MAJOR ARTICLE

The Human Fetal Immune Response to Hepatitis C Virus Exposure in Utero Jennifer M. Babik,1,2 Deborah Cohan,3 Alexander Monto,4 Dennis J. Hartigan-O'Connor,1 and Joseph M. McCune1 1Division of Experimental Medicine and 2Division of Infectious Diseases, Department of Medicine, University of California, San Francisco; 3Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco and San Francisco General Hospital; and 4Division of Gastroenterology, Department of Medicine, University of California, San Francisco and the San Francisco Veterans Affairs Medical Center, San Francisco, California

Background. Although the rate of mother-to-child transmission of hepatitis C virus (HCV) is low, the effect of HCV exposure in utero on the fetal immune system is unknown. Methods. Umbilical cord blood was obtained from 7 neonates born to HCV-seropositive, HCV RNA-positive women and 8 neonates born to HCV-seronegative women. Cord blood mononuclear cells were analyzed by immunophenotyping and by intracellular cytokine staining after HCV-specific and polyclonal stimulation. Plasma was analyzed for anti-HCV immunoglobulin M (IgM), cytokine/granzyme concentrations, and indoleamine 2,3dioxygenase (IDO) activity. Results. HCV-exposed neonates had significantly lower levels of regulatory T cells expressing HLA-DR, lower CD41 and CD81 T cell activation, and lower plasma levels of pro-inflammatory markers than did controls. However, CD41 and CD81 T cells from HCV-exposed neonates had higher IFN-c production in response to polyclonal stimulation than did T cells from controls. IDO activity was similar between groups. No HCV-specific T cell responses or anti-HCV IgM were detected in any neonates. Conclusions. HCV-exposed neonates showed a relative suppression of immune activation and proinflammatory markers, which was counterbalanced by an increased production capacity for IFN-c. These results suggest that HCV encounters the fetal immune system in utero, and alters the balance between suppressive and pro-inflammatory responses.

Hepatitis C virus (HCV) is a major cause of chronic liver disease in both children and adults worldwide [1]. Since the advent of universal screening of blood products,

Received 2 July 2010; accepted 26 August 2010. Potential conflicts of interest: none reported. Financial support: This work was supported in part by a Ruth L. Kirschstein National Research Service Award (T32 AI007641-06A2 to J.M.B), a Pilot/Feasibility grant from the UCSF Liver Center (P30 DK026743 to J.M.M.), NIH/NCRR UCSF CTSI Grant Number UL1 RR024131, a grant to the UCSF - GIVI Center for AIDS Research (P30 AI027763), and the Harvey V. Berneking Living Trust. J.M.M. is a recipient of the NIH Director's Pioneer Award Program, part of the NIH Roadmap for Medical Research, through grant DPI OD00329. The contents of this work are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. The information in this manuscript has not been presented previously at any meeting. Reprints or correspondence: Dr Joseph M. McCune, Division of Experimental Medicine, Department of Medicine, Box 1234, University of California, San Francisco, San Francisco, CA 94143-1234 ([email protected]). The Journal of Infectious Diseases 2011;203:196–206 Ó The Author 2011. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: [email protected] 1537-6613/2011/2032-0001$15.00 DOI: 10.1093/infdis/jiq044

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mother-to-child transmission (MTCT) has become the major route of HCV infection in children [2]. It is estimated that 10,000–60,000 newborns worldwide are infected with HCV by MTCT each year [3]. The rate of MTCT from HCV-seropositive, HCV RNA-positive women is 4%–6% and transmission occurs almost exclusively from women who are viremic [2]. Although the timing of transmission is not well defined, it appears that approximately one-third of transmission events occur in utero [4], with the rest occurring peripartum. Risk factors for HCV MTCT include HIV coinfection and intrapartum exposure to maternal blood [2]. Breastfeeding, HCV genotype, and mode of delivery are not associated with MTCT. There are very few studies investigating the biology of HCV MTCT, and the reason for the low rate of transmission remains unexplained. The findings that female sex [5] and the absence of HLADR13 in the infant [6] might be risk factors for transmission suggest that the fetal immune system may play a role in protection against and/or facilitation of MTCT.

Table 1. Clinical and Demographic Characteristics of Study Groups Characteristic

Control neonates (n 5 8)

HCV-exposed neonates (n 5 7)a

Pb

Neonatal characteristics: Gestational age, median (IQR), weeks

38.9 (37.9–40.9)

37.9 (36.9–39.0)

0.10

Male sex

3 (37)

2 (29)

1.0

Delivery by cesarean section

2 (25)

2 (29)

1.0

Birth weight, median (IQR), gc

3437 (2660–3878)

2680 (2440–3270)

0.10

5-minute Apgar score , 7c ALT, median (IQR), IU/Ld

0 (0) 11.0 (9.5–14.8)

1 (14) 10.0 (7.0–12.5)

1.0 0.40

Detectable HCV RNAe

0 (0)

1 (17)

1.0

Maternal characteristics: Age, median (IQR), years

26.5 (21.5–31.0)

28.5 (27.0–31.0)

Race/ethnicity White Black

0.15 0.57

2 (25)

3 (50)

3 (37)

2 (33)

Hispanic Injection drug use in pregnancy

3 (37) 1 (12)

1 (17) 2 (33)

0.54

Pregnancy complicationsf

3 (37)

3 (50)

1.0

Cirrhosis

0 (0)

0 (0)

ALT, median (IQR), IU/Lg HIV-seropositive HCV RNA, median (IQR), IU/mL

18.0 (15.3–92.5)

43.0 (23.7–67.5)

0 (0)

1 (17)

0.69 0.47

2.4 3 105(4.6 3 104 – 8.4 3 105)

.

