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Toll-like receptor (TLR) expression on CD4+ and CD8+ T-cells in patients chronically infected with hepatitis C virus
Cellular Immunology 264 (2010) 150–155
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Toll-like receptor (TLR) expression on CD4+ and CD8+ T-cells in patients chronically infected with hepatitis C virus Talia Hammond a, Silvia Lee a,b,*, Mark W. Watson e, James P. Flexman a, Wendy Cheng d, Sonia Fernandez a, Patricia Price a,c a
School of Pathology and Laboratory Medicine, University of Western Australia, Australia Department of Microbiology and Infectious Disease, Royal Perth Hospital, Australia Clinical Immunology and Immunogenetics, Royal Perth Hospital, Australia d Department of Gastroenterology and Hepatology, Royal Perth Hospital, Australia e Centre for Clinical Immunology & Biomedical Statistics, Royal Perth Hospital, Australia b c
a r t i c l e
i n f o
Article history: Received 26 March 2010 Accepted 2 June 2010 Available online 8 June 2010 Keywords: HCV TLR RIG-I Therapy
a b s t r a c t Toll-like receptor (TLR) expression on T-cells and the signalling pathways that lead to the production of cytokines may limit antigen-specific T-cell responses. Here, expression of TLR and retinoic acid inducible gene I (RIG-I) on T-cells were evaluated in patients chronically infected with hepatitis C virus (HCV), before and during pegylated interferon-a and ribavirin therapy. Expression of TLR2,3,4,7,9 and retinoic acid inducible gene (RIG)-I on different CD4+ and CD8+ T-cell sub-populations (naïve: CD45RA+CD57; central memory: TCM CD45RACD57; effector memory: TEM CD45RACD57+ and terminally differentiated effector memory: TEMRA CD45RA+CD57+) were measured by flow cytometry. TLR7, TLR9 and RIG-I expression on CD4+ T-cells and RIG-I expression on CD8+ T-cells was higher in patients than healthy controls. Therapy increased expression of TLR2, TLR4 and TLR9 and this was observed for all T-cell sub-populations. Evaluation of TLR expression at baseline did not identify patients able to achieve sustained virological response following therapy. Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved.
1. Introduction Toll-like receptors (TLR), retinoic acid inducible gene I (RIG-I)like helicases and nucleotide-binding oligodimerisation domain (NOD)-like receptors [1] recognise pathogen-associated molecular patterns found in bacteria, fungi, viruses and protozoa. TLRs are primarily expressed on dendritic cells (DCs) and are critical to their role in immune surveillance. Triggering through TLRs promotes DC maturation, production of pro-inflammatory and antiviral cytokines, up-regulation of co-stimulatory molecules and altered expression of chemokine receptors. Eleven TLRs have been identified in humans. Receptors that recognise viral products or nucleic acids are expressed in endosomal membranes (e.g. TLR3, TLR7, TLR8, TLR9). TLR2 recognises peptidoglycan and glycolipids and TLR4 recognises lipopolysaccharide. These are expressed on the cytoplasmic membrane [2].
* Corresponding author at: Level 2, MRF Building, Rear 50 Murray Street, Near Royal Perth Hospital, Perth, Western Australia 6000, Australia. Fax: +61 8 9224 0204. E-mail addresses: [email protected], [email protected] (S. Lee).
TLRs and RIG-I have potential to influence chronic HCV infection. Increased mRNA levels for several TLRs was demonstrated in unfractionated peripheral blood mononuclear cells (PBMC) [3,5], purified monocytes [4,5] and purified T-cells [4] from HCVinfected patients compared to healthy controls. RIG-I mRNA was also higher in myeloid DCs (mDCs) from HCV-infected patients than healthy individuals [6]. Production of IL-12 by mDCs stimulated with poly (I:C) (a TLR3 ligand) and production of IFNa by plasmacytoid DCs (pDCs) stimulated with CpG (a TLR9 ligand) was low in patients, despite elevated expression of TLRs and RIGI [6,7]. Furthermore, reduced IFNa production by pDCs in response to a TLR7 ligand was associated with impaired activation of naïve CD4 T-cells [7]. Rodrigue-Gervais et al. associated decreased cytokine production by mDCs with high levels of HCV RNA in unfractionated DCs [8]. Hence, HCV activates the innate immune system and inhibits TLR signalling pathways and subsequent production of cytokines. In addition to dendritic cells and monocytes, TLRs are expressed on B-cells [9–11], T-cells [12,13] and NK cells [14] and regulatory T-cells [15]. Triggering of these cells through TLRs could influence immunity and immunopathology associated with HCV disease. Here we investigated TLR and RIG-I expression on T-cell populations from chronically HCV-infected patients and healthy donors.
0008-8749/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2010.06.001
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2. Materials and methods 2.1. Patients Patients chronically infected with HCV (n = 34) were recruited from Royal Perth Hospital (Western Australia). HCV infection was diagnosed by third generation enzyme linked immunosorbent assay (ELISA) for antibodies to HCV and confirmed by qualitative reverse transcriptase polymerase chain reaction (RT-PCR) for HCV RNA in serum (Cobas Amplicor HCV Test, Roche Diagnostics, Branchburg, NJ, USA). Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll (Amersham Biosciences, Buckinghamshire, UK) density gradient centrifugation and cryopreserved in 10% DMSO and 90% fetal calf serum (FCS) for subsequent analyses. PBMC samples were also collected for 16 of the 34 patients during therapy (Pegasys, Roche, Dee Why, NSW, Australia or Pegatron, Schering-Plough, North Ryde, NSW, Australia) [median time on therapy 5 (3–10) months]. Patients with HCV genotypes 1 or 4 were treated for 48 weeks and those with genotypes 2 or 3 for 24 weeks. Healthy individuals with no evidence of exposure to HIV or HCV were included as controls and were matched by age and sex with patients [42 (21–73 years), 7males, 9 females]. The study was approved by the Royal Perth Hospital Research Ethics committee and all patients and controls gave informed consent.
Expression of CD57 and CD45RA was used to define naïve (CD45RA+CD57), central memory TCM (CD45RACD57), effector memory TEM (CD45RACD57+) and terminally differentiated effector memory TEMRA (CD45RA+CD57+) T-cells. CCR7 is more widely used to distinguish memory T-cells, but could not be detected on cryopreserved PBMC with the monoclonal antibody available commercially when this study was performed. An alternative antibody is now available. In a pilot study of seven healthy controls and five HCV-infected patients, CCR7 and CD57 defined similar percentages of naïve and CD4+ TEMRA T-cells and all CD8+ T-cell subsets. However, percentages of CD4+ TCM T-cells were lower and CD4+ TEM T-cells were higher using CCR7 rather than CD57 [19]. 2.5. Statistics All data are presented as median (range). Statistical analyses were performed using Graphpad Prism Version 5.01 (Graphpad Software, San Diego, CA, USA). Differences between study groups were evaluated using Mann–Whitney Tests. p-values <0.05 were considered statistically significant. Figures are presented as box and whisker plots showing median, 25th and 75th percentiles and range. 3. Results
2.2. Detection of HCV RNA and HCV genotyping
3.1. Patient characteristics
Sera were stored at 80 °C. Viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, USA). HCV genotype was determined using the Line Probe Assay (Inno-LiPA, Innogenetics, Gent, Belgium) or by real-time PCR and melting curve analysis using FRET probes [16]. HCV viral loads were assessed using primers binding the conserved 50 UTR region [17].
