenocytes. e, Representative flow cytometry plots (left) and cumulative composite data (right) for the percentage of endogenous CD11b+ adult and neonatal cells, and CD11b+ donor adult cells within neonates, that produce TNF-α ex vivo 48 hours after L. monocytogenes infection. f, Cytokine production by neonatal or adult mouse splenocytes after stimulation (stim) with heat-killed (HK) L. monocytogenes individually or in co-culture for 72 h as measured by enzyme-linked immunosorbent assay (ELISA). Each point represents data from an individual mouse, and the data are representative of three independent experiments. Error bars, mean±s.e.m. IFN-γ, interferon-γ.

Figure 2. Arginase inhibition overrides immunosuppression by neonatal splenocytes containing enriched CD71+ erythroid cells

a, Representative flow cytometry plots (top) and cumulative composite data (bottom) showing TNF-α production by adult CD11b+ cells co-cultured with neonatal splenocytes in the indicated ratios and stimulated (stim) with heat-killed (HK) L. monocytogenes (Lm) (bottom left), as well as CD69 expression among adult CD8+ cells co-cultured with neonatal splenocytes in the indicated ratios and stimulated with anti-CD3 antibody (bottom right). b, Normalized (percentage maximum, % max) TNF-α production by CD11b+ adult cells cultured alone (black) or co-cultured with neonatal splenocytes at a 1:1 ratio (blue), or additional supplementation (red) with the arginase inhibitors BEC or ABH, or l-arginine. c, Flow cytometric analysis showing the proportion of immune lineage (CD4+CD8+CD11b+CD11c+B220+NK1.1+) cells or CD71+TER119+ erythroid lineage cells in neonatal mouse splenocyte populations compared with adult mouse splenocyte populations, or the proportion of CD71+CD235A+ cells in human cord blood cell populations compared with adult peripheral blood mononuclear cell (PBMC) populations. d, TNF-α production by adult CD11b+ cells co-cultured with immune-lineage-cell-depleted or CD71+ cell-depleted neonatal splenocytes (left), and representative flow cytometry plots illustrating the efficiency of cell depletion (right). e, TNF-α production by unfractionated or CD71+ cell-depleted neonatal CD11b+ cells. f, TNF-α production by unfractionated or CD71+ cell-depleted CD11b+ human cord blood cells. The fold increase in TNF-α production by the CD71+ cell-depleted cell populations compared with the unfractionated controls is shown within the graph. These results are representative of three independent experiments for mice and ten individual cord blood (CB) samples. Error bars, mean±s.e.m.

Figure 3. Enriched CD71+ cells compromise neonatal host defence against infection

a, The proportion of CD71+TER119+ splenocytes and the number of recoverable bacteria (CFU) 48 h after L. monocytogenes (Lm) infection of neonatal mice treated with anti-CD71 antibody or isotype control (rat IgG) antibody. b, The number of recoverable bacteria after L. monocytogenes infection of mice during postnatal development and adult mice. c, The proportion of CD71+TER119+ splenocytes in mice during postnatal development and adult mice. d, Normalized TNF-α production (percentage maximum, % max) by adult CD11b+ responder cells co-cultured with a 1:1 ratio of splenocytes from mice in each age group. e, Representative flow cytometry plots showing the efficiency of immune lineage cell depletion (left), and cumulative composite data for TNF-α production by adult CD11b+ cells co-cultured with purified CD71+ cells from neonatal or phlebotomized adult mice (right). f, Arginase-1 and arginase-2 expression in CD71+ cells from phlebotomized adult (black) or neonatal (red) splenocytes. Each point represents data from an individual mouse, and the data are representative of three independent experiments. Error bars, mean±s.e.m. HK, heat-killed; stim, stimulation.

