Abstract
Background:
HIV-exposed uninfected (HEU) infants have increased infectious morbidity and mortality; little is known about their levels of inflammation and monocyte activation.
Methods:
Plasma samples obtained at birth and 6 months from 86 HEU mother-infant pairs enrolled in the NICHD cohorts in Brazil were compared to 88 HIV-unexposed (HU) mother-infant pairs. HIV-infected mothers received antiretroviral therapy during pregnancy, their infants received zidovudine prophylaxis and were not breastfed. IL-6, sTNFR-I and II, sCD14, sCD163, IP-10, VCAM, oxidized LDL, D-Dimer, and hsCRP were assayed by ELISA at birth and at 6 months. sTNF-RI and IL-6 were considered co-primary endpoints.
Results:
Among HIV-infected mothers, 79% had HIV-RNA <400 copies/mL prior to delivery. Compared to HU, HEU infants had a lower mean gestational age (38.7 vs. 39.3wk) and weight (3.1 vs. 3.3kg); and reached lower weight (5.9 vs. 8.5kg) and height (53.6 vs. 68.8 cm) at 6 months. With the exception of VCAM, inflammatory markers were generally higher (p≤0.005) in HEU at birth, but at 6 months only sTNFR-I and IL-6 remained higher. For HEU pairs, only IP-10 was associated with maternal levels at birth (p<0.001). In HEU, elevated levels of hsCRP and IP10 at birth were associated with lower weight at birth (p=0.04) and at 6 months (p=0.04).
Conclusions:
HIV-exposed infants have heightened inflammation and monocyte activation at birth, which for some markers persisted to 6 months of life and was not related to maternal inflammatory status. Inflammation may contribute to the increased HEU infectious morbidity and poor growth.
Keywords: HIV-exposed uninfected infants, inflammation, immune activation, morbidity, oxidative stress, growth
Introduction
Following WHO 2013 recommendations for immediate and lifelong initiation of antiretroviral therapy (ART) in all pregnant women infected with HIV, or option B+, 76% of all pregnant women living with HIV globally in 2016 were receiving ART to prevent maternal to child transmission (PMTCT)[1]. As a result of option B +, new HIV infections among infants have decreased significantly, while the number of HIV-exposed uninfected infants (HEU) has steadily increased[1].
Numerous studies have reported adverse health outcomes in HEU children including higher mortality [2], mitochondrial toxicity[3], infectious disease complications such as diarrheal disease, respiratory infections and bacterial sepsis[4], growth[5, 6], neurodevelopmental outcomes[7] and altered immunity[8]. We have previously shown increased infectious disease morbidity in HEU children from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) International Site Development Initiative (NISDI) Perinatal cohort in Latin-America[9]. This increased mortality and morbidity in HEU is undermining the efforts in PMTCT. As highlighted by Evans et al, the causes of increased adverse outcomes are likely multifactorial including socioeconomic factors, infant feeding practices, prolonged exposure to ART, co-infections and immune activation[10].
Our group and others have shown that ongoing systemic inflammation and immune activation are independently associated with co-morbidities in HIV-infected adults and children, despite virologic suppression[11-13]. Drivers of inflammation in HIV-infected adults include altered gut integrity[14], oxidized lipids[15] and viremia[16]. Multiple immunologic abnormalities have been described in HEU compared to HIV uninfected infants (HU), including impaired T and B cell activation and differentiation[12, 17-22], but little is known about the long-term effects of exposure to HIV and ART and whether HEU infants have higher levels of inflammation and monocyte activation compared to HIV uninfected infants (HU).
In this study, we used stored plasma samples from HEU mother-infant pairs enrolled in the NISDI Perinatal cohort and the Longitudinal Study in Latin American Countries (LILAC) cohort in Brazil and compared them to samples from HU mother-infant pairs from the Correlações Imunes De Infecções Respiratórias Agudas (CIRAI) cohort in Brazil. The primary objectives of this sub-study were to determine whether selected markers of systemic inflammation, monocyte activation and oxidized lipids were different between HEU and HU infants at birth and 6 months, and to correlate biomarkers in infants to those of their mothers at the time of delivery. Lastly, we sought to describe the relationship among markers of inflammation and immune activation with those of respiratory tract infections, maternal immunologic and virologic status, and infants’ growth parameters. Our primary hypothesis is that at birth and at 6 months of life, HEU infants will have higher markers of inflammation when compared to levels in HU infants. Additionally, we assessed markers in HIV-infected mothers to determine whether their heightened inflammation was associated with inflammatory responses in the newborn period and beyond.