From the 34 patients studied at baseline, samples were available for 16 patients during therapy [5 (3–10) months]. For all subsequent analyses, these two groups of patients were compared. SVR were achieved in 20 of the 34 patients (59%) and 11 of the 16 patients (69%) assessed on therapy. Serum HCV RNA (p < 0.0001) and alanine aminotransferase (ALT) levels (p < 0.0001) were lower during therapy than at baseline (Table 1).
2.3. Liver biopsy
3.2. TLR and RIG-I expression on CD4+ and CD8+ T-cells is normal or high in HCV-infected patients and may rise on therapy
All patients were biopsied as part of a routine assessment for therapy. Fibrosis was evaluated using the Scheuer [18] scoring system. Fibrosis was staged as: F0, no fibrosis; F1, portal fibrosis without septa; F2, portal fibrosis with few septa; F3, portal fibrosis with many septa; F4, cirrhosis. 2.4. Flow cytometry for analysis of TLR and RIG-I expression Cell-surface antigens to identify different T-cell subsets were identified using CD4-PerCP-Cy5.5, CD8-APC-Cy7, CD45RA-PE-Cy7, (BD Biosciences, San Jose, CA, USA) and CD57-FITC (eBioscience, USA), stained for 15 min at 22–24 °C. Extracellular TLR expression was detected with TLR2-FITC and TLR4-PE (eBioscience, San Diego, CA, USA). To detect intracellular TLR expression, cells were stained with cell-surface antigens and then washed. Cells were fixed and permeabilised using Cytofix/Cytoperm Kit™ (BD Biosciences, San Jose, CA, USA) and then incubated with TLR3-PE, TLR9-PE (eBioscience, San Diego, CA, USA), a polyclonal TLR7 (rabbit IgG) (Imgenex, San Diego, CA, USA) or a polyclonal RIG-I (rabbit IgG) (Alexis Biochemicals, Switzerland) for 30 min at 4 °C. For TLR7 and RIG-I, cells were further washed and incubated with goat anti-rabbit IgG-FITC (1:1000) (Imgenex) for 30 min at 4 °C. Stained cells were washed twice and analysed on a FACSCanto™ (Becton Dickinson, San Jose, CA, USA). At least 300,000 events were acquired and analysed using FlowJo (Treestar, San Carlos, CA, USA). Lymphocytes were identified by forward and side-scatter (FSC and SSC). Results for TLR expression are shown in terms of mean fluorescence intensity (MFI).
Several TLRs and RIG-I can be detected on CD4+ and CD8+ T-cells from healthy control donors and from untreated HCV-infected patients. Levels of TLRs and RIG-I on CD4+ and CD8+ T-cells were significantly lower, compared to monocytes and dendritic cells (p < 0.001) in controls (Supplementary Fig. 1) and patients (data not shown). In general, TLR and RIG-I expression were lower or similar in CD4+ and CD8+ T-cells compared to B-cells. Expression of RIG-I, TLR7 and TLR9 on CD4+ T-cells was higher in patients (p = 0.001, p = 0.01 and p = 0.09, respectively) (Fig. 1a). Only expression of RIG-1 on CD8+ T-cells differed between patients and controls (p = 0.004), but levels of TLR7 were marginally higher in patients (p = 0.06) (Fig. 1b). Therapy generally increased TLR expression on CD4+ T-cells, where TLR2, TLR4 and TLR9 increased statistically (p < 0.0001,
Table 1 Characteristics of HCV-infected patients studied.
Age (years) Sex (male/female) HCV genotype 1/2/3 Serum HCV RNA (log10 copies/mL) Serum ALT (U/mL) Fibrosis (Scheuer) 1/2/3/4 *
Baseline (n = 34)
During therapy (n = 16)
47 (21–66)* 20/14 20/2/12 5.4 (4.0–6.6) 122 (25–449) 12/11/4/7
40 (23–53) 7/9 10/1/5 2.5 (2.5–6.2)# 34 (10–177)# 6/7/2/5
Median (range). p < 0.05 Mann-Whitney Test relative to baseline.
#
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a) CD4+ T-cells 2000
*
Healthy controls HCV-infected patients
1600
*
During therapy
*
*
MFI
1200
* 800
400
0
TLR2
TLR3
TLR4
TLR7
TLR9
RIG-I
b) CD8+ T-cells Healthy controls
2000
HCV-infected patients
*
During therapy
*
*
*
1600
MFI
1200
*
800
400
0
TLR2
TLR3
TLR4
TLR7
TLR9
RIG-I
Fig. 1. Expression of TLR and RIG-I on CD4+ (a) and CD8+ (b) T-cells in healthy controls (diagonal), untreated HCV-infected patients (white) and patients during therapy (grey). TLR and RIG-I expression is measured by flow cytometry and data are presented as mean fluorescence intensity (MFI). p-values were determined by Mann–Whitney Test. *p < 0.05.
p = 0.004, p = 0.0005, respectively) (Fig. 1a). Expression of TLR on CD8+ T-cells also increased, reaching statistical significance for TLR2, TLR4, TLR7 and TLR9 (p < 0.0001, p = 0.01, p = 0.02 and p = 0.0004, respectively) (Fig. 1b). 3.3. TLR and RIG-I expression are generally higher on memory CD4+ Tcells than naïve CD4+ T-cells but rise on therapy in all sub-populations To determine whether the increased TLR expression associated with HCV disease reflected increased expression on sub-populations of T-cells, we assessed naïve (CD45RA+CD57), central memory TCM (CD45RACD57), effector memory TEM (CD45RACD57+) and terminally differentiated effector memory TEMRA (CD45RA+CD57+) Tcells (see Section 2). TLR and RIG-I were expressed on all naïve and memory T-cell subsets tested (see Table 2). In all donors, TLR2 and TLR4 expression was highest on CD4+ TEMRA, followed by TEM, TCM and naïve CD4+ T-cells, respectively. Expression of TLR3, RIG-I, TLR7 and TLR9 was similar in all memory CD4+ T-cell subsets, but significantly lower on naïve CD4+ T-cells (p < 0.05). TLR7 and RIG-I expression did not differ in naïve and memory CD8+ T-cell subsets. Similar to CD4+ T-cells, naïve CD8+ T-cells had the lowest TLR3 expression with no differences observed between memory subsets. In contrast, expression of TLR9 in TEMRA was similar to naïve cells (p = 0.34), which was lower compared to TCM (p = 0.0004) and TEM (p = 0.0016). We also evaluated whether increased expression observed on total CD4+ and CD8+ T-cells during therapy (Fig. 1) was due to
up-regulation on specific T-cell subsets. Therapy increased expression of TLR2, TLR4 and TLR9 on all subsets of CD4+ and CD8+ T-cells compared to baseline (p < 0.05, Table 2). Additionally, TLR7 expression on naïve and memory CD8+ T-cells was higher in treated patients. These data suggests increased TLR expression during therapy occurred on all T-cell subsets. 3.4. Expression of TLR and RIG-I on T-cells did not predict treatment outcome Sustained virological responses (SVR) after pegIFNa/ribavirin therapy were achieved in 20 of 34 patients (59%). Before therapy, expression of all TLRs and RIG-I was similar in SVR and non-SVR (Table 3). No associations were observed between expression of TLR and RIG-1 with HCV viral load, HCV genotype, serum alanine aminotransferase or fibrosis scores (data not shown). 4. Discussion Chronic HCV infection is characterized by diminished capacity for T-cells to produce IFNc against several HCV proteins when compared to patients who are able to clear the virus following acute infection [20]. These responses may be limited by TLR expression or function. In addition to their role on APC, TLR are expressed on T-cells in several infectious diseases, including lymphatic filariasis [21], tuberculosis [22] and HIV [23]. Here, we detected TLR2, TLR3, TLR4, TLR7, and TLR9 expression on CD4+ and CD8+ T-
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T. Hammond et al. / Cellular Immunology 264 (2010) 150–155 Table 2 TLR expression on CD4+ (a) and CD8+ (b) T-cells in HCV-infected patients and controls.