HCV genotype 1a Unknown

1 (17) 5 (83)

. .

NOTE. Data are shown as the number (%) of subjects, unless otherwise indicated. ALT, alanine aminotransferase; IQR, interquartile range. a

The HCV-exposed group was composed of 7 neonates born to 6 HCV-positive mothers (there was one set of dichorionic-diamniotic twins).

b

P values were calculated for the difference between groups using the Mann-Whitney test for comparison of continuous variables and the Fisher exact test or chisquared test for proportions, as appropriate. c

Birth weight and Apgar score data were available for 6 control subjects.

d

ALT testing was performed on umbilical cord blood form 6 controls and 5 HCV-exposed neonates.

e

Umbilical cord blood HCV RNA testing was performed on 6 subjects from each group. The detection limit of the assay was 615 IU/mL.

f

Maternal pregnancy complications, as determined using standardized diagnostic criteria by the obstetrics service, included preeclampsia, chronic or pregnancyinduced hypertension, and gestational diabetes. g

ALT values were available for 6 of the women in the control group.

Fetal exposure to HCV likely occurs more frequently than in utero transmission. Bidirectional trafficking of maternal and fetal cells across the placenta occurs routinely [7, 8], and it is therefore difficult to imagine a scenario whereby viral particles would not also cross the placenta with some regularity. Based on an HCV load of 105–106 copies/mL and 600 mL/min placental blood flow at term [9], an estimated 1013–1014 HCV virions access the placental bed during gestation, making it highly probable that some particles would cross the placenta even if transfer was inefficient. This led us to ask: if HCV exposure in utero is common, what is the effect of such exposure on the fetal immune system? The fetal immune environment is skewed toward tolerance and Th2 immune responses to avoid Th1 and pro-inflammatory responses that are toxic to the placental/fetal unit [10–12]. There are multiple mechanisms of maternal-fetal tolerance, including regulatory T cells (Tregs) and the suppressive enzyme indoleamine 2,3-dioxygenase (IDO) [11, 13]. Indeed, recent work

from our laboratory has shown that the fetus mounts a Treg response to noninherited maternal antigens on cells that cross into the fetal circulation [7]. We hypothesized that exposure to HCV antigens in utero might elicit a similar suppressive immune response. In this study, we aimed to determine if in utero exposure to HCV altered the fetal immune environment, with particular attention to Tregs, T cell activation and pro-inflammatory markers, IDO activity, and antigen-specific immune responses. METHODS Patients and blood samples

Umbilical cord blood (UCB) was obtained from 7 neonates born to HCV-seropositive, HCV RNA-positive women (HCVexposed group) and 8 neonates born to HCV-seronegative women (control group). All deliveries occurred at San Francisco General Hospital. Basic clinical and demographic data were Exposure to Hepatitis C Virus in Utero

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collected at the time of delivery (Table 1). All maternal laboratory values were obtained from the medical record and were performed as part of routine clinical care. HCV antibody status was determined by immunoassay (Siemens Healthcare Diagnostics), HCV RNA by the VERSANT HCV RNA 3.0 Assay (Siemens Healthcare Diagnostics), and HCV genotype by sequencing of the 5# UTR (ARUP Laboratories). All subjects were hepatitis B surface antigen negative. At delivery, UCB was collected from the umbilical vein using sterile cordocentesis to minimize the possibility of maternal blood contamination. Serum was immediately isolated and tested for the level of alanine aminotransferase and HCV RNA (as above). Blood samples from HCV-positive adults were used as positive controls in some assays and were obtained from the Liver Studies Group at the San Francisco Veterans Affairs Medical Center. All samples were collected under protocols approved by the Institutional Review Board at the University of California, San Francisco, that were in accordance with the guidelines of the US Department of Health and Human Services. Isolation of mononuclear cells

Plasma was separated from whole blood collected in sodium heparin and was stored at 280°C. Cord blood mononuclear cells (CBMC) or peripheral blood mononuclear cells (PBMC) were then isolated by Ficoll density gradient centrifugation (Histopaque-1077, Sigma-Aldrich). For UCB, nucleated red blood cells were removed by a second round of Ficoll density gradient centrifugation [14].

flow cytometer (BD Biosciences) and data were analyzed using FlowJo software (Treestar, Inc). For each experiment, LSR-II photomultiplier tube voltages were set using rainbow bead target values to minimize variability between sample runs. Intracellular cytokine staining