Methods
Study Design:
The NISDI and LILAC studies have been previously described [9, 23, 24]. Briefly, NISDI was a multi-center prospective observational cohort of HIV-infected pregnant women and their offspring, regardless of infection status conducted at clinical sites in Latin America. CIRAI was a cohort study that assessed correlates of lower respiratory infections in the first 6 months of life of Brazilian infants born to healthy mothers, as compared to those born to HIV-infected mothers.
Study population:
For the purpose of this sub-study, HEU participants in the NISDI Perinatal and LILAC protocols were included if they were born in Brazil, were the product of a singleton pregnancy and had available plasma samples from birth and 6 months of age, as well as from their mothers close to delivery. Infants were considered HIV uninfected if they had at least two negative HIV virologic assays with one test performed at ≥1 month and one at ≥4 months of age, or if they had two negative HIV antibody test results, including at least one negative test results after 6 months of age. Additional inclusion criteria included birth weight ≥ 2500 grams and gestational age of ≥37 weeks. Infants were excluded if they had birth defects, cardiovascular or pulmonary disease.
In the CIRAI study, participants included infants who were born to HIV-uninfected mothers and were receiving some artificial milk feedings or had been weaned by 4 weeks of age.
Study evaluation
In the NISDI cohort, study visits included a medical history, a physical examination and collection of laboratory samples. Women eligible to participate had access to antiretrovirals and their infants had access to formula feeding. In the NISDI Perinatal cohort, women were enrolled during pregnancy, at 8 weeks gestation or later, and followed through delivery and postpartum. Their children had study visits at birth, 6-12 weeks of age and at 6 months of age. In the LILAC cohort, women were enrolled after 22 weeks of pregnancy and followed through delivery and postpartum. All subjects (women and their infants) were followed every six months thereafter, for up to five years after delivery. During these visits, a medical history was obtained, physical examination performed, and blood samples collected, and plasma stored at −70C until analysis without prior thaw.
In the CIRAI cohort, medical history, physical examination and laboratory evaluations were performed at birth and at 6 months of age. Maternal peripheral blood and cord blood were obtained. Plasma samples were stored at −70C until analysis without prior thaw.
Inflammation, coagulation and soluble immune activation markers
The specific biomarkers selected are markers of immune activation, inflammation and coagulation which are hallmarks of HIV infection. These markers either correlate with non-AIDS co-morbidities or cardiometabolic complications, or drive other hallmarks of immune dysregulation in HIV. Interleukin 6 (IL 6), soluble TNFα receptor I and II (sTNF-RI and sTNF-RII) and D-dimer were selected as they are robust independent predictors of non-AIDS morbidities in HIV-infected subjects virologically suppressed on ART, even after adjusting for factors such obesity and smoking[25]. Soluble CD14 (sCD14), a soluble marker of monocyte activation, was measured as it has been independently associated with mortality and progression of atherosclerosis[26]. Another marker of monocyte activation, soluble CD163 (sCD163), was found by our group to be independently associated with insulin resistance in HIV-infected Ugandan children on ART. We have shown that oxidized LDL (oxLDL) upregulates monocyte activation in HIV[26], making oxLDL a potentially important mediator on the causal pathway of monocyte activation. Interferon gamma induced protein 10 (IP-10), on the other hand, has been associated with T cell activation[27].Lastly, soluble vascular cell adhesion molecule (sVCAM), a marker of vascular inflammation and cardiovascular risk, is elevated in HIV-infected children[28].
Plasma markers of monocyte activation (sCD14), systemic inflammation (sTNF-RI and sTNF-RII), high sensitivity C-reactive protein (hsCRP), IL-6, sCD163, sVCAM, IP-10and oxidized LDL were measured by ELISA (R &D Systems, Minneapolis, Minnesota, USA and ALPCO, Salem, New Hampshire, USA and Mercodia, Uppsala, Sweden). The marker of coagulation D-Dimer was measured using Asserachrom D-DI immunoassay (Diagnostica Stago, Parsippany, NJ, USA). All markers assays were done at Dr. Funderburg’s laboratory at Ohio State University, Columbus, OH.
