Research ArticleHIV

Inflammatory monocytes expressing tissue factor drive SIV and HIV coagulopathy

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Science Translational Medicine  30 Aug 2017:
Vol. 9, Issue 405, eaam5441
DOI: 10.1126/scitranslmed.aam5441
  • Fig. 1. LPS drives TF expression on human monocyte subsets.

    (A) Left: Representative fluorescence-activated cell sorting plots of TF expression on human monocytes from healthy controls upon LPS stimulation in vitro. Right: Summary data (n = 5) of frequency of TFpos monocytes. Lines represent median values. Data were analyzed using Mann-Whitney U test. SSC-H, side-scatter height. (B) Frequency of TF-expressing monocytes on PBMC from healthy controls stimulated with increasing doses of LPS in vitro (n = 8). Data were analyzed using Kruskal-Wallis test with Dunn’s multiple comparisons and linear trend ad hoc test. (C) Hierarchical cluster analysis of the expression profile (z-score–normalized) of indicated genes assessed by qPCR in monocytes (n = 6 healthy donors) sorted after 6 hours of LPS stimulation (100 ng/ml), as described in Materials and Methods. (D) PCA of the expression level of indicated genes was performed. (E) Monocyte subsets were sorted on the basis of surface expression of CD14 and CD16 (n = 6 healthy donors). Representative plots show monocytes before and after sorting (left). TF protein expression in cell lysates and TF functional activity measured by the formation of factor Xa in vitro were compared between the different monocyte subsets in vitro using Kruskal-Wallis test with Dunn’s multiple comparisons posttest. Lines represent median values. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 2. Frequency of TF-expressing monocytes is increased in chronically HIV-infected individuals and in SIV-infected NHPs despite virological suppression status.

    (A) Left: Representative plots of TFpos monocytes ex vivo in healthy controls and HIV+ patients. Right: Summary data of frequency of TFpos monocytes from a cross-sectional analysis including healthy controls (n = 12), ART-naïve HIV+ patients (n = 10), and HIV+ individuals after ART-induced virological suppression (HIV+ post-ART; n =1 0). PBMC were stimulated with LPS in vitro, and frequency of TFpos monocytes (B), TF protein expression in cell lysates (C), and TF functional activity (D) were compared between the cross-sectional study groups using Kruskal-Wallis test with Dunn’s multiple comparisons posttest. (E) Left: Representative plots of TF expression on monocytes ex vivo in chronically SIV-infected pigtail macaque (PTMs) (n = 6) and African green monkey (AGMs) (n = 6). Right: Summary data of frequency of TFpos monocytes ex vivo from both naïve and chronically SIV-infected animals. PBMC were stimulated with LPS in vitro, and frequency of TFpos monocytes (F) and TF functional activity (G) were compared between naïve or SIV-infected PTMs and AGMs. Lines represent median values. Data were analyzed using Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 3. Inflammatory mediators and microbial products may drive TF expression by circulating monocytes in the context of HIV infection.

    (A) Column-purified CD14+ monocytes from 10 healthy controls were cultured for 18 hours in the presence of RPMI supplemented with 10% manufactured human AB serum or serum isolated from healthy controls, ART-naïve HIV+ patients, or HCV+ individuals, as described in Materials and Methods. (B) Monocytes were also cultured in the presence of serum from ART-naïve HIV-infected patients at different time points after ART initiation. (C) Monocytes were cultured for 18 hours in the presence of indicated blocking antibodies (10 μg/ml) or with serum previously treated with polymyxin B (0.5 μg/μl). Cells were washed and lysed for the assessment of TF protein expression using enzyme-linked immunosorbent assay. Unmatched data were analyzed using Mann-Whitney U test, whereas matched pairs were compared using Wilcoxon matched pairs test. *P < 0.05, **P < 0.01, ***P < 0.001. ns, nonsignificant.

  • Fig. 4. TF-expressing monocytes produce multiple proinflammatory cytokines.