Fresh CBMC or PBMC were cultured in RPMI-1640 containing 10% heat-inactivated fetal bovine serum and stimulated with media alone (containing DMSO), overlapping HCV peptides, or phorbol 12-myristate 13-acetate (PMA) (0.01 lg/mL) plus ionomycin (1 lg/mL) (Sigma-Aldrich). The HCV peptides were provided by the NIH Biodefense and Emerging Infections Research Resources Repository and were 12–19-mers, overlapping by 11–12 amino acids and spanning the entire sequence of the genotype 1a H77 strain. The peptides were divided into subgenomic pools (core/E1, E2, NS2/p7, NS3, NS4A/NS4B, NS5A, and NS5B) with an individual peptide concentration of 10 lg/ mL and DMSO concentration ,0.8%. Cultures were incubated at 37°C for 16 h in the presence of anti-CD28 (2 lg/mL), antiCD49d (2 lg/mL), and GolgiPlug (BD Biosciences; concentration 1:1000). Cells were then harvested and stained with anti-CD4-PE-Texas-Red, anti-CD8-PE-Cy5.5, anti-CD27-APCAlexa-Fluor-750, anti-CD45RA-PE, and LIVE/DEAD Fixable Dead Cell Stain. Cells were fixed and permeabilized using the Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences) according to the manufacturer’s instructions and stained with anti-CD3-Pacific-Blue, anti-IL-2-APC, anti-IFNcPE-Cy7, and anti-TNFa-Alexa-Fluor-700. Samples were processed and analyzed as described above.

Antibodies

Anti-CD3-Alexa-Fluor-700, anti-CD3-Pacific-Blue, anti-CD4Pacific-Blue, anti-CD25-PE-Cy7, anti-CD45RA-PE, anti-HLADR-APC-Cy7, anti-IL-2-APC, anti-IFNc-PE-Cy7, anti-TNFaAlexa-Fluor-700, and purified anti-CD28 and anti-49d were purchased from BD Biosciences. Anti-FoxP3-Alexa-Fluor-647 and anti-CD27-APC-Alexa-Fluor-750 were purchased from eBiosciences. Anti-CD4-PE-Texas-Red, anti-CD8-PE-Cy5.5, and anti-CD38-PE-Texas-Red were purchased from Invitrogen. Immunophenotyping

Absolute T cell counts were determined by staining 50 lL of whole UCB in TruCOUNT tubes (BD Biosciences) using anti-CD3-Alexa-Fluor-700, anti-CD4-Pacific-Blue, and antiCD8-PE-Cy5.5 according to the manufacturer’s instructions. Phenotyping of fresh CBMC was performed with 5 3 105 cells using anti-CD3-Alexa-Fluor-700, anti-CD4-Pacific-Blue, antiCD8-PE-Cy5.5, anti-CD25-PE-Cy7, anti-CD38-PE-Texas-Red, anti-HLA-DR-APC-Cy7, and aqua LIVE/DEAD Fixable Dead Cell Stain (Invitrogen). Cells were then fixed, permeabilized, and stained with anti-FoxP3-Alexa-Fluor-647 using the FoxP3 Staining Buffer Set (eBiosciences) according to the manufacturer’s instructions. Samples were processed on an LSR-II 198

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Luminex assay

Plasma samples were analyzed using a custom multiplex assay (LEGENDplex, Biolegend) for granzyme A, granzyme B, interferon (IFN) a, IFN-c, tumor necrosis factor (TNF) a, interleukin (IL) 2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-17A, IL-18, IL-22, and VEGF. The assay was performed as per the manufacturer’s instructions and samples were run in duplicate on a Luminex 100 System instrument (Luminex). Analyte concentrations were determined by interpolation from a standard curve. IDO activity

Plasma tryptophan and kynurenine concentrations were measured by high-performance liquid chromatography as previously described [15]. Anti-HCV IgM ELISA

Plasma was diluted 1:20 and treated with anti-human immunoglobulin G (IgG) (RF-Absorbent, IBL International) according to the manufacturer’s instructions to remove anti-HCV IgG and rheumatoid factor. Anti-HCV immunoglobulin M (IgM) was then detected using an anti-HCV IgG enzyme-linked

Figure 1. HCV-exposed neonates had similar numbers of regulatory T cells (Tregs) as controls but lower levels of HLA-DR1 Tregs. A, Representative flow plots showing the gating strategy for Tregs from control and HCV-exposed neonates. The upper panels are gated on CD41 T lymphocytes and show CD251FoxP31CD41 T cells (Tregs). The lower panels show the HLA-DR1 subset within Tregs. B, Scatter plots showing the percentage and absolute numbers of Tregs, FoxP3 MFI on Tregs, and percentage of HLA-DR1 Tregs for both groups. Horizontal lines represent the median. The HCV-exposed neonate with a positive cord blood HCV RNA level is indicated by an open triangle (D). C, The histogram shows the FoxP3 MFI on HLA-DR1 Tregs (filled solid line) and HLA-DR– Tregs (open dotted line) from a representative HCV-exposed neonate. The bar graph shows the aggregate data from all control and HCV-exposed neonates. Bars show the median and error bars indicate the interquartile range. In all panels, P values were calculated using the MannWhitney test.

immunosorbent assay (ELISA) kit (CTK Biotech) modified for IgM detection by using a horseradish peroxidase-conjugated goat anti-human IgM antibody (Bethyl Labs; dilution 1:9000). All samples were run in triplicate and read at an optical density (OD) of 450 nm on a SpectraMax M2 microplate reader (Molecular Devices). An OD was considered positive if it was greater than the mean 1 2 SD of all seronegative samples. Plasma from an adult HCV-positive subject with anti-HCV IgM detected in preliminary testing was used as a positive control.

