Acute HIV-1 and SARS-CoV-2 infections Share Slan+ Monocyte Depletion - evidence from an hyperacute HIV-1 case report

i uses Communica ion Acu e HIV-1 and SARS-CoV-2 In ec ions Sha e Slan+ Monocy e Deple ion—E idence om an Hype acu e HIV-1 Case Repo Guilhe me B. Fa ias 1,† , Robe Badu a 1,2,†, Ca olina M. Conceição1, And éM. C. Gomes 1, Ana Godinho-San os 1, Joel Laia 1, Ped o Rosmaninho 1, Diana F. San os 1, Ca a ina Mo a 1,2, A onso R. M. Almeida 1, Susana M. Fe nandes 1,2, Amelia C. T ombe a 1,*,‡ and Ana E. Sousa 1,*,‡   Ci a ion: Fa ias, G.B.; Badu a, R.; Conceição, C.M.; Gomes, A.M.C.; Godinho-San os, A.; Laia, J.; Rosmaninho, P.; San os, D.F.; Mo a, C.; Almeida, A.R.M.; e al. Acu e HIV-1 and SARS-CoV-2 In ec ions Sha e Slan+ Monocy e Deple ion—E idence om an Hype acu e HIV-1 Case Repo . Vi uses 2021,13, 1805. h ps:// doi.o g/10.3390/ 13091805 Academic Edi o : Ana Ba oso Abecasis Recei ed: 31 July 2021 Accep ed: 6 Sep embe 2021 Published: 10 Sep embe 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 1Ins i u o de Medicina Molecula João Lobo An unes, Faculdade de Medicina, Uni e sidade de Lisboa, 1649-028 Lisboa, Po ugal; [email p o ec ed] (G.B.F.); [email p o ec ed] (R.B.); [email p o ec ed] (C.M.C.); and [email p o ec ed] (A.M.C.G.); [email p o ec ed] (A.G.-S.); [email p o ec ed] (J.L.); ped o. [email p o ec ed] (P.R.); [email p o ec ed] (D.F.S.); [email p o ec ed] (C.M.); [email p o ec ed] (A.R.M.A.); [email p o ec ed] (S.M.F.) 2Cen o Hospi ala Uni e si á io Lisboa No e, Hospi al de San a Ma ia, 1649-028 Lisboa, Po ugal *Co espondence: [email p o ec ed] (A.C.T.); [email p o ec ed] (A.E.S.); Tel.: +351-217-999-521 (A.E.S.) † Co- i s au ho s. ‡ Co-senio au ho s. Abs ac : Monocy es a e key modula o s in acu e i al in ec ions, de e mining bo h in lamma ion and de elopmen o speci ic B- and T-cell esponses. Recen ly, hese cells we e shown o be associa ed o di e en SARS-CoV-2 in ec ion ou come. Howe e , hei ole in acu e HIV-1 in ec ion emains unclea . We had he oppo uni y o e alua e he mononuclea cell compa men in an ea ly hype - acu e HIV-1 pa ien in compa ison wi h an un ea ed ch onic HIV-1 and a coho o SARS-CoV-2 in ec ed pa ien s, by high dimensional low cy ome y using an unsupe ised app oach. A dis inc pola iza ion o he monocy e pheno ype was obse ed in he wo i al in ec ions, wi h main enance o p o-in lamma o y M1-like p o ile in HIV-1, in con as o he M2-like immunosupp essi e shi in SARS-CoV-2. No iceably, bo h acu e in ec ions had educed CD14 low/− CD16 + non-classical monocy es, wi h deple ion o he popula ion exp essing Slan (6-sul o LacNac), which is hough o con ibu e o immune su eillance h ough p o-in lamma o y p ope ies. This deple ion indica es a po en ial ole o hese cells in acu e i al in ec ion, which has no p e iously been explo ed. The in lamma o y s a e accompanied by he deple ion o Slan+ monocy es may p o ide new insigh s on he c i ical e en s ha de e mine he a e o i al se -poin in acu e HIV-1 in ec ion and subsequen impac on ansmission and ese oi es ablishmen . Keywo ds: acu e HIV-1 in ec ion; COVID-19; monocy es; slan; M-DC8; dend i ic cells 1. In oduc ion The in oduc ion o combined an i e o i al he apy (cART), in 1996, d ama ically changed he p ognosis o HIV-1 in ec ion, bu soon i was ealized he need o complemen- a y app oaches o con ol i al pe sis ency. Al hough he ea ly s a o cART in he acu e HIV-1 in ec ion educes he i al se -poin and immune-ac i a ion, i does no p e en he es ablishmen o cell ese oi s o in eg a ed p o i us [ 1 ]. The e o e, i is o u mos impo ance o unco e he ini ial immune esponses wi h impac on he i al dynamics. The in lamma o y en i onmen is de e minan o he dissemina