Delayed development of systemic immunity in preterm pigs as a model for preterm infants

Abstract

Preterm neonates are highly sensitive to systemic infections in early life but little is known about systemic immune development following preterm birth. We hypothesized that preterm neonates have immature systemic immunity with distinct developmental trajectory for the first several weeks of life, relative to those born at near-term or term. Using pigs as a model, we characterized blood leukocyte subsets, antimicrobial activities and TLR-mediated cytokine production during the first weeks after preterm birth. Relative to near-term and term pigs, newborn preterm pigs had low blood leukocyte counts, poor neutrophil phagocytic rate, and limited cytokine responses to TLR1/2/5/7/9 and NOD1/2 agonists. The preterm systemic responses remained immature during the first postnatal week, but thereafter showed increased blood leukocyte numbers, NK cell proportion, neutrophil phagocytic rate and TLR2-mediated IL-6 and TNF-α production. These immune parameters remained different between preterm and near-term pigs at 2-3 weeks, even when adjusted for post-conceptional age. Our data suggest that systemic immunity follows a distinct developmental trajectory following preterm birth that may be influenced by postnatal age, complications of prematurity and environmental factors. Consequently, the immediate postnatal period may represent a window of opportunity to improve innate immunity in preterm neonates by medical, antimicrobial or dietary interventions.

Figure 1. Postnatal growth and diarrhea in preterm and near-term pigs.

(a) body weight; (b) daily weight gain; (c) diarrhea incidence (fecal score >2). (d) severe diarrhea incidence (fecal score >4). A total of 92 preterm and 14 near-term pigs were included in the analysis. * and ***P < 0.05 and 0.001, respectively, between preterm and near-term pigs.

Figure 2. Blood leukocyte counts and phagocytosis activity in cord blood and venous sow blood.

(a) Blood leukocyte counts in cord blood of preterm (n = 92), near-term (n = 21) and term (n = 4) pigs and venous sow blood (n = 11). Blood neutrophil phagocytic rate, demonstrated as proportions of neutrophils exerting phagocytosis, against E. coli (b,c) and S. aureus (d,e) in newborn preterm pigs (n = 60–67), newborn near-term pigs (n = 9–21), their mother sow (n = 3–4), or from preterm blood cells with sow plasma (P cells/S plasma, n = 3), or sow blood cells with preterm plasma (S cells/P plasma, n = 3). (b,d) Representative histogram showing higher proportions of pHrodo+ neutrophils (the gate on the right) following blood challenge with E. coli and S. aureus. Values in the same cell population not sharing similar letters are significantly different (P < 0.01).

Figure 3. Pattern recognition receptor-mediated TNF-α and IL-6 production in the blood of preterm pigs at birth and adult pigs.

For each litter (n = 3), pooled whole blood of all preterm pigs at birth and blood of the sow were stimulated with 10 TLR and NOD agonists for five hours prior to plasma collection for cytokine analysis. (a) TLR2/6. (b) TLR1/2. (c) TLR2. (d) TLR3. (e) TLR4. (f) TLR5. (g) TLR7. (h) TLR9. (i) NOD1. (j) NOD2. ^*,#P < 0.05 for comparisons of IL-6 and TNF- α between preterm and adult pigs.

Figure 4. Gating strategy.

Monocytes (CD172a⁺⁺ SSC^low) and granulocytes (CD172a⁺ SSC^high) were gated based on granularity and CD172a, then confirmed by their locations in the original FSC/SSC plot. Granulocytes were further sub-divided into immature neutrophils (CD172a⁺ SSC^high 6D10⁺ 2B2^-) and mature neutrophils (CD172a⁺ SSC^high 6D10⁺ 2B2⁺). Lymphocytes were gated based on CD172a and its location in FSC/SSC plots (CD172a⁻ FSC^medium). NK cells were defined as CD16⁺ lymphocytes.

Figure 5. Development of blood leukocyte subsets and plasma components during the first four weeks of life in preterm and near-term pigs.

(a) Absolute neutrophil counts. (b) Absolute lymphocyte counts. (c) Frequency of immature (CD172a⁺ SSC^high 6D10⁺ 2B2^-) and mature neutrophils (CD172a⁺ SSC^high 6D10⁺ 2B2⁺) in total blood leukocytes. (d) The ratio of immature neutrophils in total number of neutrophils (I/T ratio). (e,f) Plasma CRP and IgG levels. A sub-population of preterm (n = 34–92) and term pigs (n = 12–21) was analyzed. Values of the same parameter during the four weeks not sharing similar letters are significantly different (P < 0.01 for a,b, P < 0.05 for c–f). *P < 0.05, compared between preterm vs. near-term pigs at the same time period.

Figure 6. Development of systemic immune responses during the first three weeks of life in preterm and near-term pigs.

(a,b) Proportion of neutrophils exerting phagocytosis in the blood against E. coli (n = 31–32 for preterm, n = 14–21 for near-term) and S. aureus (n = 55–60 for preterm, n = 9–15 for near-term). (c,d) NET response in vitro, shown by the relative increase of plasma cfDNA in samples with stimulation vs. control, following blood stimulation with PMA 100 ng/mL or LPS 1 μg/mL for 3 h (n = 51–58 for preterm, n = 12–20 for near-term pigs). (e,f) TLR2-mediated production of IL-6 and TNF-α in whole blood (n = 12 for preterm, n = 12–21 for near-term). In (a,b,e,f) ^{#, ##, ###}P < 0.05, 0.01 and 0.001, compared with corresponding values at birth. In (c,d) ^#P < 0.05, compared with the corresponding control without stimulation, which was assigned with an arbitrary unit of 1. ** and ***P < 0.01 and 0.001, compared between preterm vs. near-term pigs at the same time period.

Figure 7. The development of NK cells (CD16⁺ lymphocytes) and CD16⁺ monocytes during the first four weeks of life in preterm pigs.

(A) Frequency of NK cells in lymphocytes. (B) Frequency of CD16+ monocytes in total monocyte number (n = 21–33). Values not sharing similar letters are significantly different (P < 0.05).