Spearman’s correlation. Univariate analysis of baseline characteristics and immunologic results was performed using linear regression. Two-sided P values were calculated for all test statistics and P , .05 was considered significant. Statistical analyses were performed using GraphPad Prism software, version 5.01 (GraphPad).

Statistical analysis

Study group characteristics

The Mann-Whitney test was used for comparison of continuous variables and the Fisher exact test or v2 test was used for proportions, as appropriate. Correlation was performed by

There were no significant differences in the clinical or demographic characteristics between study groups (Table 1). The HCV-exposed group had a trend toward a lower gestational

RESULTS

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Figure 2. HCV-exposed neonates had lower levels of T cell activation than controls. A, Representative flow plots showing the gating strategy for the activation markers CD38 and HLA-DR on CD41 and CD81 T cells. B, Scatter plots showing the absolute number of HLA-DR1, CD381, and HLADR1CD381 CD41 T cells (top panels) and CD81 T cells (bottom panels). Horizontal lines represent the median. To ensure that the apparent outlier in the HLA-DR1 and HLA-DR1CD381 control data did not exert undue influence on our results, a nonparametric test (Mann-Whitney) was used to calculate P values. The HCV-exposed neonate with a positive cord blood HCV RNA level is indicated by an open triangle (D). C, Spearman correlations of the percentage of HLA-DR1 Tregs with the percentages of HLA-DR1CD41 and HLA-DR1CD81 T cells. HCV-exposed neonates are indicated by open shapes and control neonates by closed shapes.

age and lower birth weight, but both groups were delivered at a median gestational age .37 weeks (full term), and all birth weights were appropriate for gestational age. There was one set of dichorionic-diamniotic (fraternal) twins in the HCVexposed group. One of 6 HCV-exposed neonates tested had a detectable cord blood HCV RNA level, suggesting in utero infection [2]. This neonate was born to an HIV/HCV-coinfected woman. 200

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HCV-exposed neonates had similar numbers of Tregs but lower levels of HLA-DR1 Tregs than controls

We first sought to determine the levels of Tregs in each group by immunophenotyping of CBMC. We found that HCV-exposed neonates and controls had a similar percentage and absolute number of Tregs, defined as CD251FoxP31CD41 T cells (Figure 1A, 1B). In addition, the overall FoxP3 MFI on Tregs was similar between groups. However, HCV-exposed neonates had

Figure 3. HCV-exposed neonates produced more IFN-c in response to polyclonal stimulation than controls. A, Representative flow plots showing IFN-c, TNF-a, and IL-2 production from CD41 and CD81 T cells after polyclonal stimulation and intracellular staining. 2.5 3 105 fresh cord blood mononuclear cells were stimulated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin for 16 h in the presence of purified anti-CD28, anti-CD49d, and GolgiPlug. All negative control stimulations (media containing DMSO only) had background levels less than .25%. B, Scatter plots showing the aggregate

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granzyme A P=.005

200

pg/mL

IL-12

IL-17A

P=.003

10.0

150

7.5

15

100

5.0

10

50

2.5

5

HCVexposed

Control

HCVexposed

Control

granzyme B

pg/mL

HCVexposed

P=.05

80

400

60

300

40

200

20

100

HCVexposed

Control

IL-18

P=.05

Control

P=.005

20

Control

HCVexposed

Figure 4. HCV-exposed neonates had lower granzyme and pro-inflammatory cytokine levels than controls. Scatter plots showing the levels of granzyme A, granzyme B, IL-12, IL-17A, and IL-18 determined by multiplex assay, which was performed on plasma from 7 controls and 6 HCV-exposed neonates. Horizontal lines represent the median, and P values were calculated using the Mann-Whitney test. The HCV-exposed neonate with a positive cord blood HCV RNA level is indicated by an open triangle (D).

a 3.6-fold lower percentage of HLA-DR1 Tregs than did controls (Figure 1B). HLA-DR1 Tregs are a distinct subset of Tregs that have been shown to have higher FoxP3 levels and to induce a more rapid suppression of effector T cells than do HLA-DR– Tregs [16]. Although our CBMC numbers limited our ability to perform functional Treg assays, we found that HLA-DR1 Tregs from both HCV-exposed and control neonates had a 2- to 3-fold higher FoxP3 MFI than did HLA-DR– Tregs (Figure 1C). HCV-exposed neonates had lower levels of T cell activation but a similar maturation profile as controls

Based on the finding that HCV-exposed neonates had lower levels of HLA-DR1 Tregs, we next sought to determine whether the study groups also had different levels of T cell activation (Figure 2A). We found that HCV-exposed neonates had significantly lower numbers of HLA-DR1CD41, HLA-DR1CD81,

CD381CD81, and CD381HLA-DR1CD81 T cells compared with controls (Figure 2B). Results for the percentages of activated CD41 and CD81 T cells showed similar results (data not shown). The percentage of HLA-DR1 Tregs was strongly correlated with the percentage of HLA-DR1CD41 and HLADR1CD81 T cells (Figure 2C). To determine whether these differences in T cell activation could be explained by differences in maturation profile between groups, we categorized T cell subsets using the maturation markers CD45RA and CD27 [17]. There were no significant differences between groups in the proportion of naive, central memory, or effector memory CD41 or CD81 T cell subsets (data not shown). HCV-exposed neonates produced more IFN-c after polyclonal stimulation than did controls

