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Cellular and molecular mechanisms underlying the strong neonatal IL-12 response of lamb mesenteric lymph node cells to R-848.

Ferret-Bernard S, Remot A, Lacroix-Lamandé S, Metton C, Bernardet N, Drouet F, Laurent F - PLoS ONE (2010)

Bottom Line: We used sheep as a large animal model to study TLR agonist responses in the lymph nodes draining the intestine, an organ that must adapt to profound changes after birth.This difference was age-related for both organs, but the preferential IL-12 response decreased more rapidly in the MLN, with young animals producing similar amounts of this cytokine to adults, from the age of 20 days onwards.This work provides relevant information for future vaccination or immunostimulation strategies targeting neonates.

View Article: PubMed Central - PubMed

Affiliation: Equipe Contrôle et Immunologie des Maladies Entériques du Nouveau-Né, UR1282 Infectiologie Animale et Santé Publique, INRA Nouzilly, Nouzilly, France.

ABSTRACT

Background: Comparative studies on the response of neonates and adults to TLR stimulation have been almost exclusively limited to comparisons of human neonatal cord blood cells with peripheral blood from adults, and analyses of spleen cell responses in mice. We need to extend these studies and gain further information regarding such responses at mucosal sites.

Methodology/principal findings: We used sheep as a large animal model to study TLR agonist responses in the lymph nodes draining the intestine, an organ that must adapt to profound changes after birth. In response to the imidazoquinoline compound R-848, neonatal mesenteric lymph node (MLN) and spleen cells produced more IL-12 and, consequently, more IFNγ than their adult counterparts. This difference was age-related for both organs, but the preferential IL-12 response decreased more rapidly in the MLN, with young animals producing similar amounts of this cytokine to adults, from the age of 20 days onwards. Intracellular assays and depletion experiments identified CD14(+)CD11b(+)CD40(+) cells as the main producer of IL-12. These cells accounted for a greater proportion of neonatal than of adult MLN cells, and also produced, in direct response to R-848, more IL-12 after isolation. This strong IL-12 response in neonates occurred despite the production of larger amounts of the regulatory cytokine IL-10 and the stronger upregulation of SOCS-1 and SOCS-3 mRNA levels than in adult cells, and was correlated with an increase in p38/MAPK phosphorylation.

Conclusions/significance: This is the first attempt to decipher the mechanism by which neonatal MLN cells produce more IL-12 than adult cells in response to the TLR8 agonist R-848. CD14(+)CD11b(+)CD40(+) IL-12-producing cells were more numerous in neonate than in adult MLN cells and displayed higher intracellular responsiveness upon R-848 stimulation. This work provides relevant information for future vaccination or immunostimulation strategies targeting neonates.

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Comparative IL-12 and IFNγ responses of neonatal and adult MLN cells following TLR agonist stimulation.Neonatal (closed circles) and adult (open squares) MLN cells were cultured in vitro with or without 12.5 µg/ml polyI:C, 0.5 µg/ml R-848 or 5 µg/ml Gardiquimod. Supernatants were harvested after 48 h of culture and ELISA was carried out for IL-12 (A) and IFNγ (B) secretion. Non-specific cell stimulation was also carried out with 50 ng/ml PMA combined with 500 ng/ml ionomycin, with the supernatants assayed for IFNγ detection 48 h later (C). Each circle or square represents one neonate or one adult, respectively. Medians are shown for each stimulus. Non-parametric Mann-Whitney tests were used to compare data for neonates and adults: *p≤0.01; **p≤0.005; ***p≤0.001; ****p≤0.0005.
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pone-0013705-g001: Comparative IL-12 and IFNγ responses of neonatal and adult MLN cells following TLR agonist stimulation.Neonatal (closed circles) and adult (open squares) MLN cells were cultured in vitro with or without 12.5 µg/ml polyI:C, 0.5 µg/ml R-848 or 5 µg/ml Gardiquimod. Supernatants were harvested after 48 h of culture and ELISA was carried out for IL-12 (A) and IFNγ (B) secretion. Non-specific cell stimulation was also carried out with 50 ng/ml PMA combined with 500 ng/ml ionomycin, with the supernatants assayed for IFNγ detection 48 h later (C). Each circle or square represents one neonate or one adult, respectively. Medians are shown for each stimulus. Non-parametric Mann-Whitney tests were used to compare data for neonates and adults: *p≤0.01; **p≤0.005; ***p≤0.001; ****p≤0.0005.

