Decreased production of epithelial-derived antimicrobial molecules at mucosal barriers during early life

Abstract

Young age is a risk factor for respiratory and gastrointestinal infections. Here, we compared infant and adult mice to identify age-dependent mechanisms that drive susceptibility to mucosal infections during early life. Transcriptional profiling of the upper respiratory tract (URT) epithelium revealed significant dampening of early life innate mucosal defenses. Epithelial-mediated production of the most abundant antimicrobial molecules, lysozyme, and lactoferrin, and the polymeric immunoglobulin receptor (pIgR), responsible for IgA transcytosis, was expressed in an age-dependent manner. This was attributed to delayed functional development of serous cells. Absence of epithelial-derived lysozyme and the pIgR was also observed in the small intestine during early life. Infection of infant mice with lysozyme-susceptible strains of Streptococcus pneumoniae or Staphylococcus aureus in the URT or gastrointestinal tract, respectively, demonstrated an age-dependent regulation of lysozyme enzymatic activity. Lysozyme derived from maternal milk partially compensated for the reduction in URT lysozyme activity of infant mice. Similar to our observations in mice, expression of lysozyme and the pIgR in nasopharyngeal samples collected from healthy human infants during the first year of life followed an age-dependent regulation. Thus, a global pattern of reduced antimicrobial and IgA-mediated defenses may contribute to increased susceptibility of young children to mucosal infections.

Figure 1.. Mucosal immune defenses in the URT are dampened in early life

a, Heat-map of the top 1,000 differentially expressed genes in the upper respiratory tract between 7 day-old infant mice and 8 week-old adult mice (n=5). b, Top overrepresented PANTHER pathways from significantly down-regulated genes (FDR P >0.05) and (c) Top overrepresented PANTHER pathways from significantly up-regulated genes (FDR P >0.05) in 7 day-old infant mice compared to 8 week-old adult mice (n=5). d-f, Transcript levels of (d) lysozyme, Lyz1/2, (e) lactoferrin, Ltf, and (f) the pIgR, Pigr, from 7-day old infant mice and adult 8-week old mice (n=5). Data represent log₂-transformed values and the mean ± SD. Statistical significance was determined using an unpaired Student’s t test and is indicated as **, p ≤ 0.01; ***, p ≤ 0.001. g, Protein levels of lactoferrin (LTF, top), lysozyme (LYZ1/2, middle), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, bottom) and (h) pIgR (PIGR, top) and GAPDH (bottom) in nasal lavages collected from 7 day-old infant and adult mice (n=5). Std, protein standard. Data collected from two independent experiments.

Figure 2.. Age-dependent regulation of lysozyme production and function in the URT

a, Transcript levels of lysozyme (Lyz1/2) in nasal lavages collected from infant mice at 3, 5, 7, 14 and 21 days of age compared to adult mice (n=8–12). Data represent log₂-transformed values with the mean ± SD. Statistical significance was determined using an ordinary one-way ANOVA and Dunnett’s multiple comparisons post-hoc test and is indicated as **, p ≤ 0.01; ****, p ≤ 0.0001. Data collected from two independent experiments. b, Competitive index of colony forming units (CFU) from nasal lavages of infant and adult mice 7 days after IN infection with a 1:1 mixture of wild-type S. pneumoniae and S. pneumoniae pgdA mutant (n=4–7). Data represent log₁₀-transformed values with the mean ± SD. Statistical significance was determined using an unpaired Student’s t test and is indicated as *, p ≤ 0.05. Dotted line at competitive index of 1. Data collected from one experiment. c-d, CFU of nasal lavages at either 3, 7 and 14 days or 3, 14, and 21 days, after IN single infection with either wild-type S. pneumoniae or S. pneumoniae pgdA mutant in (c) adult mice (n=6–13) and (d) infant mice (n=3–10). Data represent log₁₀-transformed values with the mean ± SD. Statistical significance was determined using an unpaired Students t test and is indicated as *, p ≤ 0.05; ***, p ≤ 0.001; ****, p ≤ 0.0001. NS, not significant. Dotted line at limit of detection. Adult data collected from 2–3 independent experiments. Infant data collected from one experiment, except D14 S. pneumoniae pgdA mutant data collected from two independent experiments. e, Competitive index of CFU in nasal lavages of wild-type or lysozyme-2 (Lyz2^−/−)-deficient infant mice 3 and 14 days after intranasal infection with a 1:1 mixture of wild-type S. pneumoniae and S. pneumoniae pgdA mutant (n=4–20). Data represent log₁₀-transformed values with the mean ± SD. Statistical significance was determined using an ordinary one-way ANOVA and Sidak’s multiple comparisons post-hoc test and is indicated by **, p ≤ 0.01; ***, p ≤ 0.001. NS, not significant. Dotted line at competitive index of 1. Day 3 data collected from one experiment. Day 14 data collected from 2–3 independent experiments.