Given the phenotypic differences in T cells from HCV-exposed and control neonates, we looked for differences in T cell

data for cytokine production from CD41 T cells (top panels) and CD81 T cells (bottom panels) after stimulation with PMA and ionomycin. Horizontal lines represent the median. The HCV-exposed neonate with a positive cord blood HCV RNA level is indicated by an open triangle (D). C, Representative flow plots showing the gating strategy for the maturation markers CD45RA and CD27 on CD41 and CD81 T cells making IFN-c. Subsets were defined as follows: na€ıve (Tna€ıve: CD271CD45RA1), central memory (TCM: CD271CD45RA-), effector memory (TEM: CD27-CD45RA-), and terminally differentiated effector (TEMRA: CD27-CD45RA1) T cells. Bar graphs show the aggregate data for the maturation subsets within IFN-c1CD41 and IFN-c1CD81 T cells. Bars show the median and error bars indicate the interquartile range. The bar graph legend indicating the different T cell subsets is shown. In all panels, P values were calculated using the Mann-Whitney test. 202

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function between groups. We measured the capacity of CD41 and CD81 T cells to make IL-2, TNF-a, and IFN-c in response to polyclonal stimulation with PMA and ionomycin (Figure 3A). HCV-exposed neonates had a 3.5-fold higher percentage of CD41 and CD81 T cells making IFN-c compared with controls, but there was no difference in the production of IL-2 or TNF-a between groups (Figure 3B). A similar pattern of results was seen for the absolute numbers of CD41 and CD81 T cells making cytokines (data not shown). The majority of CD41 and CD81 T cells making IFN-c were naive T cells, but among IFN-c1CD41 T cells, there was a 12-fold higher proportion of terminally differentiated effector memory (TEMRA) cells in the HCV-exposed group compared with controls (Figure 3C). There were no differences between groups in the maturation profile of IFN-c1CD81 T cells. HCV-exposed neonates had lower levels of pro-inflammatory cytokines and granzymes than controls, but similar levels of IDO activity

To further characterize any differences in pro-inflammatory markers between groups, we performed a multiplex analysis of UCB plasma. HCV-exposed neonates had significantly lower levels of granzyme A, IL-12, and IL-17A and a trend toward lower levels of granzyme B and IL-18 (Figure 4). There were no differences between groups in the levels of IL-4, IL-6, IL-8, IL-10, IL-22, and VEGF (data not shown). IFN-a, IFN-c, IL-2, IL-4, IL13, and TNF-a were undetectable by this assay. We next sought to determine if the immunosuppressive enzyme IDO might explain the differences in pro-inflammatory markers and T cell activation between groups. IDO is the ratelimiting enzyme in the breakdown of tryptophan through the

kynurenine pathway, and its activity can be measured by determining the plasma concentrations of these analytes [15]. We found no differences in the level of tryptophan, kynurenine, or the kynurenine/tryptophan ratio between groups (data not shown). HCV-specific immune responses were not detectable in HCVexposed neonates

To determine the antigen-specific T cell functionality in HCVexposed neonates, we stimulated CBMC with overlapping HCV peptide pools and measured T cell production of IL-2, TNF-a, and IFN-c. This assay was tested in 29 HCV-positive adults, and a CD41 or CD81 T cell cytokine response was detected in more than two-thirds of subjects, independent of HCV RNA level or genotype (Figure 5; data not shown). However, none of the HCV-exposed neonates had a detectable cytokine response (Figure 5). To determine if HCV-exposed neonates mounted an antibody response against HCV, we measured the level of anti-HCV IgM in UCB plasma by ELISA. Although only 30%–80% sensitive for HCV infection [18–20], anti-HCV IgM is very specific to the fetal compartment because IgM, unlike IgG, does not cross the placenta [21, 22]. However, no anti-HCV IgM was detected in any of the HCV-exposed neonates (data not shown). Baseline characteristics and immunologic results

There was no significant association by univariate analysis of gestational age, birth weight, or maternal HCV RNA level with any of the immunologic parameters that differed between groups (data not shown). The HCV-exposed neonate who was

Figure 5. No HCV-specific T cell responses were detected in HCV-exposed neonates. Representative flow plots from an HCV-positive adult (upper panels) and an HCV-exposed neonate (lower panels) showing IFN-c and TNF-a production from CD41 and CD81 T cells after HCV peptide pool stimulation and intracellular staining. One million fresh peripheral blood mononuclear cells were stimulated with media alone (containing DMSO) or with overlapping HCV peptides for 16 h in the presence of purified anti-CD28, anti-CD49d, and GolgiPlug. Shown here are the plots for stimulation with the HCV peptide pool core/E1 (for CD41 T cells) and pool NS3 (for CD81 T cells). After subtraction of background responses, the HCV-positive adult subject had .17% of CD41 T cells and .40% of CD81 T cells making both IFN-c and TNF-a in response to peptide stimulation. No responses were seen in the HCV-exposed neonate. Exposure to Hepatitis C Virus in Utero