Mentions: Various cytokine responses to TLR agonists have been described in cells isolated from the systemic compartment [1], [25]–[27], but these responses have not been studied in cells isolated from lymph nodes draining the intestinal mucosa with exception to our previous work on caprines [18]. We continued this work in ovine for practical reasons to further dissect the cellular mechanism involved in TLR agonist response in MLN. Cells isolated from neonatal and adult sheep were stimulated in vitro with polyI:C (TLR3 agonist), Resiquimod (also called R-848) and Gardiquimod (both agonists of TLR7/8) for 48 h, and cytokine levels were determined by ELISA. Neonatal MLN cells produced variable amounts of IL-12 (Fig. 1A) and IFNγ (Fig. 1B), but the amounts produced were significantly larger than those produced by adult MLN cells, with R-848 and Gardiquimod being the best inducers. However, adult MLN cells were not intrinsically deficient for IFNγ production compared to neonatal cells, as they produced significantly larger amounts of this cytokine in response to non-specific stimulation with PMA associated with ionomycin in vitro (Fig. 1C). Moreover, by neutralising the IL-12 in vitro, we showed that IL-12 made a major contribution to IFNγ production by neonatal cells (73±9% inhibition, p<0.05). The IL-12 ELISA used cannot distinguish between IL-12p70 and the p40 chain, however this data suggests that MLN cells of neonates produce more bioactive IL-12 in response to R-848. We therefore investigated the particular features of the neonatal cells producing IL-12 in our model.


Cellular and molecular mechanisms underlying the strong neonatal IL-12 response of lamb mesenteric lymph node cells to R-848.

Ferret-Bernard S, Remot A, Lacroix-Lamandé S, Metton C, Bernardet N, Drouet F, Laurent F - PLoS ONE (2010)

Comparative IL-12 and IFNγ responses of neonatal and adult MLN cells following TLR agonist stimulation.Neonatal (closed circles) and adult (open squares) MLN cells were cultured in vitro with or without 12.5 µg/ml polyI:C, 0.5 µg/ml R-848 or 5 µg/ml Gardiquimod. Supernatants were harvested after 48 h of culture and ELISA was carried out for IL-12 (A) and IFNγ (B) secretion. Non-specific cell stimulation was also carried out with 50 ng/ml PMA combined with 500 ng/ml ionomycin, with the supernatants assayed for IFNγ detection 48 h later (C). Each circle or square represents one neonate or one adult, respectively. Medians are shown for each stimulus. Non-parametric Mann-Whitney tests were used to compare data for neonates and adults: *p≤0.01; **p≤0.005; ***p≤0.001; ****p≤0.0005.
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Related In: Results  -  Collection