Figure 4. Neonatal CD71+ cells prevent aberrant immune cell activation in tissue that is rapidly colonized with commensal microbes

a, TNF-α production by intestinal immune cells from 8-day-old mice treated with anti-CD71 antibody (or isotype control antibody (rat IgG)) on days 5 and 6. b, TNF-α production by cells recovered from the indicated tissues after anti-CD71 antibody treatment of neonatal mice (normalized to the values from isotype-control-antibody-treated neonatal mice). c, The number of recoverable bacteria (CFU) from intestine (brown), spleen (red) and lung (blue) of fetal (embryonic day (E) 18.5) and neonatal mice in the indicated age (days post parturition (PP)) after housing with unsupplemented drinking water or drinking water containing antibiotics (ampicillin, gentamicin, metronidazole, neomycin and vancomycin) from E14.5 (n=6–18 mice per time point). d, TNF-α production by cells recovered from the indicated tissues of neonatal mice sustained on antimicrobial therapy and treated with anti-CD71 antibody (normalized to the values from isotype-control-antibody-treated neonatal mice sustained on antimicrobial therapy). e, The proportion of splenocytes that are CD71+TER119+ in 8-day-old conventional and germ-free mice. f, TNF-α production by cells recovered from the indicated tissues of germ-free neonatal mice treated with anti-CD71 antibody (normalized to the values from isotype-control-antibody-treated germ-free neonatal mice). Each point represents data from an individual mouse, and the data are representative of three independent experiments. Error bars, mean±s.e.m.

Extended Data Figure 1. Adoptively transferred splenocyte cells from adult mice are retained but do not become activated in L. monocytogenes (Lm)-infected neonatal mice

a, The proportion of adult CD45.1+ donor cells compared with endogenous neonatal CD45.2+ cells among unfractionated splenocytes or various cell subsets 48 h after transfer to 5-day-old neonatal mice. b, The proportion of TNF-α-producing CD11c+ or B220+ cells among endogenous cells in adult or neonatal mice, or among transferred adult cells within neonates. c, Restored TNF-α production among neonatal cells after transfer to adult mice. The proportion of TNF-α-producing CD11b+ cells among transferred neonatal cells compared with endogenous adult cells. Forty-eight hours after infection, splenocytes were collected from infected or control mice, cultured in medium containing brefeldin A for 5 h and then subjected to cell surface and intracellular cytokine staining. Each point represents data from an individual mouse, and the data are representative of three independent experiments. Error bars, mean±s.e.m.

Extended Data Figure 2. Diminished TNF-α and IL-6 production among human cord blood cells compared with adult peripheral blood mononuclear cells

Representative plots and cumulative data showing TNF-α and IL-6 production by CD11b+ cells among adult peripheral blood mononuclear cells (PBMCs) or cord blood (CB) cells before and after stimulation with heat-killed (HK) L. monocytogenes (Lm). These results are representative of ten individual PBMCs or cord blood samples. Error bars, mean±s.e.m.

Extended Data Figure 3. Neonatal splenocytes suppress the activation of adult cells in co-culture

a, TNF-α production by adult CD11c+ and B220+ responder cells after stimulation with heat-killed (HK) L. monocytogenes (Lm), and co-culture with the indicated ratio of splenocytes from 6-day-old neonatal mice. b, Representative plots and composite data illustrating CD69 and CD25 expression by adult CD8+ responder cells after stimulation with anti-CD3 antibody, and co-culture with the indicated ratios of neonatal splenocytes. Each point represents data from an individual mouse, and the data are representative of three independent experiments. Error bars, mean±s.e.m.

Extended Data Figure 4. Arginase inhibition overrides the immunosuppressive properties of neonatal cells

TNF-α production by adult CD11b+ responder cells after stimulation with heat-killed (HK) L. monocytogenes (Lm) alone (black), or co-culture with neonatal splenocytes at a 1:1 ratio (blue), or additional supplementation (red) with arginase inhibitors (nor-NOHA or l-NOHA) (top), or 4-aminobenzoic acid hydrazide (a myeloperoxidase inhibitor), 4′-hydroxy-3′-methoxyacetophenone (an NADPH oxidase inhibitor), sodium diethyldithiocarbamate trihydrate (a superoxide dismutase inhibitor), SB-431542 hydrate (a TGF-β receptor inhibitor), anti-TGF-β antibody (clone 1D11), Sn(iv) protoporphyrin IX dichloride (a haem oxidation inhibitor), acetylcysteine (a reactive oxygen species (ROS) scavenger), 1-methyl-d-tryptophan and 1-methyl-l-tryptophan (IDO inhibitors), and an anti-IL-10 receptor antibody (clone 1B1.3A) (bottom). Each point represents data from an individual mouse, and the data are representative of two independent experiments. Error bars, mean±s.e.m.

