Immunosuppressive tryptophan catabolism and gut mucosal dysfunction following early HIV infection

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© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e‐mail: [email protected].

Immunosuppressive tryptophan catabolism and gut mucosal dysfunction

following early HIV infection

Mohammad-Ali Jenabian1,2,*, Mohamed El-Far3, Kishanda Vyboh1,2, Ido Kema4,

Cecilia T. Costiniuk1,5, Rejean Thomas6, Jean-Guy Baril7, Roger LeBlanc1,8, Cynthia

Kanagaratham2, Danuta Radzioch2, Ossama Allam9,10, Ali Ahmad9,10, Bertrand

Lebouché1, Cécile Tremblay3,9, Petronela Ancuta3,9, Jean-Pierre Routy1,2,11, for the

Montreal Primary infection and Slow Progressor Study Groups

1Chronic Viral Illnesses Service of the McGill University Health Centre, Montreal, Quebec,

Canada

2Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada

3CHUM Research Centre, Montreal, QC, Canada

4Department of Laboratory Medicine, University Medical Center, Groningen, University of

Groningen, The Netherlands

5Division of Infectious Diseases and Lachine Campus of the McGill University Health

Centre, Montreal, QC, Canada

6Clinique Médicale l’Actuel, Montreal, QC, Canada

7Clinique médicale Quartier Latin, Montreal, QC, Canada

8Clinique médicale OPUS, Montreal, QC, Canada

9Department of Microbiology and Immunology, University of Montreal, Montreal, QC,

Canada

10CHU Ste-Justine Research Center, University of Montreal, Montreal, Quebec, Canada

11Division of Hematology, McGill University Health Centre, Montreal, QC, Canada

Journal of Infectious Diseases Advance Access published January 23, 2015 at U

niversite de Montreal on January 30, 2015

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Corresponding author: Jean-Pierre Routy, Division of Hematology & Chronic Viral Illness

Service, McGill University Health Centre, 687 Pine Avenue West, Montreal, Quebec,

Canada H3A 1A1. Tel: +1 (514) 843 15 58, Fax: +1 (514) 843 14 18, Email: jean-

[email protected]

*Present address: Département des Sciences Biologiques et Centre de recherche BioMed,

Université du Québec à Montréal (UQAM), Montreal, QC, Canada.

Alternate corresponding author: Mohammad-Ali Jenabian, Département des Sciences

Biologiques et Centre de recherche BioMed, Université du Québec à Montréal (UQAM),

Montreal, QC, Canada. 141, Ave President Kennedy, Montreal, Quebec, Canada, H2X

1Y4. Tel : +1 (514) 987- 3000, poste 6794. Fax :+1 (514) 987 4647. E-mail :

[email protected]

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Abstract:

Background: Tryptophan (Trp) catabolism into kynurenine (Kyn) contributes to immune

dysfunction in chronic HIV infection. To better define the relationship between Trp

catabolism, inflammation, gut mucosal dysfunction and the role of early antiretroviral

therapy (ART), we prospectively assessed patients following early HIV Infection.

Methods: 40 patients with early infection were longitudinally followed for 12 month after

HIV diagnosis, including 24 untreated (ART-) and 16 ART-treated (ART+) individuals.

Kyn/Trp ratio, Tregs, T-cell activation, dendritic-cells and plasma levels of gut mucosal

dysfunction markers I-FABP, sST2 and LPS were assessed.

Results: Compared with healthy subjects, patients with early infection presented with

elevated Kyn/Trp which further increased in ART-, while normalized in ART+.

Accordingly, in ART- subjects the elevated Treg frequency observed at baseline continued

to increased over-time. The highest CD8 T-cell activation was observed during early

infection and decreased in those untreated during chronic infection, while normalizing in

ART+ subjects. Kyn/Trp was positively associated with CD8 T-cell activation and

inflammatory cytokines (IL-6, IP-10, IL-18, TNF-α) and negatively with dendritic-cell

frequencies at baseline and ART- patients. However, ART did not normalize gut mucosal

dysfunction markers.

Conclusion: Early ART normalized enhanced Trp catabolism and immune activation but

did not improve markers of gut mucosal dysfunction.

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Introduction

The early phase of HIV infection is characterized by rapid depletion of the total

CD4 T-cell pool and dramatic increase in immune activation associated with high HIV

plasma viral load (VL)[1]. During early infection, massive CD4 T-cell depletion is

observed mainly in gut-associated lymphoid tissues (GALT), impairing mucosal integrity

and resulting in progressive microbial translocation from the gut to the periphery[2, 3].

