Levels of Total Antioxidant Capacity,sCD14,and TGF-β2 in Breast Milk Plasma of HIV-Infected and HIV-Uninfected Lactating Women

Abstract

Introduction:

Breast milk provides nourishment for infants and nonnutritive bioactive factors, which possess key protective and developmental benefits essential in shaping the infant immune system. However, the impact of human immunodeficiency virus (HIV) and universal antiretroviral therapy (ART) on breast milk nutritional composition and immunity status is not well documented.

Objective:

The study aimed to compare breast milk immune factors; total antioxidant capacity (TAC), soluble cluster of differentiation 14 (sCD14), and transcription growth factor-beta 2 (TGF-β2) levels between HIV-infected and HIV-uninfected lactating mothers and determine the association between breast milk parameters with HIV disease progression and duration of ART.

Methods:

Breast milk sCD14, TAC, and TGF-β2 were quantified using enzyme-linked immunosorbent assays and spectrophotometric techniques in 57 HIV-infected breast feeding mothers on option B⁺ therapy for prevention of vertical transmission of HIV and 57 HIV-uninfected mothers at 6 weeks postpartum. The plasma HIV viral load was measured on enrollment and demographic data were recorded.

Results:

Mean breast milk plasma TAC levels were significantly lower in HIV-infected mothers (1,250.5 ± 280.4 μmolTE/L) compared to the HIV-uninfected participants (1,915.4 ± 326 μmolTE/L; p < 0.001). Soluble CD14 levels in HIV-infected mothers were significantly higher (7,059.3 ± 1,604.7 ng/mL) compared to the HIV-uninfected group (5,670.7 ± 1,268.3 pg/mL; p < 0.001). Similarly, TGF-β2 concentration was also significantly elevated in the HIV-infected mothers (1,426.1 ± 695.4 pg/mL) compared to the HIV-uninfected counterparts (709.2 ± 196.8 pg/mL; p < 0.001). A positive correlation was observed between breast milk plasma sCD14 concentration and the plasma viral load (r = 0.576, p < 0.001), while a significant negative correlation was observed with the duration of ART (r = −0.285, p = 0.032). TAC and TGF-β2 concentrations were inversely correlated with plasma viral load levels.

Conclusion:

HIV-infected mothers are at risk of oxidative stress. Nutritional intervention with antioxidant rich foods is recommended for this vulnerable group during breastfeeding.

Introduction

Breastfeeding is recommended for all infants as an essential source of multiple factors acting synergistically to promote healthy immune responses, nutrition and protection against infection for the developing infant.¹ Breastfeeding offers protection against respiratory and gastrointestinal infections and significantly reduces the risk of inflammatory diseases such as asthma, obesity, inflammatory bowel disease, atopy, and diabetes.² The World Health Organization (WHO) currently recommends exclusive breastfeeding for the first 6 months before introducing solids to prevent postnatal human immunodeficiency virus (HIV) transmission.³ The relationship between HIV/antiretroviral therapy (ART) and systemic humoral factors is well documented but documentation on the impact on immunological quality and micronutrient status of breast milk is scarce.

Several cytokines, soluble receptors, and immunomodulatory factors have been identified in breast milk.⁴ Most of these factors are derived from the epithelial cells of the mammary gland or from immune cells found in the milk⁵ while others are transferred from the mother's circulation. Soluble cluster of differentiation 14 (sCD14), transcription growth factor-beta (TGF-β), interleukin (IL)-6, and IL-10 are reported to play a critical role on oral tolerance development⁶ while other soluble receptors and cytokines are of potential importance.

Breast milk contains all the isoforms of TGFβ, with TGFβ2 being the most dominant.⁷ Animal models have demonstrated the protective effects of TGFβ on gut mucosal inflammation through promotion of IgA production and induction of oral tolerance.^4,8 In a previous study of pediatric patients with Crohn's disease, supplementation with TGF-β resulted in reduced mucosal IL-1 mRNA and clinical remission in 79% of patients.^9,10 In preterm infants, reduced TGF-β levels preceded development of necrotizing enterocolitis, highlighting the significance of TGF-β in inflammation and immune regulation.⁴ Increased IL-10 or TGF-β is associated with HIV-1 disease progression and likely influences the antiviral immune responses.¹¹

Breast milk contains important and essential antioxidant composition to prevent and protect against diseases in infants. Previous studies have shown that breast milk is able to suppress the effects of oxidative stress and oxidative DNA damage in newborns compared to formula milk can.¹² It has been suggested that all the problems affecting infants, especially those born prematurely, are as a result of oxygen radical disease.¹³ Excess production of free radicals and underproduction of antioxidants, results in development of oxidative stress, which may cause serious damage in infancy.

