critical role for the adenosine pathway in controlling simian
TRANSCRIPT
Critical Role for the Adenosine Pathway in Controlling SimianImmunodeficiency Virus-Related Immune Activation andInflammation in Gut Mucosal Tissues
Tianyu He,a,b Egidio Brocca-Cofano,a,b Delbert G. Gillespie,c Cuiling Xu,a,d Jennifer L. Stock,a,e Dongzhu Ma,a,d
Benjamin B. Policicchio,a,e Kevin D. Raehtz,a,d Charles R. Rinaldo,e Cristian Apetrei,a,d,e Edwin K. Jackson,c Bernard J. C. Macatangay,f
Ivona Pandreaa,b,e
Center for Vaccine Research, University of Pittsburgh, Pittsburgh, Pennsylvania, USAa; Department of Pathology,b Department of Pharmacology and Chemical Biology,c
Department of Microbiology and Molecular Genetics,d and Division of Infectious Diseases,f School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA;
Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USAe
ABSTRACT
The role of the adenosine (ADO) pathway in human immunodeficiency virus type 1/simian immunodeficiency virus (HIV-1/SIV) infection remains unclear. We compared SIVsab-induced changes of markers related to ADO production (CD39 and CD73)and breakdown (CD26 and adenosine deaminase) on T cells from blood, lymph nodes, and intestine collected from pigtailedmacaques (PTMs) and African green monkeys (AGMs) that experience different SIVsab infection outcomes. We also measuredADO and inosine (INO) levels in tissues by mass spectrometry. Finally, we assessed the suppressive effect of ADO on proinflam-matory cytokine production after T cell receptor stimulation. The baseline level of both CD39 and CD73 coexpression on regula-tory T cells and ADO levels were higher in AGMs than in PTMs. Conversely, high INO levels associated with dramatic increasesin CD26 expression and adenosine deaminase activity were observed in PTMs during chronic SIV infection. Immune activationand inflammation markers in the gut and periphery inversely correlated with ADO and directly correlated with INO. Ex vivoadministration of ADO significantly suppressed proinflammatory cytokine production by T cells in both species. In conclusion,the opposite dynamics of ADO pathway-related markers and contrasting ADO/INO levels in species with divergent proinflam-matory responses to SIV infection support a key role of ADO in controlling immune activation/inflammation in nonprogressiveSIV infections. Changes in ADO levels predominately occurred in the gut, suggesting that the ADO pathway may be involved insparing natural hosts of SIVs from developing SIV-related gut dysfunction. Focusing studies of the ADO pathway on mucosalsites of viral replication is warranted.
IMPORTANCE
The mechanisms responsible for the severe gut dysfunction characteristic of progressive HIV and SIV infection in humans andmacaques are not completely elucidated. We report that ADO may play a key role in controlling immune activation/inflamma-tion in nonprogressive SIV infections by limiting SIV-related gut inflammation. Conversely, in progressive SIV infection, signifi-cant degradation of ADO occurs, possibly due to an early increase of ADO deaminase complexing protein 2 (CD26) and adeno-sine deaminase. Our study supports therapeutic interventions to offset alterations of this pathway during progressive HIV/SIVinfections. These potential approaches to control chronic immune activation and inflammation during pathogenic SIV infectionmay prevent HIV disease progression.
Generalized immune activation and inflammation are hall-marks of human immunodeficiency virus type 1 (HIV) and
simian immunodeficiency virus (SIV) infection, which are specif-ically associated with progression to AIDS (1–6). Both innate andadaptive antiviral immune responses contribute to the chronicimmune activation observed during pathogenic HIV/SIV infec-tion, which is associated with (i) a high turnover rate of T and Blymphocytes and natural killer (NK) and myeloid dendritic cells,(ii) increased frequency of CD4� and CD8� T cells expressingactivation/proliferation markers, (iii) expansion of effector Tcells, and (iv) elevated serum levels of proinflammatory cytokinesand immune activation markers (7–10). Conversely, natural hostsof SIVs, such as African green monkeys (AGMs), sooty mang-abeys, and mandrills, in which SIV infection generally does notprogress to AIDS, do not present chronic immune activation/in-flammation despite persistent high levels of viral replication (4, 6,11–18).
Received 11 May 2015 Accepted 6 July 2015
Accepted manuscript posted online 15 July 2015
Citation He T, Brocca-Cofano E, Gillespie DG, Xu C, Stock JL, Ma D, Policicchio BB,Raehtz KD, Rinaldo CR, Apetrei C, Jackson EK, Macatangay BJC, Pandrea I. 2015.Critical role for the adenosine pathway in controlling simian immunodeficiencyvirus-related immune activation and inflammation in gut mucosal tissues. J Virol89:9616 –9630. doi:10.1128/JVI.01196-15.
Editor: G. Silvestri
Address correspondence to Ivona Pandrea, [email protected].
T.H. and E.B.-C. contributed equally to this article.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JVI.01196-15.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JVI.01196-15
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Multiple mechanisms contribute to the excessive chronic im-mune activation and inflammation associated with chronic HIVinfection. Among these, the most important contributors appearto be virus replication (19), microbial translocation from the in-testinal lumen to the broader circulation as a consequence of mu-cosal damages inflicted by localized virus replication (3, 20–24),and dysregulation of T cell homeostasis (25).
T regulatory cells (Tregs) have an important contribution tomodulating the levels of inflammatory responses to numerouspathogens, such as herpes simplex virus, respiratory syncytial vi-rus, and Mycobacterium tuberculosis (26–30). Tregs can also in-hibit effector T (TE) cell responses in HIV infection (31–33). Theexact mechanisms by which Tregs exert their modulatory effectsremain to be determined. One of the proposed mechanisms ofTreg-mediated immune suppression is through extracellularadenosine (ADO) production via the interaction of the ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) and the 5=nucleotidase (CD73) (34). Through its binding to various catego-ries of adenosine receptors, extracellular ADO can exert eitherproinflammatory or anti-inflammatory effects (35). Alternatively,CD26 in complex with adenosine deaminase (ADA) can promotethe breakdown of adenosine to inosine (INO), thus relieving theanti-inflammatory effect mediated by extracellular adenosine(36).
