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Published in final edited form as: J Invest Dermatol. 2021 Mar 4;141(8):1906–1914.e2. doi: 10.1016/j.jid.2020.12.032

Myeloid dendritic cells are major producers of interferon-β in dermatomyositis and may contribute to hydroxychloroquine refractoriness

Kristen L Chen 1,2,*, Jay Patel 1,2,*, Majid Zeidi 1,2, Maria Wysocka 2, Muhammad M Bashir 1,2, Basil Patel 2, Spandana Maddukuri 1,2, Barbara White 3, Victoria P Werth 1,2
PMCID: PMC8316264  NIHMSID: NIHMS1680028  PMID: 33675790

Abstract

Dermatomyositis pathogenesis remains incompletely understood, however recent work suggests a predominant type 1 IFN response. We explored dermatomyositis pathogenesis by quantifying inflammatory cells in skin, comparing myeloid versus plasmacytoid dendritic cell release of IFN-β, and assessing myeloid dendritic cell contribution to hydroxychloroquine refractoriness. Immunohistochemistry was performed to assess cell type expression in lesional skin biopsies from 12 patients with moderate-severe cutaneous dermatomyositis. Immunofluorescence, laser capture microdissection, and flow cytometry were used to assess myeloid dendritic cell release of IFN-β in dermatomyositis lesional skin biopsies and blood. Immunohistochemistry was utilized to determine if myeloid and/or plasmacytoid dendritic cells were increased in HCQ-nonresponders. CD4+, CD11c+, and CD69+ cells were more populous in lesional DM skin. Myeloid dendritic cells colocalized with IFN-β via immunofluorescence and laser capture microdissection revealed increased IFN-β mRNA expression by myeloid dendritic cells in lesional DM skin. In blood, both myeloid and plasmacytoid dendritic cells were major producers of IFN-β in DM patients whereas plasmacytoid dendritic cells predominately released IFN-β in healthy controls (p < 0.01). Myeloid dendritic cells were significantly increased in skin of HCQ-nonresponders compared to responders (p < 0.05). Myeloid dendritic cells appear to play an important role in DM pathogenesis and IFN-β production.

INTRODUCTION

Dermatomyositis (DM) is a systemic autoimmune condition primarily affecting the skin, muscle, and lungs, among other organs. The estimated incidence is 9.6 per 1 million persons per year (Bendewald et al., 2010) and can significantly impair patients’ quality of life (Goreshi et al., 2011). However, the pathogenesis of DM remains incompletely understood. It is thought that predisposing genes, environmental stressors, and immune- and non-immune mediated mechanisms induce susceptibility and onset of DM (Mainetti et al., 2017).

Interferons (IFNs) that regulate innate and adaptive immunity are thought to be key players in DM pathogenesis (Kao et al., 2011), as high levels of IFN-induced gene products have been found in DM blood (Huard et al., 2017) and skin tissue (Wong et al., 2012). They have inherent amplification mechanisms as many of the IFN-induced downstream molecules are regulated by IFN itself (Hall and Rosen, 2010). With regards to autoimmunity, amplification of this response may result in increased autoantigen presentation as IFNs may increase MHC class I and maturation of antigen presenting cells (APC), contributing to increased self-targeted inflammation (Hall and Rosen, 2010).

Plasmacytoid dendritic cells (pDC) are considered major producers of the type I IFNs, IFN-α/β and have been identified to infiltrate both DM skin and muscle (Greenberg, 2010). They are thought to produce high levels of type I IFNs following stimulation of toll-like receptors (TLRs)-7 and −9 (Greenberg, 2010). There additionally appears to be an autostimulatory effect where the accumulation of pDCs amplifies production of type I IFNs (Liao et al., 2010). Little work has been done to identify the contribution of other APCs, such as myeloid dendritic cells (mDC), in the type I IFN response.

Our objectives were to quantify inflammatory cells in DM skin, compare mDC versus pDC release of IFN-β in DM skin and blood, and evaluate mDC versus pDC contribution to refractoriness to hydroxychloroquine (HCQ). Better understanding of disease pathogenesis will aid in the development of targeted therapies.

