Hyper innate responses in neonates lead to increased morbidity and mortality after infection

Abstract

Neonates suffer high morbidity and mortality in infection, presumably because of the lack of a fully developed adaptive and innate immune system. Evidence of poor innate responses in neonates has been shown by using a model that sensitizes the host to Toll-like receptor (TLR)-mediated inflammation with d-galactosamine (d-GalN). However, we show that neonatal mice demonstrate much stronger inflammatory responses than adult mice in response to LPS stimulation, and such hypersensitivity extends to other TLR agonists including actual viral infection. Our study reveals that the ensuing inflammatory reaction after d-GalN sensitization reflects preferential toxicity of d-GalN to adult liver cells, rather than accurately reflecting the TLR response to LPS. We show further that an uncontrolled proinflammatory innate response due to inadequate T cells makes neonates more vulnerable to TLR agonists or viral infection. Remarkably, through transfer of T cells into neonates or depletion of T cells in adult mice, we show that T cells are sufficient and necessary to control the early inflammatory response to LPS. Therefore, neonates might suffer from the unleashed innate responses caused by an insufficient number of T cells, which leads to increased morbidity and mortality.

Fig. 1.

Neonatal mice produce stronger inflammatory responses to high-dose LPS and other TLR stimulation. (A and B) Neonatal and adult mice were administered 10 mg/kg LPS (A) or 40 mg/kg poly(I:C) (B), and serum proinflammatory cytokines were detected at 6 h after injection (n = 3–5). (C) Splenocytes (1 × 10⁶ and 2 × 10⁶) from neonatal and adult mice were stimulated with 50 μg/ml poly(I:C), 5 × 10⁵ pfu MHV-A59, or 100 ng/ml LPS for 20 h, and the culture supernatants were harvested and analyzed for detection of cytokines. **, P < 0.01 by t test. Data are presented as mean values (±SEM).

Fig. 2.

Age-dependent sensitivity to LPS alone. Mice of different ages (1-d-, 7-d-, 2-week-, and 10-week-old; n = 6–10 for each group) were injected with 10 mg/kg LPS. Survival was monitored for 7 d (A) and serum inflammatory cytokines were detected at 6 h after injection (B). **, P < 0.01 by t test. Data are presented as mean values (±SEM).

Fig. 3.

Neonatal mice are insensitive to low-dose LPS challenge, and d-GalN induces more severe hepatotoxicity in adult mice. (A) Mortality curves of neonatal (♦, n = 6) and adult (■, n = 7) mice after injection with 0.5 mg/kg LPS and 0.35 g/kg d-GalN. (B) Serum ALT and AST were detected 6 h after injection. (C) Serum proinflammatory cytokines (TNF, MCP-1, and IL-6) were detected 2 h after injection (n = 5–6). (D) Neonatal (day 7) and adult mice were injected with 0.35 g/kg d-GalN, and glycogen in liver was detected at 6 h after injection. Data were presented by percentage of glycogen decrease after d-GalN treatment (n = 4–5). (E) Neonatal (day 7) and adult mice were injected with 3 g/kg d-GalN, and serum ALT and AST were detected at 24 h after injection (n = 4–5). (F) H&E staining of formalin-fixed sections of livers from neonates or adults treated with PBS, d-GalN, or d-Gal/LPS. *, P < 0.05; **, P < 0.01 by t test. Data are presented as mean values (±SEM).

Fig. 4.

Higher production of proinflammatory cytokines during LPS stimulation is related to low numbers of T cells in neonates. (A) The percentages of T cells in the spleens of neonatal and adult mice (n = 3). (B) TNF and IL-6 in serum of WT mice and mice depleted of T cells were detected at 2 and 6 h, respectively, after 10 mg/kg LPS injection (n = 4; **, P < 0.01). (C) Neonatal mice were adoptively transferred with 3 × 10⁷ T cells purified from adult mice. Cytokines were detected 2 h after 10 mg/kg LPS (n = 3–4; *, P < 0.05; **, P < 0.01). Data are presented as mean values (±SEM). (D) Lethal dose of LPS (20 mg/kg) was injected to WT, TNFRI KO, or TNFRI/II KO neonatal mice. Survival was recorded for 7 d.

Fig. 5.

T cells restrict inflammatory cytokine production by neonatal splenocytes. (A) Neonatal splenocytes (1 × 10⁶) were stimulated with 50 μg/ml poly (I:C), 5 × 10⁵ pfu MHV-A59, or 100 ng/ml LPS in the presence or absence of 2 × 10⁶ pan-T cells. (B) Neonatal splenocytes (1 × 10⁶) were stimulated with 100 ng/ml LPS in the presence or absence of 2 × 10⁶ T cells purified from neonatal or adult mice. The culture supernatants were harvested 20 h after coculture and analyzed with Cytometric Bead Array (CBA) (BD Biosciences) for detection of cytokines. *, P < 0.05; **, P < 0.01 by t test. Data are presented as mean values (±SEM).