(a) (% CD4+ T-cells)
TLR2
TLR4
TLR3
RIG-I
TLR7
TLR9
(b) (% CD8+ T-cells)
TLR2
TLR4
TLR3
RIG-I
TLR7
TLR9
Healthy controls
HCV-infected patients
(n = 15)
Baseline (n = 35)
During therapy (n = 16)
Naive TCM TEM TEMRA Naive TCM TEM TEMRA Naive TCM TEM TEMRA Naive TCM TEM TEMRA Naive TCM TEM TEMRA Naive TCM TEM TEMRA
231 (199–284) 256 (229–328) 309 (271–1413) 994 (314–4816) 195 (45–769) 293 (96–691) 320 (104–670) 1101 (130–10,210) 378 (158–717) 521 (351–1044) 605 (366–1558) 560 (234–5931) 586 (279–869) 741 (347–1045) 733 (377–1038) 715 (366–1045) 554 (261–732) 639 (321 - 900) 650 (360–984) 679 (321–3421) 398 (269–798) 591 (411–1063) 572 (411–1030) 664 (365–2877)
225 271 330 688 183 300 323 613 378 590 627 628 702 841 837 838 636 748 740 730 605 864 755 657
(184–400) (228–425) (249–616) (286–4411) (111–929) (205–861) (254–880) (219–9828) (183–652) (350–902) (379–956) (305–1989) (479–935)# (575–1158)# (597–1154)# (599–1150)# (451–777)# (538–871)# (559–915)# (558–964) (234–1099) (343–1410) (336–1301) (287–1408)
310 (224–380)*,# 338 (261–397)*,# 428 (286–987)*,# 1325 (448–7647)* 236 (144–1352)*, 407 (290–803)*,# 434 (310–1264)*,# 1501 (347–23,139)* 412 (168–683) 678 (319–861) 721 (246–1075) 687 (263–2842) 725 (446 -1116)# 820 (509–1315)# 882 (499–1269)# 894 (543–1165)# 665 (397–991)# 778 (426–1110)# 515 (436–1072)# 839 (410–1039)*,# 858 (556–1668)*,# 1199 (1015–1966)*,# 1088 (922–1926)*,# 975 (681–3542)*,#
Naive TCM TEM TEMRA Naive TCM TEM TEMRA Naive TCM TEM TEMRA Naive TCM TEM TEMRA Naive TCM TEM TEMRA Naive TCM TEM TEMRA
281 270 313 337 166 324 295 164 478 531 592 502 669 695 687 718 628 618 634 668 342 522 449 309
284 296 320 316 146 319 325 180 322 558 582 456 772 821 788 808 683 716 700 746 441 761 616 411
(232–469) (225–487)# (206–523) (223–590) (59–991) (202–882) (194–905) (39–1186) (96–665) (351–841) (377–862) (168–1049) (535–1012)# (585–1089)# (590–1069)# (603–1119)# (510–855) (548–893)# (555–952) (566–1015) (173–1024) (311–1320) (320–1153) (171–884)
375 (253–482)*,# 393 (288–450)*,# 442 (293–1400)*,# 541 (290–1910)*,# 199 (97–925)* 469 (328–1148)*,# 438 (318–1115)*,# 280 (120–1467)*,# 312 (91–617) 683 (380–925) 733 (448–1172) 483 (172–906) 818 (563–1183)# 847 (532–1246)# 880 (506–1166)# 956 (515–1261)*,# 787 (491–1072)*,# 800 (452–1052)*,# 837 (459–1032)*,# 938 (486–1166)*,# 720 (401–1506)*,# 1038 (860–1740)*,# 960 (696–1719)*,# 725 (424–1538)*,#
(236–490) (243–524) (261–658) (258–703) (25–595) (92–940) (99–793) (106–674) (78–934) (327–1377) (292–2390) (124–2141) (360–964) (361–960) (370–982) (379–1026) (331–896) (241–911) (360–896) (371–939) (124–697) (382–935) (363–812) (219–731)
Results are presented as median (range). TLR and RIG-I expression is measured by flow cytometry and data are presented as mean fluorescence intensity (MFI). p-values were determined by Mann–Whitney Test. * Compared to baseline. # Compared to healthy controls.
cells from healthy controls and patients chronically infected with HCV. In general, expression of TLR3 and TLR7 was similar on CD4+ and CD8+ T-cells, whilst TLR2 expression was lower and TLR9 was higher in CD4+ T-cells from healthy donors and patients. CD4+ T-cells from peripheral blood [24] and tonsil tissues [13] contained more mRNA for most TLRs (except TLR3), when compared to matched CD8+ T-cells. In this study, untreated patients had higher TLR7 expression on all CD4+ and CD8+ T-cells compared to uninfected donors. This confirms a previous report of increased TLR7 mRNA levels in purified T-cells from HCV-infected patients [4]. We show for the first time that pegIFNa/ribavirin treatment increases expression of TLR2, TLR4 and TLR9 on T-cells from HCV-infected patients. Expression of TLR3 and TLR7 in monocytes and monocyte-derived DC from pa-
tients with rheumatoid arthritis and healthy controls is increased by IFNa in vitro [25], but this has not been reported in relation to HCV therapy. Baseline TLR and RIG-I expression on CD4+ and CD8+ T-cells were similar in SVR and non-SVR, so monitoring of these receptors will not predict treatment outcome. Whilst TLRs are localised in the endosomes or on the cell membrane, RIG-I is expressed in the cytoplasm. RIG-I recognises the 50 triphosphate structure of the viral RNA [26] including the 50 and 30 untranslated regions of the HCV genome generated during replication in the cytoplasm [27]. Upon binding, RIG-I activates the IFNb promoter stimulator 1 (ISP-1) leading to activation of the transcription factors IRF-3 and NFjB. mRNA for RIG-I can be detected in monocytes and PBMC from patients with chronic HCV infection [5,28]. We found more RIG-1 protein in T-cells from un-
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Table 3 Baseline expression of TLRs and RIG-I on T-cells. SVR (n = 20)
Non-SVR (n = 14)
CD4 T-cells TLR2 TLR4 TLR3 TLR7 TLR9 RIG-I
250 242 545 709 811 794
(215–323) (177–963) (326–793) (571–832) (365–1207) (596–1040)
262 250 460 682 559 762
(222–449) (203–470) (329–781) (523–822) (300–1258) (558–1045)
CD8+ T-cells TLR2 TLR4 TLR3 TLR7 TLR9 RIG-I
296 241 520 717 695 805
(220–380) (128–969) (311–734) (604–885) (306–982) (631–1005)
300 213 397 698 440 750
(262–524) (165–405) (250–716) (543–809) (222–1074) (576–1033)
+
Results are presented as median (range). TLRs and RIG-I expression is measured by flow cytometry and data are presented as mean fluorescence intensity (MFI).