ion and he es ab- lishmen o HIV-1 in ec ion, and monocy es a e cen al in hese p ocesses [ 2 ]. In ac , monocy e esponses ha e been eme ging as main con ibu o s o he pa hogenesis o ano he acu e in ec ion, se e e acu e espi a o y synd ome co ona i us 2 (SARS-CoV-2) [ 3 ]. Vi uses 2021,13, 1805. h ps://doi.o g/10.3390/ 13091805 h ps://www.mdpi.com/jou nal/ i uses Vi uses 2021,13, 1805 2 o 12 This pa hogen causes Co ona i us Disease 2019 (COVID-19), ha can lead o espi a o y ailu e and dea h in highe isk g oups [4]. Monocy es and mac ophages play a c i ical ole in he de elopmen and homeos asis o all issues and, oge he wi h dend i ic cells, a e c i ical o he ini ia ion o an adap i e immune esponse [ 2 ]. The p ope ac i a ion o he adap i e immune esponse is associa ed wi h be e disease con ol in bo h he acu e and ch onic s ages o HIV-1 in ec ion [ 5 – 8 ]. In ci cula ion, monocy es can be di ided in o h ee subse s: classical (CD14 high CD16 − ), in- e media e (CD14 high CD16 + ), and non-classical (CD14 low/neg CD16 + ). These subse s show ansc ip omic di e ences ha ansla e in o specializa ion in o di e en unc ions [ 9 ]. Due o hei capabili y o p oduce in lamma o y cy okines, and high mobili y, non-classical monocy es a e cha ac e ized as a p o-in lamma o y popula ion [ 10 , 11 ]. This subse can hen be u he di ided in o Slan posi i e (Slan + ) and Slan nega i e popula ions [12–14] . Monocy es di e en ia e in o mac ophages and dend i ic cells in he issues [ 2 , 15 ]. Recen ly, he mac ophage/monocy e pheno ype has been be e dissec ed, leading o he es ablish- men o a p o-in lamma o y pheno ype (M1-like), exp essing highe le els o CD80, CD86, and HLA-DR, o an immune-supp essi e/wound healing pheno ype (M2-like), exp essing highe le els o CD204 and CD206, as ex emes o a di e en ia ion spec um [16,17]. Monocy e/mac ophage ac i a ion synd ome was epo ed in se e e COVID-19 cases, wi h in e e on- γ (IFN γ ), in e leukin-1 (IL-1), IL-6, umo nec osis ac o - α (TNF α ), and IL-18 ha ing cen al immunopa hogenic oles in he hype -in lamma ion [ 18 ]. Ou lab demons a ed ha he expansion o M2-like monocy es was signi ican ly highe in pa ien s wi h de ec able SARS-CoV-2 plasma i al load and ha sys emic immune- egula o y myeloid cell pheno ype was he main ea u e associa ed wi h he eco e y o hospi alized pa ien s [19]. Con e sely, he p o-in lamma o y s a e is a hallma k o ch onic HIV-1 in ec ion [ 20 ]. This is e lec ed in he up egula ion o ac i a ion ma ke s in classical monocy es and he expansion o non-classical monocy es [ 21 ]. This s a e pe sis s e en in ea ed indi iduals and is hough o con ibu e o he de elopmen o non-AIDS ela ed co-mo bidi ies [22]. The es ablishmen o he in lamma o y s a e occu s ea ly in HIV-1 in ec ion, as shown by he inc ease in ac i a ion ma ke s like HLA-DR highligh ing hei con ibu ion o CD4+ T-cell ac i a ion [ 6 , 23 – 26 ]. The da a on he monocy e pheno ype du ing he acu e HIV-1 in ec ion a e ela i ely sca ce due o he a i y o hese pa ien s. In ac , mo e han 50% o acu e HIV in ec ion [ 27 ] a e asymp oma ic, imposing se ious limi s on he clinical in es iga ion o hese ini ial s ages. The e o e, he oppo uni y o in es iga e an acu e HIV-1 in ec ed pa ien , in pa allel wi h a coho o SARS-CoV-2 in ec ed indi iduals allowed us o explo e he monocy e pheno ype upon exposu e o wo dis inc i uses. We expec ha his case epo will in o m he design o u u e s udies aiming o be e unde s and he con ibu ion o he in lamma o y esponse o he in ec ion ou come. 2. Ma e ials and Me hods 2.1. Pa ien s and Con ols The s udy in ol ed an ea ly acu e HIV-1 in ec ed pa ien a Fiebig s age II [ 28 ], a ch onic HIV-1 in ec ed indi idual a