Research ArticleFibrosis

Plasmacytoid dendritic cells promote systemic sclerosis with a key role for TLR8

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Science Translational Medicine  10 Jan 2018:
Vol. 10, Issue 423, eaam8458
DOI: 10.1126/scitranslmed.aam8458
  • Fig. 1 pDCs infiltrate the skin of SSc patients and spontaneously secrete IFN-α and CXCL4.

    Skin biopsies from (A) control subjects or (B) systemic sclerosis (SSc) patients were immunostained for CD123 (red signal; original magnification, ×200; inset, ×400), and (C) quantification of the number of CD123+ counted in five high-power microscopic fields (based on five bioptic samples of patients with SSc and of five controls) is shown. (D) Representative dot plots of plasmacytoid dendritic cells (pDCs), B cells, conventional DCs (cDCs), and monocytes from peripheral blood mononuclear cells (PBMCs) from healthy donors (HDs) or SSc patients analyzed by flow cytometry. (E) Percentages of pDCs, monocytes, B cells, and cDCs within PBMC of either HDs (open circle, n = 10 to 15) or SSc patients (n = 14 to 33) defined as early diffuse (closed circle), late diffuse (closed square), or limited (closed triangle) were quantified by flow cytometry. ns, not significant. (F) Purified pDCs from HDs (n = 8) or SSc patients (n = 20) were cultured for 24 hours without stimuli. CXCL4 and interferon-α (IFN-α) were quantified by enzyme-linked immunosorbent assay (ELISA). Results were normalized to 10,000 cells to account for the differences between donors in the number of cells put in culture. (G) The mean fluorescence intensity (MFI) of the costimulatory molecules CD83 and CD86 expressed on pDCs (n = 10 for HDs; n = 33 for SSc patients) was quantified by flow cytometry. Statistical significance was evaluated using a Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 2 TLR8 is aberrantly expressed on pDCs from SSc patients.

    (A) TLR8 expression levels [relative Ct (Rel Ct)] were quantified in purified pDCs [n = 18 HDs; n = 29 SSc patients; n = 6 systemic lupus erythematosus (SLE) patients], B cells (n = 14 HDs; n = 8 SSc patients), or monocytes (n = 17 HDs; n = 11 SSc patients) from PBMCs as indicated. (B to D) Gene expression level of TLR7, TLR9, and TLR10 was quantified in (B) purified pDCs (n = 17 to 18 HDs; n = 29 to 31 SSc patients; n = 6 SLE patients), (C) purified B cells (n = 12 to 14 HDs; n = 6 to 8 SSc patients), or (D) purified monocytes (n = 16 to 18 HDs; n = 11 SSc patients) by quantitative polymerase chain reaction (qPCR). All results are represented as means ± SEM. Statistical significance was evaluated using a Mann-Whitney U test, and only comparisons that are significant are shown. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 3 TLR8 signaling induces CXCL4 and IFN-α secretion by SSc PDCs.

    (A) Gene expression levels of CXCL4 were quantified in total PBMC or pDC–depleted (dep) PBMC prepared from either HDs (n = 6) or SSc patients (n = 23) by qPCR. Statistical significance was evaluated using the unpaired Mann-Whitney test between HD and SSc PBMC and with a paired t test between PBMC and pDC-depleted PBMC from SSc patients. (B) Purified pDCs from HDs (n = 4 to 10) were cultured either with media (Med) alone as a control or in the presence of a TLR7 agonist [heat-inactivated VR95 influenza virus at a multiplicity of infection (MOI) of 2], a TLR8 agonist (ORN-8L at 200 μg/ml), or a TLR9 agonist (CpG-C274 at 0.5 μM), and 24 hours later, CXCL4 in the supernatants was measured by ELISA. Results were normalized to the value of media alone and represented as means ± SEM. (C and D) pDCs purified from SSc patients (n = 15 and 12, respectively) were cultured for 24 hours either in media alone or with either (C) ORN-8L or (D) CpG-C274, and CXCL4 production was analyzed by ELISA. Results were normalized to control and represented as means ± SEM. (E) HD (n = 10) and SSc pDCs (n = 20) were stimulated with ORN-8L for 24 hours, and supernatants were collected for measurement of IFN-α, IP-10, IL-6, and TNF by multiplex bead assay. Results were normalized to 10,000 cells. (F) Purified pDCs from SSc patients (n = 12) were cultured either with media alone or in the presence of a TLR9 agonist for 24 hours, and supernatants were collected for measurement of IFN-α, IP-10, IL-6, and TNF by multiplex bead assay. Results were normalized to 10,000 cells. All results are represented as means ± SEM. For (B) to (E), statistical significance was evaluated using a Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 4 CXCL4 potentiates TLR8-mediated activation of SSc pDCs.