treated patients compared to controls. There was higher expression on CD8+ T-cells compared to CD4+ T-cells and on memory Tcells compared to naïve T-cells. Differential expression of TLRs and RIG-I on CD4+ and CD8+ T-cells may reflect distinct roles during infection. TLR expression on T-cells may affect cytokine production following activation by specific ligands. Babu et al. detected TLR1, TLR2, TLR4 and TLR9 on memory CD45RO+ T-cells but cell-surface expression was absent on naïve CD45RA+ T-cells [21]. However, TLR2 protein was found intracellularly in naïve CD45RA+ CD4+ Tcells isolated from cord blood [29] and TLR2 can be induced in naïve T-cells following stimulation with anti-CD3 [29,30]. We report cell-surface expression of several TLRs on naïve T-cells in the absence of any stimulation, but levels were generally lower compared to memory T-cells. Studies [21,29] using CD45RA+ alone to identify naïve T-cells may include CD45RO+ T-cells that re-express CD45RA as late effector memory T-cells (TEMRA). Expression of TLR2 and TLR4 was highest on CD4+ TEMRA, whilst expression of TLR3, TLR7, TLR9 and RIG-I was similar in CD4+ TCM, TEM and TEMRA. Upon stimulation with TLR5 and TLR7/TLR8 ligands, effector memory CD4+ T-cells up-regulate production of IFNc and proliferate [31]. Engagement of TLR2 on murine CD8+ T-cells provides a strong co-stimulatory signal in the presence of suboptimal TCR signals from antigen-presenting cells [32] and drives clonal expansion of memory T-cells following vaccinia infection [33]. Hence, TLR expression on T-cells can influence T-cell differentiation and function in various diseases. Indeed, lower expression of TLR2 on Tcells from filarial-infected individuals was directly associated with reduced activation and IFNc production by T-cells following stimulation with the TLR2 ligand Pam3Cys [21]. Availability of PBMCs limited our assessment of cytokine responses to TLR ligands. However in a pilot study using cells from two HCV-infected patients, IL2 but not IFNc was produced by CD4+ and CD8+ memory T-cells when PBMC was stimulated with ligands for TLR3, TLR7/8 and TLR9 (Poly(I:C), CL075 or CpG, respectively), for up to 72 h (data not shown). The activation marker, CD69, was up-regulated on PBMC, especially in cultures stimulated with CLO75 (22% and 25% vs. 6.9% and 2.1% in unstimulated cultures). This is consistent with studies using other TLR ligands and markers of immune activation (CD69, CD38, ICAM-1 and HLA-DR) on CD4+ and CD8+ Tcells from HIV patients and controls [34–36]. We have shown that IFNc production can be detected by ELISpot after PBMCs were incubated with TLR ligands for 24 h [Lee et al. [19]]. This may result from activation of DCs by the TLR ligands rather than direct interaction with T-cells, so studies using purified CD4+ and CD8+ T-cells are needed. The scenario is compli-
cated by the ability of HCV proteins to interact with components of the TLR signaling pathways to prevent the production of cytokines [37–39]. This could explain why HCV patients exhibit poor induction of cytokine production by DCs stimulated with TLR ligands, despite increased TLR expression on these cells [6][Lee et al. [19]]. In summary, we demonstrate increased expression of TLRs and RIG-I on T-cells from patients with chronic HCV infection compared to uninfected individuals. Therapy with pegIFNa/ribavirin further increased expression of several TLRs, but not RIG-I. Longitudinal studies are warranted to gain further insight into consequences of increased TLR expression on T-cells. Acknowledgments The authors thank the patients who donated blood for this study and Saroj Nazareth and Marion McInerney who assisted in the collection of samples. This work received support from Schering-Plough Pty Limited, North Ryde, NSW. This is publication 201002 (Clinical Immunology and Immunogenetics, RPH). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cellimm.2010.06.001. References [1] E. Seki, D.A. Brenner, Toll-like receptors and adaptor molecules in liver disease: update, Hepatology 48 (2008) 322–335. [2] A.J. Thompson, S.A. Locarnini, Toll-like receptors, RIG-I-like RNA helicases and the antiviral innate immune response, Immunol. Cell Biol. 85 (2007) 435–445. [3] Q. He, C.S. Graham, E. Durante Mangoni, M.J. Koziel, Differential expression of toll-like receptor mRNA in treatment non-responders and sustained virologic responders at baseline in patients with chronic hepatitis C, Liver Int. 26 (2006) 1100–1110. [4] A. Dolganiuc, C. Garcia, K. Kodys, G. Szabo, Distinct Toll-like receptor expression in monocytes and T cells in chronic HCV infection, World J. Gastroenterol. 12 (2006) 1198–1204. [5] K. Sato, T. Ishikawa, A. Okumura, T. Yamauchi, S. Sato, M. Ayada, et al., Expression of Toll-like receptors in chronic hepatitis C virus infection, J. Gastroenterol. Hepatol. 22 (2007) 1627–1632. [6] M. Miyazaki, T. Kanto, M. Inoue, I. Itose, H. Miyatake, M. Sakakibara, T. Yakushijin, N. Kakita, N. Hiramatsu, T. Takehara, A. Kasahara, N. Hayashi, Impaired cytokine response in myeloid dendritic cells in chronic hepatitis C virus infection regardless of enhanced expression of Toll-like receptors and retinoic acid inducible gene-I, J. Med. Virol. 80 (2008) 980–988. [7] N.L. Yonkers, B. Rodriguez, K.A. Milkovich, R. Asaad, M.M. Lederman, P.S. Heeger, D.D. Anthony, TLR ligand-dependent activation of naive CD4 T cells by plasmacytoid dendritic cells is impaired in hepatitis C virus infection, J. Immunol. 178 (2007) 4436–4444. [8] I.G. Rodrigue-Gervais, L. Jouan, G. Beaule, D. Sauve, J. Bruneau, B. Willems, et al., Poly(I:C) and lipopolysaccharide innate sensing functions of circulating human myeloid dendritic cells are affected in vivo in hepatitis C virus-infected patients, J. Virol. 81 (2007) 5537–5546. [9] S. Babu, C.P. Blauvelt, V. Kumaraswami, T.B. Nutman, Diminished expression and function of TLR in lymphatic filariasis: a novel mechanism of immune dysregulation, J. Immunol. 175 (2005) 1170–1176. [10] P. Dasari, I.C. Nicholson, G. Hodge, G.W. Dandie, H. Zola, Expression of toll-like receptors on B lymphocytes, Cell Immunol. 236 (2005) 140–145. [11] A. Månsson, M. Adner, U. Höckerfelt, L.O. Cardell, A distinct Toll-like receptor repertoire in human tonsillar B cells, directly activated by PamCSK, R-837 and CpG-2006 stimulation, Immunology 118 (2006) 539–548. [12] V. Hornung, S. Rothenfusser, S. Britsch, A. Krug, B. Jahrsdörfer, T. Giese, S. Endres, G. Hartmann, Quantitative expression of toll-like receptor 1–10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides, J. Immunol. 168 (2002) 4531–4537. [13] A. Mansson, M. Adner, L.O. Cardell, Toll-like receptors in cellular subsets of human tonsil T cells: altered expression during recurrent tonsillitis, Respir. Res. 27 (2006) 7–36. [14] K.U. Saikh, J.S. Lee, T.L. Kissner, B. Dyas, R.G. Ulrich, Toll-like receptor and cytokine expression patterns of CD56+ T cells are similar to natural killer cells in response to infection with Venezuelan equine encephalitis virus replicons, J. Infect Dis. 188 (2003) 1562–1570. [15] N.K. Crellin, R.V. Garcia, O. Hadisfar, S.E. Allan, T.S. Steiner, M.K. Levings, Human CD4+ T cells express TLR5 and its ligand flagellin enhances the suppressive capacity and expression of FOXP3 in CD4+CD25+ T regulatory cells, J. Immunol. 175 (2005) 8051–8059.