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HCV RNA-positive had the highest number of overall Tregs, the highest FoxP3 MFI, the lowest fraction of HLA-DR1 Tregs, and the lowest level of IL-18 (Figures 1B and 4). Exclusion of these data points from their respective analyses did not alter our findings (data not shown), and results of other assays for this neonate were at or near the median. DISCUSSION Our results show that HCV-exposed neonates had a relative suppression of T cell activation and pro-inflammatory markers compared with controls, which was offset by a higher production capacity for IFN-c. This indicates a balance between pro- and anti-inflammatory responses in the HCV-exposed neonate. To our knowledge, this is the first description of the immunologic characteristics of UCB from HCV-exposed neonates. Taken together, these results suggest that HCV contacts the fetal immune system in utero, likely via transplacental passage. There are several potential sites in the placenta for the passage of free or cell-associated virus [23], but it is also possible that HCV may directly infect the placenta. Many of the putative HCV receptors and attachment factors have been detected in the placenta, including claudin-1, occludin, SR-B1, LDLr, and DC-SIGN [24–26]. Alternatively, it is possible that HCV proteins cross the placenta as soluble antigens rather than as whole virions. In particular, the HCV core protein can be secreted in soluble form and has known immunomodulatory effects including the suppression of T cell proliferation and activation [27, 28]. An attractive hypothesis is that the HCV core protein may be playing a role in the alteration of fetal immunity in utero. While it seems likely that HCV crosses into the fetal circulation and contacts the fetal immune system, antigen-specific immune responses were not detected in HCV-exposed neonates. One possible explanation is that neonatal responses were below the assay limit of detection despite the excellent sensitivity of these assays for adult samples. Second, it is possible that antiHCV responses were being actively suppressed or resulted in Th2 cytokine production, and experiments to test these hypotheses are ongoing. Third, it is possible that HCV might not contact the fetal immune system directly. For example, the increased IFN-c production seen in HCV-exposed neonates could be a result of maternal pro-inflammatory cytokines [29] or antiidiotype antibodies that mimic exposure to HCV antigens [30, 31]. These possibilities seem less likely, but we were unable to exclude them entirely because we did not have paired maternal blood samples. It is also possible that maternal chronic liver disease itself, irrespective of HCV, might have indirectly affected the immune system of the HCV-exposed neonates, perhaps as a result of maternal cytokine dysregulation [32]. We did not have any women in the control group with chronic liver disease 204

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in order to test this hypothesis because liver disorders in pregnancy are rare [33], but no woman in either group had cirrhosis and there were no differences in maternal ALT values between groups. Last, it is possible that the innate immune system or unconventional T cell responses might play a larger role than classic adaptive immune responses in protecting the fetus from in utero infection [34–38]. What mechanism underlies the altered immune balance seen in HCV-exposed neonates? The levels of Tregs, IL-10, and IDO activity were not different between groups. HCV-exposed neonates did have lower levels of HLA-DR1 Tregs, a subset of Tregs that are reported to be more highly suppressive [16]. However, because the level of HLA-DR1 Tregs was highly correlated with the level of CD41 and CD81 T cell activation, the presence of HLA-DR1 Tregs might in fact be a surrogate marker of immune activation in this setting. Another possible mechanism for the altered immune balance in HCV-exposed neonates is the immunomodulatory HCV core protein, as discussed above. Finally, it is possible, although unlikely, that the frequency of unmeasured maternal coinfections was higher in the control group, thereby resulting in greater in utero antigen exposure and T cell activation. Although most of the pro-inflammatory markers and immune activation indices were lower in HCV-exposed neonates, IFN-c production capacity was higher. This balance between pro- and anti-inflammatory mechanisms in response to infectious disease exposure in utero has been described elsewhere [39–41]. On the one hand, low levels of T cell activation may be beneficial in promoting T cell function for the clearance of HCV [42]. On the other hand, IFN-c produced in response to in utero exposure might play a role in protecting infants from infection, as in the case of HIV [29, 40]. Therefore, the balance of pro- and anti-inflammatory mechanisms might serve to protect against in utero HCV infection. Indeed, 5 of 6 neonates tested did not show evidence of in utero infection. The mechanism behind the increased IFN-c production in HCV-exposed neonates remains to be determined, but one possibility is via an epigenetic process that reverses the neonatal hypermethylation of the IFN-c promoter [43]. Given our finding that HCV-exposed neonates had a higher proportion of TEMRA cells among IFN-c1CD41 T cells, it is also possible that HCV exposure alters the maturation pathway of memory T cells. The clinical effect of this altered fetal immune balance in HCV-exposed neonates remains to be determined. For example, will the increased IFN-c production decrease the incidence of allergy or augment the clinical response to vaccination and infection? The maintenance of a Th2 skew and poor IFN-c production after birth have been associated with the development of asthma and weaker responses to vaccination [44, 45]. Conversely, the presence of a Th1-biased immune response in the setting of congenital Trypanosoma cruzi infection has been shown to augment the IFN-c response to neonatal vaccination