he ime o diagnosis wi hou exposu e o an i e o i al he apy, and wen y pa ien s wi h acu e SARS-CoV-2 in ec ion (con i med by RT-PCR o nasopha yngeal swabs), all ollowed a he Cen o Hospi ala Uni e si á io Lisboa No e (CHULN, Lisboa, Po ugal). Ele en heal hy indi iduals we e included as con ols. The clinical and epidemiological de ails a e depic ed in Table 1. The COVID-19 pa ien s we e p e iously included in a ecen ly published s udy [ 19 ]. Clinical da a we e collec ed by he clinicians in eg a ing he esea ch eam. Whole blood was p ocessed immedia ely a e sampling. The e we e no di e ences in sample handling o ma e ial used among pa ien s and heal hy con ols. In o med consen was ob ained om all pa icipan s and he s udy was app o ed by he E hics commi ee o he CHULN/Faculdade de Medicina da Uni e sidade de Lisboa/Cen o Académico de Medicina de Lisboa. Vi uses 2021,13, 1805 3 o 12 Table 1. Clinical and ou ine labo a o y da a om pa ien s and heal hy con ols. HIV-1 Acu e HIV-1 Ch onic COVID-19 * Heal hy Con ols Numbe (males) 1 (0) 1 (1) 20 (17) 11 (9) Age (yea s) 22 49 55.5 (39–65) 58 (39–65) Time om symp oms s a (days) 13 NA 8.5 (5–11) NA CRP (mg/dL) 2.60 0.37 8.8 (4.85–25.5) ND PCT (ng/mL) 0.48 ULoD 0.16 (0.11–0.38) ND Fe i in (ng/mL) 412 262 939 (402–1906) ND In e leukin 6 (pg/mL) 1.5 2.8 18 (4.5–36) 0.85 (0.24–1.6) Lymphocy es/µL 1620 3130 920 (845–1662) 1940 (1423–2200) CD4+ T cells/µL 364 210 247 (133–392) 768 (544–998) CD8+ T cells/µL 503 863 145 (81.2–262) 414 (158–577) CD4/CD8 a io 0.72 0.24 1.78 (0.93–2.50) 1.95 (1.53–4.17) Neu ophils/mL 1830 2290 4251 (2413–6917) 3228 (2521–6390) Lymphocy es/neu ophils a io 0.89 1.37 0.23 (0.15–0.5) 0.51 (0.47–0.61) Monocy es/mL 810 720 349 (223–537) 398 (275–733) Basophils/mL 10 50 20 (9.7–35) 32 (16–63) Eosinophils/mL 30 200 13 (6.7–56) 115 (96–297) SARS-CoV-2 Plasma Vi al Load # (RNA cps/mL) NA NA 112 (24–498) NA HIV-1 Plasma Vi al Load (RNA cps/mL) 1,320,000 57,200 NA NA Values exp essed as medians (in e qua ile ange) unless o he wise speci ied. CRP: C eac i e p o ein; PCT: p ocalci onin; NA: no applicable; ND: no done; ULoD: unde limi s o de ec ion. * COVID-19-associa ed co-mo bidi ies: a e ial hype ension 9 pa ien s (45%); diabe es ype II 6 pa ien s (30%); obesi y 6 pa ien s (30%); lung emphysema 2 pa ien s (10%); no co-mo bidi ies in HIV-1 in ec ed pa ien s; #Quan i ied in he 13 ou o 20 COVID-19 pa ien s wi h de ec able SARS-CoV-2 plasma i al load. 2.2. Flow Cy ome y Mul i-pa ame e low cy ome y was pe o med on whole blood immedia ely a e sample collec ion. A e e y h ocy e bulk lysis, 10 million leukocy es we e incuba ed a oom empe a u e o 30 min wi h a panel o luo och ome labelled an ibodies: an i-Slan Vioblue, an i-CD141 BV510, an i-CD45 BV605, an i-HLA-DR BV650, an i-CD86 BV711, an i-PD-L1 BV785, an i-CD3 FITC, an i-CD19 FITC, an i-CD66b FITC, an i-CD14 Pe CP- Cy5.5, an i-CD80 PE, an i-CD163 PE-dazzle594, an i-CD206 PE-Cy5, an i-CD123 PE-Cy7, an i-CD204 APC, an i-CD16 Alexa Fluo 700, and an i-CD1c APC-Cy7. The lis o an ibody clones is p o ided in Table S1. A e ixa ion, cells we e e-suspended in PBS and acqui ed in a Fo essa X-20 low cy ome e . The esul ing low cy ome y da a we e compensa ed and analyzed wi h FlowJo so wa e (Ve sion 10.7; T ee S a , Inc., Ashland, OR, USA). The ga ing s a egy used is ep esen ed in Figu e S1. The monocy es, mac ophages, and dend i ic cells we e de ined by selec ing cells exp essing CD45 and no he lineage ma ke s CD3, CD19, and CD66b (CD45 + Lin − ). In pa allel, a ube o iden i y he main lymphocy e popula ions was pe o med including he ollowing luo och ome labelled an ibodies: an i-CD27 BV421, an i-IgM BV510, an i-CD4 V500, an i-IgD BV605, an i-CD8 BV605, an i-HLA-DR BV650, an i-CD21 BV711, an i-CD45RO BV785, an i-CCR7 FITC, an i-CD3 Pe CP-Cy5.5, an i-CD56 PE, an i-CXCR5 PE-dazzle594, an i-CD38 PE-Cy5, an i-CD19 PE-Cy7, an i- γδ PE-Cy7, an i-CD25 APC, an i-CD16 Alexa Fluo 700, an i-CD127 APC-e luo 780 (Table S1). 