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Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Toll-like receptor (TLR) expression on CD4+ and CD8+ T-cells in patients chronically infected with hepatitis C virus Talia Hammond a, Silvia Lee a,b,*, Mark W. Watson e, James P. Flexman a, Wendy Cheng d, Sonia Fernandez a, Patricia Price a,c a
School of Pathology and Laboratory Medicine, University of Western Australia, Australia Department of Microbiology and Infectious Disease, Royal Perth Hospital, Australia Clinical Immunology and Immunogenetics, Royal Perth Hospital, Australia d Department of Gastroenterology and Hepatology, Royal Perth Hospital, Australia e Centre for Clinical Immunology & Biomedical Statistics, Royal Perth Hospital, Australia b c
a r t i c l e
i n f o
Article history: Received 26 March 2010 Accepted 2 June 2010 Available online 8 June 2010 Keywords: HCV TLR RIG-I Therapy
a b s t r a c t Toll-like receptor (TLR) expression on T-cells and the signalling pathways that lead to the production of cytokines may limit antigen-specific T-cell responses. Here, expression of TLR and retinoic acid inducible gene I (RIG-I) on T-cells were evaluated in patients chronically infected with hepatitis C virus (HCV), before and during pegylated interferon-a and ribavirin therapy. Expression of TLR2,3,4,7,9 and retinoic acid inducible gene (RIG)-I on different CD4+ and CD8+ T-cell sub-populations (naïve: CD45RA+CD57; central memory: TCM CD45RACD57; effector memory: TEM CD45RACD57+ and terminally differentiated effector memory: TEMRA CD45RA+CD57+) were measured by flow cytometry. TLR7, TLR9 and RIG-I expression on CD4+ T-cells and RIG-I expression on CD8+ T-cells was higher in patients than healthy controls. Therapy increased expression of TLR2, TLR4 and TLR9 and this was observed for all T-cell sub-populations. Evaluation of TLR expression at baseline did not identify patients able to achieve sustained virological response following therapy. Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved.
1. Introduction Toll-like receptors (TLR), retinoic acid inducible gene I (RIG-I)like helicases and nucleotide-binding oligodimerisation domain (NOD)-like receptors [1] recognise pathogen-associated molecular patterns found in bacteria, fungi, viruses and protozoa. TLRs are primarily expressed on dendritic cells (DCs) and are critical to their role in immune surveillance. Triggering through TLRs promotes DC maturation, production of pro-inflammatory and antiviral cytokines, up-regulation of co-stimulatory molecules and altered expression of chemokine receptors. Eleven TLRs have been identified in humans. Receptors that recognise viral products or nucleic acids are expressed in endosomal membranes (e.g. TLR3, TLR7, TLR8, TLR9). TLR2 recognises peptidoglycan and glycolipids and TLR4 recognises lipopolysaccharide. These are expressed on the cytoplasmic membrane [2].
* Corresponding author at: Level 2, MRF Building, Rear 50 Murray Street, Near Royal Perth Hospital, Perth, Western Australia 6000, Australia. Fax: +61 8 9224 0204. E-mail addresses: [email protected], [email protected] (S. Lee).
TLRs and RIG-I have potential to influence chronic HCV infection. Increased mRNA levels for several TLRs was demonstrated in unfractionated peripheral blood mononuclear cells (PBMC) [3,5], purified monocytes [4,5] and purified T-cells [4] from HCVinfected patients compared to healthy controls. RIG-I mRNA was also higher in myeloid DCs (mDCs) from HCV-infected patients than healthy individuals [6]. Production of IL-12 by mDCs stimulated with poly (I:C) (a TLR3 ligand) and production of IFNa by plasmacytoid DCs (pDCs) stimulated with CpG (a TLR9 ligand) was low in patients, despite elevated expression of TLRs and RIGI [6,7]. Furthermore, reduced IFNa production by pDCs in response to a TLR7 ligand was associated with impaired activation of naïve CD4 T-cells [7]. Rodrigue-Gervais et al. associated decreased cytokine production by mDCs with high levels of HCV RNA in unfractionated DCs [8]. Hence, HCV activates the innate immune system and inhibits TLR signalling pathways and subsequent production of cytokines. In addition to dendritic cells and monocytes, TLRs are expressed on B-cells [9–11], T-cells [12,13] and NK cells [14] and regulatory T-cells [15]. Triggering of these cells through TLRs could influence immunity and immunopathology associated with HCV disease. Here we investigated TLR and RIG-I expression on T-cell populations from chronically HCV-infected patients and healthy donors.
0008-8749/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2010.06.001
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2. Materials and methods 2.1. Patients Patients chronically infected with HCV (n = 34) were recruited from Royal Perth Hospital (Western Australia). HCV infection was diagnosed by third generation enzyme linked immunosorbent assay (ELISA) for antibodies to HCV and confirmed by qualitative reverse transcriptase polymerase chain reaction (RT-PCR) for HCV RNA in serum (Cobas Amplicor HCV Test, Roche Diagnostics, Branchburg, NJ, USA). Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll (Amersham Biosciences, Buckinghamshire, UK) density gradient centrifugation and cryopreserved in 10% DMSO and 90% fetal calf serum (FCS) for subsequent analyses. PBMC samples were also collected for 16 of the 34 patients during therapy (Pegasys, Roche, Dee Why, NSW, Australia or Pegatron, Schering-Plough, North Ryde, NSW, Australia) [median time on therapy 5 (3–10) months]. Patients with HCV genotypes 1 or 4 were treated for 48 weeks and those with genotypes 2 or 3 for 24 weeks. Healthy individuals with no evidence of exposure to HIV or HCV were included as controls and were matched by age and sex with patients [42 (21–73 years), 7males, 9 females]. The study was approved by the Royal Perth Hospital Research Ethics committee and all patients and controls gave informed consent.
Expression of CD57 and CD45RA was used to define naïve (CD45RA+CD57), central memory TCM (CD45RACD57), effector memory TEM (CD45RACD57+) and terminally differentiated effector memory TEMRA (CD45RA+CD57+) T-cells. CCR7 is more widely used to distinguish memory T-cells, but could not be detected on cryopreserved PBMC with the monoclonal antibody available commercially when this study was performed. An alternative antibody is now available. In a pilot study of seven healthy controls and five HCV-infected patients, CCR7 and CD57 defined similar percentages of naïve and CD4+ TEMRA T-cells and all CD8+ T-cell subsets. However, percentages of CD4+ TCM T-cells were lower and CD4+ TEM T-cells were higher using CCR7 rather than CD57 [19]. 2.5. Statistics All data are presented as median (range). Statistical analyses were performed using Graphpad Prism Version 5.01 (Graphpad Software, San Diego, CA, USA). Differences between study groups were evaluated using Mann–Whitney Tests. p-values <0.05 were considered statistically significant. Figures are presented as box and whisker plots showing median, 25th and 75th percentiles and range. 3. Results
2.2. Detection of HCV RNA and HCV genotyping
3.1. Patient characteristics
Sera were stored at 80 °C. Viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, USA). HCV genotype was determined using the Line Probe Assay (Inno-LiPA, Innogenetics, Gent, Belgium) or by real-time PCR and melting curve analysis using FRET probes [16]. HCV viral loads were assessed using primers binding the conserved 50 UTR region [17].
