Neonate Intestinal Immune Response to CpG Oligodeoxynucleotide Stimulation

Background: The development of mucosal vaccines is crucial to efficiently control infectious agents for which mucosae are the primary site of entry. Major drawbacks of these protective strategies are the lack of effective mucosal adjuvant. Synthetic oligodeoxynucleotides that contain several unmethylated cytosine-guanine dinucleotide (CpG-ODN) motifs are now recognized as promising adjuvants displaying mucosal adjuvant activity through direct activation of TLR9-expressing cells. However, little is known about the efficacy of these molecules in stimulating the intestinal immune system in neonates. Methodology/Principal Findings: First, newborn mice received CpG-ODN orally, and the intestinal cytokine and chemokine response was measured. We observed that oral administration of CpG-ODN induces CXC and CC chemokine responses and a cellular infiltration in the intestine of neonates as detected by immunohistochemistry. We next compared the efficiency of the oral route to intraperitoneal administration in stimulating the intestinal immune responses of both adults and neonates. Neonates were more responsive to TLR9-stimulation than adults whatever the CpG-ODN administration route. Their intestinal epithelial cells (IECs) indirectly responded to TLR9 stimulation and contributed to the CXC chemokine response, whereas other TLR9-bearing cells of the lamina-propria produced CC chemokines and Th1-type cytokines. Moreover, we showed that the intestine of adult exhibited a significantly higher level of IL10 at homeostasis than neonates, which might be responsible for the unresponsiveness to TLR9-stimulation, as confirmed by our findings in IL10-deficient mice. Conclusions/Significance: This is the first report that deciphers the role played by CpG-ODN in the intestine of neonates. This work clearly demonstrates that an intraperitoneal administration of CpG-ODN is more efficient in neonates than in adults to stimulate an intestinal chemokine response due to their lower IL-10 intestinal level. In addition we report the efficiency of the oral route at inducing intestinal chemokine responses in neonate that might be taken into consideration for further vaccine development against neonatal diseases

Recruitment of inflammatory cells in the MLN after CpG-ODN treatment.

<p>Eight-day-old neonatal mice received 20 µg/g of CpG-ODN by the oral route. Twenty-four hours later, MLN from 10 neonates were removed and pooled for FACS analysis. Four pools in each group were analyzed. These data were from two representative experiments. Mann-Whitney non parametric test ** p<0.005, * p<0.05.</p

IL12p40 and IFNγ responses after CpG-ODN treatment in the intestine.

<p>CpG-ODN was administered orally in 8-day-old WT and TLR9 KO neonate mice at a dose of 20 µg/g (black boxes). Control mice received PBS orally (white boxes). N = 5 neonates from two different litter for each group. Six hours later, ilea were removed for RNA extraction. RT-PCRs were performed for amplification of IL12p40 (A) and IFNγ (B) mRNA. These data are representative of two experiments. Data were analyzed by the non-parametric Mann-Whitney test.</p

Role of IL10 in the chemokine response induced by CpG-ODN stimulation.

<p>(A) Ilea of 8-day old neonatal (n = 8) and of adult (n = 6) mice were removed for RNA extraction. IL10 and FoxP3 mRNA levels were measured by real-time RT-PCR. Data were analyzed by the non-parametric Mann-Whitney test. (B) Neonates (n = 3) and adults (n = 3) IL10-deficient mice received PBS or 20 µg/g CpG-ODN orally and 10 µg/g intraperitoneally. They were killed 6 hours later and the ilea were removed. Total RNAs were extracted and RT-PCR analyses were performed for CXCL1, CXCL10, CCL2 and CCL7.</p

Chemokine responses of intestinal epithelial cells after CpG-ODN stimulation.

<p>(A) Intestinal epithelial cells (IEC) were purified from neonatal (n = 5) or adult mice (n = 5). RNA was extracted from the purified cells for TLR9 mRNA RT-PCR analysis. (B) Two murine intestinal epithelial cell lines (ICcl2 and CMT93) and purified IECs from adults and neonates were stimulated in vitro with 5 µg/ml of CpG-ODN for 2 hours, and RNAs were extracted for CXCL1 and CXCL10 measurement. (C) Neonatal (n = 30 for each group) and adult mice (n = 3 for each group) received CpG-ODN by the oral and IP route. They were killed 6 hours later, and the IECs were purified. RNAs were extracted and RT-PCR analyses were performed for CXCL1 and CXCL10 expression. Data were submitted to the non-parametric Mann-Whitney test. (*, p<0.05). ND, Not Done.</p

Recruitment of inflammatory cells in the intestine of neonates after CpG-ODN treatment.

<p>Eight-day-old neonatal mice received 20 µg/g of CpG-ODN orally. Twenty-four hours after treatment, ilea were removed to analyze the recruitment of inflammatory cells by immunohistology. Six to ten neonates from different litters (5 sections for each animal) per group were analyzed. Data were analyzed by non-parametric Mann-Whitney test.</p