The CD39/CD73/adenosine pathway has an anti-inflamma-tory effect in a variety of disease conditions, being involved inmodulating airway inflammation and fibrosis (37, 38), gastroin-testinal inflammation (39, 40), and tumor immune evasion (41–45). However, the role of ADO in the pathogenesis of HIV infec-tion is not completely elucidated. CD39� Treg expansion iscorrelated with decreased interleukin-2 (IL-2) production (46).CD4� CD73� T cells are depleted in HIV-1-infected patients re-gardless of viral suppression (47), while peripheral CD73-express-ing CD8� T cells from HIV-infected patients can be partially re-versed by antiretroviral therapy (ART) (48). Notably, the vastmajority of these studies focused on T cells from peripheral blood.Since progressive HIV/SIV infections are characterized by pro-found mucosal dysfunction caused by increased cell death associ-ated with high virus replication and inflammatory responses (3,22, 49–51), we studied the changes in CD39 and CD73 expressionon Tregs from the gut of SIV-infected nonhuman primates(NHPs). We also aimed to better understand the role of the ADOpathway in modulating immune activation/inflammation duringHIV/SIV infection.
We assessed CD39 and CD73 expression on CD4� and CD8�
Tregs isolated from blood, lymph node (LN), and intestine fromprogressor pigtailed macaques (PTMs) (10, 24, 49) and nonpro-gressor AGMs (17, 52, 53) infected with the same SIVsab strain.We report that CD39 and CD73 are coexpressed predominantly atmucosal sites and that the coexpression levels of these two mole-cules on CD4� and CD8� Tregs are higher in AGMs than inPTMs. We also report that the AGM nonprogressive host hashigher baseline levels of ADO in mucosal tissues and that theselevels further increase upon SIVsab infection in this species. Incontrast, the pathogenic infection of PTMs is associated with adramatic increase in ADA activity and CD26 expression on intes-tinal T cells, along with significant postinfection increases of INOlevels. In vivo, ADO levels inversely correlate with immune acti-vation, inflammation, and intestinal damage markers, while exvivo, exogenous ADO inhibits production of proinflammatory cy-
tokines by stimulated T cells. Our findings suggest that the ADOpathway plays a critical role in controlling immune activation andinflammation at mucosal sites in the natural hosts of SIV andcould contribute to the lack of intestinal dysfunction observed inthis species.
MATERIALS AND METHODSAnimals and infection. Fourteen PTMs (Macaca nemestrina) and 15AGMs (Chlorocebus sabaeus) were included in the study. All the animalswere males, aged 4 to 9 years old. They were infected with plasma equiv-alent to 300 50% tissue culture infectious doses (TCID50) of SIVsab (49).The follow-up was for 180 days postinfection (dpi) or until progression toAIDS. All NHPs were clinically monitored throughout the follow-up.They were housed at the Regional Industrial Development Corporation(RIDC) Park facility of the University of Pittsburgh in accordance with therecommendations of the Association for Assessment and Accreditation ofLaboratory Animal Care (AAALAC) International Standards and with therecommendations in the Guide for the Care and Use of Laboratory Ani-mals of the National Institutes of Health (54). The Institutional AnimalUse and Care Committee (IACUC) of the University Pittsburgh approvedthese studies (protocol 09039). Efforts were made to minimize animalsuffering in agreement with the recommendations of the Weatherall re-port on the use of nonhuman primates in research (55). The RIDC Parkfacility is air-conditioned, with an ambient temperature of 21 to 25°C, arelative humidity of 40 to 60%, and a 12-h light/dark cycle. Animals weresocially housed (paired) in suspended stainless steel wire-bottomed cagesand provided with a commercial primate diet. Fresh fruit was providedonce daily, and water was freely available at all times. A variety of envi-ronmental enrichment strategies were employed, including housing ofanimals in pairs, providing toys to manipulate, and playing entertainmentvideos in the animal rooms. In addition, the animals were observed twicedaily, and any signs of disease or discomfort were reported to the veteri-nary staff for evaluation. At the end of the study, the NHPs were eutha-nized according to procedures approved in the IACUC protocol.
Tissue sampling and isolation of peripheral blood, LNs, and muco-sal mononuclear cells. Blood, LNs, and intestinal resections (jejunum)surgically collected at the time of necropsy from uninfected animals, at theviral peak (10 dpi; here, acute infection time point), and during latechronic infection (�180 dpi; here, chronic infection time point) wereavailable for this study. Additional blood and intestinal resection sampleswere collected from uninfected animals for functional studies.
Peripheral blood mononuclear cells (PBMCs) were purified fromwhole blood by density gradient centrifugation using lymphocyte separa-tion medium (LSM; Organon-Technica, Durham, NC). Lymphocyteswere separated from LNs by pressing tissue through a nylon mesh screenand then washed with RPMI medium (Cellgro, Manassas, VA) containing5% heat-inactivated newborn calf serum, 0.01% penicillin-streptomycin,0.01% L-glutamine, and 0.01% HEPES buffer, as previously described (3,23). Intestinal biopsy specimens were digested using EDTA, followed bycollagenase, and cells were then isolated using Percoll density gradientcentrifugation, as described previously (3, 23). PBMCs and lymphocytesfrom LNs and intestinal biopsy specimens were frozen at �80°C in freez-ing medium containing RPMI medium, heat-inactivated fetal bovine se-rum (60%), and 10% dimethyl sulfoxide (DMSO).
Surface and intracellular staining for flow cytometry. The expres-sion of ADO pathway-related markers was measured by flow cytometry atcritical time points during SIV infection, using mononuclear cells isolatedfrom blood, LNs, and intestine from five PTMs and five AGMs. The fol-lowing monoclonal antibodies (MAbs) were used for flow cytometrystaining: CD3-V450 (clone SP34-2), CD4-allophycocyanin (APC; cloneL200), CD25-phycoerythrin (PE)-Cy7 (clone 2A3), CD73-peridininchlorophyll protein (PerCP)-Cy5.5 (clone AD2), CD95-PE-Cy7 (cloneDX2), CD26-fluorescein isothiocyanate (FITC; clone M-A261), HLA-DR(clone L243), Ki-67-FITC (clone B56) (all from BD Pharmingen, SanDiego, CA), CD8-PE Texas Red (clone 3B5) (Invitrogen, Camarillo, CA),
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CD39-PE [clone eBioA1 (A1); eBioscience, San Diego, CA), and FoxP3-Alexa Fluor 488 (clone 259D) (BioLegend, San Diego, CA), includingtheir respective isotypes as negative controls. All MAbs were pretitratedusing AGM and PTM blood to determine the optimal staining dilution(52, 53, 56–58). Live/Dead fixable blue dead-cell stain (Life Technologies,Grand Island, NY) was used to determine the viability of the cells.
For cell surface staining, cells were first incubated with MAb for 25min at room temperature, then washed with phosphate-buffered saline(PBS), and finally fixed with 300 �l of 2% paraformaldehyde (52, 53,56–58).
For FoxP3 intracellular staining, cells were fixed with FoxP3 Fix/Permbuffer (BioLegend) for 20 min and permeabilized with FoxP3 Perm buffer(BioLegend) for 15 min at room temperature (23).
