High-fat diet exacerbates SIV pathogenesis and accelerates disease progression

The Jou nal o Clinical In es iga ion RESEARCH ARTICLE 5474 jci.o g Volume 129 Numbe 12 Decembe 2019 In oduc ion Pe sis en immune ac i a ion and in lamma ion a e hallma ks o ch onic HIV/SIV in ec ion and s ong p edic o s o disease p o- g ession, independen o plasma i al loads (pVLs), o pe iphe al CD4+ T cell coun s (1). They also pe sis in indi iduals wi h HIV/ SIV and nonhuman p ima es (NHPs) on an i e o i al he apy (ART), p edic ing poo immune es o a ion, accele a ed aging, and de elopmen o non–AIDS-associa ed como bidi ies (2–5). The mechanisms o HIV/SIV-associa ed immune ac i a ion and in lam- ma ion a e he subjec o in ense esea ch, wi h key de e minan s including pe sis en i us eplica ion, coin ec ions, dys egula ion o T cell homeos asis, and mic obial ansloca ion (4, 6–8). Mean- while, o he unknown ac o s likely con ibu e o HIV/SIV-associ- a ed ch onic immune ac i a ion and in lamma ion. Among hese, die a y habi s may a ec in lamma ion h ough mul iple pa hways (9), al e ing he pa hogenesis o ch onic HIV/SIV in ec ion. Li le is known, howe e , wi h ega d o hese e ec s. Consuming a high- a die (HFD) is a leading isk ac o o obesi y and ype 2 diabe es (10), which a e bo h cha ac e ized by ch onic in lamma ion (10), ma ked by ele a ed TNF-α, IL-1, IL-6, and accumula ion o p oin lamma o y mac ophages in he adipose issue (10). A HFD al e s he gas oin es inal (GI) mucosa, dis up s gu mic obio a, and induces in es inal damage. These condi ions all lead o mic obial ansloca ion (11–13), u he boos ing ch onic immune ac i a ion and in lamma ion, hus accele a ing HIV/SIV disease p og ession. In humans, Wes e n die s a e associa ed wi h an inc eased isk o nonalcoholic a y li e disease (NAFLD) (14). The impac o Wes e n die on li e heal h is disce nible wi hin weeks (15). In he Uni ed S a es, NAFLD became a signi ican heal h p oblem in male adolescen s who equen ly consume Wes e n die s (15). HFD can also induce NAFLD in mice and a s, along wi h signi ican ele a- ion in TNF-α, NF-κB, and oxida i e s ess. These li e al e a ions co ela e wi h hickening o he ca o id in ima (16), sugges ing a possible connec ion be ween li e pa hology and ca dio ascula (CV) disease. In humans, h ough ele a ed in lamma ion ia he TLR4/NF-κB signaling pa hway, HFD causes enal, e inal, and b ain unc ion impai men s (17). As li e , enal, and neu ological diso de s a e also common HIV-associa ed como bidi ies, HFD may inc ease he isk o o he se e i y o HIV como bidi ies. Consuming a high- a die (HFD) is a isk ac o o obesi y and diabe es; bo h o hese diseases a e also associa ed wi h sys emic in lamma ion, simila o HIV in ec ion. A HFD induces in es inal dysbiosis and impai s li e unc ion and coagula ion, wi h a po en ial nega i e impac on HIV/SIV pa hogenesis. We adminis e ed a HFD ich in sa u a ed a s and choles e ol o nonpa hogenic (A ican g een monkeys) and pa hogenic (pig ailed macaques) SIV hos s. The HFD had a nega i e impac on SIV disease p og ession in bo h species. Thus, inc eased cell-associa ed SIV DNA and RNA occu ed in he HFD- ecei ing nonhuman p ima es, indica ing a po en ial ese oi expansion. The HFD induced p ominen immune cell in il a ion in he adipose issue, an impo an SIV ese oi , and heigh ened sys emic immune ac i a ion and in lamma ion, al e ing he in es inal immune en i onmen and igge ing gu damage and mic obial ansloca ion. Fu he mo e, HFD al e ed lipid me abolism and HDL oxida ion and also induced li e s ea osis and ib osis. These me abolic dis u bances igge ed incipien a he oscle osis and heigh ened ca dio ascula isk in he SIV-in ec ed HFD- ecei ing nonhuman p ima es. Ou s udy demons a es ha die a y in ake has a disce nable impac on he na u al his o y o HIV/SIV in ec ions and sugges s ha die a y changes can be used as adju an app oaches o HIV-in ec ed subjec s, o educe in lamma ion and he isk o non- AIDS como bidi ies and possibly o he in ec ious diseases. High- a die exace ba es SIV pa hogenesis and accele a es disease p og ession Tianyu He,1,2 Cuiling Xu,1,3 Noah K ampe1, S ephanie M. Dillon,4 Paola Se e,1,3 Elizabe h Falwell,1,3 Geo ge S. Ha e -Rich e ,1,2 Ti any Bu e ield,5 Tammy L. Dunsmo e,1 William M. McFadden J .,1 Ka h yn J. Ma in,1 Benjamin B. Policicchio,1,6 Ke in D. Raeh z,1,3 Ellen P. Penn,1 Russell P. T acy,7 Ruy M. Ribei o,8,9 Daniel N. F ank,4 Ca a C. Wilson,4 Alan L. Landay,5 C is ian Ape ei,1,3,6 and I ona Pand ea1,2,6 1Cen e o Vaccine Resea ch, 2Depa men o Pa hology, and 3Di ision o In ec ious Diseases, Depa men o Medicine, School o Medicine, Uni e si y o Pi sbu gh, Pi sbu gh, Pennsyl ania, USA. 4Di ision o In ec ious Diseases, Uni e si y o Colo ado, Anschu z Medical Campus, Au o a, Colo ado, USA. 5Depa men o Mic obial Pa hogens and Immuni y, Rush Uni e si y, Chicago, Illinois, USA. 6Depa men o In ec ious Diseases and Mic obiology, G adua e School o Public Heal h, Uni e si y o Pi sbu gh, Pi sbu gh, Pennsyl ania, USA. 7Depa men o Pa hology and Labo a o y Medicine, Uni e si y o Ve mon , Bu ling on, Ve mon , USA. 8Los Alamos Na ional Labo a o y, Los Alamos, New Mexico, USA. 9Faculdade de Medicina da Uni e sidade de Lisboa, Lisbon, Po ugal. Con lic o in e es : The au ho s ha e decla ed ha no con lic o in e es exis s. Copy igh : © 2019, Ame ican Socie y o Clinical In es iga ion. Submi ed: Ma ch 20, 2018; Accep ed: Sep embe 10, 2019; Published: No embe 11, 2019. Re e ence in o ma ion: J Clin In es . 