2.3. Unsupe ised Analysis Fo he unsupe ised analysis we ollowed he wo k low desc ibed by Melsen J. e al. [29] . A e compensa ion, we selec ed he CD45 + Lin − cells and pe o med he analysis on he same numbe o e en s (72591) om each pa ien and heal hy con ol. A e ans o ma ion and no maliza ion o he da a, we pe o med dimensionali y educ ion wi h Uni o m Mani old App oxima ion and P ojec ion (UMAP) using he uwo package [ 30 ]. Fo clus- e ing, he X-shi algo i hm was applied o de ine he app op ia e numbe o clus e s o he da ase [ 31 ]. A e wa ds, he da a we e clus e ed using FlowSOM [ 32 ]. The UMAP highligh ing he 14 clus e s ob ained, as well as he exp ession o he di e en ma ke s a e Vi uses 2021,13, 1805 4 o 12 shown in he Figu e S2. All da a analysis was pe o med in R (4.1.0), using Bioconduc o (3.13) [ 33 ]. Fo da a isualiza ion he phea map and idy e se packages we e used. UMAPs we e also gene a ed on T cells, ga ing on CD3+ lymphocy es, and on B cells, ga ing on CD19+ lymphocy es, using 57,907 and 10,012 e en s om each pa ien and heal hy con ol, espec i ely. The main T- and B-cell subse s we e hen de ined manually ( Figu es S3 and S4 , espec i ely), using FlowJo. 2.4. HIV-1 Read-Ou s Vi al load was measu ed by eal- ime PCR pe o med on whole blood in EDTA, Cobas ® 6800/8000 (Roche, Basel, Swi ze land), a ge ing he LTR and gag egions o HIV-1 genome, enabling quan i ica ion and sub ypi ica ion among: HIV-1M (A-D, F-H, CRF01_AE, CRF02_AG), HIV-1O, HIV-1N. De ec ion o an ibodies o HIV-1 and HIV-2 , was made using a ou h gene a ion ELISA (Cobas 8000; Roche, Basel, Swi ze land). An igenemia o p24 was measu ed using E olis sys em (BioRad; He cules, CA, USA). Finally, wes e n blo s we e pe o med on En , Pol and Gag o HIV-1 and 2, NewLa Blo 1 and NewLa Blo 2 assays in a Au oblo 3000 (BioRad). 2.5. SARS-CoV2 Plasma Vi al Load A d ople digi al PCR (ddPCR) es ki (SARS-CoV-2 ddPCR Tes Ki , Bio-Rad) was used o quan i y SARS-CoV2 plasma i al load, on QX200 ™ ddPCR Sys em (Bio-Rad), ollowing manu ac u e ’s ins uc ions. To al RNA was ex ac ed om 560 µ L o plasma (QIAamp ® Vi al-RNA MiniKi , QIAGEN), and duplica es o 20 µ L ddPCR eac ion using 5 µ L RNA we e analyzed on Quan aSo Analysis P o (1.0.596). Plasma samples wi h N1 o N2 egions, o bo h egions de ec ed we e conside ed posi i e. SARS-CoV-2 RNA concen a ions (cp/mL) we e calcula ed conside ing he ex ac ed olume o plasma. 3. Resul s Ou aim was o compa e he monocy e and dend i ic cell pheno ype in acu e HIV-1 and SARS-CoV-2 in ec ions. We we e able o in es iga e an ea ly hype -acu e HIV-1 in ec ed indi idual, classi ied as Fiebig s age wo, possessing high le els o plasma i al load and de ec able p24 an igen- emia in he absence o speci ic HIV an ibodies [ 28 ]. The pa ien ea u ed e e and as henia p eceding 13 days he diagnosis, and ele a ed amino ans e ases, compa ible wi h a HIV- induced hepa i is and high in lamma o y esponse. The compa ison was pe o med wi h a coho o 20 acu e SARS-CoV-2 pa ien s, included in a p e ious s udy [ 19 ]. As con ols, we en olled 11 heal hy subjec s and a ea men naï e ch onic HIV-1 pa ien , asymp oma ic despi e an ad anced disease s age as illus a ed by he low CD4+ T-cell coun (Table 1). Bo h he acu e HIV-1- and SARS-CoV-2-in ec ed pa ien s showed signs o in lamma- ion, namely ele a ed C eac i e p o ein (CRP) and e i in le els, as expec ed in an acu e i al in ec ion, hough