RESULTS

CD4+ T-cells, CD11c+ cells, and CD69+ cells are the predominant inflammatory cells in moderate to severe DM skin lesions

We evaluated cellular infiltrates of DM skin in 12 patients with moderate-to-severe cutaneous DM disease despite standard background therapy. We found CD4+ T-cells (67.9 ± 62.5 cells/HPF), CD11c+ cells (34.5 ± 46.4 cells/HPF), and CD69+ cells (28.6 ± 25.6 cells/HPF) (Figures 1a–c) were more populous in DM lesional skin, compared to CD8+ T-cells (14.4 ± 13.8 cells/HPF), mast cells (13.6 ± 14.1 cells/HPF), and CD123+ cells (6.1 ± 13.3 cells/HPF) (Figure 1d–f). Isotypes illustrated in Figure 1g–h. Graphical representation can be seen in Figure 1i.

Figure 1. Inflammatory cells in moderate to severe lesional DM skin (n=12).

Figure 1.

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Immunohistochemistry (IHC) for (a) CD4, b) CD11c (mDC), c) CD69, d) CD8, e) Tryptase (mast cells), and f) CD123 (pDC). IHC (g) mouse isotype and (h) rabbit isotype. (i) Cells quantified per HPF and graphed. Imaged at 20x magnification. DM, dermatomyositis; HPF, high-power field; IHC, immunohistochemistry; mDC, myeloid dendritic cells; pDC, plasmacytoid dendritic cells. Scale bar = 50 μm. Graph shows median ± IQR.

CD11c is a reliable mDC marker in lesional skin of DM patients

To evaluate CD11c as a marker for mDCs we used Image Mass Cytometry (IMC) to triple label lesional DM skin with CD11c, CD163, and HLA-DR. We used metal conjugated antibodies CD11c (Figure 2a, red), CD163 (Figure 2b, blue), and HLA-DR (Figure 2c, green). CD11c and CD163 both overlapped with HLA-DR, identifying APCs, as seen in Figure 2d and 2e. Co-labeling of CD11c and CD163 demonstrated there is no overlap between these two cell markers (Figure 2f). Triple labeling revealed separation between CD11c+HLA-DR+ cells and CD163+HLA-DR+ cells (Figure 2g), suggesting CD11c is a reliable marker for separating positive mDC populations and negative macrophage populations in dermatomyositis skin.

Figure 2. Separation of mDCs from macrophages in moderate-severe lesional DM skin (n=7).

Figure 2.

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Image Mass Cytometry for (a) CD11c (mDCs), (b) CD163 (macrophages), (c) HLA-DR, (d) colocalization of CD11c and HLA-DR, (e) colocalization of CD163 and HLA-DR, (f) separation of CD11c and CD163, and (g) colocalization of CD11c, CD163, and HLA-DR demonstrating separation of mDCs (white arrows) from macrophages (yellow arrows). mDC, myeloid dendritic cells. Nuclei represented with Ir-DNA Intercalator (gray). Scale bars = 10 μm.

IFN-β is produced by mDCs in lesional skin of DM patients

Given the high number of CD11c+ mDCs, we evaluated their role in IFN-β production. We used immunofluorescence to identify CD11c+ mDCs (Figure 3a, red) and IFN-β (Figure 3b, green). Immunofluorescence co-labeling demonstrated co-localization of IFN-β and CD11c+ cells (Figure 3c, yellow/orange) in the skin of DM patients, suggesting mDCs produce IFN-β. To investigate IFN-β production at an mRNA level, we implemented laser capture microdissection (LCM) to capture the CD11c+ mDC cell population and compare it to the population of keratinocytes within each skin biopsy. Figure 4a shows the identification and circling of CD11c+ mDCs to be dissected (red) using rapid immunofluorescence (IF) staining. Subsequent laser dissection along the red outline and extraction of the CD11c+ mDC is shown in Figure 4b. Reverse transcriptase-PCR of the acquired CD11c+ mDCs and keratinocytes revealed an increased relative mRNA expression of IFN-β by mDCs (3.45 ± 5.64 vs 0.37 ± 0.79, p<0.05) (Figure 4c).