From the 34 patients studied at baseline, samples were available for 16 patients during therapy [5 (3–10) months]. For all subsequent analyses, these two groups of patients were compared. SVR were achieved in 20 of the 34 patients (59%) and 11 of the 16 patients (69%) assessed on therapy. Serum HCV RNA (p < 0.0001) and alanine aminotransferase (ALT) levels (p < 0.0001) were lower during therapy than at baseline (Table 1).
2.3. Liver biopsy
3.2. TLR and RIG-I expression on CD4+ and CD8+ T-cells is normal or high in HCV-infected patients and may rise on therapy
All patients were biopsied as part of a routine assessment for therapy. Fibrosis was evaluated using the Scheuer [18] scoring system. Fibrosis was staged as: F0, no fibrosis; F1, portal fibrosis without septa; F2, portal fibrosis with few septa; F3, portal fibrosis with many septa; F4, cirrhosis. 2.4. Flow cytometry for analysis of TLR and RIG-I expression Cell-surface antigens to identify different T-cell subsets were identified using CD4-PerCP-Cy5.5, CD8-APC-Cy7, CD45RA-PE-Cy7, (BD Biosciences, San Jose, CA, USA) and CD57-FITC (eBioscience, USA), stained for 15 min at 22–24 °C. Extracellular TLR expression was detected with TLR2-FITC and TLR4-PE (eBioscience, San Diego, CA, USA). To detect intracellular TLR expression, cells were stained with cell-surface antigens and then washed. Cells were fixed and permeabilised using Cytofix/Cytoperm Kit™ (BD Biosciences, San Jose, CA, USA) and then incubated with TLR3-PE, TLR9-PE (eBioscience, San Diego, CA, USA), a polyclonal TLR7 (rabbit IgG) (Imgenex, San Diego, CA, USA) or a polyclonal RIG-I (rabbit IgG) (Alexis Biochemicals, Switzerland) for 30 min at 4 °C. For TLR7 and RIG-I, cells were further washed and incubated with goat anti-rabbit IgG-FITC (1:1000) (Imgenex) for 30 min at 4 °C. Stained cells were washed twice and analysed on a FACSCanto™ (Becton Dickinson, San Jose, CA, USA). At least 300,000 events were acquired and analysed using FlowJo (Treestar, San Carlos, CA, USA). Lymphocytes were identified by forward and side-scatter (FSC and SSC). Results for TLR expression are shown in terms of mean fluorescence intensity (MFI).
Several TLRs and RIG-I can be detected on CD4+ and CD8+ T-cells from healthy control donors and from untreated HCV-infected patients. Levels of TLRs and RIG-I on CD4+ and CD8+ T-cells were significantly lower, compared to monocytes and dendritic cells (p < 0.001) in controls (Supplementary Fig. 1) and patients (data not shown). In general, TLR and RIG-I expression were lower or similar in CD4+ and CD8+ T-cells compared to B-cells. Expression of RIG-I, TLR7 and TLR9 on CD4+ T-cells was higher in patients (p = 0.001, p = 0.01 and p = 0.09, respectively) (Fig. 1a). Only expression of RIG-1 on CD8+ T-cells differed between patients and controls (p = 0.004), but levels of TLR7 were marginally higher in patients (p = 0.06) (Fig. 1b). Therapy generally increased TLR expression on CD4+ T-cells, where TLR2, TLR4 and TLR9 increased statistically (p < 0.0001,
Table 1 Characteristics of HCV-infected patients studied.
Age (years) Sex (male/female) HCV genotype 1/2/3 Serum HCV RNA (log10 copies/mL) Serum ALT (U/mL) Fibrosis (Scheuer) 1/2/3/4 *
Baseline (n = 34)
During therapy (n = 16)
47 (21–66)* 20/14 20/2/12 5.4 (4.0–6.6) 122 (25–449) 12/11/4/7
40 (23–53) 7/9 10/1/5 2.5 (2.5–6.2)# 34 (10–177)# 6/7/2/5
Median (range). p < 0.05 Mann-Whitney Test relative to baseline.
#
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a) CD4+ T-cells 2000
*
Healthy controls HCV-infected patients
1600
*
During therapy
*
*
MFI
1200
* 800
400
0
TLR2
TLR3
TLR4
TLR7
TLR9
RIG-I
b) CD8+ T-cells Healthy controls
2000
HCV-infected patients
*
During therapy
*
*
*
1600
MFI
1200
*
800
400
0
TLR2
TLR3
TLR4
TLR7
TLR9
RIG-I
Fig. 1. Expression of TLR and RIG-I on CD4+ (a) and CD8+ (b) T-cells in healthy controls (diagonal), untreated HCV-infected patients (white) and patients during therapy (grey). TLR and RIG-I expression is measured by flow cytometry and data are presented as mean fluorescence intensity (MFI). p-values were determined by Mann–Whitney Test. *p < 0.05.
p = 0.004, p = 0.0005, respectively) (Fig. 1a). Expression of TLR on CD8+ T-cells also increased, reaching statistical significance for TLR2, TLR4, TLR7 and TLR9 (p < 0.0001, p = 0.01, p = 0.02 and p = 0.0004, respectively) (Fig. 1b). 3.3. TLR and RIG-I expression are generally higher on memory CD4+ Tcells than naïve CD4+ T-cells but rise on therapy in all sub-populations To determine whether the increased TLR expression associated with HCV disease reflected increased expression on sub-populations of T-cells, we assessed naïve (CD45RA+CD57), central memory TCM (CD45RACD57), effector memory TEM (CD45RACD57+) and terminally differentiated effector memory TEMRA (CD45RA+CD57+) Tcells (see Section 2). TLR and RIG-I were expressed on all naïve and memory T-cell subsets tested (see Table 2). In all donors, TLR2 and TLR4 expression was highest on CD4+ TEMRA, followed by TEM, TCM and naïve CD4+ T-cells, respectively. Expression of TLR3, RIG-I, TLR7 and TLR9 was similar in all memory CD4+ T-cell subsets, but significantly lower on naïve CD4+ T-cells (p < 0.05). TLR7 and RIG-I expression did not differ in naïve and memory CD8+ T-cell subsets. Similar to CD4+ T-cells, naïve CD8+ T-cells had the lowest TLR3 expression with no differences observed between memory subsets. In contrast, expression of TLR9 in TEMRA was similar to naïve cells (p = 0.34), which was lower compared to TCM (p = 0.0004) and TEM (p = 0.0016). We also evaluated whether increased expression observed on total CD4+ and CD8+ T-cells during therapy (Fig. 1) was due to
up-regulation on specific T-cell subsets. Therapy increased expression of TLR2, TLR4 and TLR9 on all subsets of CD4+ and CD8+ T-cells compared to baseline (p < 0.05, Table 2). Additionally, TLR7 expression on naïve and memory CD8+ T-cells was higher in treated patients. These data suggests increased TLR expression during therapy occurred on all T-cell subsets. 3.4. Expression of TLR and RIG-I on T-cells did not predict treatment outcome Sustained virological responses (SVR) after pegIFNa/ribavirin therapy were achieved in 20 of 34 patients (59%). Before therapy, expression of all TLRs and RIG-I was similar in SVR and non-SVR (Table 3). No associations were observed between expression of TLR and RIG-1 with HCV viral load, HCV genotype, serum alanine aminotransferase or fibrosis scores (data not shown). 4. Discussion Chronic HCV infection is characterized by diminished capacity for T-cells to produce IFNc against several HCV proteins when compared to patients who are able to clear the virus following acute infection [20]. These responses may be limited by TLR expression or function. In addition to their role on APC, TLR are expressed on T-cells in several infectious diseases, including lymphatic filariasis [21], tuberculosis [22] and HIV [23]. Here, we detected TLR2, TLR3, TLR4, TLR7, and TLR9 expression on CD4+ and CD8+ T-
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T. Hammond et al. / Cellular Immunology 264 (2010) 150–155 Table 2 TLR expression on CD4+ (a) and CD8+ (b) T-cells in HCV-infected patients and controls.