2019;129(12):5474–5488. h ps://doi.o g/10.1172/JCI121208. The Jou nal o Clinical In es iga ion RESEARCH ARTICLE 5475 jci.o g Volume 129 Numbe 12 Decembe 2019 ec ed NHP species. HFD also signi ican ly inc eased sys emic immune ac i a ion and in lamma ion and al e ed he gu mucosal en i onmen , p o oundly comp omising gu in eg i y and inc eas- ing mic obial ansloca ion. Fu he mo e, HFD induced li e s e- a osis, ib osis, and al e a ions o he lipid me abolism. Finally, HFD inc eased equency and se e i y o CV lesions. Al oge he , ou esul s p o ide s ong e idence o he c i ical ole o HFD in modula ing HIV/SIV mo bidi y and mo ali y. Resul s S udy design. We used a die ha p o ided 32% calo ies om a (Table 1), as opposed o no mal chow, which only p o ides 13% calo ies om a (Table 1). The composi ion o he HFD we used closely mimicked ha o a ypical Ame ican die o he high con- en o sa u a ed a s and choles e ol (23). HFD was adminis e ed o 2 NHP models o nonpa hogenic (AGM) and pa hogenic (PTM) SIV in ec ion. The goal was no o compa e he HFD impac be ween species, bu a he o assess he HFD consequences wi hin he same species, as di ec compa isons be ween species may be lawed by majo in insic immunologic and me abolic di e ences. In he same line o eason, we did no pe ec ly ma ch he iming o HFD adminis a ion in he 2 species. Fou AGMs ecei ed HFD om 102 days be o e in ec ion and up o 200 days a e in ec ion (dpi) (Figu e 1). One HFD- ea ed AGM died du ing he ollow-up. The emaining 3 AGMs we e eleased a he s udy comple ion. Nine his o ical con ol AGMs we e used (Figu e 1). They we e SIV in ec ed in simila condi- ions, had a simila ollow-up and sampling schedule o he AGMs ecei ing HFD, bu ecei ed a egula chow die (Table 1). The AGM his o ical con ols we e no subjec ed o in es inal and li e esec ions. Fi e PTMs ecei ed HFD om 39 days be o e SIV in ec ion un il hey p og essed o AIDS (Figu e 1). Ten PTMs we e used as con ols (Figu e 1). They we e SIV in ec ed in simila condi ions bu ecei ed a egula chow die (Table 1). Th ee PTMs we e con- empo a y con ols and had a ollow-up and sampling schedule simila o ha o HFD- ecei ing PTMs (including in es inal and li e esec ions). They ei he p og essed o AIDS o we e eu ha- nized when he las HFD- ecei ing PTM died wi h AIDS. The emaining 7 PTMs we e his o ical con ols. They had a ollow-up and sampling schedule simila o ha o HFD- ecei ing PTMs, bu we e no subjec ed o in es inal and li e esec ions. They we e eu hanized when hey p og essed o AIDS. All con ols ecei ed he same chow die . HFD has a majo impac on disease p og ession and su i al o SIV-in ec ed NHPs. Fi s , we ques ioned whe he HFD a ec ed key pa ame e s o HIV/SIV in ec ion (i.e., CD4+ T cell coun s and pVLs). Pe iphe al CD4+ T cell loss was signi ican ly mo e p ominen in he SIVsab-in ec ed AGMs ecei ing HFD compa ed wi h con- ols. As such, al hough a end owa d eco e y was obse ed du ing ch onic in ec ion, he CD4+ T cell coun s we e s ill sig- ni ican ly lowe in HFD- ecei ing AGMs compa ed wi h con ols (Figu e 2A). In e es ingly, a CD4+ T cell dec ease end occu ed in AGMs a e HFD adminis a ion e en be o e SIV in ec ion, which was no due o CD4+ T cell down egula ion (24), as no inc eases in he CD8-αlow o CD4negCD8neg T cells we e obse ed in he HFD- ea ed AGMs du ing he ollow-up (da a no shown). Addi ionally, HFD is a classical isk ac o o CV diseases. HFD induces a he oscle osis (18) by enhancing dyslipidemia and mac ophage in il a ion in he ao a (18). HFD also al e s he coag- ula ion p o iles and p omo es hype coagulopa hy, by inc easing plasma le els o endogenous h ombin and p ocoagulan ac o s (e.g., FII, FVII, FVIII, and FXII), o dec eased ib inolysis (19). Hype coagulabili y is common in HIV/SIV in ec ions and may con ibu e o esidual immune ac i a ion and in lamma ion (20) e en in indi iduals wi h ART supp ession (2, 3). By al e ing he lip- id me abolism and coagula ion, HFD may hus enhance he inci- dence o CV como bidi ies, he mos equen complica ions, and he leading cause o mo ali y in HIV-in ec ed subjec s (21). As such, HFD may signi ican ly a ec he na u al his o y o HIV/SIV, limi ART e icacy, p omo e HIV/SIV- ela ed como bid- i ies, and ul ima ely inc ease