Extended Data Figure 5. Human cord blood compared with adult peripheral blood mononuclear cells is enriched with CD71+CD235A+ erythroid cells, and the depletion of these erythroid cells unleashes the activation of the remaining neonatal immune cells

a, The proportion of CD71+CD235A+ cells among human adult PBMCs and cord blood. b, The proportion of CD71+CD235A+ cells among unfractionated and CD71+ cell-depleted cord blood (top), and the proportion of IL-6-producing cells among CD11b+ cells or the proportion of CD69+ cells among CD8+ cells in ten individual unfractionated and CD71+ cell-depleted cord blood specimens. The fold increase in CD71+ cell-depleted populations compared with unfractionated controls is shown above each pair of bars. Error bars, mean±s.e.m.

Extended Data Figure 6. CD71+ cell depletion invigorates neonatal immune cell activation

TNF-α-producing CD11c+ or B220+ cells after stimulation with heat-killed (HK) L. monocytogenes (Lm), or CD69 expression by CD8+ T cells after stimulation with anti-CD3 antibody among unfractionated or CD71+ cell-depleted splenocytes from 6-day-old neonatal mice. Each point represents data from an individual mouse, and the data are representative of three independent experiments. Error bars, mean±s.e.m.

Extended Data Figure 7. Infection-induced cell activation is enhanced in the neonatal lymph node, where immunosuppressive CD71+ erythroid cell numbers are diminished

a, The proportion of CD71+TER119+ cells among splenocytes and inguinal lymph node cells in 6-day-old neonatal mice. b, Representative plots and composite data comparing TNF-α production by CD11b+ cells and CD69 expression by CD8+ cells 48 h after L. monocytogenes (Lm) infection (red line histogram) or in no infection controls (black line histogram) in neonatal mice. Each point represents data from an individual mouse, and the data are representative of three independent experiments. Error bars, mean±s.e.m.

Extended Data Figure 8. CD71+ cell depletion restores neonatal resistance to E. coli infection

a, Recoverable bacteria 48 h after infection with the indicated E. coli doses in 6-day-old (neonatal) or 8-week-old (adult) mice. b, Recoverable bacteria 48 h after E. coli infection (200 CFU) of anti-CD71-antibody-treated neonatal mice compared with isotype-control-antibodytreated neonatal mice. Anti-CD71 antibody or isotype control antibody (150 µg) was administered on days 5 and 6 after birth, corresponding to 1 day before infection and the day of infection. Each point represents data from an individual mouse, and the data are representative of three independent experiments. Error bars, mean±s.e.m.

Extended Data Figure 9. Phlebotomy-induced adultCD71+ erythroid cells have a similar subset distribution to neonatal cells but do not cause susceptibility to infection

a, The proportion of CD71+TER119+ splenocytes in adult control or phlebotomized (bled) mice 5 days after the initiation of daily phlebotomy for 3 consecutive days. b, The distribution of TER119+ erythroid cells based on CD71 expression and size (FSC, forward scatter) among splenocytes from 6-day-old (neonatal) or phlebotomized 8-week-old (adult) mice. c, The number of recoverable bacteria after L. monocytogenes (Lm) infection (1,000 CFU). Each point represents data from an individual mouse, and the data are representative of three independent experiments. Error bars, mean±s.e.m.

Extended Data Figure 10. CD71+ erythroid cells selectively restrict immune cell activation in the intestine

a, Relative CD40, CD80 and CD86 expression by CD11b+ or CD11c+ cells recovered from the indicated tissues of 8-day-old (neonatal) mice treated with 150 µg anti-CD71 antibody (red histograms gated on CD11b+ cells) or 150 µg isotype control antibody (blue histograms gated on CD11b+ cells) on days 5 and 6. The mean fluorescence intensity for each parameter in the cells from CD71+ cell-depleted mice was normalized to the levels in isotype-control-antibody-treated mice. b, CD40, CD80 and CD86 expression for the neonatal mice described in a after receiving antibiotic supplementation. c, CD40, CD80 and CD86 expression for the neonatal mice described in a after maintenance under gnotobiotic germ-free conditions. Each point represents data from an individual mouse, and the data are representative of three independent experiments. Error bars, mean±s.e.m.