Microbial translocation has been recognized as a major contributor to immune dysfunction

and persistent immune activation during HIV infection[1-3]. Moreover, this persistent

immune activation is independently associated with a greater risk of non-AIDS related

morbidity and mortality[2].

We and others have reported that tryptophan (Trp) catabolism into kynurenine

(Kyn) by indoleamine 2,3-dioxygenase-1 (IDO-1), expressed by dendritic cells (DC) and

monocytes, skews CD4 T-cell differentiation into regulatory T-cells (Tregs) instead of T-

helper (Th17) cells and directly impairs T-cell responses[4, 5]. It is well recognized that

IDO-1 induced Treg production is associated with immunosuppressive effect during

pregnancy, cancer and viral infections[6, 7]. In HIV infection, the altered Th17/Treg

balance is directly linked to increased and persistent IDO-1 activity via interferon (IFN)-γ

signaling and Toll-like receptor (TLR) stimulation[4] and Trp breakdown is associated with

immune activation [8]. Indeed, increased IDO-1 activity is associated with the degree of

microbial translocation, HIV disease progression and predicts mortality[4, 5, 9]. More

recently, it has been reported that dysbiosis of the gut microbiota is associated with IDO-1

and contributes to HIV disease progression[10]. However, the causal effect of Trp

catabolism, gut mucosal dysfunction and its impact on the inflammatory response in early

infection has not yet been addressed.

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Initiation of anti-retroviral therapy (ART) during early infection results in lower

HIV burden and reduced HIV reservoir size[11-14]. Early ART has also been associated

with reduction of anti-HIV antibody formation[15] and has a significant impact on health-

related quality of life[16]. 6 months of ART reduces Trp catabolism although not to normal

levels[17]. We and others have previously shown that long-term ART reduces both IDO-1

expression and normalizes IDO-induced Trp catabolism[4, 5, 18]. However, limited data

are available regarding the influence of early ART on immunosuppressive IDO-1 activity

and recovery of gut mucosal dysfunction.

In this study, we compared IDO-induced Trp catabolism, immune activation, markers of

myeloid-lymphoid inflammation and gut mucosal dysfunction in HIV-infected adults who

did and did not receive ART during the early phase of infection.

Material and methods

Study population

Peripheral blood mononuclear cells and plasma were longitudinally collected from HIV-

infected individuals who had an estimated date of infection of less than 180 days through

the Montreal HIV primary infection study. Diagnosis of HIV infection was established

based on a positive p24 antigen and/or a detectable HIV-RNA, subsequently confirmed by

Western blot. Some patients started ART during the first year of infection based on their

CD4 T-cell count and the decision of the physicians and patients. Samples were obtained

from the Canadian Slow Progressor Cohort for HIV elite controllers (n=12) and from the

Chronic Viral Illness Service, McGill University Health Centre (MUHC), Montreal, QC,

Canada, for healthy controls (n=12) (Table 1).

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Ethics statement

This study was conducted according to the Declaration of Helsinki and received approval

from the MUHC’s Ethical Review Board. All study subjects provided written informed

consent for participation in the study.

Measurement of the IDO enzymatic activity

Plasma levels of Trp and its catabolite, Kyn, were measured by an automated on-line solid-

phase extraction-liquid chromatographic-tandem mass spectrometric (XLC-MS/MS)

method as we previously reported and IDO-1 enzymatic activity was determined by

Kyn/Trp ratio[5, 19].

Flow cytometry

Flow cytometry analyses were performed by a four-laser LSRII flow cytometer (BD

Bioscience, Mississauga, ON, CA). The following antibodies were used: CD3-Pacific blue,

CD4-FITC, CD4-PercpCy5.5, CD4-PECy5, CD4-APC-Cy7, CD8-Alexa700, CD25-PE,

CD27-Alexa700, CD28-PECy5, CD57-APC, CD127-PECy7, CD38-APC, HLA-DR-

APCCy7, CCR5-PE, α4-FITC, β7-PECy5, Lineage-FITC (including anti-CD3, CD14,

CD19, CD20, CD56), CD11c-APC and CD123-PE) (BD Bioscience, Mississauga, Ontario,

CA); CD45RA-ECD (Beckman Coulter, Mississauga, Ontario, CA); CD8-APCeFluor780

and FOXP3Alexa488 were from (eBioscience, San Diego, USA). The viability marker

Vivid (Invitrogen, Burlington, Ontario, CA) was used to exclude dead cells from analysis.

Data was analyzed using FlowJo software v7.6.5.