Figure 3. Colocalization of CD11c and IFN-β in moderate to severe lesional DM skin (n=7).

Figure 3.

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Immunofluorescence of (a) CD11c (mDC), (b) IFN-β, and (c) colocalization of CD11c and IFN-β (yellow). IFN, interferon; mDC, myeloid dendritic cells. Scale bar = 100 μm.

Figure 4. IFN-β mRNA in CD11c+ cells in moderate to severe lesional DM skin (n=4).

Figure 4.

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(a) Rapid immunofluorescence of CD11c+ mDC (outlined in red). (b) Laser capture microdissection successfully procures outlined CD11c+ mDC, leaving a rim of fluorescence from laser mediated high intensity light absorption and reflection. (c) Graph of IFN-β mRNA expression of keratinocytes versus CD11c+ cells. mDC, myeloid dendritic cell. Scale bar = 10 μm. Graphs show median ± IQR. *p<0.05.

IFN-β is produced by both mDCs and pDCs in peripheral blood of DM patients

Flow cytometry demonstrated peripheral blood from both healthy controls (HC) (Figure 5a) and DM patients (Figure 5b) have similar percentages of mDCs (HC: 10 ± 8.5; DM: 9.9 ± 8.9) and pDCs (HC: 4.7 ± 7.0; DM: 5.6 ± 5.3). In healthy controls, IFN-β is almost exclusively produced by pDCs (mDC: 1.4 ± 3.0 vs pDC: 40.56 ± 22.1) (p<0.01) (Figure 5c) whereas in DM patients, IFN-β is produced by both mDCs and pDCs at high levels and there is no significant difference between cell source (mDC: 58.0 ± 34.6 vs pDC: 76.92 ± 25.2) (Figure 5d). Examples of our gating strategy for Figures 5c and 5d are illustrated respectively in Figures 5e and 5f.

Figure 5. IFN-β release by CD11c+ mDCs and CD123+ pDCs in peripheral blood of healthy controls (n=5) and DM patients (n=5).

Figure 5.

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Flow cytometry analyses quantifying percentages of HLA-DR+Lin- cells that are CD11c+ mDCs or CD123+ pDCs in (a) healthy controls and (b) DM patients. Flow cytometry analyses quantifying IFN-β (MFI) release by mDCs and pDCs in (c) healthy controls and (d) DM patients. Example of gating strategy on FlowJo analysis in (e) healthy controls and (f) DM patients. Note: IFN-β release stimulated with R848. IFN, interferon; Lin, Lineage; mDC, myeloid dendritic cells; MFI, mean fluorescence intensity; pDC, plasmacytoid dendritic cells. Graphs show median± IQR. **p<0.01.

HCQ-nonresponders have significantly increased numbers of mDCs in DM skin

Our analysis showed significantly increased numbers of CD11c+ mDCs in HCQ nonresponders (43.9 ± 49.2) compared to HCQ responders (17.4 ± 32.2) (p<0.05) (Figure 6a–c). There was no significant difference in number of pDCs in HCQ-nonresponders (4.2 ± 20.2) compared to HCQ-responders (2.2 ± 10.7) (Figure 6d–f). Additionally, there are significantly more CD11c+ cells compared to CD123+ cells in both HCQ-responders (p<0.05) and –nonresponders (p<0.01). Expression of IFN-α was found to be higher in HCQ-responders compared to HCQ-nonresponders (p<0.01) (Figure 6g–i), while there was no significant difference in IFN-β expression between the two populations (p>0.05) (Figure 6j–l).

Figure 6. CD11c+ mDCs and CD123+ pDCs in moderate to severe lesional DM skin from HCQ responders (n=8) and -nonresponders (n=8).

Figure 6.