    (A) Representative plots show intracellular cytokine staining for IL-1β, IL-6, and TNF-α in monocytes from healthy donors (n = 12) upon LPS stimulation in vitro. SSC-A, side-scatter area. (B) Polyfunctional analysis of TFneg and TFpos monocytes upon LPS stimulation. (C to E) The cytokine expression profiles in TFneg and TFpos monocytes were compared using χ2 tests. (F) Frequency of monocytes producing more than one cytokine ex vivo was compared between TFneg and TFpos monocytes in a prospective cohort of ART-naïve HIV+ patients (n = 15) before therapy initiation and after ART-induced virological suppression (HIV+ post-ART). (G) Frequency of monocytes producing more than one cytokine ex vivo was compared between TFneg and TFpos monocytes in chronically SIV-infected PTMs (n = 6) and AGMs (n = 6). Unmatched data were compared using the Mann-Whitney U test, whereas matched comparisons were performed using the Wilcoxon matched pairs test. **P < 0.01, ***P < 0.001.

  • Fig. 5. Ixolaris potently blocks TF activity but not protein expression in activated monocytes from HIV+ patients and chronically SIV-infected NHPs.

    (A) TF functional activity in vitro measured by factor Xa formation in elutriated monocyte cultures from healthy donors stimulated with LPS and treated with indicated doses of Ixolaris (n = 5). Data represent percentage of the positive control [LPS (1 μg/ml) without Ixolaris]. TF activity upon treatment with Ixolaris was compared between (B) healthy controls (n = 12) and unmatched ART-naïve HIV+ (n = 10) patients and those who achieved ART-induced virological suppression (post-ART; n = 10) as well as between (C) chronically SIV-infected PTMs (n = 6) and AGMs (n = 6). (D) Left: Representative plots of intracellular IL-6 and TNF-α production in monocytes from ART-naïve HIV+ individuals stimulated in the presence or absence of Ixolaris. Right: Summary data showing frequency of TFpos monocytes and percent of cytokine-producing monocytes in stimulated cultures treated or not with Ixolaris. Data were analyzed using the Wilcoxon matched pairs test. *P < 0.05, ***P < 0.001.

  • Fig. 6. Anticoagulant treatment positively affects the immune activation and systemic inflammation of highly pathogenic SIVsab infection in PTMs.

    (A) Dose-dependent dynamics of PT/INR and aPTT after Ixolaris addition to plasma isolated from uninfected human subjects. (B) Dose-dependent dynamics of PT/INR and aPTT after Ixolaris addition to plasma isolated from uninfected PTMs. (C) Dynamics of the proinflammatory cytokine IL-17 in untreated versus Ixolaris-treated, SIVsab-infected PTMs, as assessed by Luminex. Dynamics of activated double-positive HLA-DR+/CD38+ CD4+ (D) and CD8+ T cells (E) in untreated versus Ixolaris-treated, SIVsab-infected PTMs, as assessed by flow cytometry. Dynamics of TF expression (F) on CD14+ monocytes in untreated versus Ixolaris-treated, SIVsab-infected PTMs, as assessed by flow cytometry, as well as CD80 (G), CD86 (H), and Glut-1 (I) expression on CD14+ monocytes in untreated versus Ixolaris-treated, SIVsab-infected PTMs, as assessed by flow cytometry. (J) Dynamics of D-dimer (DD) in untreated versus Ixolaris-treated, SIVsab-infected PTMs, as assessed by immunoturbidimetric assay. (K) Dynamics of plasma SIVsab viremia in untreated versus Ixolaris-treated, SIVsab-infected PTMs, as assessed by a real-time PCR assay. vRNA, viral RNA. (L) Survival in untreated versus Ixolaris-treated, SIVsab-infected PTMs. Statistical analyses were performed with grouping of acute versus chronic infection time points, as described in Materials and Methods, except for TF and Glut-1 for which the statistical analyses were carried out on whole dynamics, due to limited availability of samples for flow staining. Survival analysis was performed using the Mantel-Cox test.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/405/eaam5441/DC1

    Materials and Methods

    Fig. S1. Gating strategy used to evaluate monocytes in PBMC.