Two million lymphocytes were analyzed with an LSRII flow cytometer(BD Biosciences) and FlowJo, version 7.6, software (TreeStar Inc.). CD4�
and CD8� T cell percentages were obtained by gating first on lymphocytesand then on CD3� T cells. The frequency of CD25� FoxP3� or CD26�
cells was determined by gating on CD4� or CD8� T cells, respectively.The frequencies of CD39�, CD73�, and CD39� CD73� T cells were thendetermined by gating on the CD4� and CD8� Tregs (CD25� FoxP3�).
Measuring tissue levels of adenosine using mass spectrometry. FreshLN and intestinal tissues were collected from four AGMs and five PTMs asdescribed above in the tissue sampling section. The tissues were snap-frozen in liquid nitrogen immediately after sampling and then stored at�80°C until assayed. Frozen tissue was weighed, placed in 5 ml of 1-pro-panol (�20°C), cut into small pieces, and vigorously and repeatedly ho-mogenized. 1-Propanol was employed as a solvent to both inactivate en-zymes and extract purines from the tissue. Next, each sample wascentrifuged, and the supernatant was collected. One milliliter of superna-tant was placed in a separate tube for further processing and analysis.[13C10]adenosine (internal standard) was added to this 1-ml portion ofthe supernatant, the sample was again centrifuged, and the supernatantwas recovered and taken to dryness in a sample concentrator. The residuewas reconstituted in 0.2 ml of water and filtered using a 30-kDa MicroconYM-30 centrifugal filter unit (EMD Millipore Corporation, Billerica,MA). Adenosine in the filtrate was measured using a liquid chromatogra-phy-tandem mass spectrometer by selected reaction monitoring using amodification of our previously published method (59). Briefly, sampleswere introduced into an Acquity ultraperformance liquid chromatograph(UPLC) system (Waters, Milford, MA) fitted with a Waters UPLC BEHC18 column (1.7-�m particle size; 2.1 by 100 mm). Adenosine was elutedas follows: mobile phase A, 1% acetic acid in H2O; mobile phase B, meth-anol; flow rate, 0.3 ml/min. The elution gradient (mobile phase A/mobilephase B) was 99.5%/0.5% (0 to 2 min), 98%/2% (2 to 3 min), 85%/15% (3to 4 min), and 99.5%/0.5% (4 to 5 min). The column was connected to aheated electrospray ionization source, and adenosine levels were ana-lyzed using a TSQ Quantum Ultra triple quadrupole mass spectrome-ter (ThermoFisher Scientific, Waltham, MA) operating in the positive-ion mode. The following mass-to-charge transitions were monitored:278¡141 m/z for [13C10]adenosine, 268¡136 m/z for adenosine, and269¡137 m/z for inosine. Results were computed from a standard curveand then corrected to total amount per unit weight of tissue.
IHC assessment and quantification of myxovirus resistance proteinA (MxA). Immunohistochemical (IHC) was performed on formalin-fixed, paraffin-embedded jejunum samples. Sections 4 �m thick weredeparaffinized, rehydrated, and rinsed. For antigen retrieval, the sectionswere microwaved in Vector Unmasking Solution, and endogenous per-oxidases were quenched with 3% hydrogen peroxide. Sections were incu-bated with MxA (rabbit polyclonal; Abcam, Cambridge, MA, USA). Sec-ondary antibodies and actin/biotin were from a Vector Vectastain ABCElite kit (rabbit IgG; Vector Laboratories, Burlingame, CA, USA). Forvisualization, sections were incubated with diaminobenzidine (DAB;Dako) and counterstained with hematoxylin.
Quantification was performed using Fiji image software using 10 rep-resentative images, per section, per time point, per animal. Regions of
interest (ROIs) were selected, a positive signal within ROIs was isolated viacolor threshold, and the percent area positive was measured and averaged.
Measuring adenosine deaminase activity in plasma. Adenosinedeaminase (ADA) is the enzyme that directly catalyzes the deaminationreaction from ADO to INO (60). Plasma activity of ADA was measuredwith an adenosine deaminase assay kit (Diazyme Laboratories, Poway,CA). One unit of ADA is defined as the amount of ADA that generates 1�mol of INO from ADO per min at 37°C.
Plasma testing of biomarkers for intestinal damage. Plasma lipopoly-saccharide (LPS) was quantified with a commercially available Limulus ame-bocyte lysate kit (Lonza, Walkersville, MD) (24, 49), according to themanufacturer’s protocol. The plasma intestinal fatty acid-binding protein(I-FABP) level was quantified with a monkey I-FABP enzyme-linkedimmunosorbent assay (ELISA) kit (MyBioSource, Inc., San Diego, CA).
Titration of ADO. Prior to assessing the role of ADO in cytokinesuppression on intestinal and peripheral blood T cells, we performed atitration of ADO on PBMCs from uninfected NHPs. Fresh PBMCs (2 �106) from AGMs (n � 3) and PTMs (n � 3) were activated with CD3 (1�g/ml; BD Biosciences) and CD28 (2 �g/ml; BD Biosciences) antibodiesin the presence of ADO at 0.25, 2.5, and 12.5 mM in RPMI medium for 6h at 37°C. Brefeldin A (10 mg/ml; Sigma-Aldrich) was added for the last 4h of incubation for cytokine measurements. The cells were then washedwith PBS and incubated with a cocktail of monoclonal antibodies specificfor surface molecules, such as CD3-V450 (clone SP34.2; BD BiosciencesPharmingen), CD4-APC (clone L200; BD Biosciences Pharmingen), andCD8�/-PE-Texas Red (clone 3B5; Life Technologies) for 40 min in thedark at 4°C. Once washed with 2% fetal bovine serum (FBS), the cells werefixed and permeabilized with Cytofix/Cytoperm solution (BD Biosci-ences) for 15 min in the dark at room temperature. The cells were thenwashed with Perm/Wash buffer (BD Biosciences) and stained with fluo-rescence-conjugated monoclonal antibodies to gamma interferon (IFN-)–FITC (clone 4S.B3; BD Biosciences Pharmingen), IL-2-PE (cloneMQ1-17H12; BD Biosciences Pharmingen), and TNF-� (clone MAb11;BD Biosciences Pharmingen) for 30 min in the dark at 4°C. Labeled cellswere washed once with PBS–2% FBS, fixed in 2% formaldehyde-PBS(Affimetrix, Santa Clara, CA), and then acquired the same day on a cus-tom four-laser BD LSRII instrument (BD Bioscience). Only singlet eventswere gated, and a minimum of 250,000 live CD3 cells were acquired.Populations were analyzed using FlowJo software, version 7.6.5 (TreeStar, Inc., Ashland, OR), and the graphs were generated with GraphPadPrism, version 6.04.