Statistical Analysis:
The primary objective was to compare markers of inflammation at birth and at 6 months of life between HIV exposed uninfected and HIV-unexposed healthy infants. A priori, we selected sTNF-RI and IL-6 as co-primary endpoints.
The sample size calculation was based on effect size since data on markers of inflammation and monocyte activation are not available for the studied age group. For a moderate effect size of 0.5, 86 subjects per groups were needed to achieve 90% power. Effect size of 0.5 corresponds to a difference of 407 pg/ml for sTNF-RI with standard deviation 813, and 681 ng/ml for sCD14 with standard deviation 1361. A two-sided two-sample t-test was used with a significance level of 0.05.
Descriptive statistics including mean, standard deviation, and frequency/proportion were used to characterize laboratory testing results of markers of inflammation and monocyte activation, as well as other study variables. For differences in markers and other study variables between HEU and HU subjects, continuous variables were compared using two sample t-tests, and categorical variables were compared using chi-square tests. Correlation coefficients were calculated for associations between biomarkers. Linear regression models were used to compare the difference in biomarkers between groups, adjusting for maternal biomarkers and HIV-related variables. Linear and logistic regression modeling was also used to examine the effects of the markers on infant growth parameters and clinical events (lower respiratory tract infections and hospitalizations).
Results
Baseline characteristics
Detailed demographic information and maternal characteristics of the complete NISDI and LILAC cohorts have been published previously [23, 24, 29]. Overall, most women were enrolled in Brazil and Argentina and 99% were on ART during pregnancy. The maternal to child transmission was 1%. For this particular sub-study, 86 HEU mother-infant pairs and 88 HU mother-infant pairs met the inclusion criteria and were included. As seen in Table 1, compared with HIV-uninfected mothers, HIV-infected mothers were older, more likely to be of black race, and underwent more elective C-sections (p≤0.02). HEU infants had lower gestational age, as well as lower height and weight at birth which persisted at 6 months of age (p<0.0001).
Table 1:
Baseline Characteristics
| Baseline Characteristic | Total (N=174) |
HEU (N=86) |
HU (N=88) |
p-value* |
|---|---|---|---|---|
| MOTHERS | ||||
| Mean (SD) maternal age at delivery (years) | 26.6 (6.2) | 27.7 (6.3) | 25.5 (5.9) | 0.02 |
| Maternal race: n (%) | ||||
| Black | 31 (17.8) | 24 (27.9) | 7 (8.0) | <0.0001 |
| Mestizo | 5 (2.9) | 5 (5.8) | 0 (0.0) | |
| White | 97 (55.7) | 31 (36.0) | 66 (75.0) | |
| Other | 20 (11.5) | 5 (5.8) | 15 (17.0) | |
| Unknown | 21 (12.1) | 21 (24.4) | 0 (0.0) | |
| Substance use during pregnancy: n (%) | ||||
| Alcohol | 18 (10.3) | 9 (10.5) | 9 (10.2) | 1.00 |
| Tobacco | 23 (13.2) | 11 (12.8) | 12 (13.6) | 1.00 |
| Marijuana | 1 (0.6) | 1 (1.2) | 0 (0.0) | 0.49 |
| Cocaine/crack | 2 (1.1) | 2 (2.3) | 0 (0.0) | 0.24 |
| Heroin/opiate | 0 | 0 | 0 | -- |
| Any substance | 34 (19.5) | 18 (20.9) | 6 (18.2) | 0.70 |
| Mode of delivery: n (%) | ||||
| Elective C-Section | 50 (28.7) | 36 (41.9) | 14 (15.9) | 0.0003 |
| Non Elective C-Section | 28 (16.1) | 14 (16.3) | 14 (15.9) | |
| Vaginal | 96 (55.2) | 36 (41.9) | 60 (68.2) | |
| HIV variables: | ||||
| Mean (SD) CD4+ cell count (cells/μL) at hospital discharge | 509 (263) | |||
