Human Th17 Cells Lack HIV-Inhibitory RNases and Are Highly Permissive to Productive HIV Infection

doi:10.1128/JVI.02869-15

. 2016 Aug 12;90(17):7833-47.

doi: 10.1128/JVI.02869-15. Print 2016 Sep 1.

Human Th17 Cells Lack HIV-Inhibitory RNases and Are Highly Permissive to Productive HIV Infection

Aaron Christensen-Quick¹, Mark Lafferty¹, Lingling Sun², Luigi Marchionni³, Anthony DeVico⁴, Alfredo Garzino-Demo⁵

Affiliations

¹ Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland, USA Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
² Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
³ Department of Oncology, Johns Hopkins University, Baltimore, Maryland, USA.
⁴ Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland, USA Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA.
⁵ Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland, USA Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA Department of Molecular Medicine, University of Padua, Padua, Italy agarzinodemo@ihv.umaryland.edu.

PMID: 27334595
PMCID: PMC4988157
DOI: 10.1128/JVI.02869-15

Human Th17 Cells Lack HIV-Inhibitory RNases and Are Highly Permissive to Productive HIV Infection

Aaron Christensen-Quick et al. J Virol. 2016.

. 2016 Aug 12;90(17):7833-47.

doi: 10.1128/JVI.02869-15. Print 2016 Sep 1.

Authors

Aaron Christensen-Quick¹, Mark Lafferty¹, Lingling Sun², Luigi Marchionni³, Anthony DeVico⁴, Alfredo Garzino-Demo⁵

Affiliations

¹ Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland, USA Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
² Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
³ Department of Oncology, Johns Hopkins University, Baltimore, Maryland, USA.
⁴ Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland, USA Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA.
⁵ Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland, USA Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA Department of Molecular Medicine, University of Padua, Padua, Italy agarzinodemo@ihv.umaryland.edu.

PMID: 27334595
PMCID: PMC4988157
DOI: 10.1128/JVI.02869-15

Abstract

Human immunodeficiency virus (HIV) infects and depletes CD4(+) T cells, but subsets of CD4(+) T cells vary in their susceptibility and permissiveness to infection. For example, HIV preferentially depletes interleukin-17 (IL-17)-producing T helper 17 (Th17) cells and T follicular helper (Tfh) cells. The preferential loss of Th17 cells during the acute phase of infection impairs the integrity of the gut mucosal barrier, which drives chronic immune activation-a key determinant of disease progression. The preferential loss of Th17 cells has been attributed to high CD4, CCR5, and CXCR4 expression. Here, we show that Th17 cells also exhibit heightened permissiveness to productive HIV infection. Primary human CD4(+) T cells were sorted, activated under Th17- or Th0-polarizing conditions and infected, and then analyzed by flow cytometry. Th17-polarizing cytokines increased HIV infection, and HIV infection was disproportionately higher among Th17 cells than among IL-17(-) or gamma interferon-positive (IFN-γ(+)) cells, even upon infection with a replication-defective HIV vector with a pseudotype envelope. Further, Th17-polarized cells produced more viral capsid protein. Our data also reveal that Th17-polarized cells have diminished expression of RNase A superfamily proteins, and we report for the first time that RNase 6 inhibits HIV. Thus, our findings link Th17 polarization to increased HIV replication.

Importance: Our study compares the intracellular replicative capacities of several different HIV isolates among different T cell subsets, providing a link between the differentiation of Th17 cells and HIV replication. Th17 cells are of key importance in mucosal integrity and in the immune response to certain pathogens. Based on our findings and the work of others, we propose a model in which HIV replication is favored by the intracellular environment of two CD4(+) T cell subsets that share several requirements for their differentiation: Th17 and Tfh cells. Characterizing cells that support high levels of viral replication (rather than becoming latently infected or undergoing cell death) informs the search for new therapeutics aimed at manipulating intracellular signaling pathways and/or transcriptional factors that affect HIV replication.