Figure 3.. Global reduction of lysozyme production and activity at mucosal surfaces

a, Transcript levels of lysozyme (Lyz1/2) in ileum lavages collected from infant mice at 6, 15, 21 and 28 days of age compared to adult 8 week-old mice (n=5–8). Data represent log₂-transformed values with the mean ± SD. Statistical significance was determined using an ordinary one-way ANOVA and Dunnett’s multiple comparisons post-hoc test and is indicated as **, p ≤ 0.01; ****, p ≤ 0.0001. NS, not significant. Data collected from two independent experiments, except D15 data collected from one experiment. b, Protein levels of LYZ1/2 (top) and GAPDH (bottom) in small intestine collected from 7 day-old infant and adult mice (n=4). Infant samples were pooled n=2–3. Std, protein standard. Data collected from two independent experiments. c, Competitive index of CFU from cecum of infant mice at 7, 14, and 28 days after oral infection with a 1:1 mixture of wild-type S. aureus and S. aureus oatA mutant (n=6–8). Data represent log₁₀-transformed values with the mean ± SD. Statistical significance was determined using an ordinary one-way ANOVA and Sidak’s multiple comparisons post-hoc test and is indicated by *, p ≤ 0.05. NS, not significant. Dotted line at competitive index of 1. Data collected from two independent experiments. d, Transcript levels of Pigr in ileum lavages collected from infant mice at 6, 15, 21 and 28 days of age compared to adult 8 week-old mice (n=5–8). Data represent log₂-transformed values with the mean ± SD. Statistical significance was determined using an ordinary one-way ANOVA with a Dunnett’s multiple comparisons post-hoc test and is indicated as *, p ≤ 0.05; ****, p ≤ 0.0001. NS, not significant. Data collected from two independent experiments, except D15 data collected from one experiment. e, Protein levels of PIgR (top) and GAPDH (bottom) in small intestine collected from 7 day-old infant (n=4) and adult mice (n=4). Infant samples were pooled n=2–3. Std, protein standard. Data collected from two independent experiments.

Figure 4.. Non-hematopoietic cells are responsible for reduced lysozyme expression during early life

a, Representative flow plots for frequency of macrophages (singlet live CD45⁺ CD11b⁺ F4/80⁺ Ly6G^-) and neutrophils (singlet live CD45⁺ CD11b⁺ F4/80^- Ly6G⁺) from uninfected 7 day-old infant and adult mice. b, Percentage of macrophages (singlet live CD45⁺ CD11b⁺ F4/80⁺ Ly6G^-) and (c) neutrophils (singlet live CD45⁺ CD11b⁺ F4/80^- Ly6G⁺) from naïve 7 day-old infant and adult mice (n=5–7). Data represent mean ± SD. Statistical significance was determined using a unpaired Student’s t test and is indicated as ****, p ≤ 0.0001. Data collected from two independent experiments. d, Transcript levels of Lyz1/2 in nasal lavages from infant and adult mice treated with 2 doses of either a neutrophil depleting antibody (αLy6G) or isotype control (n=5–7). Data represent log₂-transformed values with the mean ± SD. Statistical significance was determined using an ordinary one-way ANOVA with a Sidak’s multiple comparisons post-hoc test and is indicated as ****, p ≤ 0.0001. NS, not significant. Data collected from two independent experiments. e-g, Transcript levels of (e) Lyz1/2, (f) Ltf and (g) Pigr in CD45⁺ and CD45^- cells isolated from URT tissue of naïve 7 day-old infant and adult mice (n=3–6). Samples for infant mice were pooled n=3. Data represent log₂-transformed values with the mean ± SD. Statistical significance was determined using an ordinary one-way ANOVA with a Sidak’s multiple comparisons post-hoc test and is indicated as **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. Adult data collected from two independent experiments and infant data collected from one experiment using two litters. h-j, Transcript levels of (h) Lys1/2, (i) Ltf and (j) Pigr in CD45^- cells isolated from small intestine of naïve 7 day-old infant and adult mice (n=3–5). Samples for infant mice were pooled n=3–4 and samples for adult mice were pooled n=1–2. Data represent log₂-transformed values with the mean ± SD. Statistical significance was determined using a unpaired Student’s t test and is indicated as **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. Data collected from two independent experiments.