| Mean (SD) viral load (log 10 copies/mL) at hospital discharge | 2.25 (0.96) | |||
| Viral load <400 copies/mL at hospital discharge: n (%) | 68 (79.1) | |||
| ART use during pregnancy: n (%) | 86 (100) | |||
| Combination ART regimen with protease inhibitor: n (%) | 54 (62.8) | |||
| Combination ART regimen with non-nucleoside reverse transcriptase inhibitor: n (%) | 20 (23.3) | |||
| INFANTS | ||||
| Gender: n (%) | ||||
| Female | 92 (52.9) | 49 (57.0) | 43 (48.9) | 0.29 |
| Male | 82 (47.1) | 37 (43.0) | 45 (51.1) | |
| Apgar scores (at 1 min): n (%) | ||||
| <7 | 21 (12.2) | 8 (9.5) | 13 (14.8) | 0.36 |
| ≥7 | 151 (87.8) | 76 (90.5) | 75 (85.2) | |
| Mean (SD) gestational age at birth (weeks) | 39.0 (1.4) | 38.7 (1.3) | 39.3 (1.4) | 0.001 |
| Mean (SD) weight at birth (grams) | 3264 (387.3) | 3144 (346.1) | 3382 (390.9) | <0.0001 |
| Mean (SD) weight at 6 months (grams) | 7230 (2144) | 5935 (2101) | 8495 (1244) | <0.0001 |
| Mean (SD) height at enrollment (cm) | 50.5 (3.9) | 48.3 (2.0) | 55.5 (2.1) | <0.0001 |
| Mean (SD) height at 6 months (cm) | 61.3 (9.8) | 53.6 (8.6) | 68.8 (2.3) | <0.0001 |
| Mean (SD) white blood count at 6 months (103/mm3) | 10.7 (3.1) | 10.5 (2.8) | 10.8 (3.4) | 0.59 |
P-values obtained from T-test for comparisons between group means and chi-square for comparisons of categorical measures.
Comparison of inflammation and immune activation markers
HIV-infected mothers with viral load ≥ 400 copies/mL (n=17) had significantly higher levels of sCD14, IP 10, sTNF-RII and sCD163 compared to mothers with viral load <400 copies/mL (n=68). Differences in the biomarkers ranged between 0.10-0.25 (p≤0.02). With the exception of D-dimer, markers did not differ significantly at delivery between mothers receiving a PI versus a NNRTI-based ART regimen (p=0.0018).
Compared to HIV-uninfected mothers, HIV-infected mothers at delivery had significantly higher levels of sTNF-RI, IL-6. sCD14 and hsCRP (p≤ 0.001) (Table 2).
Table 2:
Comparison of markers between mothers of HEU and HU infants at delivery
| Marker (in Log10 scale) |
Mean (st.dev) | Difference (95% CI) |
p-value* | |
|---|---|---|---|---|
| HEU (n=86) | HU (n=88) | |||
| sTNF-RI (pg/ml) | 3.04 (0.12) | 2.98 (0.14) | 0.06 (0.02, 0.10) | 0.001 |
| IL-6 (pg/ml) | 0.91 (0.42) | 0.51 (0.41) | 0.40 (0.27, 0.52) | <0.0001 |
| sCD14 (pg/ml) | 3.28 (0.17) | 3.15 (0.11) | 0.13 (0.09, 0.18) | <0.0001 |
| D Dimer (ng/mL) | 3.31 (0.39) | 3.24 (0.31) | 0.07 (−0.04, 0.18) | 0.19 |
| IP10 (pg/mL) | 2.03 (0.39) | 2.08 (0.17) | −0.05 (−0.14, 0.04) | 0.27 |
| Ox LDL (mU/L) | 4.56 (0.17) | 4.75 (0.20) | −0.19 (−0.24, −0.13) | <0.0001 |
| sTNF-RII (pg/mL) | 3.44 (0.20) | 3.43 (0.16) | 0.01 (−0.04, 0.07) | 0.62 |
| VCAM (ng/mL) | 2.96 (0.14) | 2.99 (0.14) | −0.03 (−0.07, 0.01) | 0.17 |
| hsCRP (ng/mL) | 3.86 (0.35) | 3.67 (0.42) | 0.19 (0.07, 0.31) | 0.001 |
| sCD163 (ng/mL) | 2.81 (0.22) | 2.89 (0.19) | −0.09 (−0.15, −0.02) | 0.006 |
P-values obtained from T-test for comparisons between group means.
As highlighted in Table 3, at birth, systemic inflammation markers sTNF-RI, IL-6, IP-10, oxidized LDL, sCD14 and hsCRP were higher in HEU infants (p≤0.001). By 6 months of age, only IL-6 and sTNF-RI remained significantly higher in HEU infants (p<0.005). In order to adjust for differences seen in biomarkers between infected and uninfected mothers, we adjusted for maternal biomarkers and HIV variables at delivery in a linear regression analysis. The same biomarkers at birth remained significantly different between HEU vs HU infants after adjusting for maternal markers (p<0.001).