mo ali y (2, 3). Only 1 s udy p e i- ously epo ed accele a ed disease p og ession in SIV-in ec ed hesus macaques ecei ing HFD (22). The unde lying mecha- nisms and he po en ial impac on SIV- ela ed como bidi ies we e no in es iga ed in de ail, howe e , and only he model o pa ho- genic HIV in ec ion was e alua ed. To ho oughly assess he impac o HFD on HIV/SIV na u al his o y and associa ed como bidi ies, we conduc ed a comp e- hensi e s udy in which we adminis e ed a HFD ich in sa u a ed a s and choles e ol o 2 NHP models o SIVsab in ec ion: A i- can g een monkeys (AGMs), in which in ec ion is nonpa hogenic (i.e., lacking gu damage, immune ac i a ion and in lamma ion, como bidi ies and disease p og ession) and pig ailed macaques (PTMs), in which in ec ion is pa hogenic (i.e., associa ing massi e gu damage, immune ac i a ion and in lamma ion, como bidi ies, p og ession o AIDS and dea h). We epo ha HFD adminis a ion had a majo impac on i al ese oi seeding and disease p og ession in bo h SIV-in- Table 1. Compa ison o ND and HFD composi ion ND HFD Composi ionA Fa , % 5.0 15.2 Choles e ol, ppm 80 946 Linoleic acid, % 1.46 3.02 Linolenic acid, % 0.09 0.09 A achidonic acid, % <0.01 0.01 Omega-3 a y acids, % 0.15 0.02 To al sa u a ed a y acids, % 1.36 6.05 To al monounsa u a ed a y acids, % 1.72 4.85 P o ein, % 15.6 18 Ca bohyd a es, % 60 55.6 Calo ies p o ided by P o ein, % 17.96 16.7 Fa , % 12.95 31.8 Ca bohyd a es, % 69.09 51.5 HFD, high- a die ; ND, no mal die ; ppm, pa s pe million. ANu ien s a e exp essed as pe cen age o a ion excep whe e o he wise indica ed. Mois u e con en is assumed o be 10.0% o he pu pose o he calcula ions. Bolded alues ep esen majo di e ences be ween HFD and no mal chow. The Jou nal o Clinical In es iga ion RESEARCH ARTICLE 5476 jci.o g Volume 129 Numbe 12 Decembe 2019 (Figu e 2I). AIDS-de ining his ological lesions also occu ed ea lie in HFD- ecei ing PTMs (Supplemen al Table 1). A nec opsy, he HFD- ecei ing PTMs p esen ed wi h mo e lymphoid deple ion, oppo unis ic in ec ions, and mac ophage in il a ion in issues compa ed wi h con ols sac i iced a he same ime poin s a e in ec ion. The CV lesions we e also mo e se e e in he HFD- ecei - ing PTMs (Supplemen al Table 2). Al oge he , hese ea u es indi- ca e an ea ly p og ession o AIDS and de elopmen o mo e se e e SIV- ela ed como bidi ies in he HFD- ea ed animals. Mos s ikingly, despi e he nonpa hogenic na u e o SIVsab in ec ion in AGMs, 1 HFD- ecei ing AGM died du ing ollow-up (Figu e 2J), wi h mul iple bioma ke s p edic ing HIV/SIV disease p og ession inc easing in he ange ound in PTMs wi h end-s age disease: mucosal (Figu e 3A) o sys emic (Figu e 3B) T cell ac i a- ion, LPS (Figu e 3C), hyalu onic acid (Figu e 3D), soluble in e - cellula adhesion molecule-1 (Figu e 3E); and D-dime (Figu e 3F). A nec opsy, his AGM p esen ed wi h lesions ep esen a i e o AIDS (oppo unis ic in ec ions) and SIV- ela ed como bidi ies: en e i is cha ac e ized by a se e e dis up ion o he small in es ine a chi ec u e wi h epi helial lesions (Figu e 3G) and lamina p op ia in il a ion wi h oamy cells (Figu e 3H). Such shee s o oamy mac ophages in he lamina p op ia a e cha ac e is ic o a ypical mycobac e ia in ec ion o he se e ely immunosupp essed hos s. Al hough he acid- as s aining could no iden i y he bacilli in he gu , occu ence o li e g anulomas in his animal also suppo he diagnosis o a ypical mycobac e ia in ec ion (Figu e 3I). Se e e in il a i e cys oisospo iasis was also diagnosed in his AGM (Figu e 3J) and con i med h ough Giemsa s aining (Figu e 3K). The lesions ound in he in es inal epi helium and lamina p o- p ia a e pa hognomonic o in ec ion wi h Cys oisospo a belli ( o - HFD- ecei ing AGMs had highe plasma VLs, which eached signi icance du ing ch onic in ec ion (Figu e 2B), along wi h signi i- can ly highe PBMC-associa ed SIV DNA le els du ing acu e in ec- ion (Figu e 2C), sugges ing ha HFD may po en ia e ea ly ese - oi seeding. This e ec was con i med on he gu samples om he HFD- ecei ing AGMs, in which acu e p o i al DNA ( DNA) le els (Figu e 2D), along wi h acu e and ch onic i al RNA ( RNA) le els (Supplemen al Figu e 1A; supplemen al ma e ial a ailable online wi h his a icle; h ps://doi.o g/10.1172/JCI121208DS1) we e signi ican ly highe han in con ols. The acu e RNA le els we e also highe in he lymph nodes (LNs) o he HFD- ecei ing AGMs han in con ols (Supplemen al Figu e 1B). In SIVsab-in ec ed PTMs, which expe ience se e e CD4+ T cell deple ion h oughou in ec ion, HFD induced u he sig- ni ican CD4+ T cell loss a la e ime poin s (Figu e 2E). Nei he pVLs no PBMC o in es ine cell-associa ed DNA le els we e signi ican ly inc eased in he HFD- ecei ing PTMs compa ed wi h con ols (Figu e 2, F–H). We de ec ed signi ican ly highe cell-associa ed RNA le els du ing ch onic in ec ion in bo h in es- ine (Supplemen