    (A and B) Purified pDCs from SSc patients (n = 8) were cultured in media alone (control), with ORN-8L (200 μg/ml), or with ORN-8L and CXCL4 (10 μg/ml) for 24 hours, and (A) IFN-α and IP-10 or (B) IL-6 and TNF were quantified in the supernatants by ELISA. Values were normalized to ORN-8L alone and represented as means ± SEM. (C to E) Purified pDCs from HDs were cultured alone, and purified pDCs from SSc patients were cultured for 24 hours either alone or in the presence of the PI3Kδ inhibitor CAL-101 (10 μM); (C) CXCL4, (D) IFN-α, or (E) IL-6 levels were quantified by ELISA. Results are normalized to 10,000 cells. All results are represented as means ± SEM, and statistical significance was evaluated using a Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 5 CXCL4 potentiates TLR9-mediated activation but has minimal effect on TLR7-mediated activation of pDCs purified from SSc or HDs.

    Purified pDCs from HDs (A to C) or SSc patients (D and E) were cultured in media alone (control), either (A and D) with a CpG-C TLR9 agonist (0.1 μM) or with CpG-C and CXCL4 (10 μg/ml), or (B) with a CpG-B TLR9 agonist (0.25 μM) or with CpG-B and CXCL4, and (C and E) with a TLR7 agonist [heat-inactivated VR95 influenza virus at a MOI of 0.5 (FLU)] or with FLU and CXCL4 for 24 hours (n = 6 to 18 as indicated). IFN-α was quantified in the supernatants by ELISA. Values were normalized to 10,000 cells. All results are represented as means ± SEM, and statistical significance was evaluated using a Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 6 pDCs infiltrate the skin of BLM-treated mice, and their depletion attenuates skin fibrosis.

    pDC depletion was achieved as described in Materials and Methods. (A to C) Representative images of Siglec-H+ cells (red signal; original magnification, ×400) and (D) average (value based on five skin sections with n = 6 to 13 mice per group) in wild-type (WT) mice injected with (A) PBS and (B) with BLM and of (C) CLEC4C-DTR mice injected with DT and BLM. (E) Representative images of Masson’s trichrome–stained skin sections at a ×10 magnification, arrow indicates the area used to quantify skin thickness, and (F) average of skin thickness of WT mice receiving PBS [left panel in (E); open circle in (F)], WT mice injected with DT and treated with BLM [middle panel in (E); closed circle in (F)], and CLEC4C-DTR mice injected with DT and BLM [right panel in (E); closed square in (F)] is shown. (G) Collagen content in the skin expressed as a ratio of collagen per total protein (tot prot.) is shown. (H and I) Representative images of α–smooth muscle actin (α-SMA) immunohistochemistry of the dermis at a ×40 magnification and averages of the number of α-SMA–positive cells per surface area in dermis are shown. (J to L) Relative gene expression of the indicated genes using the NanoString technology. All values represent means ± SEM; n = 6 to 14 mice for each group. Data are cumulative of three independent experiments, and statistical significance was evaluated using a Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 7 pDCs are critical for the maintenance of skin fibrosis and for the presence of CXCL4 in the skin.

    Fibrosis was induced by injection of BLM in WT mice for either 3 or 5 weeks (wks) or in CLEC4C-DTR mice for 5 weeks as described in Materials and Methods. At 3 weeks of BLM treatment, pDC depletion was achieved in CLEC4C-DTR mice by DT injection (in red) while fibrosis induction was sustained for 2 more weeks. (A) Average of skin thickness and (B) of the number of Siglec-H+ cells in the skin. (C) Representative images of the cxcl4 transcript by RNA in situ hybridization (brown signal; original magnification, ×400; inset, ×630) and (D) average of the number of CXCL4+ cells. Experiment was performed twice (n = 4 to 10 per group). All results are represented as means ± SEM, and statistical significance was evaluated using a Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 8 TLR8 exacerbates disease in the BLM-induced fibrosis model.

    (A) Representative images of Masson’s trichrome–stained skin sections at a magnification of ×10 of WT mice injected with PBS or WT mice, Tg8 mice, TLR7KO mice, and Tg8 × TLR7KO (Tg8 × 7KO) mice injected with BLM. (B) Average of skin thickness (n = 11 to 18 per group). (C) Representative images of Siglec-H+ cells in the skin of WT mice injected with PBS or with BLM and of Tg8 mice, TLR7KO mice, and Tg8 × TLR7KO mice injected with BLM. (D) Histograms represent number of Siglec-H+ cells and are cumulative of at least 10 mice from three independent experiments. (E and F) Relative expression of indicated genes regulated by TLR8 and/or TLR7 using the NanoString technology. (G and H) Gene expression levels of huTLR8 by qPCR for (G) macrophages (F4/80+, CD11b+) or (H) pDCs (F4/80, CD11b and B220+, Siglec-H+, BST2+) purified from WT or Tg8 mice (n = 3 to 4) that received PBS or 4 weeks of BLM as indicated (see Materials and Methods). All results are represented as means ± SEM from three to six independent experiments for each group, and statistical significance was evaluated using a Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Table 1 Clinical and demographic characteristics of the SSc patients.