Table 3:
Comparison of all infant markers according to age
| Marker | Birth | Six Months of Age | ||||
|---|---|---|---|---|---|---|
| Mean (SD) | p- value* | Mean (SD) | p- value* | |||
| HEU | HU | HEU | HU | |||
| sTNF--RI (pg/mL) | 3.32 (0.16) | <0.0001 | <0.0001 | <0.0001 | 2.96 (0.13) | <0.0001 |
| IL-6 (pg/mL) | 0.78 (0.44) | <0.0001 | <0.0001 | <0.0001 | 0.13 (0.51) | <0.0001 |
| D Dimer (ng/mL) | 3.17 (0.47) | 0.56 | 0.56 | 0.0003 | 2.95 (0.27) | 0.0003 |
| IP10 (pg/mL) | 2.06 (0.45) | 0.001 | 0.001 | 0.001 | 2.21 (0.36) | 0.001 |
| Ox LDL (mU/L) | 4.40 (0.28) | <0.0001 | <0.0001 | 0.003 | 4.57 (0.18) | 0.003 |
| sTNF-RII (pg/mL) | 3.64 (0.17) | 0.21 | 0.21 | 0.02 | 3.62 (0.18) | 0.02 |
| VCAM (ng/mL) | 3.22 (0.11) | <0.0001 | <0.0001 | <0.0001 | 3.23 (0.16) | <0.0001 |
| hsCRP (ng/mL) | 3.27 (0.49) | <0.0001 | <0.0001 | 0.16 | 2.93 (0.57) | 0.16 |
| sCD14 (pg/mL) | 3.04 (0.14) | <0.0001 | <0.0001 | 0.03 | 3.26 (0.12) | 0.03 |
| sCD163 (ng/mL) | 2.72 (0.23) | 0.45 | 0.45 | 0.86 | 2.90 (0.21) | 0.86 |
SD = Standard deviation; CI = Confidence interval
P-values obtained from T-test for comparisons between group means.
Association between maternal variables and infant markers
With the exception of IP-10 (p<0.001), maternal markers were not associated with those of HEU infants, but several associations were observed between biomarkers of HU mothers and their infants. IL-6, sTNF-RI and II, sCD14 and sCD163 measures among HU mothers were associated with that of their infants at delivery (p≤0.04).
Few maternal HIV-related variables were associated with infant biomarkers. Maternal CD4+ cell count was only associated with infant sTNF-RII at birth (p=0.03); maternal HIV-RNA was not associated with any infant biomarkers at birth, and only sTNF-RII at 6 months was significantly associated with maternal ART regimen (p=0.004).
Correlation between oxidized LDL and inflammation markers
Since our group found that oxLDL is a main drier of systemic inflammation and monocyte activation in adults[26, 30], we assessed the correlation between oxLDL and measured biomarkers. Oxidized LDL positively correlated with D Dimer, IL-6, sTNF-RI, hsCRP and sCD14, and negatively with VCAM (Table 4).