Figure 5.. Delayed development of specialized epithelial cells in the URT during early life

a, Heatmap of log_2-transformed gene expression values in nasal lavages collected from infant (7 day old), juvenile (25 day old) and adult (8 week old) mice. Genes are representative of specific epithelial subsets from the respiratory tract and include: serous (Ltf, Lyz1, Lyz2, Pip, and Azgp1), mucous (Azgp1, Tspan8, Muc5b and Bpifb2), secretory (Muc5ac, Tfcp2l1, Muc1 and Psca), multiciliated (Foxj1, Pifo, Muc1, Palld, and Cyp24a1), deuterosomal (Cdc20b, Hes6, Ccdc67, and Foxj1), suprabasal (Serpinb3a, Serpinb3c, Krt19, Fabp5, Tfcp2l1 and S100a9) and basal (Krt5, Trp63 and Dlk2). Genes found to be statistically different between infant and adult mice are designated *, p-adjusted value <0.05. b, Representative coronal URT tissue sections of a naive infant (6 day old) and adult (8 week old) mouse. Sections were stained with hematoxylin and eosin (H&E; top row), anti-pIgR antibody (middle row) or anti-lactoferrin antibody (bottom row). Levels of pIgR and lactoferrin were detected by IHC using DAB substrate (brown precipitate) and counterstained with hematoxylin.

Figure 6.. Maternal milk partially compensates for reduced lysozyme production in infant mice

a, Experimental design for assessment of maternal milk-derived lysozyme in clearance of a lysozyme sensitive S. pneumoniae strain (pgdA). At 1–2 days after birth, half of the pups from a wild-type dam and half of the pups from a lysozyme-2 deficient dam were switched. After 7 days all pups (8–9 days-old) were infected with S. pneumoniae pgdA mutant. b, CFU of nasal lavages at 7 days after IN infection with S. pneumoniae pgdA mutant in wild-type and lysozyme-2 (Lyz2^−/−)-deficient infant mice (n=7–10). Data represent log₁₀-transformed values with the mean ± SD. Statistical significance was determined using an ordinary one-way ANOVA and Dunnett’s multiple comparisons post-hoc test and is indicated as *, p ≤ 0.05.

Figure 7.. Temporal increase in lysozyme and pIgR expression during the first year of life in children

a, Transcript levels of lysozyme (LYZ; left), the pIgR (PIGR; middle) and lactoferrin (LTF; right) in nasopharyngeal samples from healthy infants at 7 days (w1) and 12 months (m12) of age. b, Transcript levels of LYZ and PIGR in nasopharyngeal samples from healthy infants at 11 time-points (2 hours after birth (d0), at 24 hours (d1), at 7 (w1) and 14 days (w2) and at 1, 2, 3, 4, 6, 9, and 12 months (m1, m2, m3, m4, m6, m9 and m12)). n = 286 samples. Data represent log₂-transformed intensity values generated using microarray. Statistical significance was determined using a linear mixed effects model to correct for repeated measures (lmerTest R-package) and included time point as fixed effect and subject as a random intercept. c, Heatmap of the Z-transformed mean gene expression values at each time-point for serous (LTF, LYZ, LYZL1, LYZL2, PIP and AZGP1), suprabasal (TFCP2L1, SERPINB3, KRT19, FABP5 and S100A9) and basal (KRT5, TP63 and DLK2) specific genes. The log₂-transformed gene expression values were averaged per time-point and then Z-scores for these mean expression values were determined.