al Figu e 1C) and LNs (Supplemen al Figu e 1D) in he HFD- ecei ing PTMs, howe e , sugges ing highe i al eplica ion in issues. No e ha SIVsab-in ec ed PTMs unde go massi e i al eplica ion and yield e y high VLs (25), which likely masked he HFD e ec on VL. These da a sugges ha HFD inc eases i us eplica ion, pa icu- la ly in issues and may hus ha e an impac on he i al ese oi size. HFD a ec ed SIV su i al in bo h species (Figu e 2, I and J). Al hough he limi ed sample size p ecluded eaching s a is ical signi icance, he e was a clea end owa d as e p og ession o AIDS in HFD- ecei ing PTMs compa ed wi h con ols (P = 0.0522) Figu e 1. The s udy design o SIVsab: HFD adminis a ion and sampling schedule. A o al o 13 AGMs and 15 PTMs we e in ec ed wi h 300 TCID50 o SIVsab. Fou AGMs ini ia ed HFD a 102 days be o e in ec ion and main ained i un il 200 days a e in ec ion (dpi). Nine his o ical AGM con ols we e included and ecei ed no mal chow. Fi e PTMs ini ia ed HFD a 39 days be o e in ec ion and main ained i un il nec opsy (128–218 days a e in ec ion). Th ee con empo a y and 7 his o ical con ols we e also included and ecei ed no mal chow. Sampling o he AGMs and PTMs in he s udy and con ol g oups was pe o med as shown. The Jou nal o Clinical In es iga ion RESEARCH ARTICLE 5477 jci.o g Volume 129 Numbe 12 Decembe 2019 i ied in he colon by immunohis ochemis y (Figu e 3N, uppe pan- els). The CMV in ec ion in he colon was also con i med h ough PCR (Supplemen al Figu e 2). La ge, some imes mul inuclea ed cells wi h i al cy oplasmic o nuclea inclusions su ounded by a clea halo (Figu e 3N, lowe panels) also indica i e o CMV in ec ion we e p esen in he lung along wi h in e s i ial in lamma ion (Figu e 3O). CMV in ec ion o he lung was con i med by PCR (Supplemen- al Figu e 2). The same echnique iden i ied CMV in he spleen and submandibula LNs (Supplemen al Figu e 2), and he i us could be quan i ied in blood (a 67 DNA copies/mL o plasma), hus con- i ming he dissemina ed CMV in ec ion. Finally, ch onic glome uloneph i is and h ombo ic mic oan- giopa hy (TMA), ha is, nume ous mic o h ombi in he kidney and o he issues (Figu e 3P), was also ound in he AGM p og esso . me ly known as Isospo a belli) and we e iden i ied, as usually done in a ou ine his opa hological exam, using HE s aining (Figu e 3J). Di e se e olu iona y o ms o he pa asi e we e easily ecog- nizable in acuoles loca ed in he cy oplasm o he gu epi helial cells (Figu e 3, J and K). The acuoles con aining he pa asi es we e mos equen ly loca ed unde he hos cells’ nuclei ha we e some imes pushed ho izon ally c ea ing he classical T-shape o m. The pa asi es we e associa ed wi h eosinophil in il a ion in he lamina p op ia (Figu e 3L). Cys oisospo iasis is an oppo unis- ic in ec ion ound in pa ien s in ec ed wi h HIV who ha e se e e immunosupp ession (26). Fu he mo e, he AGM p og esso also p esen ed wi h coli is ha associa ed c yp a ophy and in il a ion o all in es inal laye s wi h mononuclea cells (Figu e 3M). CMV-posi i e cells we e iden- Figu e 2. HFD accele a es disease p og ession and a ec s su i al o SIV-in ec ed NHPs. Pe iphe al CD4+ T cell deple ion in SIV-in ec ed AGMs (A) and PTMs (E) a e shown as index o baseline le els and compa ed a key ime poin s o SIV in ec ion wi hin HFD g oup wi h he F iedman es co ec ed o mul iple compa isons and be ween HFD and con ol g oups wi h K uskal-Wallis es . Plasma i al loads (B), PBMC-associa ed i al DNA (C), and in es- inal cell-associa ed DNA (D) le els in SIV-in ec ed AGMs, and in SIV-in ec ed PTMs (F–H) we e compa ed a acu e and ch onic in ec ion be ween HFD and con ol g oups wi h K uskal-Wallis es . Da a a e p esen ed as indi idual alues wi h medians. Sample size (n) and P alues a e p esen ed on g aphs. BL, baseline (p ein ec ion p e-HFD); Ac, acu e in ec ion; Ch , ch onic in ec ion; Fa , p ein ec ion pos -HFD; Nx, nec opsy. Kaplan–Meie su i al cu es o SIV-in ec ed PTMs (I) and AGMs (J) and PTMs a e illus a ed. Compa ison be ween su i al cu es o HFD and con ol g oups is pe o med wi h Man el-Cox me hod. P alues a e p esen ed on g aphs. The Jou nal o Clinical In es iga ion RESEARCH ARTICLE 5478 jci.o g Volume 129 Numbe 12 Decembe 2019 Figu e 3. Ele a ed bioma ke s and pa hologic changes o he HFD- ecei ing AGMs ha p og essed o AIDS-like disease. HFD- ecei ing AGM HFD1 de eloped an AIDS-like disease, wi h dis inc ly ele a ed le els o HLA-DR and CD38 coexp essing CD8+ T cells in he in es ine (A) and pe iphe y (B), LPS (C), hyalu onic acid (D), sICAM-1 (E), and D-dime (F), as compa ed wi h he emaining 3 AGMs on HFD. Ac, acu e in ec ion; BL, baseline (p ein ec ion p e-HFD); Ch , ch onic in ec ion; Fa , p ein ec ion pos -HFD. Rep esen a i e H&E images o lesions o AIDS and SIV-associa ed como bidi ies om HFD1. (G) En e i is, wi h dis up ed in es i- nal a chi ec u e, i egula c yp s, enla