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The C-Type Lectin Receptor DC-SIGN Has an Anti-Inflammatory Role in Human M(IL-4) Macrophages in Response to Mycobacterium tuberculosis - PubMed

  • ️Mon Jan 01 2018

The C-Type Lectin Receptor DC-SIGN Has an Anti-Inflammatory Role in Human M(IL-4) Macrophages in Response to Mycobacterium tuberculosis

Geanncarlo Lugo-Villarino et al. Front Immunol. 2018.

Abstract

DC-SIGN (CD209/CLEC4L) is a C-type lectin receptor (CLR) that serves as a reliable cell-surface marker of interleukin 4 (IL-4)-activated human macrophages [M(IL-4)], which historically represent the most studied subset within the M2 spectrum of macrophage activation. Although DC-SIGN plays important roles in Mycobacterium tuberculosis (Mtb) interactions with dendritic cells, its contribution to the Mtb-macrophage interaction remains poorly understood. Since high levels of IL-4 are correlated with tuberculosis (TB) susceptibility and progression, we investigated the role of DC-SIGN in M(IL-4) macrophages in the TB context. First, we demonstrate that DC-SIGN expression is present both in CD68+ macrophages found in tuberculous pulmonary lesions of non-human primates, and in the CD14+ cell population isolated from pleural effusions obtained from TB patients (TB-PE). Likewise, we show that DC-SIGN expression is accentuated in M(IL-4) macrophages derived from peripheral blood CD14+ monocytes isolated from TB patients, or in macrophages stimulated with acellular TB-PE, arguing for the pertinence of DC-SIGN-expressing macrophages in TB. Second, using a siRNA-mediated gene silencing approach, we performed a transcriptomic analysis of DC-SIGN-depleted M(IL-4) macrophages and revealed the upregulation of pro-inflammatory signals in response to challenge with Mtb, as compared to control cells. This pro-inflammatory gene signature was confirmed by RT-qPCR, cytokine/chemokine-based protein array, and ELISA analyses. We also found that inactivation of DC-SIGN renders M(IL-4) macrophages less permissive to Mtb intracellular growth compared to control cells, despite the equal level of bacteria uptake. Last, at the molecular level, we show that DC-SIGN interferes negatively with the pro-inflammatory response and control of Mtb intracellular growth mediated by another CLR, Dectin-1 (CLEC7A). Collectively, this study highlights a dual role for DC-SIGN as, on the one hand, being a host factor granting advantage for Mtb to parasitize macrophages and, on the other hand, representing a molecular switch to turn off the pro-inflammatory response in these cells to prevent potential immunopathology associated to TB.

Keywords: C-type lectin receptors; DC-SIGN; Mycobacterium tuberculosis; anti-inflammatory; macrophages.

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Figures

Figure 1
Figure 1

DC-SIGN-expressing macrophages are present in pulmonary lesions of non-human primates (NHP) infected with Mtb. (A) Representative immunohistochemical images illustrating the distribution of CD68 (middle row) and DC-SIGN (bottom row), among areas where leukocyte infiltration (top row) is detected by hematoxylin and eosin (HE), in pulmonary tissue and granulomas of NHP with very mild (left columns) and severe (right columns) pathology. (B) Representative immunohistochemical images illustrating the distribution of DC-SIGN, CD163, and MerTK in areas where leukocyte infiltration is detected by HE, such as in granulomas of NHP with severe pathology. (C) Left panel: immunostaining of DC-SIGN (green: Alexa-488) and CD68 (red: Alexa-555) in lung tissue of NHP with severe pathology; right panel: immunostaining of DC-SIGN (green: Alexa-488) and CD163 (red: Alexa-555) in lung tissue of NHP with severe pathology. Green arrow points out a cell positive for DC-SIGN only; red arrow a cell positive for CD68 or CD163 only; and yellow arrow for a cell positive for both DC-SIGN and CD68/CD163. Scale bar = 10 µm.

Figure 2
Figure 2

Human tuberculosis-associated microenvironment induces DC-SIGN expression in macrophages. (A) DC-SIGN expression is induced in M(IL-4) macrophages from TB patients. Freshly isolated monocytes from healthy subjects (HS, white) and TB patients (TB, black) were differentiated into macrophages using M-CSF. At day 5, the cells were activated with IL-4 (20 ng/ml) for 48 h to induce the M(IL-4) macrophage program. Otherwise, macrophages were kept under M-CSF to fully establish the M(M-CSF) program. The cells were harvested and the DC-SIGN expression was analyzed by flow cytometry. Vertical bar graphs depicting the median fluorescent intensity (MFI) of DC-SIGN expression in the different cell populations. Results are expressed as mean ± SD (n = 10 donors). (B) DC-SIGN expression is induced by pleural fluid from TB patients. Freshly isolated monocytes from HS were differentiated into macrophages using M-CSF. At day 5, the cells were activated for 48 h with sera from HS (HS-S, black) and TB patients (TB-S, gray), acellular pleural fluid from TB patients (TB-PE, white), and IL-4 (20 ng/ml, vertical stripes). The cells were harvested and the DC-SIGN expression was analyzed by flow cytometry. Vertical bar graphs depicting the MFI of DC-SIGN expression in the different cell populations. Results are expressed as mean ± SD (n = 13 donors). (C) DC-SIGN-expressing macrophages are present in the pleural cavity of TB patients. Mononuclear cells were isolated either from peripheral blood from HS (HS-PB) and TB patients (TB-PB), or from the pleural effusions from TB patients (TB-PE), and the expression of DC-SIGN was analyzed on the CD14+ population by flow cytometry. Results are expressed as vertical scatter plots showing the MFI of DC-SIGN for each population; each individual symbol represents a single donor. Two-tailed t-test (unpaired/parametric): *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS = not significant.