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pone-0013705-g001: Comparative IL-12 and IFNγ responses of neonatal and adult MLN cells following TLR agonist stimulation.Neonatal (closed circles) and adult (open squares) MLN cells were cultured in vitro with or without 12.5 µg/ml polyI:C, 0.5 µg/ml R-848 or 5 µg/ml Gardiquimod. Supernatants were harvested after 48 h of culture and ELISA was carried out for IL-12 (A) and IFNγ (B) secretion. Non-specific cell stimulation was also carried out with 50 ng/ml PMA combined with 500 ng/ml ionomycin, with the supernatants assayed for IFNγ detection 48 h later (C). Each circle or square represents one neonate or one adult, respectively. Medians are shown for each stimulus. Non-parametric Mann-Whitney tests were used to compare data for neonates and adults: *p≤0.01; **p≤0.005; ***p≤0.001; ****p≤0.0005.
Mentions: Various cytokine responses to TLR agonists have been described in cells isolated from the systemic compartment [1], [25]–[27], but these responses have not been studied in cells isolated from lymph nodes draining the intestinal mucosa with exception to our previous work on caprines [18]. We continued this work in ovine for practical reasons to further dissect the cellular mechanism involved in TLR agonist response in MLN. Cells isolated from neonatal and adult sheep were stimulated in vitro with polyI:C (TLR3 agonist), Resiquimod (also called R-848) and Gardiquimod (both agonists of TLR7/8) for 48 h, and cytokine levels were determined by ELISA. Neonatal MLN cells produced variable amounts of IL-12 (Fig. 1A) and IFNγ (Fig. 1B), but the amounts produced were significantly larger than those produced by adult MLN cells, with R-848 and Gardiquimod being the best inducers. However, adult MLN cells were not intrinsically deficient for IFNγ production compared to neonatal cells, as they produced significantly larger amounts of this cytokine in response to non-specific stimulation with PMA associated with ionomycin in vitro (Fig. 1C). Moreover, by neutralising the IL-12 in vitro, we showed that IL-12 made a major contribution to IFNγ production by neonatal cells (73±9% inhibition, p<0.05). The IL-12 ELISA used cannot distinguish between IL-12p70 and the p40 chain, however this data suggests that MLN cells of neonates produce more bioactive IL-12 in response to R-848. We therefore investigated the particular features of the neonatal cells producing IL-12 in our model.

Bottom Line: We used sheep as a large animal model to study TLR agonist responses in the lymph nodes draining the intestine, an organ that must adapt to profound changes after birth.This difference was age-related for both organs, but the preferential IL-12 response decreased more rapidly in the MLN, with young animals producing similar amounts of this cytokine to adults, from the age of 20 days onwards.This work provides relevant information for future vaccination or immunostimulation strategies targeting neonates.

View Article: PubMed Central - PubMed

Affiliation: Equipe Contrôle et Immunologie des Maladies Entériques du Nouveau-Né, UR1282 Infectiologie Animale et Santé Publique, INRA Nouzilly, Nouzilly, France.

ABSTRACT

Background: Comparative studies on the response of neonates and adults to TLR stimulation have been almost exclusively limited to comparisons of human neonatal cord blood cells with peripheral blood from adults, and analyses of spleen cell responses in mice. We need to extend these studies and gain further information regarding such responses at mucosal sites.

Methodology/principal findings: We used sheep as a large animal model to study TLR agonist responses in the lymph nodes draining the intestine, an organ that must adapt to profound changes after birth. In response to the imidazoquinoline compound R-848, neonatal mesenteric lymph node (MLN) and spleen cells produced more IL-12 and, consequently, more IFNγ than their adult counterparts. This difference was age-related for both organs, but the preferential IL-12 response decreased more rapidly in the MLN, with young animals producing similar amounts of this cytokine to adults, from the age of 20 days onwards. Intracellular assays and depletion experiments identified CD14(+)CD11b(+)CD40(+) cells as the main producer of IL-12. These cells accounted for a greater proportion of neonatal than of adult MLN cells, and also produced, in direct response to R-848, more IL-12 after isolation. This strong IL-12 response in neonates occurred despite the production of larger amounts of the regulatory cytokine IL-10 and the stronger upregulation of SOCS-1 and SOCS-3 mRNA levels than in adult cells, and was correlated with an increase in p38/MAPK phosphorylation.

Conclusions/significance: This is the first attempt to decipher the mechanism by which neonatal MLN cells produce more IL-12 than adult cells in response to the TLR8 agonist R-848. CD14(+)CD11b(+)CD40(+) IL-12-producing cells were more numerous in neonate than in adult MLN cells and displayed higher intracellular responsiveness upon R-848 stimulation. This work provides relevant information for future vaccination or immunostimulation strategies targeting neonates.

Show MeSH
Related in: MedlinePlus