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Multiplex quantification of plasma inflammatory markers

Plasma levels of inflammatory soluble factors interleukin (IL)-6, IL-18, interferon-induced

protein (IP)-10 and tumor necrosis factor (TNF)-α were measured in duplicate using a

ProcartaPlex Multiplex Immunoassays according to the manufacturer’s instructions

(eBioscience). Mean fluorescence intensities for each analyte in each sample were detected

using the MAGPIX instrument (Luminex, Austin, Texas) and the results were analyzed

using the xPONENT 4.2 software (Millipore) to obtain the protein concentration of each

soluble factor in each sample.

Measurement of plasma levels of I-FABP, sST2 and LPS

Plasma levels of markers of the Intestinal-type fatty acid-binding protein (I-FABP) and the

soluble Suppression of Tumorigenicity-2 (sST-2) were measured by ELISA using

commercially available kits from Hycult Biotech (Uden, the Netherlands) and R&D system

(Minneapolis, MN), respectively. Plasma levels of the marker of microbial translocation,

lipopolysaccharide (LPS), were measured using commercially available kits from Cusabio

(Wuhan, China).

Statistical analysis

Statistical analyses were performed using GraphPad Prism software version#5. Kruskal-

Wallis tests were performed for comparisons between study groups. Unpaired t-tests or

Mann-Whitney U tests were used for comparisons of two non-paired study variables

according to the sample size. Wilcoxon matched pairs test was used for comparisons of two

paired study variables. The Spearman rank correlation test was used to identify associations

amongst study variables.

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Results

Study population

Forty-eight patients diagnosed during early infection (<180 days since infection) were

enrolled. Baseline and one-year follow-up patient clinical characteristics are described in

Table 1. The estimated dates of infection were as follows: less than 30 days (n=4), 31-90

days (n=10) and 91-180 days (n=34). Twenty-four patients remained untreated (ART-)

after one year of follow-up. Seventeen patients were ART-treated (ART+) during the first

year of infection including seven within 3 months, five between 3-6 months and five

between 6-12 months following the estimated infection date. One patient who’s VL

remained elevated despite ART was excluded from longitudinal analysis. Samples from

seven patients were not available for longitudinal assessment at the 12-month follow-up

time-point. Therefore 40 patients (24 ART-, 16 ART+) were assessed longitudinally. ART

significantly improved CD4 T-cell count, and VL was below the level of detection

compared to baseline (626±205 versus 456±192cells/mL, p=0.003 and <1.7 versus 6.3±6.8

log10 copies/mL, p=0.0005, not shown).

Early ART initiation rapidly normalized immunosuppressive Trp catabolism and

halts Treg expansion.

Short-term ART can reduce Trp catabolism, although not to normal levels [17]. We have

previously reported that Trp catabolism was normalized to levels similar to healthy subjects

after a mean 8 years of successful ART[5]. Here, we assessed the Trp catabolism following

ART during early infection. Patients in the early phase of infection displayed elevated

plasma levels of Kyn compared to healthy subjects (2.58±0.67 versus 1.86±0.52μmol/L,

Figure 1A). Early ART-treated patients (ART+) presented decreased levels of Kyn whereas

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in ART- patients levels of Kyn remained high (2.05±0.50 and 2.62±0.60μmol/L).

Consistently, the Kyn/Trp ratio was elevated at baseline, normalized in ART+, and

remained high in ART- patients when compared to healthy subjects (0.05±0.02, 0.04±0.01,

0.05±0.02 and 0.04±0.01, respectively, Figure 1B). Kyn levels decreased following ART

compared to baseline (2.77±0.71 versus 2.06±0.51μmol/L, Figure 1C) and further increased

over time for those who remained untreated (2.33±0.55 versus 2.64±0.60μmol/L, Figure

1E). Accordingly, the Kyn/Trp ratio followed similar trend as it decreased longitudinally

following ART and increased when patients remained untreated (Figures 1D,F). These

results demonstrate that Trp catabolism rapidly normalized following early ART and

continued to increase in untreated patients.

As Trp catabolism by IDO-1 contributes to the expansion of Tregs in cancer and

HIV infection [4-6], we evaluated the peripheral frequency of Tregs following early-

infection. At baseline, the frequency of CD4+CD25highCD127low Tregs was similar in early

infection and healthy subjects (not shown), while it was significantly increased over time

during the longitudinal follow-up in ART- patients but not ART+ compared to baseline

(6.22±1.53 versus 5.6±1%, p=0.01and 6.22±1.6 versus 5.7±1.46, p=0.14 respectively, not

shown). When transcription factor FoxP3 was included in the analysis, the expression of

CD4+CD25highCD127lowFoxP3high Tregs also did not differ between early infection and

healthy subjects (Figure 2A). However, we observed a significant increase in the frequency

of FoxP3+Tregs in ART- but not in ART+ patients compared to healthy subjects (Figure

2A). When assessed longitudinally, an increasing trend in the proportion of FoxP3+Tregs

was observed in ART- patients but remained unchanged following ART (Figures 2B,C).