Enzymes with antioxidant effects include superoxide dismutase and catalase, which prevent the formation of radicals and other peroxides, as well as glutathione peroxidase, which catalyses the reduction of different peroxides, have been demonstrated in milk. In addition, human milk contains nonenzymatic antioxidants such as vitamin C, lactoferrin, and vitamin E.¹⁴ Therefore, maintaining the balance between free radical production and antioxidant defense systems within the body has important oral and systemic health implications. In the context of HIV infection, changes in levels of antioxidants in people living with HIV is usually attributed to nutrient malabsorption, increased nutrient expenditure, urinary loss, loss of appetite, and complex metabolic alterations that result in severe weight loss and muscle wasting.¹⁵

Soluble CD14 is among the innate immunity factors present in breast milk. It is part of a complex that recognizes lipopolysaccharide, a main component of gram-negative bacterial cell envelope.¹⁶ The majority of sCD14 resides on monocytes and other immune cells, but it can also be found in soluble form in human serum and body secretions. Jones et al., reported an association between reduced sCD14 levels in breast milk 3 months postpartum with development of eczema in infants after 6 months.¹⁷ Serum sCD14, is one of the clinically relevant biomarkers in microbial translocation and HIV-associated immune activation.¹⁸ Previous studies have indicated that children affected by HIV have poor intestinal integrity, increased microbial translocation, and monocyte activation.¹⁹ The present study determined the levels of Total Antioxidant capacity (TAC), sCD14, and transcription growth factor-beta 2 (TGF-β2) in the breast milk plasma of HIV-infected and -uninfected lactating Zimbabwean mothers.

Methods

Study participants

A cross sectional study of 114 HIV breast feeding women enrolled in a prevention of mother to child transmission program, receiving universal ART and HIV-uninfected lactating women, was conducted. The participants were stratified equally by HIV infection status thus, HIV-infected (n = 57) and HIV-uninfected (n = 57). Participants were recruited from Gweru district polyclinics in Zimbabwe at 6 weeks postdelivery after obtaining written informed consent from each participant.

On recruitment, participants were subjected to HIV antibody testing as per national testing algorithm using Determine test kits (Abbott Diagnostics, Abbott Park, IL) to confirm infection status. HIV-negative mothers were retested after a further 6 weeks using the same protocol to detect possible seroconversion. Venous blood (5 mL) was collected from HIV-infected mothers for determination of baseline plasma HIV RNA viral load. Matured breast milk samples were collected by manual expression into labeled sterile conical tubes from all the participants. Breast milk plasma was used for determination of TAC, while milk serum obtained after centrifugation at 1006 g was used for quantification of sCD14 and TGF-β2. Milk samples were stored at −80°C pending analysis.

Information regarding maternal HIV status, antiretroviral use, duration, and regimen was obtained and recorded. Anthropometric measurements, including mid-upper arm circumference (MUAC), height, and weight, were recorded at enrollment. Ethical approval was granted by the Medical Research Council of Zimbabwe (MRCZ/A/2466) before commencement of study.

Determination of sCD14 and TGF-β2

TGF-β2 and sCD14 concentrations were measured in breast milk using Quantikine enzyme-linked immunosorbent assays (R&D Systems, Minneapolis) following the manufacturer's instructions. All assays were run in duplicate. The lower detection limits for TGF-β2 and sCD14 assays were 7 and 0.125 pg/mL, respectively.

Determination of TAC

Ferric reducing antioxidant power assay was used to determine breast milk total antioxidant potential.²⁰ The principle of the method is based on the reduction of the iron III-2,4,6-tripyridyl-s-triazine (Fe3+-TPTZ) complex to the ferrous form. The absorbance of the reaction mixture was measured at 593 nm and the reducing capacity expressed as μmol of Trolox-equivalents/L. The TAC in each sample was obtained by plotting a standard curve against absorbance values of serial standard samples.

Determination of HIV viral load

Maternal plasma was quantified for HIV-1 RNA load using an automated Roche COBAS^® TaqMan^® 96 Analyzer (Roche Diagnostics Ltd, Rotkruez, Switzerland) with a lower detection limit of 20 copies/mL.

Statistical analysis

Statistical analysis was performed using STATA version 14.2 software (Stata Corporation, College Station, TX) and GraphPad Prism 7 (GraphPad Software, Inc., CA). Demographic, laboratory, and clinical characteristics were compared between HIV-infected and HIV-uninfected lactating mothers using the Student's t-test. Pearson's correlation coefficient was used to assess the association between the breast milk immunological parameters and the plasma viral load/duration of ART. A p-value <0.05 was considered to be statistically significant.

