Cellular metabolism constrains innate immune responses in early human ontogeny

Abstract

Pathogen immune responses are profoundly attenuated in fetuses and premature infants, yet the mechanisms underlying this developmental immaturity remain unclear. Here we show transcriptomic, metabolic and polysome profiling and find that monocytes isolated from infants born early in gestation display perturbations in PPAR-γ-regulated metabolic pathways, limited glycolytic capacity and reduced ribosomal activity. These metabolic changes are linked to a lack of translation of most cytokines and of MALT1 signalosome genes essential to respond to the neonatal pathogen Candida. In contrast, they have little impact on house-keeping phagocytosis functions. Transcriptome analyses further indicate a role for mTOR and its putative negative regulator DNA Damage Inducible Transcript 4-Like in regulating these metabolic constraints. Our results provide a molecular basis for the broad susceptibility to multiple pathogens in these infants, and suggest that the fetal immune system is metabolically programmed to avoid energetically costly, dispensable and potentially harmful immune responses during ontogeny.

Fig. 1

Responses to Candida spp. in neonatal immune cells. a Phagocytosis of Candida in monocytes (boxes and whiskers), including a representative flow microscopy diagram (white bar = ∼10 µm). Data pooled from multiple experiments over 14 months (9 to 17 subjects per age group; see Supplemental Data for clinical information on preterm subjects); b IL-1β and c IL-6 response (blood mononuclear cells) to C. albicans or C. parapsilosis (24 h stimulation; 10 to 18 subjects per age group; boxes and whiskers); (d) IL-1β (24 h; 11 to 21 subjects per age group; boxes and whiskers) and e representative gating for pro-IL-1β (5 h LPS stimulation), gated on CD14-expressing cells (black = fluorescent-minus one control; orange = unstimulated; blue = LPS; representative preterm sample is from a 26 weeks’ infant); f pro-IL-1β (5 h) or g IL-6 (24 h) in response to LPS, zymosan or curdlan (mononuclear cells; 11 to 21 subjects per age group; boxes and whiskers); for b and c, data was pooled from multiple experiments assayed in four ELISA batches with similar distribution of samples per age group

Fig. 2

Importance of dectin-1 in response to Candida in neonatal monocytes. a Antibody blocking of dectin-1 reduces cytokine production to C. albicans (C_a) in adult mononuclear cells (one-sided t-test with Welch’s correction for unequal variance; 6 to 12 subjects per condition; boxes and whiskers); b Phagocytosis of C. albicans upon blocking with anti-dectin-1 receptor antibody, same-isotype control or cytochalasin-D (Cyto-D); (11 to 18 subjects per age group; boxes and whiskers); Blocking of c C. albicans (C_a) or d C. parapsilosis (C_p) phagocytosis using laminarin (blocking dectin-1), mannan (blocking dectin-2 and mannose receptor), anti-DC-SIGN, and anti-CD206 antibodies, or a combination of all three antibodies (bar graph with mean ± standard deviation (SD); 2 to 3 subjects per age group)

Fig. 3

Transcriptome analysis in neonatal monocytes. a Gene ontology analysis of differentially expressed genes (FDR < 0.01) between unstimulated preterm, and combined term and adult samples (monocytes; n = 8 to 12 subjects/age group); Expression heatmap and unsupervised clustering for genes involved in b glycolysis, c oxidative phosphorylation and fatty acid beta-oxidation and d ribosomal proteins. Scale represents z-score

Fig. 4

Transcriptome and cytokine responses to LPS in neonatal monocytes. a Principal component (PC) analysis of unstimulated versus LPS-stimulated monocytes and b Venn diagram of differentially expressed genes (LPS-stimulated samples) overlapping between age groups (FDR 5%). c Expression heatmap of cytokine genes (5 h LPS; scale = z-score) Data from same subjects as in Fig. 3, except for 1 adult and 2 preterm subjects with insufficient cells for LPS condition; d Production of cytokines following LPS stimulation (24 h, mononuclear cells) by ELISA (effect of age by two-way ANOVA; mean ± SD; n = 3 to 6 subjects per age group)