Table 4:
Correlation among markers of inflammation, immune activation and oxidized lipids in infants
| Correlatio n coefficient (p-value) |
D Dimer | IL6 | IP10 | Ox_L DL_ |
sTNF- RI |
sTNF- RII |
VCAM | hsCRP | sCD14 | sCD163 |
|---|---|---|---|---|---|---|---|---|---|---|
| D Dimer | 0.00 (0.99) |
0.07 (0.36) |
0.05 (0.49) |
0.21
(0.01) |
0.19
(0.01) |
0.16
(0.04) |
0.11 (0.14) |
0.17 (0.03) |
0.09 (0.26) |
|
| IL6 | 0.04 (0.64) |
0.19
(0.01) |
−0.01 (0.86) |
0.10 (0.18) |
0.04 (0.62) |
0.19
(0.01) |
0.34
(<.001) |
0.33
(<.001) |
0.18
(0.02) |
|
| IP10 | −0.10 (0.22) |
0.16
(0.05) |
−0.03 (0.74) |
0.24
(0.002) |
0.31
(<.001) |
0.48
(<.001) |
−0.04 (0.60) |
0.27
(<.001) |
0.13 (0.10) |
|
| Ox_LDL |
0.20
(0.01) |
0.18
(0.02) |
0.14 (0.08) |
−0.12 (0.13) |
−0.01 (0.87) |
−0.08 (0.27) |
−0.00 (0.99) |
0.12 (0.11) |
−0.04 (0.60) |
|
| sTNF-RI | 0.13 (0.11) |
0.37
(<.001) |
0.27
(<.001) |
0.23
(0.002) |
0.33
(<.001) |
0.34
(<.001) |
0.13 (0.08) |
0.19
(0.01) |
0.36
(<.001) |
|
| sTNF-RII | 0.10 (0.20) |
−0.02 (0.76) |
0.08 (0.31) |
0.07 (0.36) |
0.22
(0.004) |
0.46
(<.001) |
0.15 (0.06) |
0.28
(<.001) |
0.26
(<.001) |
|
| VCAM |
0.18
(0.03) |
0.12 (0.12) |
−0.13 (0.09) |
−0.17
(0.03) |
0.08 (0.29) |
0.12 (0.12) |
0.05 (0.51) |
0.25
(<.001) |
0.32
(<.001) |
|
| hsCRP | 0.11 (0.18) |
0.16
(0.03) |
0.31
(<.001) |
0.32
(<.001) |
0.36
(<.001) |
0.14 (0.07) |
−0.03 (0.70) |
0.38
(<.001) |
0.21
(0.01) |
|
| sCD14 | 0.05 (0.55) |
0.14 (0.07) |
0.27
(<.001) |
0.37
(<.001) |
0.27
(<.001) |
0.07 (0.35) |
−0.19
(0.01) |
0.48
(<.001) |
0.32
(<.001) |
|
| sCD163 | −0.06 (0.45) |
0.00 (0.98) |
0.12 (0.11) |
0.03 (0.67) |
−0.01 (0.94) |
0.20
(0.01) |
0.05 (0.53) |
0.20
(0.01) |
0.24
(0.002) |
Pearson correlation coefficients were calculated between all pairs of markers, both at infants’ birth (results reported below diagonal) and the 6 month visit (results reported above diagonal), with HEU and HU infants combined.
Relationship between biomarkers and infant outcomes
Among HEU infants, elevated markers of inflammation at birth were associated with lower weight. Specifically, elevated birth hsCRP and IP10 in infants were associated with lower weight at birth (p=0.04) and at 6 months (p=0.04), respectively.
Except for sTNF-RII at birth in HEU (p=0.02), there was no association between any other markers and odds of lower respiratory tract infection or hospitalization.
Discussion
In this study of Brazilian mother-infant pairs, we found that HIV-exposed infants have higher levels of inflammation, monocyte activation, and oxidized LDL when compared to levels in unexposed infants at birth and some inflammation markers remained higher at 6 months of life. Importantly, this higher inflammation milieu is not merely the result of passive maternal transfer. Lastly, we found associations between biomarkers and HIV-exposed infants’ impaired growth.
Several hypotheses have been proposed relating to the causes of increased infectious disease complications and impaired growth in HEUs compared to HUs, including epigenetic changes, HIV viral particles, ART, maternal immune suppression and activation, reduced maternal antibody transfer, distorted maternal microbiota, maternal co-infections, poor fetal growth and preterm [10]delivery[4, 10, 31]. In this study, we specifically assessed the role of maternal and infant alteration in inflammation and immune activation as it relates to these complications.
Previous reports have described increased immune activation in HEU infants compared to HU infants[18, 32]. In a recent finding from Zimbabwe, HEU had higher levels of hsCRP at birth and at 6 months, but not IL-6 or sCD14 when compared to levels in HU infants[33]. In our analysis, we extended these findings by evaluating the effects of maternal inflammation, as well as assessing a broad range of markers of systemic inflammation, immune activation, and coagulation. We also measured lipid oxidation which has been known to be associated with non-AIDS complications in HIV-infected adults[34-39], as well as in children[13, 40, 41] and lastly known to drive inflammation overall[15, 26, 30]. Notable differences that could account for the heterogeneity in the results between the studies are that mothers in the Zimbabwean cohort were not on ART and all infants were breastfed.