PubMed Disclaimer

Figures

**FIG 1**
Effects of T_h17-polarizing cytokines on the percentage of HIV-infected, CD4 T cells. (A) Human CD4⁺ T cells were isolated from peripheral blood by negative selection and then mitogenically activated with phytohemagglutinin (PHA; 5 μg/ml) and interleukin-2 (IL-2; 10 ng/ml) for 3 days prior to infection. Cells were then washed, infected with the indicated isolates of HIV for 2.5 h, washed again, and then resuspended in medium containing IL-2 or the T_h17-polarizing cytokines IL-1β, IL-6, and IL-23 (10 ng/ml) and TGF-β (2 ng/ml). (B and C) Time course (B) and day 5 comparison (C) of the total percentages of p24⁺ cells from IL-2 and from T_h17-polarizing conditions. D₀, D₃, D₅, and D₇, days 0, 3, 5, and 7, respectively. (D) Representative plots of cells infected for 5 days with CCR5-tropic HIV_BaL, CXCR4-tropic HIV_NL4-3, or CCR5-tropic, transmitted-founder isolate HIV_AD17. (E) The indicated cytokines were added after infection with HIV_BaL. The IL-17 expression shown was in uninfected samples. *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001.

**FIG 2**
Comparison of total and infected IL-17- or IFN-γ-producing cells. PHA-activated CD4⁺ T cells were infected with HIV_BaL for 5 days in IL-2 or the T_h17-polarizing cytokines IL-1β, IL-6, and IL-23 (10 ng/ml) and TGF-β (2 ng/ml). (A and B) Comparison of p24 expression among total, IL-17⁻ IFN-γ⁻, IL-17⁺, and IL-17⁻ IFN-γ⁺ T cells. (C and D) Comparison of IL-17 expression among total, p24⁻, or p24⁺ T cells. (E) Fold enrichment of p24⁺ cells among cells that either express or lack IFN-γ or IL-17. (F) Fold enrichment of IFN-γ- or IL-17-single-positive cells among p24⁺ cells, relative to p24⁻ cells. (G) Percentages of IL-17- and IFN-γ-expressing cells were measured by flow cytometry. Blue depicts the proportion of IL-17⁻ IFN-γ⁻ cells, red shows IL-17⁺ cells, and yellow shows IFN-γ⁺ IL-17⁻ cells. *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001.

**FIG 3**
HIV infections of CCR6-sorted, prepolarized cells. (A) Cells were sorted according to their expression of CCR6 and then TCR stimulated in the presence of T_h0- or T_h17-polarizing cytokines (IL-2 and IL-1β, IL-6, IL-23, and TGF-β, respectively) for 3 days. Cells were then infected with the indicated isolates of HIV, washed, resuspended in medium with IL-2/IL-23, and analyzed by flow cytometry 5 days postinfection. *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001. (B) Representative flow cytometry plots depicting p24 and IL-17 expression in T_h0- or T_h17-polarized cells infected with the indicated isolates of HIV. (C) Percentages of IL-17- and IFN-γ-expressing cells were measured by flow cytometry. Blue depicts the proportion of IL-17⁻ IFN-γ⁻ cells, red shows IL-17⁺ cells, and yellow shows IFN-γ⁺ IL-17⁻ cells. (D) Five nanograms of p24/ml from the supernatant of HIV_BaL-infected T cells under the indicated conditions (or the equivalent volume from condition-matched, uninfected cells) was added to preseeded TZM-BL cells in triplicate. Luciferase activity was measured 48 h later, and relative light unit (RLU) measurements from infected supernatants were adjusted by subtracting the RLU from their corresponding uninfected supernatants.

**FIG 4**
IL-17 and IFN-γ expression among CCR6-sorted, prepolarized cells infected with a pseudotype HIV vector, HIV_AMLV. (A) Pie charts of average percentages of IL-17- and IFN-γ-expressing cells among CCR6-sorted cells that were T_h0 or T_h17 polarized and then infected with an *Env*-deficient HIV vector in an amphotropic murine leukemia virus (AMLV) envelope for 4 days. Total and p24⁺-gated cells from 5 donors are shown. (B) Flow cytometry plots from the donor whose cells became most T_h17 polarized. (C) Representative plots of IL-17 and p24 expression in phytohemagglutinin (PHA)-activated CD4 T cells infected for 4 days with HIV_AMLV in the presence of IL-2 or T_h17-polarizing cytokines.

**FIG 5**
p24 mean fluorescence intensities (MFIs) among infected IL-17⁺ and IL-17⁻ cells and supernatant p24 from sorted, polarized cells infected with HIV_BaL. (A and B) Representative histograms (A) and donor-matched plots (B) of p24 geometric MFIs among IL-17⁺ and IL-17⁻, PHA-activated cells that were infected with HIV_BaL for 5 days in the presence of IL-2 or T_h17-polarizing cytokines. (C and D) p24 MFI plots from sorted, polarized cells that were infected with HIV_BaL (C) or HIV_AMLV (D). (E) Supernatant levels of p24 5 days postinfection, as measured by enzyme-linked immunosorbent assay (ELISA; n = 5). *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001. n.s., not significant.