Figure 3
Figure 3

DC-SIGN regulates the pro-inflammatory response by M(IL-4) macrophages against Mtb. Human monocytes were differentiated into macrophages using M-CSF. At day 5, macrophages were transfected with siRNA targeting DC-SIGN (siDC-SIGN) or a non-targeting siRNA (siControl). The following day, the cells were activated with IL-4 (20 ng/ml) for 48 h to induce M(IL-4) program. The cells were then infected with Mtb at multiplicity of infection (MOI) of 3 bacteria to 1 cell. At 18 h p.i., the cells were harvested and their supernatant collected. Assessment of (A) gene mRNA expression by qRT-PCR analysis, and (B) cytokine and chemokine content by semi-quantitative antibody array. Vertical bar graphs illustrate the fold change of mRNA/protein levels in siDC-SIGN over siControl macrophages; “0” was set arbitrarily to represent no change. Results are expressed as mean ± SD (n = 4 donors). One-tailed Mann–Whitney (unpaired/nonparametric): *P < 0.05. (C) Assessment of cytokines by ELISA analysis. Results are expressed as before-and-after plot for the indicated genes (n = 11 donors). Each circle within the plots represents a single donor. Two-tailed Wilcoxon (matched-paired/nonparametric): P < 0.05 was considered as the level of statistical significance.

Figure 4
Figure 4

Different roles for DC-SIGN in the M(IL-4) macrophage–Mtb interaction. Human monocytes were differentiated into macrophages using M-CSF. At day 5, macrophages were transfected with siRNA targeting DC-SIGN (siDC-SIGN, white) or a non-targeting siRNA (siControl, black). The following day, the cells were activated for 48 h with IL-4 (20 ng/ml) to induce M(IL-4) program. (A) DC-SIGN expression in M(IL-4) macrophages is redundant for the binding and internalization of Mtb. Control and DC-SIGN-depleted macrophages were tested for the capacity to bind (at 4°C, left) or phagocytose (at 37°C, right) the Mtb strain expressing GFP during 4 h of challenge. Bar graphs illustrate the median fluorescent intensity (MFI) of cells positive for GFP, as measured by flow cytometry analysis. Results are expressed as mean ± SD (n = 4 donors). NS, not significant. (B) DC-SIGN influences the capacity of M(IL-4) macrophages to control the Mtb intracellular burden. The cells were infected with Mtb (MOI of 0.2 bacteria to 1 cell) and the intracellular growth of the bacteria was followed at 4 h (day 0), 72 h (day 3), 120 h (day 5), and 168 h (day 7), as measured by colony forming unit (CFU) assays. Results are expressed as vertical scatter plots showing the CFU scoring per ml; each circle represents a single donor. Two-tailed Wilcoxon (matched-paired/nonparametric): *P < 0.05; NS, not significant.

Figure 5
Figure 5

DC-SIGN interferes negatively with the activation of M(IL-4) macrophages triggered by Dectin-1. Human monocytes were differentiated into macrophages using M-CSF. At day 5, the macrophages were transfected with siRNA targeting DC-SIGN (siDC-SIGN, squares) and Dectin-1 (siDectin-1, upward triangle), both siRNAs (siDKO, downward triangle), or a non-targeting siRNA (siControl, circles). The following day, the cells were activated with IL-4 (20 ng/ml) for 48 h to induce M(IL-4) program and the C-type lectin receptor expression. (A) Assessment of IL-6 secretion by ELISA analysis. The cells were infected with Mtb at MOI of 3 bacteria to 1 cell. At 18 h p.i., the supernatant from these cells was collected. Results are expressed as vertical scatter plots, and as mean ± SD (n = 7 donors). (B) Assessment of Mtb intracellular growth by colony forming unit (CFU) assay. The cells were infected with Mtb (MOI of 0.2 bacteria to 1 cell), and the intracellular growth of the bacteria was followed at 4 h (day 0, left) or 120 h (day 5, right). Results are expressed as vertical scatter plots, and as mean ± SD (n = 8 donors). Two-tailed Wilcoxon (matched-paired/nonparametric): *P < 0.05, **P < 0.01; NS, not significant. (C,D) Upon differentiation until day 5, macrophages were then activated with IL-4 for 48 h. Prior to stimulation, M(IL-4) macrophages were pre-treated for 30 min with blocking antibodies for either DC-SIGN or Dectin-1, or both. An irrelevant antibody was used as a control. M(IL-4) macrophages were then treated with either (C) cytochalasin D (1 μg/ml), β-glucan (10 µg/ml) and ManLAM (10 µg/ml), or (D) LPS (1 µg/ml) and ManLAM (10 µg/ml). After 24 h, the supernatants were collected and the production of IL-6 was measured by for ELISA analysis.

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