Assessment of cell survival after ADO treatment. To ascertain thepossibility of ADO toxicity, frozen PBMCs (2 � 106) from one PTM wereactivated with CD3 (1 �g/ml; BD Biosciences) and CD28 (2 �g/ml; BDBiosciences) antibodies in the presence of ADO at 0.25, 2.5, and 12.5 mMin RPMI medium for 6 h at 37°C. Brefeldin A (10 mg/ml; Sigma-Aldrich)was added for the last 4 h of incubation for cytokine measurements. Then,stimulated PBMCs were stained for viability (blue dye; Life Technologies)and incubated for 10 min in the dark at room temperature. The percent-age of PBMC survival was calculated as the percentage of the ratio betweenviable cells.
Ex vivo suppressive activity of ADO on intestinal T cells andPBMCs. To assess the role of ADO in the suppression of cytokine pro-duction by T cells in the intestine and peripheral blood, 2 � 106 frozenintestinal cells from 6 uninfected AGMs and 5 uninfected PTMs, as well as2 � 106 PBMCs from 9 uninfected AGMs and 12 uninfected PTMs wereisolated and activated with CD3 (1 �g/ml; BD Biosciences) and CD28 (2�g/ml; BD Biosciences) antibodies in the presence of 0.25 mM ADO(Sigma-Aldrich) in RPMI medium for 6 h at 37°C. Brefeldin A (10 mg/ml;Sigma-Aldrich) was added for the last 4 h of incubation for cytokinemeasurements. Then lymphocyte suspensions were processed as de-scribed above.
Statistical analysis. Statistical analyses were carried out using Prism,version 6, software (GraphPad Software, San Diego, CA). Data were ex-pressed as means � standard errors of the means (SEM) unless otherwise
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specified. To compare the absolute count and frequency of cells betweendifferent stages of infection, different lymphoid compartments and dif-ferent animal models, a nonparametric Mann-Whitney test was used tocalculate the exact P value. The correlations between markers were eval-uated using a Spearman rank correlation test. A P value of �0.05 wasconsidered significant.
RESULTSSignificantly higher CD39 and CD73 coexpression in the muco-sal compartment than in circulation. It has been previously re-ported that CD39 and CD73 are coexpressed on murine Tregs andthat they comediate immune suppression (27). However, in hu-mans, the coexpression of these ectoenzymes is low on the surfaceof circulating CD4� T cells (47). Moreover, classic Tregs (definedbased on Fox-P3� CD25� expression) do not express CD73 (47).
Previous studies in humans focused only on circulating T cells. Wecomparatively assessed for the first time coexpression of CD39and CD73 on CD4� and CD8� Tregs collected from blood, LNs,and intestine of NHPs. Furthermore, to better understand thecontribution of this cell subset to the pathogenesis of AIDS, wecompared and contrasted the dynamics of CD39 and CD73 coex-pression on Tregs in pathogenic (PTMs) and nonpathogenic(AGMs) SIV hosts (Fig. 1).
The average frequency of circulating CD4� Tregs coexpressingCD39 and CD73 was low in both AGMs and PTMs (1.095% versus1.198% for AGMs and PTMs, respectively) (Fig. 1a and c). Simi-larly, the frequency of circulating CD8� Tregs coexpressing CD39and CD73 was low in both species (1.035% versus 1.234% forAGMs and PTMs, respectively) (Fig. 1b). In the LNs, CD39 and
FIG 1 CD39 and CD73 coexpression on Tregs isolated from blood, lymph nodes, and intestine in NHPs. (a) Significantly higher levels of CD39 and CD73coexpression were observed on CD4� Tregs in the intestine (INT) than in lymph nodes (LNs) or blood of AGMs and PTMs. Significantly higher preinfectionlevels of CD39 and CD73 coexpression were observed on CD4� Tregs in intestine in AGMs than in PTMs. (b) Significantly higher levels of CD39 and CD73coexpression were observed on CD8� Tregs in the intestine than in other tissue compartments of AGMs and PTMs. (c) Representative fluorescence-activated cellsorter plots of CD39 and CD73 coexpression on CD4� Tregs in different compartments in AGMs and PTMs prior to infection. Data are presented as individualvalues with the group means (long solid lines) and standard errors of the means (short solid lines). A Mann-Whitney test was used to calculate the exact P value.
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CD73 coexpression on CD4� and CD8� Tregs was significantlyhigher than on circulating Tregs in both AGMs and PTMs (CD4�
Tregs, 12.23% versus 5.72% for AGMs and PTMs, respectively;CD8� Tregs, 13.04% versus 8.65% for AGMs and PTMs, respec-tively) (Fig. 1a to c). Furthermore, the fractions of CD4� andCD8� Tregs coexpressing CD39 and CD73 were significantlyhigher in the intestine than in circulation in both models (CD4�
Tregs, 54.48% versus 12.19% for AGMs and PTMs, respectively;CD8� Tregs, 29.14% versus 14.94% for AGMs and PTMs, respec-tively) (Fig. 1a to c).
Interestingly, coexpression levels of CD39 and CD73 on theintestinal CD4� Tregs isolated from AGMs were significantlyhigher than those in PTMs (P � 0.0079) (Fig. 1a and c), suggestinga higher production of ADO in the natural host of SIVsab whichcontrols chronic immune activation and inflammation and doesnot develop intestinal dysfunction during SIV infection.
Altogether, these results showed that Tregs coexpressing CD39and CD73 are mainly present in the intestine and suggest that theycould impact HIV/SIV pathogenesis at this major site of virusreplication and T cell depletion.
CD39 and CD73 coexpression on CD4� Tregs increases sig-nificantly post-SIV infection in PTMs. Prompted by the findingof major differences in CD39 and CD73 expression on Tregs iso-lated from uninfected AGMs and PTMs, we next monitored thedynamics of these immuno-phenotypic markers on mucosalTregs at critical time points (i.e., preinfection, acute infection, andchronic infection) in NHPs in which SIV infection has oppositeclinical outcomes. The primary limiting factor for assessing theimpact of SIV infection on cell function is the massive mucosalCD4� T cell depletion which is a prominent feature of acute SIVinfection (see Fig. S1 in the supplemental material) (3, 49, 61–64).The maximal CD4� T cell depletion is achieved between 14 and 35dpi (3, 49, 61). To avoid this limitation, the acute mucosal sampleswere collected at 10 days postinfection (dpi) (see Fig. S1), whichcorresponds to the peak of viral replication and acute immuneactivation; this is also a time point at which mucosal CD4� T cellsare already impacted by the virus, but depletion is not complete(see Fig. S1), allowing the study of CD4� T cell subsets. Further-more, in this study only normal-progressor PTMs that usuallypartially restore and maintain up to 10 to 20% of their mucosalCD4� T cells for a relatively long period of time (49) were in-cluded. As such, our sampling time combined with the samplingstrategy of collecting intestinal resections gave us an abundant cellyield to perform the analyses described below.