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Immunohistochemistry of CD11c+ cells in (a) HCQ responders, (b) HCQ nonresponders. (c) Graph quantifying CD11c+ cells per HPF in HCQ responders versus nonresponders. Immunohistochemistry of CD123+ cells in (d) HCQ responders and (e) HCQ nonresponders. (f) Graph quantifying CD123+ cells per HPF in HCQ responders versus nonresponders. Immunofluorescence (IF) staining of IFN-α in (g) HCQ responders, (h) HCQ nonresponders. (i) Graph quantifying IFN-α mean fluorescence intensity (MFI) in HCQ responders versus nonresponders. IF staining of IFN-β in (j) HCQ responders, (k) HCQ nonresponders. (l) Graph quantifying IFN-β MFI in HCQ responders versus nonresponders. DM, dermatomyositis; HCQ, hydroxychloroquine; HPF, high-power field; IFN, interferon; mDC, myeloid dendritic cell; MFI, mean fluorescence intensity; pDC, plasmacytoid dendritic cell. Scale bars = 30 μm. Graphs show median ± IQR. *p<0.05.

DISCUSSION

DM pathogenesis is poorly understood. In order to better understand this disease, we began by quantifying several cell types in moderate-severe DM lesional skin. Caproni and colleagues reported CD4+ T-lymphocytes as predominant infiltrating cells (Caproni et al., 2004), which we similarly found. However, we also identified CD11c+ mDCs and CD69+ cells as major cell types in moderate-to-severe DM skin.

Given the high number of mDCs in DM skin, particularly juxtaposed to the scarce presence of pDCs, we hypothesized that mDCs may play a larger role in DM pathogenesis than previously known. Recent studies have implicated type 1 IFNs, in particular IFN-β as a driver of disease pathogenesis (Huard et al., 2017, Wong et al., 2012), which is generally assumed to be derived from pDCs (Fitzgerald-Bocarsly et al., 2008). Previous literature has suggested the likelihood of other cellular sources of IFN-β, including keratinocytes and/or endothelial cells, as DM skin has shown discordant expression of pDCs and type I IFNs, with some showing impressive type I IFN expression and rare pDCs, and vice versa (Magro et al., 2010). We considered that mDCs may additionally be significant producers of IFN-β as they too are capable of expressing a variety of pattern recognition receptors (PRR) that may stimulate type I IFN response (Ali et al., 2019).

Immunofluorescence imaging of CD11c and IFN-β showed colocalization in lesional DM skin, suggesting mDCs produce IFN-β at the protein level. LCM showed increased IFN-β relative mRNA expression from mDCs compared to keratinocytes. These findings suggest mDCs play an important role in the overall type I IFN signature in the skin. Our analysis via flow cytometry of DM versus healthy control blood supports this finding. We additionally demonstrated that in the peripheral blood of healthy controls, pDCs are almost exclusive producers of IFN-β, but in DM patients IFN-β was produced by both mDCs and pDCs. The percentage of mDCs versus pDCs in the peripheral blood of DM patients compared to healthy controls was not significantly different. To our knowledge this is the first study directly comparing IFN-β production from mDCs versus pDCs in DM patients. This suggests that mDCs, in addition to pDCs, are important in DM disease pathogenesis.

Given these results, we further hypothesized that DM patients may not respond to HCQ due to high numbers of mDCs. A previous report found only approximately 25% of DM patients respond to HCQ, with the remainder requiring second and third line therapies (Anyanwu et al., 2017). We found significantly higher numbers of CD11c+ mDCs in HCQ-nonresponders compared to HCQ-responders, similar to our previously reported findings in CLE (Zeidi et al., 2019). Interestingly, IFN-α was lower in HCQ-nonresponders and there was no difference in IFN-β expression between the two groups. This suggests HCQ may have effects on high IFN-α expressing responders. HCQ-nonresponders may not respond due to differences in cell sources such as mDCs that alter the type I IFN composition. Other inflammatory stimuli may drive refractory disease as well, but it is unclear which cytokines drive the HCQ-nonresponder population and future studies will aim to elucidate this. Our finding of increased mDCs in DM HCQ-nonresponders compared to HCQ-responders again suggests these cells are important in DM disease pathogenesis.