IL-6 was only ele a ed in SARS-CoV-2-in ec ed pa ien s. COVID-19 was associa ed wi h neu ophilia wi h lymphopenia, as epo ed be o e [ 3 ], which was no obse ed in he acu e HIV-1-in ec ed pa ien (Table 1). Despi e a no mal lymphocy e coun , we obse ed a educ ion o ci cula ing CD4+ T cells in he acu e HIV-1 pa ien , ye in he absence o CD8+ T-cell expansion (Table 1). These ea u es we e obse ed in he ch onically HIV-1-in ec ed pa ien , who showed he highes in e sion o he CD4/CD8 a io (Table 1). Monocy e coun s we e highe in HIV-1 as compa ed o SARS-CoV-2-in ec ed indi iduals and he con ols (Table 1). In o de o explo e he impac o acu e HIV-1 in ec ion on he ci cula ing monocy es and dend i ic cells, we pe o med an unsupe ised analysis o a panel o M1 and M2 ma k- e s by low cy ome y, using UMAP and clus e ing wi h he FlowSOM algo i hm, ob aining 14 clus e s (Figu e 1, Figu es S1 and S2). To acili a e he in e p e a ion he 14 clus e s ob ained we e u he anno a ed in o 10 subse s based on ma ke exp ession shown in Figu e S2, de ining he majo monocy e and dend i ic cell popula ions (Figu e 1A). Vi uses 2021,13, 1805 5 o 12 Figu e 1. P o iling o he mononuclea cells compa men in HIV-1 and SARS-CoV-2 in ec ions using unsupe ised analysis. ( A ) UMAP o he CD45 + Lin − da ase o all samples wi h he hea map showing ela i e ma ke exp ession o he clus e s ob ained wi h he FlowSOM clus e ing algo i hm and he manual anno a ion o he main subse s. ( B ) Rela i e subse dis ibu ion in ep esen a i e indi iduals om he heal hy con ol and COVID-19 g oups, as well as in he un ea ed HIV-1 acu e and HIV-1 ch onic pa ien s. Classical monocy es ep esen a key popula ion o in e es o be s udied in acu e i al in ec ions, gi en hei wide spec um o in lamma o y and homeos a ic unc ions [ 34 ]. The ela i e popula ion dis ibu ion in he acu e HIV-1 pa ien e ealed a wo- old inc ease in he p o-in lamma o y M1-like pheno ype in classical monocy es, de ined as CD14 high CD16 − CD86 + CD80 + CD163 low , when compa ed o con ols (Figu es 1B and 2A). COVID- 19 pa ien s showed he opposi e end, ha ing an inc ease in he mo e immunomodula o y M2-like classical monocy es, de ined as CD14 high CD16 − CD163 high CD204 + CD206 + , wi h a educ ion in he M1-like pheno ype (Figu es 1B and 2B). The ch onic HIV-1 pa ien ollowed he end o he acu e HIV-1 pa ien , wi h a mo e p onounced inc ease in M1-like classical monocy es (Figu es 1B and 2A). This shi in he balance be ween M1 and M2 pheno ypes highligh s he high plas ici y o esponses in hese cells, as HIV-1 in ec ion was associa ed wi h inc eased exp ession o immune ac i a ion and in lamma ion ma ke s, while SARS-CoV-2 inc eased he ma ke s associa ed wi h immunomodula ion. In e es ingly, ano he popula ion ha seemed o be highly a ec ed in acu e HIV-1 in ec ion was he non-classical (CD14 low/− CD16 + ) monocy es. Non-classical monocy es a e known o be eadily able o lea e ci cula ion o exe se e al p o-in lamma o y unc ions in he issues. This popula ion was dec eased in bo h acu e HIV-1 and SARS-CoV-2 in ec ions, wi h he Slan+ subse being he mos a ec ed (Figu es 1B and 2B). Using manual analysis, we con i med ha he e was a no iceable loss o non-classical monocy es in bo h acu e HIV-1 and SARS-CoV-2 in ec ions (Figu e 2C), wi h he Slan+ popula ion being deple ed in bo h cases (Figu e 2D). I is impo an o no e ha he ch onic HIV-1 pa ien did no show a dec ease o non-classical monocy es, and p esen ed an expansion o he Vi uses 2021,13, 1805 6 o 12 Slan+ subse , which is in acco dance wi h p e ious li e a u e on ch onic i emic HIV-1 pa ien s [35,36]. Figu e 2. Shi o M1-like p o ile and loss o Slan+ Monocy es in acu e HIV-1 in ec