We explored several potential causes of HEU infants’ increased inflammation: exposure to a pro-inflammatory environment in utero; passive transfer of maternal inflammation or exposure to ART. Although infected mothers had significantly elevated systemic inflammation and immune activation compared to levels in uninfected mothers, our findings remained unchanged after adjusting for maternal inflammation. This suggests that inflammation and immune activation in these infants are likely not solely the result of exposure to a pro-inflammatory fetal environment. In addition, maternal markers did not correlate with infant markers, suggesting that in HIV, inflammatory biomarkers likely do not cross the placenta. This has been previously demonstrated in uninfected mothers[42, 43]. Furthermore, we explored the associations between maternal HIV factors, such as viremia, CD4+ cell count and ART regimen, and infants’ inflammation. Only sTNF-RII was associated with CD4+ cell count and maternal ART regimen. In our study, 80% of infected mothers had viral loads below the limit of detection, therefore it is difficult to ascertain the contribution of direct HIV exposure in utero to infants’ inflammation. In summary, antenatal factors such as pro-inflammatory milieu, antiretroviral therapy, and placental transfer do not seem to contribute greatly to the inflammation and immune activation seen in HIV-exposed uninfected infants.
Oxidized LDL, a marker of oxidative stress, is a known driver of immune activation in HIV[26, 30]. HEU infants had higher levels of oxidized LDL levels than unexposed infants. In addition, oxidized LDL correlated with several markers of inflammation and immune activation. We hypothesize that oxidative stress may be a potential cause of elevated and persistent inflammation in HEU infants. Although ART itself may not directly be associated with increased inflammation in HEUs, maternal ART may cause mitochondrial toxicity leading to oxidative stress. We have previously reported on mitochondrial toxicity in HEU infants[44]. Recent findings suggest that evidence of mitochondrial toxicity can persist up to one year after in utero exposure to pre-exposure prophylaxis or PrEP regimens in HEU infants[45].
The clinical significance of the elevated biomarkers at birth and 6 months is unclear. It is unknown whether the increase in selected markers in the newborn period are associated with the increased mortality and morbidity seen in HEUs in resource limited settings. We attempted to identify whether perturbations in inflammation could on its own lead to increased susceptibility to infection and altered growth patterns in this vulnerable population. Data from resource limited settings, before the widespread use of ART for PMTCT, have clearly shown that HEU infants are smaller for gestational age and remain underweight or stunted [33, 46, 47]. Similarly, in the context of maternal ART with combination therapy and suppressed viral load, we found that HEU infants were born significantly earlier, had lower birth weight, and remained smaller through 6 months. This is concerning as in HEU infants, preterm birth and low birth weight are associated with higher mortality through the first year of life[48]. Our findings suggest that underlying systemic inflammation may hinder normal growth in HEU infants.
Lower respiratory tract infections are common in HEUs in Latin America [9] and risk of hospitalizations for lower respiratory tract infections is also increased in HEU children[49]. Only one marker, sTNF-RII, was associated with increased risk of hospitalization due to a lower respiratory tract infection in this study. To our knowledge, no studies have previously assessed the impact of ongoing inflammation in HEUs to their risk of infectious disease complications. Prendergast et al, found that despite elevated biomarkers in HEUs compared to HUs; markers of immune activation and gut integrity were not associated with breast-milk HIV transmission in Zimbabwean HEU infants[33]. In HIV-infected adults, sTNF-RII is associated with diabetes[35] and pre-cancerous lesions[50]. Further studies are warranted to further evaluate the consequences of inflammation on the incidence and complications of infectious diseases in HEUs.
Our study has several strengths including the extensive immunophenotyping of maternal-infant pairs, as well as the longitudinal design. Second, we included a well-characterized unexposed birth cohort from the same country for comparison. Third, the majority of infected mothers were on combination ART and virally suppressed, which is very relevant to both domestic and international HIV research agendas as the majority of pregnant women with HIV are currently on ART. Lastly, for adequate comparison with the HEU cohorts, where breastfeeding was avoided, the HU cohort retained infants that were weaned or receiving artificial milk by 4 weeks of age. However, our study includes several limitations. First, we did not assess maternal co-infections, perform nutritional assessment or examine infant enteropathy, all of which could also be contributing factors to ongoing immune activation. Second, there are demographic differences between the HIV-infected and HIV-uninfected mothers which may affect our results.