While mDCs have not been described previously in DM, there are some reports of similar infiltrating cells in psoriasis, rhinitis, and inflammatory bowel disease (Beitnes et al., 2012, Eguíluz-Gracia et al., 2016, Grimm et al., 1995, Jenner et al., 2014, Zaba et al., 2009). These cells have been termed monocyte-derived DCs and are not normally present in healthy controls (Collin and Bigley, 2018). They may play an important role in antigen presentation and amplification of the adaptive immune system (Collin and Bigley, 2018). The type I IFN response has pleiotropic effects promoting maturation, survival and differentiation of dendritic cells, T cells, and B cells (Crouse et al., 2015, Gallucci et al., 1999, Kiefer et al., 2012). Thus, initial type I IFN signatures may promote further inflammation and augment the type I IFN response, leading to an amplification loop as implicated in other autoimmune diseases (Crow and Ronnblom, 2019). Further work will need to be done to identify the initiating insult for IFN production and origin of these cells specifically in DM.

There are a few limitations to the study. First, we have a relatively limited sample size. Second, while we found both mDCs and pDCs are major sources of IFN-β, we did not survey other cell types or markers and there may be overlapping phenotypes and other cellular sources of IFN-β in DM. Other type I IFNs such as α, κ, and ω exist and may also contribute to disease with similar or distinct expression patterns. We will explore this in future studies. Third, using LCM we were unable to measure mRNA of HC mDCs or pDCs due to low cell counts in the tissues. pDCs were also too scarce in DM patients to extract RNA. Therefore, only IFN-β of mDCs and keratinocytes in DM biopsies could be compared. Finally, in comparing mDCs and pDCs in HCQ responders versus nonresponders, there were more patients with systemic disease in the HCQ nonresponder group compared to the responder group.

Several studies have illustrated that IFN-β is an important driver in DM disease pathogenesis (Chen et al., 2018, Huard et al., 2017, Wong et al., 2012). Our findings suggest mDCs, in addition to pDCs, are major producers of IFN-β in DM patients but not in healthy controls. Increased numbers of mDCs were found in HCQ-nonresponders. This poor response to HCQ may be a result of high IFN-β production by mDCs, however further investigation is warranted.

MATERIALS & METHODS

Ethics Statement

The University of Pennsylvania Institutional Review Board approved human subject involvement in this study. All subjects in this study signed an Informed Consent document before donating blood or tissue.

Patients

All patients were diagnosed with DM (classic or amyopathic) by VPW by either Bohan and Peter criteria (Bohan and Peter, 1975) or Sontheimer criteria (Concha et al., 2019, Sontheimer, 2002). In our IHC quantification of inflammatory cells, IF of mDC and IFN-β, LCM of mDCs, and mass cytometry experiments, lesional skin biopsies came from 12 patients with moderate-severe cutaneous DM despite standard of care background therapy. Demographics detailed in Table S1. In our flow cytometric analysis of mDC versus pDC production of IFN-β, we compared peripheral blood mononuclear cells (PBMCs) from 5 HC and 5 DM (3 amyopathic, 2 classic) patients from a longitudinal DM database. Demographics detailed in Table S2. Finally, in our evaluation of mDCs, pDCs, IFN-α, and IFN-β expression in HCQ responders versus nonresponders, lesional DM skin was obtained from biopsies of patients subsequently characterized subsequently characterized in terms of responsiveness to HCQ, based on disease activity data prospectively collected in a longitudinal database. Demographics detailed in Table S3. Response to HCQ was defined as sufficient improvement in skin lesions so that further escalation of therapy was not needed.