ion. ( A , B ) Boxplo s showing he equencies and he medians o he iden i ied subse s using FlowSOM, as shown in Figu e 1. Each do ep esen s one subjec . ( C , D ) Illus a i e do plo s o he manual analysis pe o med in ep esen a i e indi iduals om he heal hy con ol and COVID-19 g oups, as well as in he HIV-1 acu e and un ea ed HIV-1 ch onic pa ien s showing he exp ession o CD14 and CD16 in monocy es (C); and he exp ession o HLA-DR and Slan in he non-classical (CD14low/−CD16+) monocy es (D). Rega ding myeloid dend i ic cells (mDC), he myeloid subse exp essing CD141 (CD141+ mDC), was educed in bo h acu e HIV-1 and SARS-CoV-2 in ec ions, whe eas he CD1c+ mDC subse was ela i ely p ese ed (Figu es 1B and 3A). Howe e , he e we e di e ences in pheno ype o CD1c+ mDCs, namely highe le els o ac i a ion/co- s imula o y ma ke s CD80 and CD86 in he acu e HIV-1 as compa ed o SARS-CoV-2- Vi uses 2021,13, 1805 7 o 12 in ec ed pa ien s (Figu e 3B). The plasmacy oid dend i ic cells (pDC) we e also educed in acu e HIV-1 and SARS-CoV-2 pa ien s (Figu e 3A), wi hou any no iceable al e a ions in ma ke exp ession (Figu e 3B). The educ ion in ci cula ing dend i ic cells and he educed exp ession o ac i a ion ma ke s a e in ag eemen wi h p e ious s udies ha e ealed an impai ed unc ion o dend i ic cells in COVID-19 [37]. Figu e 3. Ci cula ing dend i ic cells in acu e HIV-1 in ec ion. ( A ) Boxplo s showing he equencies and he medians o he iden i ied popula ion. Each do ep esen s one subjec . ( B ) Hea map showing he exp ession o co-s imula o y and ac i a ion ma ke s wi hin CD1c+ mDCs and pDCs o he HIV-1 acu e and un ea ed HIV-1 ch onic pa ien s, as well as he heal hy con ol and COVID-19 g oups. CD141+ mDC ma ke exp ession is no shown gi en he low numbe o e en s. Bo h monocy es and dend i ic cells play an impo an ole in he ini ia ion o he adap i e immune esponses. The e o e, we pe o med a pa allel e alua ion o he T- and B-cell compa men s wi h a dimensionali y educ ion by UMAP on he CD3+ (Figu e S3) and CD19+ lymphocy es (Figu e S4), espec i ely. We documen ed CD4+ and CD8+ T-cell ac i a ion in bo h acu e in ec ions, associa ed wi h expansion o cen al and e ec o memo y subse s, and inc eased exp ession o CD38 and HLA-DR in he CD8+ T cells, pa icula ly in he hype -acu e HIV-1 in ec ed pa ien (Figu e S3). As, expec ed he un ea ed HIV-1 in ec ed pa ien ea u ed a ma ked deple ion o CD4+ T cells and expansion o CD8+ T cells, accompanied by con ac ion o he naï e compa men and e minally-e ec o T-cell di e en ia ion (Figu e S3). Impo an ly, we obse ed a clea expansion o ci cula ing plasmablas s in bo h acu e HIV-1 and SARS- CoV-2 in ec ions, suppo ing he moun ing o speci ic an ibody esponses, which we e no de ec ed in ch onic HIV-1 in ec ion and heal hy con ols as expec ed. Mo eo e , bo h acu e in ec ions ea u ed expansion o memo y swi ched and unswi ched B-cell popula ions (Figu e S4). The memo y B-cell subse s we e al eady los in he ad anced ch onically in ec ed HIV-1 in ec ed pa ien , as p e iously desc ibed [38]. Vi uses 2021,13, 1805 8 o 12 4. Discussion Ou da a illus a e he plas ici y o he monocy e and dend i ic cell pheno ype in acu e i al in ec ions, as seen by he di e en changes in he M1-M2-like balance be ween HIV-1 and SARS-CoV-2 in ec ion in classical monocy es. A he same ime, a non-p e iously desc ibed deple ion o he Slan+ subse o non-classical monocy es was ound in he wo acu e in ec ions. Thus, his HIV-1 acu e case epo p o ides new lines o u he in es iga e he myeloid compa men in u u e coho s udies. M1-like classical monocy e expansion was obse ed in he acu e HIV-1 pa ien , whe eas in SARS-CoV-2 in ec ion he immunomodula o y M2-like