No significant changes in CD39 and CD73 expression on in-testinal CD4� Tregs were detected in AGMs upon SIVsab infec-tion (Fig. 2a). Conversely, the frequency of intestinal CD4� Tregscoexpressing CD39 and CD73 increased dramatically upon SIVinfection in PTMs: 64.78% during acute infection (P � 0.0159)and 65.12% during chronic infection (P � 0.0317). These in-creased levels observed in PTMs were in the range of those ob-served in AGMs (Fig. 2a and b).
The significant increase of CD39 and CD73 expression on in-testinal Tregs of PTMs suggests a potential increase in ADO pro-duction at the mucosal site upon SIV infection.
Divergent dynamics of ADO and INO occur upon SIVsabinfection in nonprogressive and progressive NHP hosts. To con-firm that ADO expression is indeed increased upon SIV infectionin progressive hosts, we directly measured the levels of ADO andits metabolites (i.e., INO) in the LNs and intestine in PTMs prior
to infection and at critical time points postinfection. Results werecontrasted with those obtained by measuring the same markers insimilar tissues collected from AGMs. ADO/INO measurementswere not possible in blood due to the high instability of ADO,which is lost during blood processing. However, tissues snap-fro-zen in liquid nitrogen allowed ADO/INO measurements at tissuesites using mass spectrometry.
No significant change in the levels of ADO or its deaminationproduct INO was observed in the LNs of either species (Fig. 3a andb). Conversely, significant differences between PTMs and AGMswith regard to ADO/INO production were observed at the muco-sal site after SIVsab infection. Thus, intestinal levels of ADO in-creased during acute SIVsab infection in AGMs (P � 0.0317) butnot in PTMs (Fig. 3c). Conversely, while INO levels did notchange in AGMs throughout SIV infection, they significantly in-creased in chronically SIV-infected PTMs (P � 0.0317) (Fig. 3d).
Our results identified a trend of higher baseline levels of ADOin the gut of AGMs than those in PTMs (Fig. 3a and c). ADO levelsfurther increased upon SIVsab infection, becoming significantlyhigher in the intestine of AGMs than in those of PTMs duringacute infection (P � 0.0286) (Fig. 3c). In contrast to observationsin AGMs, ADO appears to be rapidly deaminized into INO inSIV-infected PTMs, causing the significant differences observedbetween the two species during chronic SIVsab infection (P �0.0286) (Fig. 3d). As such, our results suggest that ADO may con-tribute to the different outcomes of SIV infection in progressiveand nonprogressive hosts.
The main difference between pathogenic and nonpathogenicSIV infection relies on the levels of chronic immune activationand inflammation. We hypothesized that the ADO pathway isinvolved in keeping intestinal immune activation and inflamma-tion at bay during nonprogressive SIV infection. This could effec-tively protect this species against developing gut dysfunction de-spite the severe CD4 T� cell depletion experienced during SIVacute infection (3, 65). To test our hypothesis, we correlated ADOand INO levels with markers of immune activation and cell pro-liferation in the gut.
While no clear correlation was observed between ADO andthe immune activation markers, probably due to the rapid me-tabolization of ADO and the relatively limited number of ani-mals included in the study, our analyses clearly showed that thelevels of INO and intestinal immune activation markers aredirectly correlated in chronically SIVsab-infected PTMs (Fig.3e). These correlations are either significant or show cleartrends toward significance (Fig. 3e).
We further confirmed these flow cytometry results by assessingthe correlations between the ADO pathway and myxovirus resis-tance protein A (MxA), a marker previously validated for moni-toring inflammation in the gut (66). MxA is a protein with highlyconserved sequences across species that showed excellent andconsistent reactivity in previous studies (67). The levels of MxAexpression were measured by IHC and quantitative image analysistools. These analyses showed that inflammatory responses to SIVinfection mirrored the levels of immune activation identifiedthrough flow cytometry in both PTMs and AGMs. MxA tran-siently increased in AGMs during acute infection (P � 0.0286)and returned to preinfection levels during chronic infection inboth lamina propria (Fig. 4a and b) and Peyer’s patches (see Fig.S2 in the supplemental material). Conversely, MxA increases atthe same sites were not resolved at the transition from acute to
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chronic infection and remained significantly elevated from thebaseline throughout the follow-up in PTMs (P � 0.0286) (Fig. 4aand b; see also Fig. S2) and were directly correlated with INO levelsin this species (Fig. 4c).
Gut inflammation leads to severe cell death and release of mi-crobial products in circulation (20, 24, 68, 69). We therefore cor-related the levels of ADO/INO with those of markers of intestinaldamage. Using ELISAs, we monitored plasma levels of two bio-markers of intestinal damage: intestinal fatty acid binding protein(I-FABP), a protein released by necrotic enterocytes, and lipo-polysaccharide (LPS), a biomarker of microbial translocation.I-FABP levels were inversely correlated with ADO in AGMs,which maintain mucosal barrier function during chronic SIV in-fection (3, 70) (Fig. 5a). Conversely, LPS levels were directly cor-related with INO (Fig. 5b) in PTMs in which extensive mucosaldamage occurs during SIV infection (49, 71).
Our results provide compelling evidence that the ADO/INOpathway is related to the levels of mucosal inflammation and gut
damage during SIV infection. Furthermore, comparison betweenpathogenic and nonpathogenic SIV infections clearly relates thechanges in the ADO pathway to key features leading to differentoutcomes of infection.
The pathways involved in ADO breakdown are dramaticallyupregulated after SIV infection in PTMs. To further investigatethe pathways involved in ADO production and breakdown, weassessed CD26 and ADA dynamics in AGMs and PTMs. CD26 isstrongly upregulated after T cell activation (72, 73) and can bindto ADA to promote ADO breakdown, thus attenuating the effectsmediated by ADO signaling (36). Therefore, in addition to assess-ing the ectoenzymes that produce ADO (CD39 and CD73), wecompared and contrasted changes in the frequency of CD4� andCD8� T cells expressing CD26 in blood, LNs, and intestine inSIVsab-infected PTMs and AGMs. We did not find any significantchange in the frequencies of CD4� and CD8� T cells expressingCD26 in PBMCs from either AGMs or PTMs (see Fig. S3a and b inthe supplemental material). In the mesenteric LNs, significant in-
FIG 2 Dynamics of CD39 and CD73 coexpression on mucosal CD4� Tregs in SIVsab-infected NHPs. (a) A significant increase in the levels of CD39 and CD73coexpression was observed on CD4� Tregs in the intestine of PTMs upon SIV infection. (b) Representative fluorescence-activated cell sorter plots of CD39 andCD73 coexpression on mucosal CD4� Tregs from PTMs prior to infection and at critical time points postinfection. Data are presented as individual values withthe group means (long solid lines) and standard errors of the means (short solid lines). A Mann-Whitney test was used to calculate the exact P value. Pre,preinfection.