Immunohistochemistry

Formalin-fixed and paraffin embedded (FFPE) 4-mm skin biopsies were cut into 5-μm sections and placed onto glass slides. Slides were incubated at 60°C overnight, deparaffinized in Citrisolv® (Fischer Scientific, Hampton, NH) and rehydrated with serial ethanol dilutions. Antigen retrieval was achieved in EDTA in a 95°C pressure cooker for 20 minutes. Peroxidase was deactivated with 0.3% H2O2 in deionized H2O for 5 minutes. Tissues were blocked in 5% BSA protein block (Dako, Carpintera, CA) for 4 hours and incubated with primary antibody at 4°C overnight. Tissues were stained for CD4 T-cells, CD8 T-cells, mast cells (tryptase), CD69 cells, mDCs (CD11c), and pDCs (CD123). Antibodies and dilutions are detailed in Table S4. Samples were probed with biotinylated anti-goat/anti-mouse/anti-rabbit (Abcam, ab64257) for 50 minutes and streptavidin-HRP (Dako) for 30 minutes. Tissues were developed using NOVAred® chromogen (Vector Laboratories, Burlingame, CA) for 5–10 minutes. Tissues were dehydrated using ethanol serial dilution and Citrisolv®. Tissues were mounted with Permount (Fisher Scientific) and glass coverslips. Sections were analyzed with the Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan). Cells in the dermis were quantified in five nonoverlapping 40x objective magnification fields.

Image Mass Cytometry triple-labeling

FFPE 4-mm biopsies prepared, deparaffinized, and antigen retrieved as above. Metal conjugated antibodies were prepared using three Maxpar X8 antibody labeling kits for metals: 173Yb, 174Yb, and 175Lu (Fluidigm, San Francisco, CA). Tissues were blocked in 3% BSA for 1 hour at room temperature and then incubated with a cocktail of metal conjugated CD11c, HLA-DR, and CD163 antibodies. Antibodies and dilutions detailed in Table S5. Tissues were washed and incubated with Intercalator-Ir for 30 minutes at room temperature (Fluidigm). Final wash was done in deionized water and slides were subsequently air dried for 30 minutes. Regions of interest (2mm x 1mm) were ablated at a frequency of 200Hz on the Hyperion Imaging System (Fluidigm). Images extracted using MCDViewer (Fluidigm) and composites created in ImageJ.

Immunofluorescence

FFPE 4-mm skin biopsies were prepared on glass slides, deparaffinized, and antigen retrieved as above. Tissues were blocked in 5% BSA for 1 hour and incubated with CD11c, IFN-α, and IFN-β primary antibodies (Table S4) at 4°C overnight. Tissues were washed and incubated with secondary antibodies either goat-anti-mouse-594 or goat-anti-rabbit-488 (Fischer Scientific). Tissues were washed again and treated with TrueView (Vector Laboratories, Burlingame, CA) for 2 minutes to reduce autofluorescence after which they were washed, stained with DAPI, and mounted using Vector antifade mounting medium (Vector Laboratories). Composite images created using ImageJ.

Laser Capture Microdissection

FFPE slides were cut RNase free onto polyethylene napthalate (PEN) membrane slides and deparaffinized in Xylene 3 times for 20 seconds each and serial dilutions of ethanol for 30 seconds two times each. Slides were blocked with 10% normal goat serum (NGS) and incubated with CD11c (Abcam, ab52632) in 5% NGS/2%BSA/0.05%Tween-20/RNase inhibitor for 10 minutes and then incubated with secondary antibody goat-anti-rabbit-594 (Fischer Scientific) for 15 minutes. All steps were used with RNase free agents. CD11c+ cells were identified and laser cut into adhesive caps (Zeiss, Germany). Keratinocytes were captured for control comparison and identified by morphology and location. RNA was extracted from laser captured cells using the RNeasy FFPE Kit (Qiagen, Valencia, CA) and converted into cDNA using the High-Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, Foster City, CA). cDNA was amplified by QT-PCR using the TaqMan custom designed array card assay and mRNA was measured via the VIIA 7 Real Time PCR (Applied Biosystems, Foster City, CA). mRNA levels were normalized to PPIA (Applied Biosystems, Hs99999904_m1); relative IFN-β mRNA expression was identified using the following probe: Hs01077958_s1 (Applied Biosystems; IFN-β). Relative gene expression was calculated using the comparative CT method (ΔΔCt).