These results indicate that early ART administration can breakdown the progressive

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expansion of Tregs ultimately observed during the chronic phase of infection in ART-

patients.

Kyn/Trp ratio is associated with generalized immune activation.

As HIV infection is associated with persistent immune activation, we first evaluated CD8

T-cell activation by measuring CD38/HLA-DR co-expression. Patients at baseline

displayed the most elevated frequency of activated CD8 T-cells compared to any other

group which normalized rapidly following ART (17.40±13.86 versus 2.64±1.89%, Figure

3A). CD8 T-cell activation was lower in the chronic phase in ART- patients compared to

baseline although activation continued to remain elevated compared to healthy subjects

(10.40±7.40 versus 1.95±1.66%, Figure 3A). Longitudinal assessment revealed a drastic

reduction in CD8 T-cell activation from baseline to ART+ (22.41±17.99 versus

2.64±1.89%, Figure 3B), whereas there was slight decrease in CD8 T-cell activation in

ART- patients (14.70±9.37 versus 10.40±7.40%, Figure 3C). Interestingly, positive

correlations were observed between Kyn/Trp and CD8 T-cell immune activation at baseline

and for ART- patients (Figures 3D,E) but not in ART+ (not shown). Consistent with our

previous studies[5], a strong correlation was observed between VL and Kyn/Trp ratio at

baseline and in ART- patients (p=0.017, R=0.34 and p=0.02, R=0.47 respectively, not

shown).

Senescent cells, defined as CD8+CD28-CD57+, are notably increased in chronic

HIV infection as a marker of immune dysfunction[20]. We therefore assessed their levels in

association with Kyn/Trp ratio. Levels of senescent cells were elevated at baseline

compared to HS (34.35±9.90 versus 12.80±12.37%, not shown) and treatment status did

not affect senescence expression within study groups (not shown). When assessed

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longitudinally, no significant difference from baseline to ART+ was observed (36.33±9.36

versus 35.90±12.36%, not shown), indicating a halt in further increases to senescence

although ART- displayed a marked increase in senescence expression (33.17±10.23 versus

38.20±11.19, p=0.02, not shown). Importantly, a strong positive correlation was only

observed between senescent cells and Kyn/Trp ratio in chronic infection for ART- patients

(Figure 3F,G).

Low CD4/CD8 ratio in HIV-infected patients is related to both innate and adaptive

immune activation and is associated with a higher risk of non-AIDS events even with

ART[21]. In our cohort, we observed an increase in CD4/CD8 ratio only for ART+ but not

in ART- when compared to baseline (0.97±0.39 versus 0.58±0.28, p=0.0009 and 0.82±0.63

versus 0.85±0.57, p=0.2, not shown). Importantly, we observed a negative correlation

between CD4/CD8 ratio and Kyn/Trp ratio at baseline and ART- groups (Figures 3H,I).

Furthermore, Kyn/Trp ratio was positively associated with HIV disease progression

markers IL-6 and IP-10 and inflammatory cytokines IL-18 and TNF-α (Table 2).

Collectively, the results indicate that Trp catabolism is associated with generalized immune

activation, immunosenescence and HIV-mediated inflammation, which could revert back to

normal following early ART initiation.

Lower circulating DC frequency is associated with higher Kyn/Trp ratio

As DCs constitute the main cell types which express IDO-1 enzyme, we evaluated

the changes in DC frequency following early infection in relation to Trp catabolism. At key

study visits, no differences were observed between the frequency of CD11c+ myeloid DC

(mDC) and CD123+ plasmacytoid DC (pDC) at baseline and for ART- patients compared

to healthy subjects (not shown). However, overtime when patients were compared to

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themselves, we observed a significant increase in peripheral mDC and pDC frequency

following early ART compared to baseline (0.31±0.13% versus 0.50±0.17% and 0.15±0.1

versus 0.22±0.12% , Figures 4A,B) while remaining unchanged in ART- patients (0.4±0.19

versus 0.41±0.18% and 0.2±0.1 versus 0.18±0.1 respectively, Figures 4C,D). These results

suggest that inflammation observed at baseline and in ART- patients might contribute to the

migration of DCs into inflammatory sites, such as the gut mucosa, resulting in a decrease in

their relative frequency in the periphery[22]. Importantly, we observed that lower

peripheral DC frequency was negatively associated with Kyn/Trp ratio at baseline and in

ART- patients (Figures 4E-H). This is in line with higher expression of IDO-expressing

DCs in the gut mucosa and higher dietary Trp catabolism observed in experimental

intestinal inflammation[23].