Fig. 5

Gene expression and translation of dectin-1 signaling proteins. a Illustration of selected signaling molecules downstream of dectin-1; b Polysome profiles and c quantification of signalosome genes (qPCR) in monosome, disome, and light and heavy polysome fractions (monocytes). Data are from 4 subjects per age group (boxes and whiskers; RQ = relative quantification); d Quantification of signalosome genes (qPCR) in total RNA fractions (4 to 5 subjects/age group; mean ± SD); e Surface expression of dectin-1 (flow cytometry, mononuclear cells, gated on CD14-expressing cells; data pooled from 10 to 23 subjects per age group; boxes and whiskers); f Representative (cropped) Western blot of MALT1 and Bcl10 protein expression in monocytes after 0 to 60 min LPS stimulation. Representative blot is from a 29 weeks gestation sample. Images cropped from same blot probes with each antibody; cumulative quantification of 4 independent Western blot experiments for g MALT1 and h Bcl10 (mean ± SD)

Fig. 6

MALT1 is essential for Candida recognition and curdlan signaling in human monocytes. IL-1β production after stimulation (24 h) with a curdlan or b LPS, or with c C. albicans or d C. parapsilosis, and upon inhibition (i) of MALT1 (using mepazine hydrochloride), MyD88 (ST2825), Raf (GW5074) or Syk (Piceatannol), or blocking of dectin-1 or dectin-2 using antibodies (data pooled from 6 experiments (6 subjects); mean ± SD); e Effect of MALT1 inhibition, dectin-1-blocking antibody or actin polymerization inhibition (using cytochalasin-d) on phagocytosis of Candida by monocytes (boxes and whiskers; data also from 6 experiments). Statistical significance was calculated using 2-sided paired t-tests

Fig. 7

Impaired glycolysis and mTOR activity in preterm neonatal monocytes. a Effect of blocking glycolysis on cytokine responses to LPS stimulation (24 h) in mononuclear cells (2-sided paired t-tests; boxes and whiskers; 4 to 9 samples per condition; 2-DG = 2-deoxy-d-glucose); b Extracellular acidification rates (ECAR, normalized to total protein content), under baseline glucose-free conditions, after addition of glucose, oligomycin, and 2-DG (representative experiment from a 29 week gestation preterm sample); c Glycolytic capacity (cumulative data pooled from 4 independent experiments; 3–9 subjects per age group; mean ± SD; p value by Mann–Whitney U test); d Lactate production in mononuclear cells after LPS stimulation (24 h); p value = effect of age by 2-way ANOVA. Data from 9 to 10 subjects per age group; boxes and whiskers; e Effect of blocking glycolysis or translation (using cycloheximide, CHX) on the phagocytosis of C. albicans (C_a); boxes and whiskers; 3 subjects; Cyto-D = cytochalasin-D; f Depiction of signaling events between Toll-like receptor and Raf-1-mediated dectin-1 activation, mTOR phosphorylation, and increased glycolysis and protein synthesis; g Western blot image (cropped) of mTOR/4EBP1 expression and mTOR phosphorylation (monocytes; preterm sample from 29 weeks gestation; cropped images from same blot probed for mTOR, β-actin, and 4EBP1, and stripped/re-probed for phospho-mTOR); h Expression of mTOR-related genes (monocytes; 6–12 per age group; boxes and whiskers; 2-way ANOVA across age groups, only significant p values are shown)

Fig. 8

Inhibition glycolysis results in loss of MALT1 protein expression. Effect of blocking glycolysis (using 2-DG) or of blocking translation (using cycloheximide, as control) on MALT1 protein expression (monocytes). a MALT1 protein was detected by Western blot (left panel; representative of two experiments; cropped images from same blot probed with each antibody) at 8 h and 19 h. Lymphoblastoid cell line (LCL) lysate used as positive control for MALT1 protein expression; MALT1 protein detection (b) at 16 h and (c) over time (intracellular staining by flow cytometry, gated on CD14-expressing cells; MFI mean fluorescence intensity; dotted line: signal for fluorescence-minus-one staining control MFI level; boxes and whiskers with a paired 2-sided t-test in b; mean ± SD in c and d; d corresponding cell viability over time (mean ± SD); 6 subjects. e Effect of MALT1 inhibition on IL-1β, IL-6, and lactate production at rest and following LPS (mononuclear cells; boxes and whiskers with 2-sided paired t-tests); f correlation between LPS-induced IL-1β and IL-6, and lactate production (Spearman’ r; *p < 0.05; with dotted regression line); 8 subjects. All experiments were conducted in adult cells