(a) (% CD4+ T-cells)
TLR2
TLR4
TLR3
RIG-I
TLR7
TLR9
(b) (% CD8+ T-cells)
TLR2
TLR4
TLR3
RIG-I
TLR7
TLR9
Healthy controls
HCV-infected patients
(n = 15)
Baseline (n = 35)
During therapy (n = 16)
Naive TCM TEM TEMRA Naive TCM TEM TEMRA Naive TCM TEM TEMRA Naive TCM TEM TEMRA Naive TCM TEM TEMRA Naive TCM TEM TEMRA
231 (199–284) 256 (229–328) 309 (271–1413) 994 (314–4816) 195 (45–769) 293 (96–691) 320 (104–670) 1101 (130–10,210) 378 (158–717) 521 (351–1044) 605 (366–1558) 560 (234–5931) 586 (279–869) 741 (347–1045) 733 (377–1038) 715 (366–1045) 554 (261–732) 639 (321 - 900) 650 (360–984) 679 (321–3421) 398 (269–798) 591 (411–1063) 572 (411–1030) 664 (365–2877)
225 271 330 688 183 300 323 613 378 590 627 628 702 841 837 838 636 748 740 730 605 864 755 657
(184–400) (228–425) (249–616) (286–4411) (111–929) (205–861) (254–880) (219–9828) (183–652) (350–902) (379–956) (305–1989) (479–935)# (575–1158)# (597–1154)# (599–1150)# (451–777)# (538–871)# (559–915)# (558–964) (234–1099) (343–1410) (336–1301) (287–1408)
310 (224–380)*,# 338 (261–397)*,# 428 (286–987)*,# 1325 (448–7647)* 236 (144–1352)*, 407 (290–803)*,# 434 (310–1264)*,# 1501 (347–23,139)* 412 (168–683) 678 (319–861) 721 (246–1075) 687 (263–2842) 725 (446 -1116)# 820 (509–1315)# 882 (499–1269)# 894 (543–1165)# 665 (397–991)# 778 (426–1110)# 515 (436–1072)# 839 (410–1039)*,# 858 (556–1668)*,# 1199 (1015–1966)*,# 1088 (922–1926)*,# 975 (681–3542)*,#
Naive TCM TEM TEMRA Naive TCM TEM TEMRA Naive TCM TEM TEMRA Naive TCM TEM TEMRA Naive TCM TEM TEMRA Naive TCM TEM TEMRA
281 270 313 337 166 324 295 164 478 531 592 502 669 695 687 718 628 618 634 668 342 522 449 309
284 296 320 316 146 319 325 180 322 558 582 456 772 821 788 808 683 716 700 746 441 761 616 411
(232–469) (225–487)# (206–523) (223–590) (59–991) (202–882) (194–905) (39–1186) (96–665) (351–841) (377–862) (168–1049) (535–1012)# (585–1089)# (590–1069)# (603–1119)# (510–855) (548–893)# (555–952) (566–1015) (173–1024) (311–1320) (320–1153) (171–884)
375 (253–482)*,# 393 (288–450)*,# 442 (293–1400)*,# 541 (290–1910)*,# 199 (97–925)* 469 (328–1148)*,# 438 (318–1115)*,# 280 (120–1467)*,# 312 (91–617) 683 (380–925) 733 (448–1172) 483 (172–906) 818 (563–1183)# 847 (532–1246)# 880 (506–1166)# 956 (515–1261)*,# 787 (491–1072)*,# 800 (452–1052)*,# 837 (459–1032)*,# 938 (486–1166)*,# 720 (401–1506)*,# 1038 (860–1740)*,# 960 (696–1719)*,# 725 (424–1538)*,#
(236–490) (243–524) (261–658) (258–703) (25–595) (92–940) (99–793) (106–674) (78–934) (327–1377) (292–2390) (124–2141) (360–964) (361–960) (370–982) (379–1026) (331–896) (241–911) (360–896) (371–939) (124–697) (382–935) (363–812) (219–731)
Results are presented as median (range). TLR and RIG-I expression is measured by flow cytometry and data are presented as mean fluorescence intensity (MFI). p-values were determined by Mann–Whitney Test. * Compared to baseline. # Compared to healthy controls.
cells from healthy controls and patients chronically infected with HCV. In general, expression of TLR3 and TLR7 was similar on CD4+ and CD8+ T-cells, whilst TLR2 expression was lower and TLR9 was higher in CD4+ T-cells from healthy donors and patients. CD4+ T-cells from peripheral blood [24] and tonsil tissues [13] contained more mRNA for most TLRs (except TLR3), when compared to matched CD8+ T-cells. In this study, untreated patients had higher TLR7 expression on all CD4+ and CD8+ T-cells compared to uninfected donors. This confirms a previous report of increased TLR7 mRNA levels in purified T-cells from HCV-infected patients [4]. We show for the first time that pegIFNa/ribavirin treatment increases expression of TLR2, TLR4 and TLR9 on T-cells from HCV-infected patients. Expression of TLR3 and TLR7 in monocytes and monocyte-derived DC from pa-
tients with rheumatoid arthritis and healthy controls is increased by IFNa in vitro [25], but this has not been reported in relation to HCV therapy. Baseline TLR and RIG-I expression on CD4+ and CD8+ T-cells were similar in SVR and non-SVR, so monitoring of these receptors will not predict treatment outcome. Whilst TLRs are localised in the endosomes or on the cell membrane, RIG-I is expressed in the cytoplasm. RIG-I recognises the 50 triphosphate structure of the viral RNA [26] including the 50 and 30 untranslated regions of the HCV genome generated during replication in the cytoplasm [27]. Upon binding, RIG-I activates the IFNb promoter stimulator 1 (ISP-1) leading to activation of the transcription factors IRF-3 and NFjB. mRNA for RIG-I can be detected in monocytes and PBMC from patients with chronic HCV infection [5,28]. We found more RIG-1 protein in T-cells from un-
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Table 3 Baseline expression of TLRs and RIG-I on T-cells. SVR (n = 20)
Non-SVR (n = 14)
CD4 T-cells TLR2 TLR4 TLR3 TLR7 TLR9 RIG-I
250 242 545 709 811 794
(215–323) (177–963) (326–793) (571–832) (365–1207) (596–1040)
262 250 460 682 559 762
(222–449) (203–470) (329–781) (523–822) (300–1258) (558–1045)
CD8+ T-cells TLR2 TLR4 TLR3 TLR7 TLR9 RIG-I
296 241 520 717 695 805
(220–380) (128–969) (311–734) (604–885) (306–982) (631–1005)
300 213 397 698 440 750
(262–524) (165–405) (250–716) (543–809) (222–1074) (576–1033)
+
Results are presented as median (range). TLRs and RIG-I expression is measured by flow cytometry and data are presented as mean fluorescence intensity (MFI).