Absence of change in the frequency of T-cells expressing α4β7 gut homing receptor,

following early ART

It has been shown the loss of circulating CD4 T-cells expressing gut homing marker α4β7

integrin mirrors the CD4 depletion in the GALT and occurs within days after SIV

infection[24]. In all study groups, CD4 T-cell subsets expressing α4β7 were lower than

CD8 T-cells (primary-infection: 9.2±3.3 versus 20.67±8.8%, ART+: 9.37±3.6 versus

24±9.8%, ART-: 9±3.2 versus 18.35±7.5; p<0.0001 for all comparisons, not shown) and no

correlation was observed between VL or Kyn/Trp and α4β7+ T-cell frequency (not shown).

These α4β7+ T-cells expressed higher levels of CCR5 compared to total CD4 T-cells

(early-infection 12.85±7.6% versus 6.9±4.3%, p<0.0001; ART+: 6.03±4% versus

4.46±2.5%, p=0.02; ART-: 10.3±7.1 versus 6±4.4%, p<0.0001, not shown). A significant

decrease compared to baseline was longitudinally observed on CCR5+α4β7+ and memory

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α4β7+CD4+ T-cells in both ART- (13±8.7 versus 10.3±7.1%, p=0.03 and 92.2±4.2%

versus 90.3±6.1%, p=0.02) and ART+ patients (90±5.6 versus 86.1±8.7%, p=0.002 and

12.6±6 versus 6±3.9%, p=0.002) which favor the preferential depletion of α4β7+ cells

resulting in damage to gut mucosal immunity (not shown).

Early treatment with ART did not improve markers of gut mucosal damages and

microbial translocation

Massive CD4 T-cell depletion in the GALT causes impaired mucosal integrity resulting in

microbial translocation[2, 3]. We therefore evaluated the changes in plasma markers

associated with gut mucosal damage, including I-FABP [25], sST-2 [26-28], and the

marker of microbial translocation, LPS[29]. Our results showed an increase in plasma

levels of both I-FABP and sST-2 at baseline compared to healthy subjects which remained

high even in patients receiving ART (Figures 5A,B). No improvement was observed in

levels of these two markers when assessed overtime following early ART (I-FABP:

734.7±461.5 versus 1074.0±613.1pg/ml; sST-2: 20334±6549 versus 20843±8513pg/ml, not

shown). Accordingly, an increase in plasma levels of microbial product LPS was observed

at baseline, which remained high during the chronic phase in both ART+ and ART- patients

(107.3±42.2 versus 113.1±32.4; 116.0±40.7 respectively, Figure 5C).

Discussion

We previously reported that Trp catabolism into Kyn, in association with HIV VL

and lower CD4 T-cell count, is immunosuppressive in HIV infection via alteration of

Th17/Treg balance[5, 19]. Kyn/Trp ratio is also recognised as an independent predictor of

HIV disease progression and mortality[21, 30]. In this longitudinal assessment of patients

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since early infection, we observed that early ART initiation had a beneficial impact by

normalizing Trp catabolism to levels observed in HS and decreasing various markers of

myeloid and lymphoid inflammation. Several studies on Austrian, Ugandan and Chinese

cohorts of HIV-infected patients showed that 6-12 months of successful ART can reduce

Kyn/Trp ratios by only 50%[17, 18, 31]. In contrast with these studies, we observed a rapid

normalization of Kyn/Trp ratio by early ART following early infection indicating the

importance of the ART initiation timing on Trp catabolism[31]

As we and others have previously shown that IDO-induced Trp catabolism

contributes to the generation of Tregs in HIV-infected patients[4, 5, 19], we evaluated

peripheral Tregs frequency. In relation to the Kyn/Trp ratio, we observed a continuous

increase in Tregs frequency in ART- patients compared to baseline or healthy subjects.