Results

Demographic and clinical characteristics of HIV-infected and HIV-uninfected lactating mothers

Demographic and clinical characteristics of the participants are shown in Table 1. The mean age of HIV-infected mothers was significantly higher than that of HIV-uninfected mothers. However, there were no significant differences in terms of weight, height, and MUAC between groups by HIV status. Of the HIV-infected mothers, 50 (87.8%) were on Tenolam E ART regimen, while only 7 (12.3%) were on Tenolam N. On the contrary, 10.5% of the participants had viral load levels >1,000 copies/mL while 89.5% had viral loads <1,000 copies/mL. The median duration on ART was 48 months, interquartile range (IQR): 8–60 months (Table 1).

Table 1.

Demographic and Clinical Characteristics of Participants

Characteristic	HIV-infected (n = 57)	HIV-uninfected (n = 57)	p
Mean age (years) ± SD	30.6 ± 5.4	26.5 ± 5.9	<0.001
Mean weight (kg) ± SD	64.2 ± 12.6	63.4 ± 12	0.728
Mean height (cm) ± SD	163.3 ± 5.8	164.3 ± 5.9	0.366
Mean MUAC (cm) ± SD	26.4 ± 3.4	26.2 ± 3.6	0.847
ART regimen, n %
TDF+3TC+EFV600: Tenolam E	50 (87.8)	NA	NA
TDF+3TC+NVP: Tenolam N	7 (12.3)	NA	NA
Viral load copies/mL, median (IQR)	56 (0–215)	NA	NA
Viral load >1,000 copies/mL, n %	6 (10.5)	NA	NA
Duration of ART in months, median (IQR)	48 (8–60)	NA	NA

3TC, lamivudine; ART, antiretroviral therapy; EFV600, Efavirenz 600 mg; HIV, human immunodeficiency virus; IQR, interquartile range; MUAC, mid-upper arm circumference; NA, nonapplicable; NVP, Nevirapine; SD, standard deviation; TDF, tenofovir disoproxil fumarate.

Breast milk plasma concentrations of TAC, sCD14, and TGF-β2 in HIV-infected and HIV-uninfected groups

The mean (standard deviation [SD]) breast milk plasma TAC in HIV-infected participants (1,250.5 ± 280.4 μmolTE/L) were significantly lower compared with HIV-uninfected participants (1,915.4 ± 326 μmolTE/L). In contrast, mean (SD) sCD14 levels in HIV-infected mothers were significantly higher (7,059.3 ± 1,604.7 ng/mL) compared to the HIV-uninfected group (5,670.7 ± 1,268.3 pg/mL). The mean (SD) breast milk plasma levels of TGF-β2 were also significantly elevated in the HIV-infected group (1,426.1 ± 695.4 pg/mL) compared to the HIV-uninfected group (709.2 ± 196.8 pg/mL) (Table 2).

Table 2.

Mean Levels of Breast Milk Total Antioxidant Capacity, Soluble Cluster of Differentiation 14, and Transcription Growth Factor-Beta 2 in Human Immunodeficiency Virus-Infected and -Uninfected Mothers

Biochemical parameter	HIV-infected (n = 57)	HIV-uninfected (n = 57)	t	p
Biochemical parameter	Mean ± SD	Mean ± SD	t	p
TAC (μmolTE/L)	1,250.5 ± 280.4	1,915.4 ± 326	−11.7	<0.001
sCD14 (ng/mL)	7,059.3 ± 1,604.7	5,670.7 ± 1,268.3	5.1	<0.001
TGF-β2 (pg/mL)	1,426.1 ± 695.4	709.2 ± 196.8	−7.5	<0.001

sCD14, soluble cluster of differentiation 14; SD, standard deviation; TAC, total antioxidant capacity; TE, trolox equivalent; TGF-β2, transcription growth factor beta 2.

Breast milk plasma correlations of TAC, sCD14, and TGF-β2 with plasma HIV viral load and duration of ART treatment

A significant positive correlation was observed between breast milk plasma sCD14 concentration and the plasma viral load (r = 0.576, p < 0.001), while a significant negative correlation was observed with the duration of ART treatment (r = −0.285, p = 0.032). However, TAC (r = −0.118, p = 0.384) and TGF-β2 (r = −0.130, p = 0.334) concentrations were inversely correlated with plasma viral load levels. In addition, TAC (r = 0.091, p = 0.502) and TGF-β2 (r = −0.030, p = 0.825) concentrations were not associated with the duration of ART treatment (Table 3).

Table 3.

Correlation of Breast Milk Total Antioxidant Capacity, Soluble Cluster of Differentiation 14, and Transcription Growth Factor-Beta 2 with Plasma Human Immunodeficiency Virus Viral Load and Duration of Antiretroviral Therapy Treatment

Biochemical parameter	Plasma viral load		Duration of ART
Biochemical parameter	r-values	p	r	p
TAC (μmolTE/L)	−0.118	0.384	0.091	0.502
sCD14 (ng/mL)	0.576	<0.001	−0.285	0.032^*
TGF-β2 (pg/mL)	−0.130	0.334	−0.030	0.825

Statistically significant at p < 0.05.