    Age—years, mean (SD)51.32 (12.82)
    Sex—n, % female61, 77.2%
    Race—n, %55, 69.6% Caucasian
    18, 22.8% African American
    5, 6.3% Asian
    Disease duration—years,
    mean (SD)
    5.6 (4.9)
    Scleroderma subtype
    n, % diffuse
    n, % limited
    62, 78.5%
    13, 16.5%
    MRSS—mean (SD)16.4 (11)
    Autoantibody n, % Scl70
    n, % RNA polymerase 3
    n, % centromere
    n, % RNP
    28, 35.4% Scl70
    21, 26.6% POL3
    11, 13.9% CENB
    4, 5.06% RNP
    Interstitial lung disease
    present—n, %
    43, 54.4%
    Pulmonary hypertension
    present—n, %
    8, 10.1%
  • Table 2 Clinical and demographic characteristics of the SLE patients.

    Age—years, mean (SD)40.67 (10.98)
    Sex—n, % female5, 83.33%
    Race—n, %2, 33.3% Caucasian
    2, 33.3% African American
    2, 33.3% Asian
    Disease duration—years,
    mean (SD)
    15 (3.29)
    ACR criteria—n, %
      Malar rash3, 50%
      Discoid rash0, 0%
      Photosensitivity3, 50%
      Oral ulcers3, 50%
      Arthritis5, 83%
      Serositis3, 50%
      Renal disorder2, 33%
      Neurologic disorder0, 0%
      Hematologic disorder6, 100%
      Immunologic disorder5, 83%
      Positive ANA6, 100%
    Positive titer of
    anti-dsDNA—n, % total
    3, 50%
      1+ positive1
      2+ positive1
      3+ positive1
      4+ positive0
    History of positive
    anti-RBP—n, % total
    4, 66.67%
      Anti-Ro3
      Anti-La1
      Anti-Sm0
      Anti-RNP3
    SLEDAI—mean (SD)4.5 (2.43)

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/423/eaam8458/DC1

    Materials and Methods

    Fig. S1. BDCA4-positive cells express BDCA2, and both are specific markers for pDC.

    Fig. S2. Monocytes of SSc do not express BDCA4.

    Fig. S3. Reduction of the expression levels of TLRs in pDC-depleted PBMC.

    Fig. S4. Monocytes are not depleted in pDC-depleted PBMC.

    Fig. S5. CAL-101 specifically inhibits IFN-α but not IL-6 production by TLR9-activated pDCs.

    Fig. S6. pDCs purified from HDs or SSc patients have similar expression of CXCR3B.

    Fig. S7. Injection of DT leads to the specific depletion of pDCs in CLEC4C-DTR mice.

    Table S1. Clinical and demographic characteristics of the 79 individual SSc patients as well as the overall description of the patient population are shown.

    Table S2. Clinical and demographic characteristics of the six individual SLE patients as well as the overall description of the patient population are shown.

    Table S3. Primary data.

  • Supplementary Material for:

    Plasmacytoid dendritic cells promote systemic sclerosis with a key role for TLR8

    Marie Dominique Ah Kioon, Claudio Tripodo, David Fernandez, Kyriakos A. Kirou, Robert F. Spiera, Mary K. Crow, Jessica K. Gordon, Franck J. Barrat*

    *Corresponding author. Email: barratf{at}hss.edu

    Published 10 January 2018, Sci. Transl. Med. 10, eaam8458 (2018)
    DOI: 10.1126/scitranslmed.aam8458

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. BDCA4-positive cells express BDCA2, and both are specific markers for pDC.
    • Fig. S2. Monocytes of SSc do not express BDCA4.
    • Fig. S3. Reduction of the expression levels of TLRs in pDC-depleted PBMC.
    • Fig. S4. Monocytes are not depleted in pDC-depleted PBMC.
    • Fig. S5. CAL-101 specifically inhibits IFN-α but not IL-6 production by TLR9-activated pDCs.
    • Fig. S6. pDCs purified from HDs or SSc patients have similar expression of CXCR3B.
    • Fig. S7. Injection of DT leads to the specific depletion of pDCs in CLEC4C-DTR mice.
    • Table S1. Clinical and demographic characteristics of the 79 individual SSc patients as well as the overall description of the patient population are shown.
    • Table S2. Clinical and demographic characteristics of the six individual SLE patients as well as the overall description of the patient population are shown.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S3 (Microsoft Excel format). Primary data.

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