ged illi, and al e a ion o he su ace epi helium. (H) Nume ous oamy mac ophages in he lamina p op ia o he small in es ine sugges i e o ac i e a ypical mycobac e ial disease. (I) Hepa ic g anulomas, sugges i e o a ypical mycobac e ial disease. (J) Cys oisospo a belli p esen in acuoles in he in es inal epi helium. (K) Giemsa-s aining con i ma ion o small in es ine C. belli in ec ion. (L) Eosinophil in il a ion in he small in es ine, cha ac e is ic o pa asi ic in ec ions. (M) Coli is, wi h dis up ed in es inal a chi ec u e, a ophic c yp s and mononuclea cell in il a ion o he mucosa, submuco- sa, and o he muscle laye s. (N) Uppe panels: CMV immunohis ochemis y, wi h posi i e cells in he colon. Lowe panels: Nume ous la ge cells in he lung, wi h nuclea and cy oplasmic i al inclusions cha ac e is ic o CMV in ec ion (de ail o O). (O) In e s i ial pneumonia, wi h hickened al eola walls due o in il a ion wi h mononuclea cells. (P) Nume ous mic o h ombi in he kidney glome uli indica i e o TMA (solid a ow). Dis up ed kidney co ex a chi ec u e wi h i egula , small ib o ic glome uli, pa hognomonic o ch onic glome uloneph i is (dashed a ow). (Q) Enla ged capilla ies in he kidney pa enchyma indica i e o ch onic s asis associa ed wi h hea ailu e. (R) Nume ous hemoside in-laden mac ophages in he lung, indica i e o ch onic s asis associa ed wi h hea ailu e. O iginal magni ica ions: ×200 (G–I, M, and O–R); ×400 (L); ×600 (J, K, and N). The Jou nal o Clinical In es iga ion RESEARCH ARTICLE 5479 jci.o g Volume 129 Numbe 12 Decembe 2019 TMA occu s in p og essi e HIV/SIV in ec ions, whe e i is associa - ed wi h inc eased le els o D-dime (3). The AGM p og esso had D-dime le els in he ange o PTMs wi h end-s age disease (Fig- u e 3F). Ch onic s asis (cha ac e is ic o conges i e hea ailu e) was ound in he li e , kidneys (Figu e 3Q), and lungs (Figu e 3R) and was cha ac e ized by dila ed capilla ies, inc eased blood con- en , and nume ous hemoside in-laden mac ophages (Figu e 3R). HFD exace ba es sys emic immune ac i a ion and in lamma ion. Immune ac i a ion and in lamma ion a e hallma ks o ch onic pa hogenic HIV/SIV in ec ions (1). As HFD also inc eases in lam- ma ion independen ly (10), we nex asked whe he HFD and SIV in ec ion can syne gis ically d i e immune ac i a ion and in lam- ma ion. While SIVsab-in ec ed AGMs ypically do no exhibi sys emic immune ac i a ion and in lamma ion (1), he equency o CD8+ (Figu e 4A) and CD4+ (Supplemen al Figu e 3A) T cells exp essing HLA-DR+ and CD38+ inc eased immedia ely in AGMs a e s a ing he HFD, emaining signi ican ly ele a ed h ough- ou he ollow-up pe iod, wi h he highes le els o CD38+ HLA- DR+ T cells being obse ed in he AGM p og esso (Figu e 3, A and B). The Ki-67+ CD4+ T cells we e also signi ican ly inc eased du ing bo h acu e and ch onic in ec ion (Supplemen al Figu e 3B) in HFD- ecei ing AGMs compa ed wi h con ols. SIV-in ec ed PTMs ecei ing HFD simila ly expe ienced a sig- ni ican inc ease o he HLA-DR+ CD38+ CD8+ T cells du ing bo h acu e and ch onic in ec ion (Figu e 4D). The le els o he sys emic in lamma ion bioma ke C eac- i e p o ein (CRP) we e signi ican ly highe in HFD- ecei ing SIV-in ec ed AGMs and PTMs compa ed wi h con ols (Figu e 4, B and E). The le els o RANTES, a bioma ke o mac ophages and T cell ec ui men a a he oscle o ic lesion si es (27), we e also signi ican ly ele a ed compa ed wi h con ols in bo h spe- cies (Figu e 4, C and F). HFD induces in lamma ion and immune in il a ion in he adi- pose issue. Adipose issue is an impo an HIV ese oi (28). P omp ed by ou esul s showing bo h highe le els o cell- associa ed RNA as well as DNA and inc eased acu e and ch on- ic immune ac i a ion and in lamma ion in he HFD- ecei ing NHPs, we u he in es iga ed he in lamma o y s a us o he adi- pose issue. In SIV-in ec ed PTMs, HFD was associa ed wi h mo e se e e in lamma o y in il a es in he adipose issue su ounding c i ical o gans (GI ac and he hea ), compa ed wi h SIV-in ec - ed con ols. High-deg ee in lamma o y in il a es, ep esen ed by mononuclea cells, we e obse ed in he pe i oneal a (Figu e 5, le panel) and he epica dial adipose issue (Figu e 5, igh pan- el). Se e e in lamma o y in il a es in he epica dium pene a - ed in he unica ad en i ia o he adjacen co ona y a e ies. In 1 HFD- ea ed PTM, in lamma o y in il a es pene a ed he en i e co ona y wall, inducing signi ican damage and p omo ing leuko- cy e adhesion (Figu e 5, igh panel, a ow). Inc eased me abolic ac i i y may be a sou ce o he immune ac i- a ion obse ed in HFD- ecei ing NHPs. Inc eased cell me abolism may d i e immune ac i a ion. Using he seaho se echnology, we assessed he impac o HFD on he me abolic s a us in PTMs and showed ha HFD al e ed he PBMC me abolic s a e e en be o e SIV in ec ion. We quan i ied ae obic glycolysis by moni o ing he ex acellula acidi ica ion a e (ECAR) a basal espi a ion and in esponse o oligomycin ( o measu e ATP p oduc ion), luo oca - bonyl cyanide phenylhyd azone ( o measu e maximal espi a ion), Figu e 4. HFD induces ele a ed sys emic immune ac i a ion and in lamma ion in SIV-in ec ed NHPs. F equencies o CD38 and HLA-DR coexp essing CD8+ T cells (A), CRP le els (B) and RANTES le els (C) o baseline le els in AGMs, as well as in PTMs (D–F), a e compa ed a key ime poin s o SIV in ec ion wi hin HFD g oup wi h F iedman es co ec ed o mul iple compa isons, and be ween HFD and con ol g oups wi h he K uskal-Wallis es . Da a a e p e- sen ed as indi idual alues wi h medians. Sample size (n) and P alues a e p esen ed on g aphs. Ac, acu e in ec ion; BL, baseline (p ein ec ion p e-HFD); Ch , ch onic in ec ion; Fa , p ein ec ion pos -HFD; Nx, nec opsy. The Jou nal o Clinical In es iga ion RESEARCH ARTICLE 5480 jci.o g Volume 129 Numbe 12 Decembe 2019 and o enone addi ion ( o measu e nonmi ochond ial espi a ion) (Supplemen al Figu e 4A). PBMCs collec ed a e adminis e ing he HFD had signi ican ly highe AUC o ECAR compa ed wi h PBMCs collec ed be o e HFD (Supplemen al Figu e 4B). The oxygen consump ion a e (OCR) was also measu ed o quan i y mi ochond ial oxida i e phospho yla ion. A e HFD, PBMCs had a end o a lowe OCR/ECAR a io a basal espi a ion (Supple- men al Figu e 4C), indica ing a me abolic shi owa d ae obic glycolysis. A e HFD, PBMCs also showed mode a ely inc eased maximal espi a ion OCR, spa e espi a o y capaci y and ATP p oduc ion, albei wi hou eaching signi icance (P > 0.1) (Supple- men al Figu e 4D). These esul s collec i ely indica e a pos -HFD PBMC me abolic shi owa d ae obic glycolysis, e lec ing and con ibu ing o a heigh ened ac i a ion s a us. HFD comp omises gu immune in eg i y, p omo ing mic obial ansloca ion. A majo con ibu o o HIV-/SIV- ela ed immune ac i a ion and in lamma ion is gu dys unc ion and he conse- quen mic obial ansloca ion (6). Because he HFD-induced changes could po en ially dis up he in es inal immune homeo- s asis and epi helial ba ie in eg i y, we nex assessed HFD impac on he in es inal immune en i onmen and mic obial ansloca ion. In HFD- ecei ing AGMs, in es inal CD4+ T cells we e los e en be o e in ec ion, ye wi hou eaching s a is ical signi icance (P = 0.4029), p obably due o limi ed sample size (Figu e 6A). Simila o blood, his dec ease was no due o CD4+ T cell down egula ion (24), as no inc ease o he mucosal CD8-αlow/ CD4negCD8neg T cells occu ed in he HFD- ea ed AGMs (da a no shown). CD4+ T cell loss in ol ed all memo y subse s (Figu e 6B). In es inal CD4+ T cell eco e y was minimal in ch onically SIV- in ec ed HFD- ecei ing AGMs ( o 22.5% ± 4.5% o baseline le - els), while being mode a e in con ols ( o 47.5% ± 5.9% o baseline le els) (Figu e 6A), as epo ed (8). In PTMs, he magni ude o he SIVsab-induced mucosal CD4+ T cell deple ion obscu ed any di - e ence be ween HFD- ea ed PTMs and con ols. Among he mucosal CD4+ T cell subse s, T egs showed a dec easing end in HFD- ed AGMs e en be o e in ec ion (P = 0.125) (Supplemen al Figu e 5A). In PTMs, mucosal CD4+ T egs we e signi ican ly deple ed h oughou SIV in ec ion (Supplemen- al Figu e 5B), being signi ican ly lowe in he HFD- ea ed PTMs compa ed wi h con ols du ing bo h acu e and ch onic in ec- ion. T eg dec eases could diminish mucosal ole ance o in es- inal pa hogens and dis up local in lamma ion in he gu in he HFD- ecei ing NHPs (29). HFD also induced inc eased ac i a ion o mucosal immune cells in HFD- ecei ing PTMs, wi h inc eases o Glu -1+ CD38+ HLA-DR+ CD4+ T cells (Figu e 6C) and mac ophage ac i a ion, indica ed by he inc eased exp ession o CD80 (Figu e 6D) and CD86 (Figu e 6E), especially du ing ch onic SIV in ec ion. Fu he mo e, HFD also signi ican ly inc eased myelope - oxidase+ neu ophil in il a es associa ed wi h mo e p onounced epi helial lesions and a highe equency o c yp abscesses in ch onically SIV-in ec ed PTMs (Figu e 7A), sugges ing g ea e in es inal ba ie damage and s onge in lamma o y esponses due o heigh ened mic obial ansloca ion. Sys emic bioma ke s o mic obial ansloca ion and he asso- cia ed mucosal damage inc eased pos -HFD adminis a ion. Plasma LPS le els inc eased in HFD- ecei ing AGMs du ing bo h acu e and ch onic SIV in ec ion (Figu e 7B) and we e highes in he AGM p og esso (Figu e 3C). No di e ences in LPS le els we e obse ed be ween he HFD- ecei ing and con ol PTMs, likely due o he massi e SIV-induced in es inal damage, o o in insi- cally ele a ed LPS le els in PTMs (30). Addi ionally, in es inal a y acid-binding p o ein (I-FABP), which is eleased by nec o ic en e ocy es, inc eased e en p io SIV in ec ion and emained inc eased h oughou he ollow-up in he HFD- ecei ing AGMs (Figu e 7C). I-FABP was also signi ican ly ele a ed in he HFD- ecei ing PTMs, especially du ing acu e SIV in ec ion and a