ART, antiretroviral therapy; sCD14, soluble cluster of differentiation 14; TAC, total antioxidant capacity; TE, trolox equivalent; TGF-β2, transcription growth factor beta 2.

Discussion

In the current study, significant differences were observed in breast milk immunity profiles between HIV-infected and HIV-uninfected lactating mothers in the era of prolonged ART. Elevated sCD14 and TGF-β2 levels were observed in HIV-infected subjects as opposed to uninfected counterparts. Systemic immune activation and inflammation are critical features of chronic HIV infection and have been associated with HIV disease progression and subsequent HIV-related morbidity and mortality among adults.^18,21

ART has demonstrated success in suppressing virus replication, extending lifespan, and improving standard of life for people living with HIV, but has failed to completely eliminate chronic immune activation and inflammation.^22,23 Significantly higher sCD14 levels in HIV-positive patients compared to HIV-negative controls have also been observed in other studies.²⁴ In a recent study, elevated sCD14 levels in breast milk 3 months postpartum were associated with development of an IgE-mediated allergic disorder.²⁵ However, in another study, reduced breast milk sCD14 levels at 3 months postpartum increased the risk of development of eczema in 6 months old infants.¹⁷ In addition, allergic symptoms were evident by 4 years of age where low sCD14 levels were observed in mother's colostrum.²⁶ The differences observed in previous studies could be due to variations in time points of breast milk collections as well as variation in follow-up periods. Generally, both serum and breast milk concentrations of sCD14 tend to be higher in atopic mothers than nonatopic mothers.²⁷

In this study, high milk sCD14 concentrations among the HIV-infected participants correlated positively with plasma HIV viral load and correlated negatively with the duration of ART treatment. Similarly, in a study conducted in Malawi, increased plasma and breast milk levels of sCD14 were associated with a compromised maternal immunological status and lower infant birth weight.²⁸ However, a study conducted in Australia revealed no significant influence of plasma sCD14 on virological status below 10,000 copies/mL.²⁹ In contrast, a study conducted in Zimbabwe, found no significant correlation of plasma sCD14 with either plasma viral load or CD4 count.³⁰

Numerous lines of evidence have indicated that HIV infection triggers pronounced oxidative stress. HIV-induced oxidative stress plays an important role in the development of a wide spectrum of virus-associated pathologies such as neurotoxicity, lung, and cardiovascular disorders. HIV-positive individuals exhibit enhanced reactive oxygen species production, in monocytes and severely increased concentrations of oxidized nucleic bases and lipid peroxidation products.^31–33 In the current study, breast milk TAC in HIV-infected mothers was significantly lower than that of HIV-uninfected counterparts. This is supported by a Nigerian study, which exhibited the same pattern of oxidative stress by maternal HIV status.³⁴ However, a similar, although statistically insignificant, reduction of TAC was observed in maternal plasma of the HIV-infected lactating mothers. These observations were attributed to reduced concentrations of antioxidant trace metals in milk such as copper and iron. In a previous observational study, HIV-positive groups taking ART for above or below 5 years showed higher concentrations of malondialdehyde and glutathione peroxidase activity and reduced concentrations of glutathione than the HIV-negative control group.³¹

In the current study, elevated breast milk levels of TGF-β2 were observed in HIV-infected mothers compared to HIV-uninfected counterparts. A previous study demonstrated the bifunctional role of TGF-β in mother to child transmission of HIV-1 infection where it favored HIV-1 infection of macrophages, but suppressed that of mammary epithelial MCF-7 cells.³⁵ Therefore, it can be assumed that the witnessed elevated TGF-β2 levels under universal ART account for the current reduction in HIV postnatal infant infection rates as a result of the HIV infection suppressive effects. Furthermore, it can be speculated that persistent inflammation associated with HIV infection can trigger a counter regulatory anti-inflammatory response that increases maternal production of TGF-β2. This is further supported by the inverse correlation observed between the cytokine and the HIV viral load in the current study.

Therefore, further studies are required to better understand the interaction between HIV infection and breast milk cytokines. In infants, increased levels of TGF-β2 in breast milk were reportedly related to higher risk of development eczema.³⁶ However, in a review by Oddy and Rosales,¹⁹ high levels of TGF-β2 in breast milk were inversely correlated with the incidence of allergic diseases in childhood. The factors influencing the levels of TGF-β in breast milk are not well known. TGF-β levels in breast milk often differ between countries and races.^37,38

Conclusion

Oxidative stress is a key feature of HIV infection and therefore highlights the need for nutrient supplementation particularly antioxidant rich foods during breast feeding.

Footnotes

Disclosure Statement

No competing financial interests exist in this study.

Funding Information

No funding was received for this research.

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