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FIG 3 Adenosine (ADO) and inosine (INO) dynamics in the lymph nodes and intestine during progressive and nonprogressive SIVsab infection and correlationwith the immune activation in the intestine. No significant increase of either ADO (a) or INO (b) in the lymph nodes throughout the follow-up was observed.(c) A significant increase of ADO levels was observed in the intestine (INT) of AGMs during acute infection, and there was no significant change in levels in PTMthroughout infection. ADO levels were significantly higher during acute SIV infection in AGMs than in PTMs. (d) A significant increase was observed in the INOlevel in the intestine in PTMs but not in AGMs. Significantly higher INO levels were observed in PTMs than in AGMs during chronic SIV infection. ADO andINO levels are presented as individual values (ng/mg wet weight) with the group means (long solid lines) and standard errors of the means (short solid lines). AMann-Whitney test was used to calculate the exact P values. (e) Correlations between INO levels and the levels of chronic immune activation markers in the gutin PTMs were evaluated using the Spearman rank correlation test. r and P values are shown as exact values. Significant correlation is illustrated as a solid line.
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creases of CD26 expression were observed only in PTMs duringboth acute and chronic SIVsab infection, while no significantchanges were observed in the mesenteric LNs collected fromAGMs (see Fig. S3c and d). A dramatic increase of CD26 expres-sion (�20-fold) was observed on both CD4� and CD8� T cells
isolated from the intestine of PTMs during both acute (P � 0.0159and 0.0317, respectively) and chronic (P � 0.0079 and 0.0159,respectively) infection (Fig. 6a and b). These changes occurredearly in infection and were maintained throughout the follow-up(Fig. 6c). In contrast, CD26 expression in AGMs increased tran-
FIG 4 Dynamics of MxA expression in the lamina propria of SIVsab-infected NHPs and correlation with INO. (a) Immunohistochemistry for MxA expression(brown) in the jejunum of AGMs and PTMs prior to infection and during acute and chronic infection. (b) Fold increase of MxA expression during acute andchronic infection. A significant but transient increase of MxA expression was seen during acute infection in AGMs, while the increase in MxA expression in PTMswas persistent throughout SIV infection. During chronic infection PTMs showed significantly higher MxA expression than AGMs. (c) Positive correlationbetween MxA expression and INO levels in SIVsab-infected PTMs. r and P values are shown as exact values. Data are presented as individual values with the groupmeans (long solid lines) and standard errors of the means (short solid lines). A Mann-Whitney test was used to calculate the exact P value. The dotted linerepresents the basal level of MxA expression in AGMs and PTMs. Relationships between MxA expression and INO were evaluated using a Spearman rankcorrelation test.
FIG 5 Correlations between ADO and INO levels and intestinal damage markers during progressive and nonprogressive SIVsab infections. (a) Inversecorrelation between ADO levels and intestinal fatty acid binding protein (I-FABP) in AGMs. (b) Direct correlation between INO levels and lipopolysaccharide(LPS) in PTMs. r and P values are shown for AGMs (green) and PTMs (blue). Significant correlations are represented by solid lines. Relationships between ADO,INO, and intestinal damage markers were evaluated using a Spearman rank correlation test.
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siently during the acute SIV infection and reached significanceonly for mucosal CD8� T cells (P � 0.0286) (Fig. 6a). In PTMs,the frequency of CD4� and CD8� T cells expressing CD26strongly correlated with INO levels in the intestine duringchronic infection (P � 0.0214 and P � 0.0108, respectively)(Fig. 6d and e).
Due to the multiple factors that can interfere with CD26 ex-pression, thus impacting the results of CD26 testing and prevent-ing a definitive conclusion with regard to its role in modulatingthe effects of ADO on the immune activation and inflammation,we next measured the plasma ADA activity. This enzyme directly
catalyzes ADO deamination to INO. Our results showed that ADAactivity remained low throughout infection in AGMs while in-creasing dramatically during acute SIV infection in PTMs (P �0.0317) (Fig. 7a). In PTMs, ADA activity was inversely correlatedwith ADO levels (P � 0.0499) (Fig. 7b). Notably, ADA activity wassignificantly lower in AGMs than in PTMs both prior to andthroughout the infection (Fig. 7a).
Our results point to a robust and consistent increase in CD26expression on CD4� and CD8� T cells collected from effector andinductive mucosal lymphoid sites in PTMs. Similarly, pathogenicSIVsab infection in PTMs was associated with increases in the
FIG 6 CD26 expression in the intestine in SIVsab-infected NHPs and correlation with ADO and INO. (a) A significant increase of CD26 expression was observedon CD4� T cells during acute and chronic infection in PTMs. (b) A transient increase of CD26 expression was observed on CD8� T cells during acute infectionin AGMs, and a persistent increase was observed throughout the follow-up in PTMs. (c) Representative fluorescence-activated cell sorter plots of CD26expression on CD4� T cells in the intestine of PTMs preinfection and during acute and chronic SIV infection. While CD26 levels increased on CD4� and CD8�
T cells throughout infection, a positive correlation between CD26 expression on CD4� (d) and CD8� T cells (e) and INO levels could be established duringchronic SIVsab infection in PTMs only. Data are presented as individual values with the group means (long solid lines) and standard errors of the means (shortsolid lines). A Mann-Whitney test was used to calculate the exact P value. Relationships between INO levels and CD26 expression were evaluated using aSpearman rank correlation test.
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plasma ADA activity. These changes, which are specifically asso-ciated with pathogenic SIV infection, may explain the lack of ADOincrease in this species and thus reconcile our previous findings ofdiscordant increases in CD39 and CD73 expression on Tregs inthe absence of concomitant elevations of ADO in tissues collectedfrom PTMs. Furthermore, this rapid breakdown of ADO intoINO may prevent ADO from exerting its anti-inflammatory effectand contribute to the pathogenic outcome of SIV infection inPTMs.