Flow cytometry

To analyze the cellular source of IFN-β, blood samples were obtained by venous puncture from 5 healthy controls and 5 patients with cutaneous dermatomyositis. PBMCs were isolated by density gradient over Ficoll-Hypaque by standard procedures. Isolated PBMCs at 1×106 cells/mL were cultured in the presence of R848 (1ug/ml) (Invivogen, San Diego, CA) and Brefeldin A (Biolegend, San Diego, CA) for a total of 5 hours. 2×106 cells were aliquoted into each FACS tube, pretreated with staining buffer (2% FCS), blocked with mouse IgG2b (Abcam, ab18428), and stained with surface antibodies (Lineage cocktail, HLA-DR, CD11c, and CD123) for 25 minutes. Conjugated antibodies are detailed in Table S6. Cells were washed in PBS, fixed and permeabilized, stained for IFN-β for 20 minutes, and resuspended in 0.2 ml PBS. Single-cell suspensions underwent flow cytometric analysis on an LSR Fortessa B flow cytometer (BD Biosciences, San Jose, CA) and analyzed with FlowJo software. A total of 150,000–200,000 events were collected for each analysis. Dendritic cells were identified by gating on leukocytes and identifying the HLA-DR+ and Lineage negative (Lin-) sub-population. This excluded CD3+ T cells, CD14+ monocytes/macrophages, CD16+/CD56+ NK cells, CD16+ neutrophils, CD19+ and CD20+ B cells. Using this sub-population, mDCs and pDCs were then identified by CD11c and CD123, respectively. To control for experimentally observed nonspecific binding of the IFN-β antibody, we established gates based on IFN-β positive cells using mean fluorescence intensity on unstimulated cells. These gates were applied to R848 stimulated cells to identify true IFN-β positive cells.

Statistics

Statistical analysis was performed using GraphPad-Prism7 software. Mann-Whitney test was used to compare relative IFN-β mRNA expression from keratinocytes and CD11c+ cells, percentages of HLA-DR+Lin- cells that expressed CD11c or CD123 measured by flow cytometry, IFN-β release from either mDCs or pDCs measured by flow cytometry, and expression of CD11c+ and CD123+ cells in HCQ-responders versus –nonresponders. All statistics were reported as median ± IQR as the data was nonparametric.

Data Availability Statement

There are no data sets available for this submission

Supplementary Material

1

ACKNOWLEDGEMENTS

This project is supported by the Department of Veterans Affairs Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development, the Myositis Association (TMA), Core A of the Penn Skin Biology and Diseases Resource-based Center, funded by 1P30AR069589-01 (Millar), and the National Institutes of Health (NIAMS) [R21 AR066286].

Abbreviations:

APC

antigen presenting cell

DM

dermatomyositis

HCQ

hydroxychloroquine

IF

immunofluorescence

IHC

immunohistochemistry

IFN

interferon

mDC

myeloid dendritic cell

MFI

mean fluorescence intensity

pDC

plasmacytoid dendritic cell

Footnotes

CONFLICTS OF INTEREST

BW: Employee and stock holder of Corbus Pharmaceuticals, Inc, a company that provided lenabasum for use in this study

VPW: University of Pennsylvania owns the copyright for the CDASI.

The remaining authors state no conflict of interest.