**FIG 6**
Comparison of RNase expression and HIV inhibition in T_h17-polarized cells with those in T_h0-polarized cells. (A) Representative flow cytometry plots depicting IL-17 expression among T_h0-polarized, CCR6⁻ cells and T_h17-polarized, CCR6⁺ cells. SSC, side scatter. (B) Box plot showing differential gene expression between T_h0- and T_h17-polarized cells. Total RNA from T_h0- or T_h17-polarized cells was analyzed by using microarrays. Relative levels of gene expression in T_h17-polarized cells and T_h0-polarized cells are shown. (C) Thirty micrograms of total protein from cell lysates of CCR6⁻, T_h0-polarized and CCR6⁺, T_h17-polarized CD4 T cells from three donors was denatured and separated according to size by SDS-PAGE, transferred to a PVDF membrane, and then probed for the indicated RNase expression with anti-RNase antibodies. The loading control was glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (D) T_h0- and T_h17-polarized CD4 T cells were infected with HIV_IIIB, washed, and then resuspended in medium containing the indicated RNases. Percent inhibition was calculated from the percentage of p24⁺ cells, as measured by flow cytometry.

**FIG 7**
Potential mechanisms and effects of T_h17 permissiveness during acute HIV infection. Heightened permissiveness to HIV in response to T_h17- or T_fh-polarizing cytokines may promote viral replication by direct transcriptional modulation of proviral and/or antiviral response genes. The subsequent loss of T_h17 cells could be attributable to programmed cell death, conversion to T_fh cells, or the cytopathic effects of high virus production. The loss of T_h17 cells then promotes the disruption of mucosal immunity. APC, antigen-presenting cell.

See this image and copyright information in PMC

Cited by

The transcriptome of HIV-1 infected intestinal CD4+ T cells exposed to enteric bacteria.
Yoder AC, Guo K, Dillon SM, Phang T, Lee EJ, Harper MS, Helm K, Kappes JC, Ochsenbauer C, McCarter MD, Wilson CC, Santiago ML. Yoder AC, et al. PLoS Pathog. 2017 Feb 27;13(2):e1006226. doi: 10.1371/journal.ppat.1006226. eCollection 2017 Feb. PLoS Pathog. 2017. PMID: 28241075 Free PMC article.
Th17 CD4+ T-Cell as a Preferential Target for HIV Reservoirs.
Renault C, Veyrenche N, Mennechet F, Bedin AS, Routy JP, Van de Perre P, Reynes J, Tuaillon E. Renault C, et al. Front Immunol. 2022 Feb 7;13:822576. doi: 10.3389/fimmu.2022.822576. eCollection 2022. Front Immunol. 2022. PMID: 35197986 Free PMC article. Review.
A Lachnospiraceae-dominated bacterial signature in the fecal microbiota of HIV-infected individuals from Colombia, South America.
San-Juan-Vergara H, Zurek E, Ajami NJ, Mogollon C, Peña M, Portnoy I, Vélez JI, Cadena-Cruz C, Diaz-Olmos Y, Hurtado-Gómez L, Sanchez-Sit S, Hernández D, Urruchurtu I, Di-Ruggiero P, Guardo-García E, Torres N, Vidal-Orjuela O, Viasus D, Petrosino JF, Cervantes-Acosta G. San-Juan-Vergara H, et al. Sci Rep. 2018 Mar 14;8(1):4479. doi: 10.1038/s41598-018-22629-7. Sci Rep. 2018. PMID: 29540734 Free PMC article.
Functional roles of the human ribonuclease A superfamily in RNA metabolism and membrane receptor biology.
Lee HH, Wang YN, Hung MC. Lee HH, et al. Mol Aspects Med. 2019 Dec;70:106-116. doi: 10.1016/j.mam.2019.03.003. Epub 2019 Mar 25. Mol Aspects Med. 2019. PMID: 30902663 Free PMC article. Review.
HIV replication is associated to inflammasomes activation, IL-1β, IL-18 and caspase-1 expression in GALT and peripheral blood.
Feria MG, Taborda NA, Hernandez JC, Rugeles MT. Feria MG, et al. PLoS One. 2018 Apr 19;13(4):e0192845. doi: 10.1371/journal.pone.0192845. eCollection 2018. PLoS One. 2018. PMID: 29672590 Free PMC article.