    Fig. S2. Detailed phenotyping of TF-expressing monocytes.

    Fig. S3. Frequency of TF-expressing monocytes in patients prospectively undergoing ART and its relationship with CRP and D-dimer.

    Fig. S4. Circulating CD14+ monocytes, but not myeloid dendritic cells, express TF in chronically SIV-infected PTMs.

    Fig. S5. Induction of TF expression in the gut of PTMs infected with SIVab.

    Fig. S6. Intracellular cytokine production and TF expression in HIV-infected patients and in SIVab-infected NHPs.

    Fig. S7. Thrombin induces TF expression on CD14high monocytes via PAR-1.

    Fig. S8. Cell viability upon treatment with Ixolaris and/or LPS in vitro.

    Fig. S9. Activated monocytes expressing TF represent a link between coagulation and inflammation.

    Table S1. Characteristics of the NHPs used for the in vitro studies.

    Table S2. Characteristics of HIV-infected individuals included in the cross-sectional analysis.

    Table S3. Characteristics of HIV-infected individuals included in the prospective analyses.

    Table S4. List of antibodies used in the flow cytometry experiments in both human and NHP samples.

    Table S5. List of human primers.

    Table S6. Primary data.

    References (5962)

  • Supplementary Material for:

    Inflammatory monocytes expressing tissue factor drive SIV and HIV coagulopathy

    Melissa E. Schechter, Bruno B. Andrade,* Tianyu He, George Haret Richter, Kevin W. Tosh, Benjamin B. Policicchio, Amrit Singh, Kevin D. Raehtz, Virginia Sheikh, Dongying Ma, Egidio Brocca-Cofano, Cristian Apetrei, Russel Tracy, Ruy M. Ribeiro, Alan Sher, Ivo M. B. Francischetti, Ivona Pandrea,* Irini Sereti*

    *Corresponding author. Email: bruno.andrade{at}bahia.fiocruz.br (B.B.A.); isereti{at}niaid.nih.gov (I.S.); pandrea{at}pitt.edu (I.P.)

    Published 30 August 2017, Sci. Transl. Med. 9, eaam5441 (2017)
    DOI: 10.1126/scitranslmed.aam5441

    This PDF file includes:

    • Materls and Methods
    • Fig. S1. Gating strategy used to evaluate monocytes in PBMC.
    • Fig. S2. Detailed phenotyping of TF-expressing monocytes.
    • Fig. S3. Frequency of TF-expressing monocytes in patients prospectively undergoing ART and its relationship with CRP and D-dimer.
    • Fig. S4. Circulating CD14+ monocytes, but not myeloid dendritic cells, express TF in chronically SIV-infected PTMs.
    • Fig. S5. Induction of TF expression in the gut of PTMs infected with SIVab.
    • Fig. S6. Intracellular cytokine production and TF expression in HIV-infected patients and in SIVab-infected NHPs.
    • Fig. S7. Thrombin induces TF expression on CD14high monocytes via PAR-1.
    • Fig. S8. Cell viability upon treatment with Ixolaris and/or LPS in vitro.
    • Fig. S9. Activated monocytes expressing TF represent a link between coagulation and inflammation.
    • Table S1. Characteristics of the NHPs used for the in vitro studies.
    • Table S2. Characteristics of HIV-infected individuals included in the crosssectional analysis.
    • Table S3. Characteristics of HIV-infected individuals included in the prospective analyses.
    • Table S4. List of antibodies used in the flow cytometry experiments in both human and NHP samples.
    • Table S5. List of human primers.
    • References (5962)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table6 (Microsoft Excel format). Primary data)).

    [Download Table S6]

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