classical monocy es domina ed in ou s udy. The la e migh be a nega i e eedback loop o an ea lie excessi e in lamma o y esponse seen in SARS-CoV2 [ 19 ]. Impo an ly, his was no he case in HIV-1, whe e he M1-like expansion u he inc eased in he case o un ea ed ch onic HIV-1 in ec ion. Mo eo e , his p o-in lamma o y s a e pe sis s e en a e s a ing cART and is hough o con ibu e o non-AIDS ela ed co-mo bidi ies [ 20 , 39 ]. I is likely ha he pe sis en in lamma ion du ing he acu e HIV-1 in ec ion con ibu es o he dissemina ion and pe sis ence o HIV-1 [ 7 , 40 ]. Monocy es ha e been shown o up egula e ac i a ion ma ke s du ing he sha p inc ease in i emia obse ed e y ea ly upon HIV-1 in ec ion, such as in Fiebig s age II [ 24 ]. Gi en he ole o hese cells as in lamma o y cy okine p oduce s [ 41 ] and s imula o s o he adap i e immune esponses, he M1-like popula ion could con ibu e o he pe sis en immune ac i a ion ha leads o he deple ion o CD4+ T cells h oughou he ch onic in ec ion [42]. The ise in M2-like pheno ype in SARS-CoV-2 in ec ion has been p e iously desc ibed, pa icula ly in pa ien s equi ing hospi al admission [ 43 , 44 ]. This has been mainly as- socia ed wi h se e e disease [ 43 , 44 ], al hough he expansion o his immunomodula o y popula ion migh be a esponse o he ex ensi e issue damage and in lamma ion ha occu in se e e SARS-CoV-2 disease. As such, we ecen ly epo ed ha COVID-19 eco e y is associa ed wi h he main enance o his M2-like pheno ype expansion [19]. In spi e o hese di e ences, ou s udy e ealed ha a educ ion o non-classical monocy es was sha ed by bo h acu e HIV-1 and SARS-CoV-2 in ec ions, wi h a s iking deple ion o Slan+ non-classical monocy es. Non-classical monocy es a e able o quickly lea e ci cula ion o si es o in lamma ion, whe e hey p oduce in lamma o y cy okines such as TNF- α , IL-1 β , and IL-12 [ 45 ] and a e in ol ed in i al esponse. The Slan+ subse o non- classical monocy es has been shown o p oduce high amoun s o TNF- α and can show a DC like pheno ype wi h a high capaci y o co-s imula ion o CD4+ T cells [ 13 , 46 , 47 ]. Al hough hese cells ha e been s udied in he con ex o ch onic in lamma ion, as in pso iasis [ 48 ], hei po en ial ole in ea ly esponses such as in acu e i al in ec ion is s ill unclea . Using bo h unsupe ised and supe ised analysis, we show he e, o he i s ime, ha Slan+ monocy es a e deple ed in acu e HIV-1 in ec ion. The loss o his popula ion could be due o he apid ec ui men o hese cells o si es o in ec ion, and he pe sis ence o his deple ion may be due o he slow econs i u ion a e o hese cells [ 49 ]. The Slan+ monocy e deple ion was no appa en ly ela ed o he plasma i al load, since i occu ed in bo h i emic and non- i emic COVID-19 pa ien s in ou s udy. O no e, he deple ion o his popula ion does no seem o be ela ed o age, since ou acu e HIV-1 pa ien is younge han ou acu e SARS-CoV-2 in ec ed pa ien s and he same Slan+ monocy e deple ion was obse ed. Addi ionally, an expansion o his subse was obse ed in ou i emic ch onic HIV-1 in ec ed pa ien . In SARS-CoV-2, hese cells we e sugges ed o play a ole in he ampan in lamma ion ha leads o he wo sening o COVID-19, and ha e been shown o be educed by us and in ano he s udy [ 19 , 44 ]. In acu e HIV-1 in ec ion, hese cells migh pa ake in he complex se o immune esponses and ac i a ion, con ibu ing o he i al se -poin , h ough hei abili y o ac i a e CD4+ T cells [ 13 ], he p ima y a ge s o HIV-1 in ec ion. On he o he hand, hese cells ha e been con i med o ha e a ole in ch onic HIV-1 in ec ion, whe e hey we e ound o be expanded and esponsible o he o e p oduc ion o TNF-αand Il-β[35,36], which was con i med in he cu en s udy. Vi uses 2021,13, 