Importantly, this increase was halted by early ART. Our results showed that Kyn/Trp ratio

was associated with immune activation as well as HIV disease progression predictors IL-6

and IP-10 (CXC10)[32, 33] and inflammatory cytokines IL-18 and TNF-α. Another

enzyme, tryptophan 2,3-dioxygenase (TDO) mainly expressed by the liver, is also able to

catabolize Trp[34]. However, liver TDO activity is suppressed when extra-hepatic IDO

activity is induced during inflammation[34]. Correlation between Kyn/Trp and immune

activation in our study suggests IDO as the key player of Trp catabolism in our setting. We

showed that early ART, can rapidly reduce immune activation in association with

decreased Kyn/Trp. A persistently low CD4/CD8 ratio during virally-suppressive ART is

associated with increased innate and adaptive immune activation, immunosenescence,

Kyn/Trp ratio and higher risk of morbidity/mortality[21]. It has been shown that patients

who started early ART within 6 months of infection had greater CD4/CD8 ratio increase

versus patients who started ART more than two years after infection[21]. Interestingly, we

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showed that early ART initiation improved CD4/CD8 ratio and prevented CD8+ T-cell

senescence population in line with the decreased Kyn/Trp ratio.

DCs are one of the main IDO-expressing cell types. We observed that lower

peripheral mDC and pDC frequency were negatively associated with higher IDO enzyme

activity. In HIV infection, DCs migrate from peripheral blood to the GALT, where they

contribute to immune activation[22]. Furthermore, frequency of IDO-expressing DCs in gut

mucosa results in a higher immunosuppressive catabolism of dietary Trp observed in

intestinal inflammation[23]. Therefore, our results indicate that early ART could contribute

to lower mucosal inflammation, thereby resulting in a higher frequency of peripheral DCs

and a decrease in IDO-induced catabolism of dietary Trp in the gut.

Disruption of gut mucosal integrity mediates persistent immune activation in HIV

infection[1-3, 35]. In acute infection, gut memory CD4 T-cells expressing CCR5 are

preferentially infected and depleted, with 60% of CD4 T-cells being lost within 2-3 weeks

of HIV infection [35-37]. Indeed, loss of mucosal CD4 T-cells expressing gut homing

marker α4β7 occurs within a few days of SIV infection[24]. We observed decreases in

CCR5+ and memory α4β7+ T-cells in both ART- and ART+ patients compared to baseline

which favor preferential depletion of α4β7 cells, resulting in reduced gut mucosal

immunity. However, early ART initiation did improve recovery of circulating α4β7+ T-

cells.

Limited T-cell access to IL-7 in GALT due to fibrosis and architectural distortion of

mucosa represents a major limit of T-cell reconstitution[3, 38]. Fibrogenesis in gut mucosa

starts during early infection due to HIV and cytokine-mediated immune activation (eg. IL-

6, TGF-β, hyaluronic acid) and contributes to collagen deposition, increased mucosal

permeability and microbial translocation[1-3, 9, 35, 38]. Indeed, gut epithelial dysfunction

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associated with Kyn/Trp ratio and innate immune activation are independent predictors of

mortality in ART-treated patients[9]. We therefore assessed the changes in plasma markers

on gut mucosal injury following early ART. I-FABP, a cytosolic enterocyte protein[39], is

a marker of enterocyte damage[9, 40, 41] and strongly correlates with mortality in HIV

patients[9]. We also evaluated plasma levels of sST-2, a recently identified marker of gut

mucosal damages. ST-2 is the receptor for IL-33 from the IL-1 family which is involved in

pro-inflammatory reactions and Th2 immune responses [42]. Inflammatory sST2 [27, 43]

binds with IL-33 to sequestrate its effects and has been recently described as a biomarker of

intestinal inflammatory disorders[26, 28]. Our results showed an early increase in plasma

levels of I-FABP and sST-2 in EHI which remained high even in patients receiving early

ART. Accordingly, an increase in LPS plasma levels was observed at baseline which

remained high in the chronic phase even in ART+ patients. LPS stimulates monocytes

differentiation into DCs and is a microbial product and a marker of microbial

translocation[3, 44, 45]. The stable increase in levels of sST-2 and LPS at all stages of

infection, despite early ART, indicates that gut musocal damage starts in early infection and

results in microbial translocation that cannot be rapidly repaired by ART. Further studies

are needed to assess the possible role of early ART in longer follow-up time-points. Our

observations are consistent with prior studies showing that ART initiation in primary

infection leads to partial rather than complete reconstitution of gut mucosa[37, 46, 47]. To

reconcile the persistent gut mucosal damage and microbial translocation with rapid

normalization of Kyn/Trp ratio and immune activation by early ART, we propose the “liver

firewall” hypothesis. Likely early ART leads to decreased HIV-mediated immune

activation by controlling viral replication, while relatively low levels of microbial products

(LPS) may not trigger systemic activation as phagocytic kupffer cells in the liver are at

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work[48, 49]. Indeed, the liver serves as a “firewall” to filter gut microbial products that

have penetrated systemic vascular circuits, and kupffer cell exhaustion may only occur late

in HIV infection or in cirrhotic patients[3]. The mechanism of incomplete GALT

reconstitution is unclear and may be due to either viral or immune-mediated accelerated T-

cell destruction, or potentially to alterations in T-cell homing to the gut[37]. Combinations

of immunotherapies with ART could be beneficial as our group has shown that

administration of recombinant IL-7 in chronically infected ART-treated patients with low

CD4 T-cell recovery results in partial gut mucosal recovery[50].