he ime o p og ession o AIDS (Figu e 7D). These esul s demons a e ha HFD exace ba ed in es inal dys unc ion in bo h NHP models. HFD induces pa hological al e a ions o he li e . P e ious s ud- ies showed ha HFD a ec s li e in eg i y. In pa icula , HFD-in- duced low sys emic endo oxemia is esponsible o NAFLD (31). In ou s udy, s ea ohepa i is occu ed in AGMs 82 days a e HFD adminis a ion, p io o SIV in ec ion (Figu e 8A). Li e s ea osis also occu ed in all he HFD- ecei ing PTMs (Figu e 8A) only 49 days a e HFD (a 9 dpi). Because hese PTMs we e a a e y ea - ly s age o SIV in ec ion, NAFLD was due o die a he han SIV in ec ion. Con e sely, NAFLD occu ed in only a small ac ion o con ols and only du ing a la e s age o SIV in ec ion. The a d ople in il a es we e di usely dis ibu ed h oughou he hepa - ic lobules o he HFD- ecei ing PTMs (Figu e 8A), whe eas in con- ols, hey mos equen ly occu ed a he pe iphe y o he hepa ic lobule, pa hognomonic o hypoxia. The di e en dis ibu ion o a accumula ion in he li e poin s o di e en mechanisms o li e s ea osis in ch onically SIV-in ec ed HFD- ecei ing PTMs and con ols. NAFLD associa ed mode a e sinusoidal ib osis ha became signi ican du ing ch onic in ec ion, as his ological- ly shown by collagen quan i ica ion (Figu e 8B), and con i med by measu ing plasma hyalu onic acid (32), which signi ican ly inc eased in HFD- ecei ing AGMs and PTMs compa ed wi h con ols (Figu e 8, C and D). No ably, he AGM p og esso had Figu e 5. HFD esul s in immune cell in il a ion in adipose issue in SIV- in ec ed PTMs. Rep esen a i e H&E images o pe i oneal adipose issue a ound GI ac (le panel) and epica - dial adipose issue ( igh panels). No e p ominen lymphocy e in il a ion om he epica dial adipose issue pene a ing h ough he co ona y wall indica ed by he a ows. O iginal magni ica ions: ×200. The Jou nal o Clinical In es iga ion RESEARCH ARTICLE 5481 jci.o g Volume 129 Numbe 12 Decembe 2019 In he pa hogenic SIV in ec ion o PTMs, which al eady asso- cia es hype coagulabili y (3), HFD adminis a ion did no u he inc ease he oxidized HDL o sTF, bu induced signi ican ly highe FVII le els du ing SIV in ec ion (Figu e 9F). Fu he mo e, opo- nin-I was signi ican ly ele a ed in 3 ou o 5 ch onically SIV-in ec ed PTMs on HFD, while emaining unde ec able in con ols (Figu e 9G), di ec ly con i ming ha HFD induced se e e ca diac inju y. HFD inc eases he equency and se e i y o CV lesions in SIV- in ec ed PTMs. To assess he equency o CV issue lesions in HFD- ea ed NHPs, we conduc ed a his opa hological diagnos- ic on he nec opsy samples collec ed om he PTMs. Al hough he CV lesions ound in he HFD- ea ed PTMs we e simila o hose in ch onic SIV-in ec ed con ols, hey we e mo e se e e and occu ed ea lie (Supplemen al Table 2). CV lesions included mononuclea cell in il a ion in myoca dium (Figu e 10A) and epi- ca dium (Figu e 10B), which o med lymphoid agg ega es (Figu e 10C), se e e myoca dial cy olysis (Figu e 10D), myoca dial a y in il a ion (Figu e 10E), and myoca dial mic o h ombi (Figu e 10F). Two HFD- ecei ing PTMs had incipien ao ic dissec ion (blood pene a ion unde nea h he endo helium) (Figu e 10G). These lesions usually e ol e in o ao a aneu ysms ha may up- u e and induce a al hemo hages. Di use o localized myoca di- al ib osis also occu ed (Figu e 10H), as well as se e e pe ica dial ib osis, indica i e o ch onic pe ica di is complica ed wi h colla- he highes le els o plasma hyalu onic acid (~5.5- old inc ease om baseline) (Figu e 3D), poin ing o a signi ican ole o HFD- induced li e pa hology on he ch onic SIV in ec ion ou come. HFD inc eases he isk o CV como bidi ies in SIV-in ec ed NHPs. HFD, especially wi h high sa u a ed a , is a majo isk ac o o CV disease (33). We in es iga ed he e ec s o HFD on he coagula ion s a us and CV heal h o SIV-in ec ed NHPs, o assess he ela i e con- ibu ion o die a y habi s o he HIV-associa ed CV como bidi ies. The choles e ol (Figu e 9A) and oxidized HDL (Figu e 9B) le els inc eased in HFD- ecei ing, SIV-in ec ed AGMs, while emaining i ually unchanged in con ols. Oxidized HDL di ec - ly co ela es wi h accele a ed a he oscle osis (34); he e o e, i s pos -HFD inc ease in SIV-in ec ed AGMs (which do no de elop CV disease) indica es ha m ul e ec s o HFD on CV heal h. Solu- ble issue ac o (sTF) also inc eased in HFD- ecei ing, ch onically SIV-in ec ed AGMs (Figu e 9C), poin ing o ac i a ion o he ex in- sic coagula ion pa hway and a highe h ombo ic isk. The inc eased CV isk was also suppo ed by signi ican ly ele a ed le els o soluble p-selec in (Figu e 9D) and soluble in e cellula adhesion molecule-1 (sICAM-1) (Figu e 9E) h oughou SIV in ec ion in HFD- ecei ing AGMs compa ed wi h con ols, demons a ing inc eased pla e- le and endo helial ac i a ion, along wi h inc eased immune cell ec ui men o he blood essel walls. The AGM p og esso exhibi - ed he highes sICAM-1 (Figu e 3E), and D-dime le els (Figu e 3F). Figu e 6. HFD al e s gu immune en i onmen and ac i a ion s a es o he immune cells. (A) Mucosal CD4+ T cell deple ion in HFD- ecei ing and con ol AGMs is shown as an index o baseline le els and compa ed a key ime poin s o SIV in ec ion wi hin he HFD g oup wi h F iedman es co ec ed o mul- iple compa isons, and be ween HFD and con ol g oups wi h K uskal-Wallis es . (B) Mucosal-naï e, cen al memo y, and e ec o memo y CD4+ T cells a e shown as an index o o al baseline mucosal CD4+ T cell le els and compa ed be o e and a e HFD in p ein ec ion AGMs wi h F iedman es . F equencies o Glu -1, HLA-DR, and CD38 coexp essing CD4+ T cells (C), as well as CD80-exp essing (D) and CD86-exp essing (E) mac ophages in he in es ine o PTMs a e compa ed a key ime poin s o SIV in ec ion wi hin HFD g oup wi h F iedman es co ec ed o mul iple compa isons and be ween HFD and con ol g oups wi h K uskal-Wallis es . Da a a e p esen ed as indi idual alues wi h medians. Sample size (n) and P alues a e p esen ed on g aphs. Ac, acu e in ec ion; BL, baseline (p ein ec ion p e-HFD); Ch , ch onic in ec ion; Fa , p ein ec ion pos -HFD. The Jou nal o Clinical In es iga ion RESEARCH ARTICLE 5482 jci.o g Volume 129 Numbe 12 Decembe 2019 immune ac i a ion and in lamma ion a e being ex ensi ely in es- iga ed. Thus, in es inal dys unc ion was shown o play a cen al ole in d i ing disease p og ession (and o be po en ially d i en) in addi ion o in ec ion- ela ed causes, by en i onmen al ac o s, such as li es yle and die a y in ake. Li le is known, howe e , abou he syne gy be ween HIV/SIV in ec ion and en i onmen al ac o s in d i ing pa hogenesis. Die e ec s on HIV in ec ion a e di icul o s udy in humans, due o con ounding ac o s (ART- ela ed dyslipidemia and oxici- y, alcohol, obacco and ec ea ional d ug use, seden a y li es yle), and e hical limi a ions (expe imen al al e a ions o die may ha e unknown e ec s, posing isks o human subjec s; in asi e sampling needed o assess die e ec s on issues is impossible in humans). NHPs may be employed o ci cum en hese e hical issues, while enabling he assessmen o he impac o di e en die a y compo- nen s on SIV pa hogenesis h ough adminis a ion o pu i ied die s wi h a speci ic composi ion, s aged in oduc ion o a ious die a y elemen s, and es ing in a s ic ly con olled en i onmen . Mans ield e al. p e iously epo ed ha an a he ogenic die led o accele a ed disease p og ession in hesus macaques and highligh ed he ole o IL-18 in enhancing he disease onse (22). gen deposi ion (Figu e 10I), which may es ic ca diac unc ion. Myoca dial ich ome quan i ica ion showed signi ican inc eased ib osis o he ca diac muscle in he HFD- ea ed PTMs compa ed wi h con ols (Figu e 10, J and K). Incipien a he oscle osis lesions ( a y s eaks) in co ona ies, abdominal ao a, and ca o id a e ies we e mo e equen in he HFD- ea ed PTMs compa ed wi h con- ols (Figu e 10L). Fa y s eaks we e composed mainly by oamy cells (mac ophages ha engul ed choles e ol and iglyce ides) and mononuclea cells, wi h T cells likely accumula ing unde - nea h he ascula endo helium (Figu e 10L, le ), esul ing in a hickened blood essel in ima (Figu e 10L, middle). O ganized mic o h ombi ha igge addi ional leukocy e and pla ele adhe- sion also occu ed in he HFD- ea ed animals (Figu e 10L, igh ). Discussion Al hough he li e span o HIV-in ec ed subjec s d ama ically imp o ed wi h ART, a highe mo ali y a e han in unin ec ed indi- iduals s ill pe sis s (2). The e o e, he s udy o non-AIDS como - bidi ies in long- e m ART-supp essed subjec s has gained ac ion (2). As p omo e s o non-AIDS como bidi ies and he accele a ed aging (5), he unde lying mechanism(s) o HIV-/SIV-associa ed Figu e 7. HFD induces in es inal epi helial ba ie damage and inc eases mic obial ansloca ion. (A) Rep esen a i e images o jejunum s ained o myelope oxidase (b own) collec ed be o e in ec ion, a 9 days a e in ec ion, and a nec opsy om con empo a y con ols and HFD- ecei ing PTMs. No e subs an ially inc eased myelope oxidase unde nea h he damaged epi helium and adjacen o he c yp abscess. O iginal magni ica ions: ×200. Quan- i ica ion o he pe cen a ea o he jejunum posi i e o myelope oxidase is shown in he igh panel. Fold inc ease o LPS (B) and in es inal a y-acid binding p o ein (C) o baseline le els in AGMs, as well as old inc ease o in es inal a y-acid binding p o ein (D) o baseline le els in PTMs a e compa ed a key ime poin s o SIV in ec ion wi hin HFD g oup wi h F iedman es co ec ed o mul iple compa isons, and be ween HFD and con ol g oups wi h K uskal-Wallis es . Da a a e p esen ed as indi idual alues wi h medians. Sample size (n) and P alues a e p esen ed on g aphs. Ac, acu e in ec ion; BL, baseline (p ein ec ion p e-HFD); Ch , ch onic in ec ion; Fa , p ein ec ion pos -HFD; Nx, nec opsy.