[46]. Longitudinal studies that follow the clinical outcomes of HCV-exposed infants are needed. This study has several limitations. Our small sample size may have limited our ability to find subtle differences in immune parameters with considerable biological variation, such as Tregs. However, our results showed a consistent pattern across multiple assays and for both absolute numbers and percentages, thereby lending increased credibility to these findings. That said, our conclusions are still preliminary and should be confirmed in a larger study of HCV-exposed neonates. Another potential limitation is that we could not compare maternal immune responses between groups, which could have provided insight into differences seen in the neonates. This is an important component to include in future studies of HCV-exposed neonates. Finally, it is possible that the inclusion of one neonate born to an HIV/HCV-co-infected mother could have influenced the results of our HCV-exposed group. However, in the assays in which this neonate’s results were not near the group median, exclusion from the analysis did not alter our results. Our findings indicate that neonates exposed to HCV in utero have an altered immune reactivity manifest as a relative suppression of immune activation and pro-inflammatory markers that was counterbalanced by an increased production capacity for IFN-c. These results suggest that HCV encounters the fetal immune system in utero and alters the balance between suppressive and pro-inflammatory responses. More research on the mechanisms of HCV transmission across the placenta and its effects on the fetal immune system are needed. These results have important implications for our understanding of the fetal immune response to antigens encountered in utero and may contribute to the development of interventions aimed at interrupting the MTCT of infectious agents. Acknowledgments We would like to thank the patients and staff at the Women’s Health Center and Labor and Delivery Ward at San Francisco General Hospital for their participation in the study. We also thank Trevor Burt and Paul Baum for technical assistance and valuable discussions.

References 1. Wasley A, Alter MJ. Epidemiology of hepatitis C: Geographic differences and temporal trends. Semin Liver Dis 2000; 20:1–16. 2. Indolfi G, Resti M. Perinatal transmission of hepatitis C virus infection. J Med Virol 2009; 81:836–43. 3. Yeung LT, King SM, Roberts EA. Mother-to-infant transmission of hepatitis C virus. Hepatology 2001; 34:223–9. 4. Mok J, Pembrey L, Tovo PA, Newell ML. European Paediatric Hepatitis C Virus Network. When does mother to child transmission of hepatitis C virus occur? Arch Dis Child Fetal Neonatal Ed 2005; 90:F156–60. 5. European Paediatric Hepatitis C Virus Network. A significant sex-but not elective cesarean section-effect on mother-to-child transmission of hepatitis C virus infection. J Infect Dis 2005; 192:1872–9. 6. Bosi I, Ancora G, Mantovani W, et al. HLA DR13 and HCV vertical infection. Pediatr Res 2002; 51:746–9.

7. Mold JE, Michae¨lsson J, Burt TD, et al. Maternal alloantigens promote the development of tolerogenic fetal regulatory T cells in utero. Science 2008; 322:1562–5. 8. Gammill HS, Nelson JL. Naturally acquired microchimerism. Int J Dev Biol 2010; 54:531–43. 9. Browne JC, Veall N. The maternal placental blood flow in normotensive and hypertensive women. J Obstet Gynaecol Br Emp 1953; 60:141–7. 10. Holt P. Functionally mature virus-specific CD8 T memory cells in congenitally infected newborns: Proof of principle for neonatal vaccination? J Clin Invest 2003; 111:1645–7. 11. Trowsdale J, Betz AG. Mother’s little helpers: Mechanisms of maternalfetal tolerance. Nat Immunol 2006; 7:241–6. 12. Levy O. Innate immunity of the newborn: Basic mechanisms and clinical correlates. Nat Rev Immunol 2007; 7:379–90. 13. Munn DH, Zhou M, Attwood JT, et al. Prevention of allogeneic fetal rejection by tryptophan catabolism. Science 1998; 281:1191–3. 14. Fuss IJ, Kanof ME, Smith PD, Zola H. Isolation of whole mononuclear cells from peripheral blood and cord blood. Curr Protoc Immunol 2009; 85:7.1.1–.8. 15. Favre D, Mold J, Hunt PW, et al. Tryptophan catabolism by indoleamine 2,3-dioxygenase 1 alters the balance of TH17 to regulatory T cells in HIV disease. Sci Transl Med 2010; 2:32ra36. 16. Baecher-Allan C, Wolf E, Hafler DA. MHC class II expression identifies functionally distinct human regulatory T cells. J Immunol 2006; 176:4622–31. 17. Emu B, Sinclair E, Favre D, et al. Phenotypic, functional, and kinetic parameters associated with apparent T-cell control of human immunodeficiency virus replication in individuals with and without antiretroviral treatment. J Virol 2005; 79:14169–78. 18. Quinti I, Hassan NF, El Salman D, et al. Hepatitis C virus-specific B cell activation: IgG and IgM detection in acute and chronic hepatitis C. J Hepatol 1995; 23:640–7. 19. Pawlotsky JM, Darthuy F, Re´mire´ J, et al. Significance of anti-hepatitis C virus core IgM antibodies in patients with chronic hepatitis C. J Med Virol 1995; 47:285–91. 20. Nikolaeva LI, Blokhina NP, Tsurikova NN, et al. Virus-specific antibody titres in different phases of hepatitis C virus infection. J Viral Hepat 2002; 9:429–37. 21. Avrech OM, Samra Z, Lazarovich Z, Caspi E, Jacobovich A, Sompolinsky D. Efficacy of the placental barrier for immunoglobulins: Correlations between maternal, paternal and fetal immunoglobulin levels. Int Arch Allergy Immunol 1994; 103:160–5. 22. Landor M. Maternal-fetal transfer of immunoglobulins. Ann Allergy Asthma Immunol 1995; 74:279–83. 23. McDonagh S, Maidji E, Ma W, Chang HT, Fisher S, Pereira L. Viral and bacterial pathogens at the maternal-fetal interface. J Infect Dis 2004; 190:826–34. 24. Dye JF, Jablenska R, Donnelly JL, et al. Phenotype of the endothelium in the human term placenta. Placenta 2001; 22:32–43. 25. Ethier-Chiasson M, Duchesne A, Forest JC, et al. Influence of maternal lipid profile on placental protein expression of LDLr and SR-BI. Biochem Biophys Res Commun 2007; 359:8–14. 26. Soilleux EJ, Barten R, Trowsdale J. DC-SIGN; a related gene, DCSIGNR; and CD23 form a cluster on 19p13. J Immunol 2000; 165:2937–42. 27. Yao ZQ, Eisen-Vandervelde A, Waggoner SN, Cale EM, Hahn YS. Direct binding of hepatitis C virus core to gC1qR on CD41 and CD81 T cells leads to impaired activation of Lck and Akt. J Virol 2004; 78:6409–19. 28. Kimball P, Verbeke S, Shiffman M. HCV core protein augments cyclosporine immunosuppression. Transplant Proc 2005; 37:652–3. 29. Hygino J, Lima PG, Filho RG, et al. Altered immunological reactivity in HIV-1-exposed uninfected neonates. Clin Immunol 2008; 127:340–7. 30. Vanderbeeken Y, Sarfati M, Bose R, Delespesse G. In utero immunization of the fetus to tetanus by maternal vaccination during pregnancy. Am J Reprod Immunol Microbiol 1985; 8:39–42.