Collectively, our findings show that early ART initiation has a beneficial impact in

HIV patients by normalizing enhanced Trp catabolism and decreasing various markers of

myeloid and lymphoid inflammation, without impacting gut mucosal dysfunction.

Competing interests: The authors have declared that no competing interests exist.

Funding: This work was supported by the Canadian Institutes of Health Research (grant

MOP #103230 and CTN #257), CANFAR and Fonds de la Recherche Québec-Santé (FRQ-

S): Réseau SIDA/Maladies infectieuses et thérapie cellulaire, Québec, Canada. For Dr.

M.A. Jenabian, this work was part of his CANFAR/CTN Postdoctoral Fellowship. Dr. J.P.

Routy is the holder of the Louis Lowenstein Chair in Hematology & Oncology, McGill

University.

These results were presented in part at the 20th International AIDS conference, Melbourne,

Australia in July 2014.

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Legends for the Figures.

Figure 1. Immunosuppressive catabolism of Trp was decreased following early ART.

(A) Plasma levels of Kyn and (B) marker of IDO enzyme activity (Kyn/Trp ratio) in a cross

sectional analysis within all study groups. Whisker box showing the median and the 25th

and 75th percentiles. The whiskers of the graph show the largest and smallest values. (C)

Plasma levels of Kyn and (D) Kyn/Trp ratio in a longitudinal analysis of patients from early

infection and following early ART. (E) Plasma levels of Kyn and (F) Kyn/Trp ratio in a

longitudinal analysis in patients in early infection compared to the chronic phase in ART-

patients.

Figure 2. Early treatment with ART halts the expansion of peripheral Tregs. (A)

Cross-sectional comparison of the peripheral frequency of CD25highCD127lowFoxP3high

Tregs within study groups. Whisker box showing the median and the 25th and 75th

percentiles. The whiskers of the graph show the largest and smallest values. Changes in the

peripheral frequency of CD25highCD127lowFoxP3high in a longitudinal analysis of patients

from EHI and (B) the chronic phase in ART- patients or (C) following early ART.

Figure 3. IDO enzyme activity was associated with CD8 T-cell immune activation and

CD8/CD4 ratio in both EHI and acute phases of HIV infection. (A) Cross-sectional

comparison of the co-expression of immune activation markers HLA-DR and CD38 on

CD8 T cells within study groups. Whisker box showing the median, and the 25th and 75th

percentiles. The whiskers of the graph show the largest and smallest values. Decrease of

CD8 T cell immune activation in (B) ART+ and (C) ART- patients compared to early

infection. Positive correlation between CD8 T-cell activation and IDO enzyme activity

(Kyn/Trp ratio) in (D) early infection and (E) ART- patients. Correlation between

CD8+CD28-CD57+ frequency and Kyn/Trp ratio in (F) early infection and (G) ART-

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patients. Negative correlation between CD8/CD4 T-cells ratio and Kyn/Trp ratio in (H)

early infection and (I) ART- patients.

Figure 4. Lower circulating DCs frequency is associated with higher Kyn/Trp ratio

following primary HIV infection. Frequency of (A) mDC and (B) pDC in a longitudinal

analysis of patients in early infection and then following ART. Frequency of (C) mDC and

(D) pDC in a longitudinal analysis of early infection patients and then in chronic phase

(ART-). Negative correlations between the peripheral frequency of (E) mDCs and (F) pDC

with IDO enzyme activity in patients in early infection. Negative correlations between the

peripheral frequency of (G) mDCs and (H) pDCs and IDO enzyme activity in ART-

patients.

Figure 5. Increase in plasma markers of gut mucosal damages and microbial

translocation activity following primary HIV infection. A cross sectional analysis within

all study groups for the levels of plasma markers of intestinal mucosal damages: (A)

Intestinal-type fatty acid-binding protein (I-FABP) (B) soluble suppression of

tumorigenicity 2 (sST-2), and (C) marker of microbial translocation, lipopolysaccharide

(LPS). Whisker box showing the median and the 25th and 75th percentiles. The whiskers of

the graph show the largest and smallest values.

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Acknowledgement: Subjects in early infection were screened, recruited and followed by

Drs S. Vézina, L. Charest, M. Milne, E. Huchet, S. Lavoie, J. Friedman, M. Duchastel, F.