ADO suppresses inflammation in both AGMs and PTMs.The identified correlations between the ADO pathway and im-mune activation, inflammation, and intestinal damage markerssuggest that the ADO pathway could significantly contribute tothe control of inflammation during SIV infection. Therefore, wenext directly evaluated the potential role of ADO in suppressingproduction of inflammatory cytokines by CD4� and CD8� T cellsisolated from the intestine and peripheral blood after ex vivo T cellreceptor (TCR) stimulation with purified CD3 and CD28 anti-bodies.
In a preliminary assay, we titrated the amount of ADO neededto effectively suppress cytokine production by T cells from theintestine and peripheral blood of AGMs and PTMs. Cell incuba-tion with ADO concentrations of 0.25, 2.5, and 12.5 mM showedsimilar inhibitory effects on suppression of the production of cy-tokines by CD4� and CD8� T cells from the peripheral blood ofAGMs (see Fig. S4a in the supplemental material) and PTMs (seeFig. S4b). We further performed a survival test on frozen PBMCsfrom one PTM and identified a toxic effect of ADO: while cellsurvival was 99% after treatment with 0.25 mM ADO, survivalrates dropped after treatment with 2.5 mM (78%) and 12.5 mM(62%) ADO. Since the lowest ADO concentration showed com-parable suppression of cytokine production by T cells in the rangeof levels observed with higher concentrations, while clearly dis-playing less toxicity, the concentration of 0.25 mM was used toassess the adenosine-induced suppression of inflammatory cyto-kines by intestinal T cells and PBMC.
Administration of exogenous ADO significantly suppressedIFN- production in both CD4� (Fig. 8a and b) and CD8� (Fig.8c and d) T cells isolated from the intestine of AGMs and PTMs(see Fig. S6b in the supplemental material). Moreover, IL-2 pro-duction by the intestinal CD4� (Fig. 8e and f) and CD8� (Fig. 8g
and h) T cells from both species was also significantly suppressedby administration of exogenous ADO.
We also tested the impact of ADO on inflammatory cytokinesby CD4� and CD8� T cells isolated from blood and observed asignificant suppression of cytokine production by CD4� andCD8� T cells isolated from both AGMs and PTMs (see Fig. S5 andS6a in the supplemental material).
These results directly demonstrate that ADO has a significantimpact on the production of inflammatory cytokines by CD4�
and CD8� T cells from both PTMs and AGMs. Altogether, ourresults indicate that the CD39/CD73/ADO pathway plays a criticalrole in modulating immune activation/inflammation during SIV/HIV pathogenesis.
DISCUSSION
Increased immune activation and inflammation are the most im-portant determinants of HIV/SIV disease progression and areconsidered to be largely responsible for the lack of immune resto-ration in patients receiving antiretroviral therapy, development ofcomorbidities, and poor prognosis (74). It is therefore critical todevelop strategies to control immune activation and inflamma-tion, with the goal of improving response to treatment, clinicaloutcome, and survival in HIV-infected patients.
However, therapeutic attempts to reduce immune activationand inflammation in HIV-infected patients have not been success-ful, probably due to the fact that HIV/SIV-related immune acti-vation and inflammation are complex, multifactorial processes.Identifying the multiple immunoregulatory pathways of immuneactivation and inflammation associated with HIV/SIV infection istherefore critical for deciphering their mechanisms and for target-ing these pathways in specific therapeutic approaches.
Here, we thoroughly assessed the role of the CD39/CD73/ADOimmunosuppression pathway in modulating immune activationand inflammation during both pathogenic and nonpathogenicSIV infections.
Previous studies suggested that CD39� Tregs correlate withprogressive HIV infection (75). CD39� Treg expansion stronglycorrelates with high levels of immune activation and low CD4� Tcell counts (46), while downmodulation of CD39 expression isassociated with a slower disease progression in HIV-infected pa-tients (76). On the other hand, CD73� CD4� T cell depletion
FIG 7 Adenosine deaminase (ADA) levels in plasma in SIVsab-infected NHPs and correlation with ADO. (a) A significant increase of ADA was observed duringacute and chronic infection in PTMs. (b) Inverse correlation between ADO levels and ADA activity in PTMs. A Mann-Whitney Test was used to calculate theexact P value. Relationships between ADA levels and ADO activity were evaluated using a Spearman rank correlation test.
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correlates with increased numbers of activated T cells duringchronic HIV infection (47). These findings corroborate reportsshowing a strong correlation between downregulation of CD73expression on CD8� T cells and high levels of immune activationin HIV-infected patients (48). Altogether, these seminal studies
point to a previously unrecognized functional role of the ADOpathway in modulating HIV-1-associated immune activation.
However, these previous studies focused exclusively on the pe-ripheral blood compartment, while the bulk of virus replication,CD4� T cell depletion, and severe HIV/SIV-related inflammation
FIG 8 ADO-induced suppression of cytokine production by CD4� and CD8� T cells in the intestine. (a to d) Exogenous ADO significantly suppressed IFN-production from both CD4� (a) and CD8� T (c) cells isolated from AGMs and by CD4� (b) and CD8� T (d) cells isolated from PTMs, following in vitrostimulation with purified CD3 and CD28 antibodies. (e to h) Exogenous ADO significantly suppressed IL-2 production from both CD4� (e) and CD8� T (g) cellsisolated from AGMs and from CD4� (f) and CD8� T (h) cells isolated from PTMs, following in vitro stimulation with purified CD3 and CD28 antibodies. Dataare presented as individual values with the group means (long solid lines) and standard errors of the means (short solid lines). A Wilcoxon signed-rank test wasused to calculate the exact P value.
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occur predominantly at mucosal sites. Also, all the studies per-formed so far focused only on progressive infections that are as-sociated with severe CD4� T cell depletion (including that ofCD73� CD4� T cells), which prevents a clear understanding ofthe role of the loss of CD73� CD4� T cells in HIV pathogenesis.Finally, while previous studies assessed CD39 and CD73 expres-sion on either conventional T cells or Tregs, none assessed theADO suppression pathway as a whole. Since the components ofthis pathway work in concert to modulate the extracellular ADOlevel, investigating only individual components may preclude de-finitive conclusions with regard to the role of the ADO pathway inthe pathogenesis of AIDS.
To overcome these limitations and to fill the gaps in the scien-tific knowledge, we employed a complex strategy to investigate therole of ADO in modulating SIV/HIV-related inflammation.