1805 9 o 12 In bo h acu e in ec ions we obse ed a loss o ci cula ing CD141+ mDC and pDC, as p e iously epo ed [ 37 , 44 , 50 ], hough he acu e HIV-1 pa ien appea ed o ea u e a mo e co- s imula o y p o ile, in con as wi h he COVID-19 coho . Ne e heless, we ound e idence o moun ing o B- and T-cell esponses in bo h acu e in ec ions, as expec ed [8,51–54]. Monocy es, mac ophages, and DC esponses a e conside ed he main de e minan s o he i al se -poin upon HIV-1 in ec ion, being associa ed o di e en disease ou come [ 24 , 55 , 56 ]. Ea ly hype -acu e and un ea ed ch onic HIV-1-in ec ed indi iduals a e inc easingly a e due o he 40 decades o wo k o con ol HIV-1 ansmission. The e o e, ou s udy is limi ed by he educed numbe o hese samples and should be u he alida ed in la ge acu e HIV-1 coho s. Ne e heless, i does sugges a ole o Slan+ monocy es in acu e i al in ec ion pa hogenesis and highligh he capaci y o monocy es o espond speci ically o di e en i al in ec ions. In conclusion, ou s udy e ealed deple ion o Slan+ monocy es in acu e HIV-1 and SARS-CoV-2 in ec ions, despi e he opposi e shi s in classical monocy e pola iza ion. These esul s p omp u u e s udies on he po en ial ole ha his popula ion migh ha e in he complex immune ac i a ion s a e go e ning he p og ession o HIV-1 in ec ion and o he i al in ec ions. Supplemen a y Ma e ials: The ollowing a e a ailable online a h ps://www.mdpi.com/a icle/ 10.3390/ 13091805/s1. Figu e S1: Flow cy ome y ga ing s a egy. Figu e S2: Unsupe ised analysis o monocy es and dend i ic cells. Figu e S3: Unsupe ised analysis o T cells. Figu e S4: Unsupe - ised analysis o B cells. Table S1: Flow cy ome y an ibodies. Au ho Con ibu ions: Concep ualiza ion, G.B.F., R.B., A.C.T. and A.E.S.; me hodology, G.B.F., A.M.C.G., C.M.C., J.L., A.G.-S., P.R., D.F.S. and A.R.M.A.; alida ion, G.B.F., C.M.C., A.M.C.G. and A.C.T.; o mal analysis, G.B.F., C.M.C. and A.M.C.G.; esou ces, R.B., C.M. and S.M.F.; da a cu a ion, A.C.T. and R.B.; w i ing—o iginal d a p epa a ion, G.B.F., R.B. and A.E.S.; w i ing— e iew and edi ing, A.C.T.; isualiza ion, G.B.F., C.M.C. and A.E.S.; supe ision, A.C.T. and A.E.S.; p ojec adminis a ion, A.C.T. and A.E.S.; unding acquisi ion, A.C.T. and S.M.F. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: This wo k was unded by he ollowing g an s om Fundação pa a a Ciência e a Tecnologia (FCT), Po ugal, h ough “Apoio Especial Resea ch4COVID-19”, p ojec numbe s 125 o S.M.F. and 803 o A.C.T. Fellowships unded by FCT (Doc o a es4COVID-19, 2020.10202.BD), and Janssen-Cilag Fa macêu ica we e ecei ed by A.M.C.G. and G.B.F., espec i ely. Ins i u ional Re iew Boa d S a emen : The s udy was conduc ed acco ding o he guidelines o he Decla a ion o Helsinki and app o ed by he E hical Boa d o Cen o Hospi ala Uni e si á io Lisboa No e/Faculdade de Medicina da Uni e sidade de Lisboa/Cen o Académico de Medicina de Lisboa (Re e ence 148/20, 3 d o Ap il o 2020). In o med Consen S a emen : In o med consen was ob ained om all subjec s in ol ed in he s udy. Da a A ailabili y S a emen : The da a ha suppo he indings o his s udy a e a ailable on eques o he co esponding au ho . Acknowledgmen s: We hank all he pa icipa ing pa ien s and he heal h ca e p o essionals in ol ed in pa ien ca e, pa icula ly Rena o Cos a-Reis, Ma ia Adão-Se ano, as well as he eam membe s o Flow Cy ome y acili y a Ins i u o de Medicina Molecula João Lobo An unes (Lisboa, Po ugal) o echnical assis ance. Con lic s o In e es : The au ho s decla e no con lic o in e es . The unde s had no ole in he design o he s udy; in he collec ion, analyses, o in e p e a ion o da a; in he w i ing o he manusc ip , o in he decision o publish he esul s.