In conclusion, we have found heightened inflammation, immune activation and oxidative stress in HEU infants, indices which appear to be deleterious to growth. This raises significant concerns about the sequelae of HIV/ART exposure in utero, despite having averted infant HIV infection. The consequences of this heightened inflammation state could be more devastating than those in adults due to the presence of inflammation during the years of growth, organ maturation and neurocognitive development. Future studies are warranted to assess whether this persists beyond infancy, and if so, what are the long-term consequences.
Acknowledgements
The authors would like to thank the patients who participated in this research.
Funding: This work was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) contracts N01-HD-3-3345 (2002–2007), HHSN267200800001C (2007–2012), and HHSN275201300003C (2012–2017) ; by internal funds from University Hospitals Cleveland Medical Center (to GM), and by NICHD to SDF (K23HD088295-01A1).
Conflicts of Interest and Sources of Funding: GAM served as a consultant for Gilead, BMS, GSK/Viiv, and Merck, and has received research funding from Gilead, Merck, GSK/Viiv, and BMS. NF serves as a consultant for Gilead. NF serves as a consultant for Gilead. All other authors had no conflict of interest.
Principal investigators, co-principal investigators, study coordinators, coordinating center representatives, and NICHD staff include: Argentina: Buenos Aires: Marcelo H. Losso, Irene Foradori, Alejandro Hakim, Erica Stankievich, Silvina Ivalo (Hospital General de Agudos José María Ramos Mejía); Brazil: Belo Horizonte: Jorge A. Pinto, Victor H. Melo, Fabiana Kakehasi, Beatriz M. Andrade (Universidade Federal de Minas Gerais); Caxias do Sul: Rosa Dea Sperhacke, Nicole Golin, Sílvia Mariani Costamilan (Universidade de Caxias do Sul/ Serviço Municipal de Infectologia); Nova Iguacu: Jose Pilotto, Luis Eduardo Fernandes, Gisely Falco (Hospital Geral Nova de Iguacu - HIV Family Care Clinic); Porto Alegre: Rosa Dea Sperhacke, Breno Riegel Santos, Rita de Cassia Alves Lira (Universidade de Caxias do Sul/Hospital Conceição); Rosa Dea Sperhacke, Mario Ferreira Peixoto, Elizabete Teles (Universidade de Caxias do Sul/Hospital Fêmina); Regis Kreitchmann, Luis Carlos Ribeiro, Fabrizio Motta, Debora Fernandes Coelho (Irmandade da Santa Casa de Misericordia de Porto Alegre); Ribeirão Preto: Marisa M. Mussi-Pinhata, Geraldo Duarte, Adriana A. Tiraboschi Bárbaro, Conrado Milani Coutinho, Fabiana Rezende Amaral, Anderson Sanches de Melo (Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo); Rio de Janeiro: Ricardo Hugo S. Oliveira, Elizabeth S. Machado, Maria C. Chermont Sapia (Instituto de Puericultura e Pediatria Martagão Gesteira); Esau Custodio Joao, Leon Claude Sidi, Maria Leticia Santos Cruz, Maria Isabel Gouvêa, Mariza Curto Saavedra, Clarisse Bressan, Fernanda Cavalcanti A. Jundi (Hospital dos Servidores do Estado); São Paulo: Regina Celia de Menezes Succi, Prescilla Chow (Escola Paulista de Medicina-Universidade Federal de São Paulo); Peru: Lima: Jorge O. Alarcón Villaverde (Instituto de Medicina Tropicalal “Daniel Alcides Carrión”-Sección de Epidemiología, UNMSM), Carlos Velásquez Vásquez (Instituto Nacional Materno Perinatal), César Gutiérrez Villafuerte (Instituto de Medicina Tropicalal “Daniel Alcides Carrión”- Sección de Epidemiología, UNMSM); Data Management and Statistical Center: Yolanda Bertucci, Rachel Cohen, Laura Freimanis Hance, René Gonin, D. Robert Harris, Roslyn Hennessey, James Korelitz, Margot Krauss, Sue Li, Karen Megazzini, Orlando Ortega, Sharon Sothern de Sanchez, Sonia K. Stoszek, Qilu Yu (Westat, Rockville, MD, USA); NICHD: George K. Siberry, Rohan Hazra, Lynne M. Mofenson (Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland, USA). Supported by NICHD Contract # N01-HD-3-3345 (2002-2007) and by NICHD Contract # HHSN267200800001C (NICHD Control #: N01-HD-8-0001) (2007-2012).
Footnotes
Disclaimer: The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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