First, we compared and contrasted SIV-associated changes inmarkers linked to the ADO pathway in two different NHP modelsdeveloped in our lab (24, 49, 52, 53). In these two models, infec-tions with the same viral strain (SIVsab) have opposite clinicaloutcomes despite similar levels of viral replication: SIVsab-in-fected PTMs develop a progressive infection characterized by highlevels of immune activation and gut damage/dysfunction associ-ated with inflammation, cell death, and gut permeabilization, re-sulting in severe microbial translocation (49). Conversely,SIVsab-infected AGMs develop a persistent nonprogressive infec-tion in which immune activation and inflammation are kept atbay during chronic infection (3, 12, 17, 23, 52, 53, 56). As such,AGMs have the ability to maintain gut integrity despite similarlevels of acute mucosal CD4� T cell depletion (3). These differentpathogenic outcomes enabled us to correlate changes in ADO lev-els and in ADO-associated markers with markers of disease pro-gression in these two NHP models to better understand the role ofthis pathway in SIV pathogenesis.
Second, recognizing that one of the major sources of inflam-mation during SIV/HIV infection is the gut, we focused our inves-tigations on this compartment to assess if ADO plays a significantrole in the development of SIV-related gut dysfunction. Use ofNHPs enabled us to perform invasive gut studies at critical timepoints of progressive and nonprogressive infections. A major lim-itation for the immuno-phenotypic studies in the gut is that HIV/SIV infection induces massive mucosal CD4� T cell depletion (3,49, 50, 61–64, 77, 78). We minimized this constraint by analyzingacute mucosal samples collected at the peak of acute viral replica-tion when the mucosal CD4� T cell depletion is incomplete (3, 49,61) (see Fig. S1 in the supplemental material). Our combinedapproach of sampling prior to the achievement of complete CD4�
T cell depletion and collection of intestinal resections that yieldedlarge number of mucosal cells enabled us to investigate if there is apreferential impact of SIV infection on a given CD4� T cell subsetat the mucosal sites. The use of snap-frozen NHP tissues allowedus to directly measure ADO by mass spectrometry. Such measure-ments are not possible in cells isolated from blood or in plasmadue to low levels of ADO and to the fact that, being unstable,adenosine is easily lost during blood processing (47). Finally, incontrast to previous studies, we assessed the expression of bothmarkers (CD39 and CD73) related to ADO production on Tregsisolated from the same individuals. While murine Tregs coexpressCD39 and CD73 (34, 79), few human circulating CD4� T cellscoexpress both ectoenzymes. Only one prior study (48) reportedincreased CD39/CD73 coexpression on T cells isolated from LNs
from HIV patients. This prompted us to assess the frequency ofthe coexpression of these ADO production markers in order tomore extensively characterize this pathway.
We found that the frequency of CD39 and CD73 coexpressionby Tregs is higher in NHP tissues, particularly in the gut, than incirculation (Fig. 1), suggesting higher production of ADO at mu-cosal sites. We also found that the nonpathogenic AGM host has ahigher baseline frequency of CD39� CD73� Tregs than the pro-gressive PTM host. Together with a higher constitutive level ofADO, this feature may protect the AGMs from the negative con-sequences of increased acute virus replication and CD4� T celldepletion occurring in the gut by limiting the mucosal inflamma-tion. In this context, our results show that the baseline levels ofCD39 and CD73 coexpression could play a critical role in control-ling gut damage and subsequent systemic chronic immune acti-vation and in avoiding the disease progression associated withHIV/SIV infection via effective regulation of the inflammatoryresponse at the main sites of viral replication during the acuteinfection.
While the levels of CD39 and CD73 did not significantly vary inAGMs upon SIVsab infection, they remained high throughout thefollow-up (Fig. 2a). Furthermore, mass spectrometry detected sig-nificantly increased ADO production in the gut of AGMs earlyafter infection, further supporting a critical role of the ADO path-way in controlling mucosal inflammation and protecting againstdevelopment of intestinal dysfunction during nonpathogenic in-fection.
In the progressive PTM host, the baseline frequency of CD39�
CD73� Tregs was low prior to infection. While the expression ofboth CD39 and CD73 ectoenzymes dramatically increased uponSIVsab infection to levels comparable to those found in AGMs,PTMs still developed high mucosal inflammation and gut dys-function. Therefore, it is likely that the increased expression ofCD39 and CD73 markers upon SIV infection is too little and toolate to compensate for their intrinsically low baseline levels andthus cannot effectively control mucosal immune activation andinflammation. Mass spectrometry also confirmed the low ADOlevels in PTMs both prior to and early after infection, likely due toboth intrinsically low frequencies of CD39� CD73� Tregs andpostinfection increases of CD26 expression and ADA activitywhich promote ADO breakdown. This scenario is also supportedby the high intestinal levels of the ADO breakdown product INOin chronically infected PTMs, which were significantly higher thanin AGMs. With the levels of ADO inversely correlated with ADAactivity and the levels of INO correlated with the expression ofCD26 on both CD4� and CD8� T cells at the mucosal sites, it istempting to speculate that the ADO breakdown in PTMs occursearly on during SIV infection and results in high levels of immuneactivation, inflammation, and disease progression in this species.
In further support of the role of the ADO pathway in intestinaldysfunction, we established a direct correlation between the levelsof intestinal INO and common immune activation markers (i.e.,Ki-67 and CD38 HLA-DR) and mucosal inflammation (immuno-histochemical measurements of MxA) in SIV-infected PTMs. Wealso established an inverse correlation in PTMs between the ADOpathway and the biomarkers of intestinal damage, I-FABP andLPS. Altogether, these results point to the involvement of the ADOpathway in the control of mucosal inflammation and mainte-nance of intestinal integrity, suggesting that the ADO pathwaymight be a key player in driving HIV/SIV disease progression. In
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support of this conclusion, we provided direct evidence that, sim-ilar to results of some previous human studies (47), ADO has asignificant role in suppressing proinflammatory cytokine produc-tion by intestinal and peripheral T lymphocytes.
In conclusion, our findings indicate that the CD39/CD73/ADO pathway plays a key role in the control of immune activationand inflammation in nonprogressive SIV infections and is pre-dominantly active at mucosal sites of viral replication and CD4� Tcell depletion. Our study therefore supports therapeutic interven-tions aimed at offsetting the alterations of this pathway duringprogressive HIV/SIV infections as potential approaches to controlchronic immune activation and inflammation during pathogenicSIV infection and, ultimately, to prevent HIV disease progression.
ACKNOWLEDGMENTS
We thank Theresa Whiteside and Patrick Schuler for helpful discussion.This work was supported by NIH/NIBHL/NCRR/NIDDK RO1 grants
HL117715 (IP), R01 RR025781 (C.A. and I.P.), R21 AI069935 (I.P.),DK091190 (E.K.J.), HL109002 (E.K.J.), DK068575 (E.K.J.), andDK079307 (E.K.J.). K.D.R. is supported by NIH training grantT32AI065380-10.
The funders had no role in study design, data collection and analysis,decision to publish, or preparation of the manuscript.
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