Differences in the innate immune environment of human breast milk of HIV-infected and -uninfected mothers in Tshwane, South Africa

Abstract

The human immunodeficiency virus (HIV) has long been considered one of the most devastating diseases in human history, killing an estimated 35.4 million people to date. Nonetheless, the number of newly reported HIV cases has decreased significantly since the implementation of antiretroviral treatment. Over the past few decades, various prevention of mother-to-child transmission strategies, including treatment of the mother and prophylaxis of the neonate, have been implemented in an effort to decrease the number of children who are infected due to virus particles spreading from mother to baby. Although there has been a decrease in the number of children newly infected with HIV, there is also a significant increase in the number of children who are exposed to HIV but remain uninfected. It is becoming increasingly apparent that these infants face other complications, thought to be associated with suboptimal transfer of immune factors from mothers during pregnancy and breastfeeding. These complications include diseases such as diarrhoea, pneumonia, infection with Mycobacterium tuberculosis and other opportunistic organisms. The aim of this study was to determine whether differences exist in the innate immune environment of human breast milk between HIV-infected and HIV-uninfected mothers. This was achieved by performing different ELISA tests (multiplex and sandwich) on breast milk samples obtained from an umbrella study, Siyakhula. Various cytokines and chemokines (interleukin [IL]-6, IL-8, IL-10, IL-12p70, IL-1β, tumour necrosis factor-alpha [TNF-α], transforming growth factor-beta 1 [TGF-β1] and soluble cluster of differentiation 14 [sCD14]), defensins (Def) α and β, immune checkpoint molecules (T-cell Ig and mucin domain-3 protein [TIM]-3, lymphocyte-activation gene [LAG]-3, programmed cell death protein [PD]-1, cytotoxic T-lymphocyte-associated protein [CTLA]-4, programmed cell death ligand [PDL]-1] and key innate immune factors (lactoferrin, lysozyme and tenascin C [TNC]) were evaluated. HIV-infected mothers were significantly older and their infants had a lower gestational age at birth when compared to HIV-uninfected mothers. Levels of the pro-inflammatory cytokines, IL-12p70 and IL-1β, were higher in mothers who were HIV-uninfected in comparison to mothers who were HIV-infected. No significant differences were detected in the other cytokines, defensins, lactoferrin, lysozyme, or immune checkpoint molecules. Although these differences missed statistical significance, more HIV-infected mothers had levels of TNC in the lower quartile, while the three mothers with levels of the ICM CTLA-4, in the highest quartile, were all HIV-infected. All of these findings lead back to HIV-infected mothers having a lower inflammatory profile. In addition to the primary objectives, exploratory analysis of clinical biomarkers showed significant differences when looking at gestational age at birth, maternal weight, infant weight, and placental weight. It was found that mothers who gave birth at and below 37 weeks of gestation were older and gave birth to lower birthweight infants. It was also seen that maternal weight as well as placenta weight was lower in mothers who gave birth at and below 37 weeks. When assessing gestational age at 38 weeks, it was again seen that mothers with babies with a gestation age at birth at and below 38 weeks were older and gave birth to lower birthweight infants. Maternal weight was positively correlated with a higher Mid-upper arm circumference (MUAC), placental weight as well as sCD14 and PD-L1 concentration in the breast milk. Infant weight was dichotomised into two categories: firstly, a birth weight of 2,500 g and below was associated with a lower gestational age at birth, lower placental weight and a higher maternal age. It was also seen that more infants with TIM-3 values in the 4th quartile had a weight at or below 2,500 g. Secondly, when looking at babies born at and below the median weight of 2,890 g, it was seen that this weight category was associated with lower placental weight as well as gestational age at birth. Lastly, it was found that a direct correlation exists between placental weight and maternal weight as well as placental weight and infant weight at birth. Levels of TNC proved to be lower in mothers with a lower placental weight. Correlations between continuous variables showed significant association between HIV status and gestational age at birth, Def-α and IL-1β. In conclusion, the general innate immune environment of breast milk seems to stay relatively constant between HIV-infected and -uninfected mothers if the HIV-infected individuals remain on a stringent antiretroviral (ARV) regimen as the patients in our study did. Some differences in biomarkers were found that were contrary to what was expected: for instance, breastmilk of HIV-infected mothers had lower levels of some pro-inflammatory cytokines despite previous studies demonstrating higher background levels of systemic immune activation. Due to most previous studies being done on blood and not breast milk, further investigation is needed to determine the exact cause of this. It is recommended that breast milk samples should be taken at different time points in order to compare how innate immune factors differ at each specific time point as well as to incorporate more in-depth studies on the individual biomarkers studied.