See all "Cited by" articles

References

1. Brenchley JM, Paiardini M, Knox KS, Asher AI, Cervasi B, Asher TE, Scheinberg P, Price DA, Hage CA, Kholi LM, Khoruts A, Frank I, Else J, Schacker T, Silvestri G, Douek DC. 2008. Differential Th17 CD4 T-cell depletion in pathogenic and nonpathogenic lentiviral infections. Blood 112:2826–2835. doi:10.1182/blood-2008-05-159301. - DOI - PMC - PubMed
1. Raffatellu M, Santos RL, Verhoeven DE, George MD, Wilson RP, Winter SE, Godinez I, Sankaran S, Paixao TA, Gordon MA, Kolls JK, Dandekar S, Baumler AJ. 2008. Simian immunodeficiency virus-induced mucosal interleukin-17 deficiency promotes Salmonella dissemination from the gut. Nat Med 14:421–428. doi:10.1038/nm1743. - DOI - PMC - PubMed
1. Schuetz A, Deleage C, Sereti I, Rerknimitr R, Phanuphak N, Phuang-Ngern Y, Estes JD, Sandler NG, Sukhumvittaya S, Marovich M, Jongrakthaitae S, Akapirat S, Fletscher JL, Kroon E, Dewar R, Trichavaroj R, Chomchey N, Douek DC, O'Connell RJ, Ngauy V, Robb ML, Phanuphak P, Michael NL, Excler JL, Kim JH, de Souza MS, Ananworanich J, RV254/SEARCH 010 and RV304/SEARCH 013 Study Groups. 2014. Initiation of ART during early acute HIV infection preserves mucosal Th17 function and reverses HIV-related immune activation. PLoS Pathog 10:e1004543. doi:10.1371/journal.ppat.1004543. - DOI - PMC - PubMed
1. Hartigan-O'Connor DJ, Abel K, Van Rompay KK, Kanwar B, McCune JM. 2012. SIV replication in the infected rhesus macaque is limited by the size of the preexisting TH17 cell compartment. Sci Transl Med 4:136ra169. doi:10.1126/scitranslmed.3003941. - DOI - PMC - PubMed
1. Cecchinato V, Trindade CJ, Laurence A, Heraud JM, Brenchley JM, Ferrari MG, Zaffiri L, Tryniszewska E, Tsai WP, Vaccari M, Parks RW, Venzon D, Douek DC, O'Shea JJ, Franchini G. 2008. Altered balance between Th17 and Th1 cells at mucosal sites predicts AIDS progression in simian immunodeficiency virus-infected macaques. Mucosal Immunol 1:279–288. doi:10.1038/mi.2008.14. - DOI - PMC - PubMed

Publication types

Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Substances

Actions

Grants and funding

LinkOut - more resources

Full Text Sources
Other Literature Sources
- scite Smart Citations
Research Materials
- NCI CPTC Antibody Characterization Program

[1] Brenchley JM, Paiardini M, Knox KS, Asher AI, Cervasi B, Asher TE, Scheinberg P, Price DA, Hage CA, Kholi LM, Khoruts A, Frank I, Else J, Schacker T, Silvestri G, Douek DC. 2008. Differential Th17 CD4 T-cell depletion in pathogenic and nonpathogenic lentiviral infections. Blood 112:2826–2835. doi:10.1182/blood-2008-05-159301. - DOI - PMC - PubMed

[2] Brenchley JM, Paiardini M, Knox KS, Asher AI, Cervasi B, Asher TE, Scheinberg P, Price DA, Hage CA, Kholi LM, Khoruts A, Frank I, Else J, Schacker T, Silvestri G, Douek DC. 2008. Differential Th17 CD4 T-cell depletion in pathogenic and nonpathogenic lentiviral infections. Blood 112:2826–2835. doi:10.1182/blood-2008-05-159301. - DOI - PMC - PubMed

[3] Raffatellu M, Santos RL, Verhoeven DE, George MD, Wilson RP, Winter SE, Godinez I, Sankaran S, Paixao TA, Gordon MA, Kolls JK, Dandekar S, Baumler AJ. 2008. Simian immunodeficiency virus-induced mucosal interleukin-17 deficiency promotes Salmonella dissemination from the gut. Nat Med 14:421–428. doi:10.1038/nm1743. - DOI - PMC - PubMed

