Age-dependent maturation of Toll-like receptor-mediated cytokine responses in Gambian infants

Abstract

The global burden of neonatal and infant mortality due to infection is staggering, particularly in resource-poor settings. Early childhood vaccination is one of the major interventions that can reduce this burden, but there are specific limitations to inducing effective immunity in early life, including impaired neonatal leukocyte production of Th1-polarizing cytokines to many stimuli. Characterizing the ontogeny of Toll-like receptor (TLR)-mediated innate immune responses in infants may shed light on susceptibility to infection in this vulnerable age group, and provide insights into TLR agonists as candidate adjuvants for improved neonatal vaccines. As little is known about the leukocyte responses of infants in resource-poor settings, we characterized production of Th1-, Th2-, and anti-inflammatory-cytokines in response to agonists of TLRs 1-9 in whole blood from 120 Gambian infants ranging from newborns (cord blood) to 12 months of age. Most of the TLR agonists induced TNFα, IL-1β, IL-6, and IL-10 in cord blood. The greatest TNFα responses were observed for TLR4, -5, and -8 agonists, the highest being the thiazoloquinoline CLO75 (TLR7/8) that also uniquely induced cord blood IFNγ production. For most agonists, TLR-mediated TNFα and IFNγ responses increased from birth to 1 month of age. TLR8 agonists also induced the greatest production of the Th1-polarizing cytokines TNFα and IFNγ throughout the first year of life, although the relative responses to the single TLR8 agonist and the combined TLR7/8 agonist changed with age. In contrast, IL-1β, IL-6, and IL-10 responses to most agonists were robust at birth and remained stable through 12 months of age. These observations provide fresh insights into the ontogeny of innate immunity in African children, and may inform development of age-specific adjuvanted vaccine formulations important for global health.

Figure 1. TLR-mediated cytokine production in cord blood.

100 µl of whole cord blood was cultured overnight with each of the TLR agonists and supernatants recovered for measurement of (A) TNFα and (B) IFNγ as described in Materials and Methods (pg/mL) and presented as box and whisker plots illustrating 10 and 90 percentile error bars, n = 12–15. Comparisons between cytokine levels in the stimulated samples and the unstimulated samples were analysed using a paired Sign test at 5% significance (* represent significant differences).

Figure 2. TLR7 and 8 agonist-induced cytokine induction in cord blood.

100 µl of whole cord blood was cultured overnight with each of the TLR agonists and supernatants recovered for measurement of IFNγ, TNFα, IL-10, IL-1β and IL-6 as described in Materials and Methods (pg/mL), n = 12–15. Unstimulated values are subtracted from stimulated values and presented as box and whisker plots, n = 12–15.

Figure 3. Net TLR-mediated TNFα responses differ between agonists and across the first year of life.

100 µl of whole blood was cultured overnight with each of the TLR agonists at birth, 1, 2, 3, 4, 6, 9 and 12 months of age (x axis) and TNFα cytokine production (y axis) was measured as described in the Materials and Methods (pg/mL). Unstimulated values are subtracted from stimulated values and presented as box and whisker plots, n = 12-15.

Figure 4. TLR agonists induce greater cytokine production in Gambian newborns at 1 month compared to at birth.

100 µl whole blood was cultured overnight with each of the TLR agonists and cytokine production (pg/mL) in supernatants was measured as described in the Materials and Methods. Comparisons between cytokine levels at birth and 1 month of age were calculated using a non-parametric Mann Whitney test. *(light grey square) p = 0.05 – 0.019, **(medium grey square) p = 0.01 – 0.001, ***(dark grey square) p<0.001. Pam3.  = Pam₃CysSerLys_4, Gard.  = Gardiquimod.

Figure 5. Net TLR-mediated IFNγ responses differ between agonists and across the first year of life.

100 µl of whole blood was cultured overnight with (A) Pam3CSK4 (TLR1/2 agonist), (B) Gardiquimod™ (TLR7 agonist), (C) ssRNA (TLR8 agonist) and (D) CL075 (TLR7/8 agonist) at birth (cord), 1, 2, 3, 4, 6, 9 and 12 months of age (x axis) and IFNγ cytokine production (y axis) was measured in supernatants (pg/mL) as described in Materials and Methods. Unstimulated values are subtracted from stimulated values and data were presented as box and whisker, n = 12–15.

Figure 6. TLR8 agonists induce distinct patterns of TNFα and IFNγ production across the first year of life.

100 µl of whole blood was cultured overnight with ssRNA (TLR8 agonist) and CL075 (TLR7/8) at birth, 1, 2 and 12 months of age. (A) TNFα and (B) IFNγ cytokine (pg/mL) was measured in supernatants as described in the Materials and Methods. Unstimulated values are subtracted from stimulated values and data are presented as box and whisker plots, n = 12–15.

Figure 7. TLR7- and TLR8-mediated cytokine mRNA expression varies between agonists and across the first year of life.

300 µl of whole blood was cultured for 4 hours with Gardiquimod™ (TLR7), ssRNA (TLR8 agonist) and CL075 (TLR7/8) at (A) birth, (B) 1 month and (C) 12 months of age. RNA was purified and cytokine mRNA was quantified by real time PCR as described in Materials and Methods. Ct values were normalised against the housekeeping genes and compared to the unstimulated control (ΔCt) and fold differences of ΔCt values between unstimulated and stimulated cultures were calculated (ΔΔCt). Fold changes are presented as box and whisker plots, n = 5.

Figure 8. Correlations between ssRNA-induced cytokine mRNA and protein expression.

300 µl of whole blood was cultured for 4 hours with (A) Gardiquimod™ (TLR7), (B) ssRNA (TLR8) and (C) CL075 (TLR7/8). RNA was purified and cytokine mRNA was quantified by real time PCR as described in Materials and Methods. Comparisons between IFNγ protein and IFNG mRNA levels were calculated where stimulated values (Ct and cytokine concentrations) were divided by unstimulated values. The values were log transformed and compared using Pearson correlation test, linear regression line presented in graph, n =  5. At birth (black circles), 1 month (open circles), or 12 months (crosses) of age.

Figure 9. Age-dependent effects of cytokine transcription in response to TLR agonists.

300 µl whole blood was cultured for 4 hours with Gardiquimod™ (TLR7 agonist), ssRNA (TLR8 agonist) and CL075 (TLR7/8 agonist) at birth (cord), 1 and 12 months of age and IL-12A, IL-12B, IFNG,IL-6, IL-10 and TNF cytokine mRNA gene transcription was measured. Ct values were normalised against the housekeeping genes and compared to the unstimulated control (ΔCt). Comparisons of responses from birth, 1 and 12 months of age were made using the Kruskall Wallis test. Grey squares represent significant effects with age, p>0.05. Gard.  = Gardiquimod.