Villielm at l'Actuel Medical Clinic, M. Potter, B. Lessard, MA Charron, S. Dufresne, ME

Turgeon at Quartier Latin Medical Clinic, Drs D. Rouleau, L. Labrecque, C. Fortin, A de

Pokomandy, B. Trottier, V. Hal-Gagné, M. Munoz, B. Deligne, V. Martel-Laferrière at

UHRESS CHUM Hôtel-Dieu and Notre-Dame, N. Gilmore, M. Fletcher, J. Szabo at

MUHC Chest Institute. We are thankful for their collaboration. We are grateful for help and

administrative support from Ms AF Vassal and M Legault, V Lafontaine for lab processing

and shipment, and the members of the Network Lab Spec Committee for overall approval

of the project. We are thankful to Mrs. Angie Massicotte for her clerical assistance and

coordination. We also thank Ms. Stephanie Matte from the Canadian HIV-1 slow

progressor cohort, and Ryhan Pineda from the MUHC for coordination and blood banking.

We are thankful to Jacquie Sas and Jim Pankovich from the CIHR Canadian HIV Trials

Network (CTN) for study implementation and coordination. The authors acknowledge Dr.

Dominique Gauchat and Annie Gosselin from the flow cytometry core of the CHUM-

Research Centre, Saint-Luc Hospital, Montréal, QC, Canada, for technical assistance.

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Table 1. Clinical characteristics of study groups. These include early HIV infection,

ART treated (ART+), non-treated patients (ART-), Elite controllers and Healthy subjects.

Results are shown as mean ± standard deviation (SD) and (Range), NA: not applicable.

Characteristics Study population N=116

Early infection (n=48)

ART+ (n=16)

ART- (n=24)

Elite controllers (n=12)

Healthy subjects (n=12)

Age (years)

[Mean ± SD (range)]

36.06 ± 10.29

(19-57)

36.61 ± 10.47

(21-57)

38.08 ± 9.21

(21-56)

51.67 ± 11.58

(37-72)

47.83 ± 7.78

(35-60)

Male [n (%)] 47 (98%) 16 (100%) 23 (96%) 7 (58%) 7 (58%)

CD4 T-cell count (cells/µL)


544 ± 269.0

(220-1680)

614± 202

(366-1100)

559 ± 266

(210-1110)

756 ± 157

(510-1040)

869± 306

(281-1360)

CD8 T-cell count (cells/µL)


927± 504

(279-2590)

727± 317

(268-1325)

870± 421

(280-2180)

778± 302

(315-1211)

470± 178

(227-843)

CD4:CD8 ratio

[Mean ±SD (range)]

0.70 ± 0.47

(0.16-2.76)

0.94 ± 0.37

(0.53-1.85)

0.79 ± 0.63

(0.20-3.25)

1.13 ± 0.59

(0.70-2.65)

2.11 ± 0.99

(0.38-3.97)

Viral load (log10copies/mL)

[Mean ±SD (range)]

4.28 ± 1.08

(1.25-7.48)

1.69 ± 0.20

(1.60-2.28)

4.38 ± 0.76

(2.83-5.47) < 1.6 NA

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Table 2. Inflammatory cytokines changes following early ART in association with Trp/Kyn

ratio. Early HIV infection, ART treated (ART+), non-treated patients (ART-). Results are shown

as mean ± standard deviation (pg/mL) and (Range), NS: not significant.

Inflammatory

Cytokines

Early infection ART+ ART- All

Plasma level

(pg/ml)

Correlation

with

Kyn/Trp

Plasma level

(pg/ml)

Correlation

with

Kyn/Trp

Plasma level

(pg/ml)

Correlation

with

Kyn/Trp

Plasma

level

(pg/ml)

Correlation

with

Kyn/Trp

IL-6 1.3±2

(0-11)

p=0.06

R=0.3463

0.6±0.65

(0-2) NS

0.9±0.77

(0.1-3.5) NS NA

p =0.0002

R=0.35

IL-18 207±215

(16-1339)

p=0.03

R=0.32

158±185

(45-861) NS

158±74.8

(49-322)

p=0.002

R=0.60 NA

p <0.0001

R=0.44

IP-10 194 ± 111

(60-580)

p <0.0001

R = 0.60

155±85

(9-345) NS

217±220

(1.2-1023)

p=0.0005

R=0.66 NA

p <0.0001

R=0.57

TNF-α 44.7±37 (7-

145) NS 35±21 (7-79) NS

45.54±43.19

(7-152) NS NA NS

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Immunosuppressive tryptophan catabolism and gut mucosal dysfunction following early HIV infection

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