treated patients compared to controls. There was higher expression on CD8+ T-cells compared to CD4+ T-cells and on memory Tcells compared to naïve T-cells. Differential expression of TLRs and RIG-I on CD4+ and CD8+ T-cells may reflect distinct roles during infection. TLR expression on T-cells may affect cytokine production following activation by specific ligands. Babu et al. detected TLR1, TLR2, TLR4 and TLR9 on memory CD45RO+ T-cells but cell-surface expression was absent on naïve CD45RA+ T-cells [21]. However, TLR2 protein was found intracellularly in naïve CD45RA+ CD4+ Tcells isolated from cord blood [29] and TLR2 can be induced in naïve T-cells following stimulation with anti-CD3 [29,30]. We report cell-surface expression of several TLRs on naïve T-cells in the absence of any stimulation, but levels were generally lower compared to memory T-cells. Studies [21,29] using CD45RA+ alone to identify naïve T-cells may include CD45RO+ T-cells that re-express CD45RA as late effector memory T-cells (TEMRA). Expression of TLR2 and TLR4 was highest on CD4+ TEMRA, whilst expression of TLR3, TLR7, TLR9 and RIG-I was similar in CD4+ TCM, TEM and TEMRA. Upon stimulation with TLR5 and TLR7/TLR8 ligands, effector memory CD4+ T-cells up-regulate production of IFNc and proliferate [31]. Engagement of TLR2 on murine CD8+ T-cells provides a strong co-stimulatory signal in the presence of suboptimal TCR signals from antigen-presenting cells [32] and drives clonal expansion of memory T-cells following vaccinia infection [33]. Hence, TLR expression on T-cells can influence T-cell differentiation and function in various diseases. Indeed, lower expression of TLR2 on Tcells from filarial-infected individuals was directly associated with reduced activation and IFNc production by T-cells following stimulation with the TLR2 ligand Pam3Cys [21]. Availability of PBMCs limited our assessment of cytokine responses to TLR ligands. However in a pilot study using cells from two HCV-infected patients, IL2 but not IFNc was produced by CD4+ and CD8+ memory T-cells when PBMC was stimulated with ligands for TLR3, TLR7/8 and TLR9 (Poly(I:C), CL075 or CpG, respectively), for up to 72 h (data not shown). The activation marker, CD69, was up-regulated on PBMC, especially in cultures stimulated with CLO75 (22% and 25% vs. 6.9% and 2.1% in unstimulated cultures). This is consistent with studies using other TLR ligands and markers of immune activation (CD69, CD38, ICAM-1 and HLA-DR) on CD4+ and CD8+ Tcells from HIV patients and controls [34–36]. We have shown that IFNc production can be detected by ELISpot after PBMCs were incubated with TLR ligands for 24 h [Lee et al. [19]]. This may result from activation of DCs by the TLR ligands rather than direct interaction with T-cells, so studies using purified CD4+ and CD8+ T-cells are needed. The scenario is compli-
cated by the ability of HCV proteins to interact with components of the TLR signaling pathways to prevent the production of cytokines [37–39]. This could explain why HCV patients exhibit poor induction of cytokine production by DCs stimulated with TLR ligands, despite increased TLR expression on these cells [6][Lee et al. [19]]. In summary, we demonstrate increased expression of TLRs and RIG-I on T-cells from patients with chronic HCV infection compared to uninfected individuals. Therapy with pegIFNa/ribavirin further increased expression of several TLRs, but not RIG-I. Longitudinal studies are warranted to gain further insight into consequences of increased TLR expression on T-cells. Acknowledgments The authors thank the patients who donated blood for this study and Saroj Nazareth and Marion McInerney who assisted in the collection of samples. This work received support from Schering-Plough Pty Limited, North Ryde, NSW. This is publication 201002 (Clinical Immunology and Immunogenetics, RPH). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cellimm.2010.06.001. References [1] E. Seki, D.A. Brenner, Toll-like receptors and adaptor molecules in liver disease: update, Hepatology 48 (2008) 322–335. [2] A.J. Thompson, S.A. Locarnini, Toll-like receptors, RIG-I-like RNA helicases and the antiviral innate immune response, Immunol. Cell Biol. 85 (2007) 435–445. [3] Q. He, C.S. Graham, E. Durante Mangoni, M.J. Koziel, Differential expression of toll-like receptor mRNA in treatment non-responders and sustained virologic responders at baseline in patients with chronic hepatitis C, Liver Int. 26 (2006) 1100–1110. [4] A. Dolganiuc, C. Garcia, K. Kodys, G. Szabo, Distinct Toll-like receptor expression in monocytes and T cells in chronic HCV infection, World J. Gastroenterol. 12 (2006) 1198–1204. [5] K. Sato, T. Ishikawa, A. Okumura, T. Yamauchi, S. Sato, M. Ayada, et al., Expression of Toll-like receptors in chronic hepatitis C virus infection, J. Gastroenterol. Hepatol. 22 (2007) 1627–1632. [6] M. Miyazaki, T. Kanto, M. Inoue, I. Itose, H. Miyatake, M. Sakakibara, T. Yakushijin, N. Kakita, N. Hiramatsu, T. Takehara, A. Kasahara, N. Hayashi, Impaired cytokine response in myeloid dendritic cells in chronic hepatitis C virus infection regardless of enhanced expression of Toll-like receptors and retinoic acid inducible gene-I, J. Med. Virol. 80 (2008) 980–988. [7] N.L. Yonkers, B. Rodriguez, K.A. Milkovich, R. Asaad, M.M. Lederman, P.S. Heeger, D.D. Anthony, TLR ligand-dependent activation of naive CD4 T cells by plasmacytoid dendritic cells is impaired in hepatitis C virus infection, J. Immunol. 178 (2007) 4436–4444. [8] I.G. Rodrigue-Gervais, L. Jouan, G. Beaule, D. Sauve, J. Bruneau, B. Willems, et al., Poly(I:C) and lipopolysaccharide innate sensing functions of circulating human myeloid dendritic cells are affected in vivo in hepatitis C virus-infected patients, J. Virol. 81 (2007) 5537–5546. [9] S. Babu, C.P. Blauvelt, V. Kumaraswami, T.B. Nutman, Diminished expression and function of TLR in lymphatic filariasis: a novel mechanism of immune dysregulation, J. Immunol. 175 (2005) 1170–1176. [10] P. Dasari, I.C. Nicholson, G. Hodge, G.W. Dandie, H. Zola, Expression of toll-like receptors on B lymphocytes, Cell Immunol. 236 (2005) 140–145. [11] A. Månsson, M. Adner, U. Höckerfelt, L.O. Cardell, A distinct Toll-like receptor repertoire in human tonsillar B cells, directly activated by PamCSK, R-837 and CpG-2006 stimulation, Immunology 118 (2006) 539–548. [12] V. Hornung, S. Rothenfusser, S. Britsch, A. Krug, B. Jahrsdörfer, T. Giese, S. Endres, G. Hartmann, Quantitative expression of toll-like receptor 1–10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides, J. Immunol. 168 (2002) 4531–4537. [13] A. Mansson, M. Adner, L.O. Cardell, Toll-like receptors in cellular subsets of human tonsil T cells: altered expression during recurrent tonsillitis, Respir. Res. 27 (2006) 7–36. [14] K.U. Saikh, J.S. Lee, T.L. Kissner, B. Dyas, R.G. Ulrich, Toll-like receptor and cytokine expression patterns of CD56+ T cells are similar to natural killer cells in response to infection with Venezuelan equine encephalitis virus replicons, J. Infect Dis. 188 (2003) 1562–1570. [15] N.K. Crellin, R.V. Garcia, O. Hadisfar, S.E. Allan, T.S. Steiner, M.K. Levings, Human CD4+ T cells express TLR5 and its ligand flagellin enhances the suppressive capacity and expression of FOXP3 in CD4+CD25+ T regulatory cells, J. Immunol. 175 (2005) 8051–8059.
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