[4] Raffatellu M, Santos RL, Verhoeven DE, George MD, Wilson RP, Winter SE, Godinez I, Sankaran S, Paixao TA, Gordon MA, Kolls JK, Dandekar S, Baumler AJ. 2008. Simian immunodeficiency virus-induced mucosal interleukin-17 deficiency promotes Salmonella dissemination from the gut. Nat Med 14:421–428. doi:10.1038/nm1743. - DOI - PMC - PubMed

[5] Schuetz A, Deleage C, Sereti I, Rerknimitr R, Phanuphak N, Phuang-Ngern Y, Estes JD, Sandler NG, Sukhumvittaya S, Marovich M, Jongrakthaitae S, Akapirat S, Fletscher JL, Kroon E, Dewar R, Trichavaroj R, Chomchey N, Douek DC, O'Connell RJ, Ngauy V, Robb ML, Phanuphak P, Michael NL, Excler JL, Kim JH, de Souza MS, Ananworanich J, RV254/SEARCH 010 and RV304/SEARCH 013 Study Groups. 2014. Initiation of ART during early acute HIV infection preserves mucosal Th17 function and reverses HIV-related immune activation. PLoS Pathog 10:e1004543. doi:10.1371/journal.ppat.1004543. - DOI - PMC - PubMed

[6] Schuetz A, Deleage C, Sereti I, Rerknimitr R, Phanuphak N, Phuang-Ngern Y, Estes JD, Sandler NG, Sukhumvittaya S, Marovich M, Jongrakthaitae S, Akapirat S, Fletscher JL, Kroon E, Dewar R, Trichavaroj R, Chomchey N, Douek DC, O'Connell RJ, Ngauy V, Robb ML, Phanuphak P, Michael NL, Excler JL, Kim JH, de Souza MS, Ananworanich J, RV254/SEARCH 010 and RV304/SEARCH 013 Study Groups. 2014. Initiation of ART during early acute HIV infection preserves mucosal Th17 function and reverses HIV-related immune activation. PLoS Pathog 10:e1004543. doi:10.1371/journal.ppat.1004543. - DOI - PMC - PubMed

[7] Hartigan-O'Connor DJ, Abel K, Van Rompay KK, Kanwar B, McCune JM. 2012. SIV replication in the infected rhesus macaque is limited by the size of the preexisting TH17 cell compartment. Sci Transl Med 4:136ra169. doi:10.1126/scitranslmed.3003941. - DOI - PMC - PubMed

[8] Hartigan-O'Connor DJ, Abel K, Van Rompay KK, Kanwar B, McCune JM. 2012. SIV replication in the infected rhesus macaque is limited by the size of the preexisting TH17 cell compartment. Sci Transl Med 4:136ra169. doi:10.1126/scitranslmed.3003941. - DOI - PMC - PubMed

[9] Cecchinato V, Trindade CJ, Laurence A, Heraud JM, Brenchley JM, Ferrari MG, Zaffiri L, Tryniszewska E, Tsai WP, Vaccari M, Parks RW, Venzon D, Douek DC, O'Shea JJ, Franchini G. 2008. Altered balance between Th17 and Th1 cells at mucosal sites predicts AIDS progression in simian immunodeficiency virus-infected macaques. Mucosal Immunol 1:279–288. doi:10.1038/mi.2008.14. - DOI - PMC - PubMed

[10] Cecchinato V, Trindade CJ, Laurence A, Heraud JM, Brenchley JM, Ferrari MG, Zaffiri L, Tryniszewska E, Tsai WP, Vaccari M, Parks RW, Venzon D, Douek DC, O'Shea JJ, Franchini G. 2008. Altered balance between Th17 and Th1 cells at mucosal sites predicts AIDS progression in simian immunodeficiency virus-infected macaques. Mucosal Immunol 1:279–288. doi:10.1038/mi.2008.14. - DOI - PMC - PubMed

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Human Th17 Cells Lack HIV-Inhibitory RNases and Are Highly Permissive to Productive HIV Infection

Affiliations

Human Th17 Cells Lack HIV-Inhibitory RNases and Are Highly Permissive to Productive HIV Infection

Authors

Affiliations

Abstract

Figures

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Research Materials

Abstract

Figures

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Related information

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Research Materials