Exposure to Hepatitis C Virus in Utero

d

JID 2011:203 (15 January)

d

205

31. Hahn-Zoric M, Carlsson B, Bjo¨rkander J, Osterhaus AD, Mellander L, Hanson LA. Presence of non-maternal antibodies in newborns of mothers with antibody deficiencies. Pediatr Res 1992; 32:150–4. 32. Fainboim L, Chern˜avsky A, Paladino N, Flores AC, Arruvito L. Cytokines and chronic liver disease. Cytokine Growth Factor Rev 2007; 18:143–57. 33. Joshi D, James A, Quaglia A, Westbrook RH, Heneghan MA. Liver disease in pregnancy. Lancet 2010; 375:594–605. 34. Vermijlen D, Brouwer M, Donner C, et al. Human cytomegalovirus elicits fetal cd T cell responses in utero. J Exp Med 2010; 207:807–21. 35. Fievet N, Varani S, Ibitokou S, et al. Plasmodium falciparum exposure in utero, maternal age and parity influence the innate activation of foetal antigen presenting cells. Malar J 2009; 8:251. 36. Hermann E, Alonso-Vega C, Berthe A, et al. Human congenital infection with Trypanosoma cruzi induces phenotypic and functional modifications of cord blood NK cells. Pediatr Res 2006; 60:38–43. 37. Ricci E, Malacrida S, Zanchetta M, et al. Toll-like receptor 9 polymorphisms influence mother-to-child transmission of human immunodeficiency virus type 1. J Transl Med 2010; 8:49. 38. Renneson J, Dutta B, Goriely S, et al. IL-12 and type I IFN response of neonatal myeloid DC to human CMV infection. Eur J Immunol 2009; 39:2789–99. 39. Vekemans J, Truyens C, Torrico F, et al. Maternal Trypanosoma cruzi infection upregulates capacity of uninfected neonate cells to produce pro- and anti-inflammatory cytokines. Infect Immun 2000; 68:5430–4.

206

d

JID 2011:203 (15 January)

d

Babik et al.

40. Kuhn L, Coutsoudis A, Moodley D, et al. Interferon-gamma and interleukin-10 production among HIV-1-infected and uninfected infants of HIV-1-infected mothers. Pediatr Res 2001; 50:412–6. 41. Flanagan KL, Halliday A, Burl S, et al. The effect of placental malaria infection on cord blood and maternal immunoregulatory responses at birth. Eur J Immunol 2010; 40:1062–72. 42. Falconer K, Gonzalez VD, Reichard O, Sandberg JK, Alaeus A. Spontaneous HCV clearance in HCV/HIV-1 coinfection associated with normalized CD4 counts, low level of chronic immune activation and high level of T cell function. J Clin Virol 2008; 41:160–3. 43. White GP, Watt PM, Holt BJ, Holt PG. Differential patterns of methylation of the IFN-c promoter at CpG and non-CpG sites underlie differences in IFN-c gene expression between human neonatal and adult CD45RO- T cells. J Immunol 2002; 168:2820–7. 44. Prescott SL, Holt PG. Abnormalities in cord blood mononuclear cytokine production as a predictor of later atopic disease in childhood. Clin Exp Allergy 1998; 28:1313–6. 45. Kondo N, Kobayashi Y, Shinoda S, et al. Reduced interferon gamma production by antigen-stimulated cord blood mononuclear cells is a risk factor of allergic disorders – 6-year follow-up study. Clin Exp Allergy 1998; 28:1340–4. 46. Dauby N, Alonso-Vega C, Suarez E, et al. Maternal infection with Trypanosoma cruzi and congenital Chagas disease induce a trend to a type 1 polarization of infant immune responses to vaccines. PLoS Negl Trop Dis 2009; 3:e571.