Maternal BCG scar is associated with increased infant proinflammatory immune responses

Patrice Akusa Mawa, Emily L Webb, Abdelali Filali-Mouhim, Gyaviira Nkurunungi, Rafick-Pierre Sekaly, Swaib Abubaker Lule, Sarah Prentice, Stephen Nash, Hazel M Dockrell, Alison M Elliott, Stephen Cose, Patrice Akusa Mawa, Emily L Webb, Abdelali Filali-Mouhim, Gyaviira Nkurunungi, Rafick-Pierre Sekaly, Swaib Abubaker Lule, Sarah Prentice, Stephen Nash, Hazel M Dockrell, Alison M Elliott, Stephen Cose

Abstract

Introduction: Prenatal exposures such as infections and immunisation may influence infant responses. We had an opportunity to undertake an analysis of innate responses in infants within the context of a study investigating the effects of maternal mycobacterial exposures and infection on BCG vaccine-induced responses in Ugandan infants.

Material and methods: Maternal and cord blood samples from 29 mother-infant pairs were stimulated with innate stimuli for 24h and cytokines and chemokines in supernatants were measured using the Luminex® assay. The associations between maternal latent Mycobacterium tuberculosis infection (LTBI), maternal BCG scar (adjusted for each other's effect) and infant responses were examined using linear regression. Principal Component Analysis (PCA) was used to assess patterns of cytokine and chemokine responses. Gene expression profiles for pathways associated with maternal LTBI and with maternal BCG scar were examined using samples collected at one (n=42) and six (n=51) weeks after BCG immunisation using microarray.

Results: Maternal LTBI was positively associated with infant IP-10 responses with an adjusted geometric mean ratio (aGMR) [95% confidence interval (CI)] of 5.10 [1.21, 21.48]. Maternal BCG scar showed strong and consistent associations with IFN-γ (aGMR 2.69 [1.15, 6.17]), IL-12p70 (1.95 [1.10, 3.55]), IL-10 (1.82 [1.07, 3.09]), VEGF (3.55 [1.07, 11.48]) and IP-10 (6.76 [1.17, 38.02]). Further assessment of the associations using PCA showed no differences for maternal LTBI, but maternal BCG scar was associated with higher scores for principal component (PC) 1 (median level of scores: 1.44 in scar-positive versus -0.94 in scar-negative, p=0.020) in the infants. PC1 represented a controlled proinflammatory response. Interferon and inflammation response pathways were up-regulated in infants of mothers with LTBI at six weeks, and in infants of mothers with a BCG scar at one and six weeks after BCG immunisation.

Conclusions: Maternal BCG scar had a stronger association with infant responses than maternal LTBI, with an increased proinflammatory immune profile.

Keywords: BCG immunisation; Heterologous effects; Infant innate responses; Latent Mycobacterium tuberculosis infection; Maternal BCG scar; Maternal infections; Tuberculosis.

Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Figures

Fig. 1
Fig. 1
The association between maternal BCG scar and infant innate responses. Combined median cytokine or chemokine production following overnight stimulation with lipopolysaccharide (LPS) (toll-like receptor (TLR) 4 agonist), FSL-1 (TLR2/6 agonist), CpG-ODN2006 (TLR9 agonist), PAM3Cys-Ser (TLR1/2 agonist), CL097 (TLR7/8 agonist), Mannan (DC-SIGN agonist) and Curdlan (Dectin-1 agonist). Cytokines representing Th1/proinflammatory (IFN-γ, IL-12p70, TNF-α and IL-1β), immunoregulatory responses (IL-10) and chemokines/growth factors (IP-10, VEGF and GM-CSF) measured by Luminex® assay are shown for the mothers’ blood (Fig. 1A) and for infants’ cord blood (Fig. 1B). Data presentation was performed using GraphPad Prism.
Fig. 2
Fig. 2
Scatterplots of first and second factor loadings for maternal and cord blood, derived from Principal Component Analysis of 17 analytes, showing cytokines and chemokines (A and C), individual mothers (B) and neonates (D). For mothers, the first principal component (PC) was characterized by a mixture of cytokines and the second PC consisted of chemokines. For neonates, the first PC was characterized by proinflammatory cytokines and the second PC consisted of chemokines, based on factor loadings >0.1. Red circles represent BCG scar-positive (Scar+) mothers and their infants. BCG scar-negative (Scar−) mothers and their infants are represented by blue triangles. One infant had overall high background responses (unstimulated samples) for most cytokines/chemokines measured. Subtracting the unstimulated values from antigen stimulated values gave overall low net values, thus the negative PC scores (−6.311 for PC1 and −6.228 for PC2). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3
Fig. 3
The association between maternal LTBI, maternal BCG scar and the innate immune responses in mothers and neonates. PCA was used to assess the association between maternal LTBI, maternal BCG scar and infant responses. The association between maternal LTBI and infant innate responses (A and B), and the association between maternal BCG scar and maternal (C and D) and infant (E and F) responses are shown. Two PCs that explained 43% and 53% of the variance in the dataset for mothers and neonates, respectively, were identified. The box plots represent the median and the interquartile range of the levels of the two PCs. The whiskers show the minimum and maximum values. P values are from Wilcoxon rank sum test.
Fig. 4
Fig. 4
Cluster analysis of the innate cytokines and chemokines using the average linkage distance between clusters using R. Clusters go from root to leaf node for each cytokine and for the individual infants. Clusters in between are based on their agglomerative value. The branch shows the similarity, the shorter the branch, the more similar. Expression levels of individual cytokines (log10 [pg/ml]) are represented by shades of blue to red based on their correlations according to the dendrogram on the left, with highest values in dark red and the lowest in dark blue. Three distinct sets of correlated cytokines “clusters” are indicated as C1, C2 and C3 on the left. In addition, eleven cytokines (C4) form a cluster that has mainly inflammatory cytokines. Most infants of mothers with a BCG scar (top, green) clustered together in one discrete group, distinct from infants of mothers without a BCG scar (top, light blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5
Fig. 5
Gene Set Enrichment Analysis for the comparison of infants of mothers with and without LTBI. A checkerboard map showing top enriched pathways on y-axis and top leading edge genes (gene members contributing most to the enrichment score) on the x-axis. Scale at the right represents the gene expression fold change (log2 (exposed/unexposed)). Red (blue) indicates genes that are up-regulated (down-regulated) among infants of mothers with LTBI mothers. Interferon and inflammation response pathways were up regulated in infants of mothers with LTBI at six weeks. FDR adjusted p-value cut off of <0.25 was applied for pathways significance. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6
Fig. 6
Gene Set Enrichment Analysis for the comparison of infants of mothers with and without a BCG scar. A checkerboard map is presented showing top enriched pathways on y-axis and top leading edge genes (gene members contributing most to the enrichment score) on the x-axis. Scale at the right represents the gene expression fold change (log2 (scar+/scar−). Red (blue) indicates genes that are up-regulated (down-regulated) among infants of scar-positive mothers. Interferon and inflammation response pathways are up regulated in infants of mothers with a BCG scar at one and six weeks after BCG immunisation. FDR adjusted p-value cut off of

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