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REFERENCES

  1. Ali S, Mann-Nüttel R, Schulze A, Richter L, Alferink J, Scheu S. Sources of Type I Interferons in Infectious Immunity: Plasmacytoid Dendritic Cells Not Always in the Driver’s Seat. Front Immunol 2019;10:778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anyanwu CO, Chansky PB, Feng R, Carr K, Okawa J, Werth VP. The systemic management of cutaneous dermatomyositis: Results of a stepwise strategy. Int J Womens Dermatol 2017;3(4):189–94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beitnes AC, Ráki M, Brottveit M, Lundin KE, Jahnsen FL, Sollid LM. Rapid accumulation of CD14+CD11c+ dendritic cells in gut mucosa of celiac disease after in vivo gluten challenge. PLoS One 2012;7(3):e33556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bendewald MJ, Wetter DA, Li X, Davis MD. Incidence of dermatomyositis and clinically amyopathic dermatomyositis: a population-based study in Olmsted County, Minnesota. Arch Dermatol 2010;146(1):26–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bohan A, Peter JB. Polymyositis and dermatomyositis (first of two parts). N Engl J Med 1975;292(7):344–7. [DOI] [PubMed] [Google Scholar]
  6. Caproni M, Torchia D, Cardinali C, Volpi W, Del Bianco E, D’Agata A, et al. Infiltrating cells, related cytokines and chemokine receptors in lesional skin of patients with dermatomyositis. Br J Dermatol 2004;151(4):784–91. [DOI] [PubMed] [Google Scholar]
  7. Chen M, Quan C, Diao L, Xue F, Xue K, Wang B, et al. Measurement of cytokines and chemokines and association with clinical severity of dermatomyositis and clinically amyopathic dermatomyositis. Br J Dermatol 2018;179(6):1334–41. [DOI] [PubMed] [Google Scholar]
  8. Collin M, Bigley V. Human dendritic cell subsets: an update. Immunology 2018;154(1):3–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Concha JSS, Tarazi M, Kushner CJ, Gaffney RG, Werth VP. The diagnosis and classification of amyopathic dermatomyositis: a historical review and assessment of existing criteria. Br J Dermatol 2019;180(5):1001–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Crouse J, Kalinke U, Oxenius A. Regulation of antiviral T cell responses by type I interferons. Nat Rev Immunol 2015;15(4):231–42. [DOI] [PubMed] [Google Scholar]
  11. Crow MK, Ronnblom L. Type I interferons in host defence and inflammatory diseases. Lupus Sci Med 2019;6(1):e000336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eguíluz-Gracia I, Bosco A, Dollner R, Melum GR, Lexberg MH, Jones AC, et al. Rapid recruitment of CD14(+) monocytes in experimentally induced allergic rhinitis in human subjects. J Allergy Clin Immunol 2016;137(6):1872–81.e12. [DOI] [PubMed] [Google Scholar]
  13. Fitzgerald-Bocarsly P, Dai J, Singh S. Plasmacytoid dendritic cells and type I IFN: 50 years of convergent history. Cytokine Growth Factor Rev 2008;19(1):3–19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gallucci S, Lolkema M, Matzinger P. Natural adjuvants: endogenous activators of dendritic cells. Nat Med 1999;5(11):1249–55. [DOI] [PubMed] [Google Scholar]
  15. Goreshi R, Chock M, Foering K, Feng R, Okawa J, Rose M, et al. Quality of life in dermatomyositis. J Am Acad Dermatol 2011;65(6):1107–16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Greenberg SA. Dermatomyositis and type 1 interferons. Curr Rheumatol Rep 2010;12(3):198–203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Grimm MC, Pullman WE, Bennett GM, Sullivan PJ, Pavli P, Doe WF. Direct evidence of monocyte recruitment to inflammatory bowel disease mucosa. J Gastroenterol Hepatol 1995;10(4):387–95. [DOI] [PubMed] [Google Scholar]
  18. Hall JC, Rosen A. Type I interferons: crucial participants in disease amplification in autoimmunity. Nat Rev Rheumatol 2010;6(1):40–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Huard C, Gullà SV, Bennett DV, Coyle AJ, Vleugels RA, Greenberg SA. Correlation of cutaneous disease activity with type 1 interferon gene signature and interferon β in dermatomyositis. Br J Dermatol 2017;176(5):1224–30. [DOI] [PubMed] [Google Scholar]
  20. Jenner W, Motwani M, Veighey K, Newson J, Audzevich T, Nicolaou A, et al. Characterisation of leukocytes in a human skin blister model of acute inflammation and resolution. PLoS One 2014;9(3):e89375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kao L, Chung L, Fiorentino DF. Pathogenesis of dermatomyositis: role of cytokines and interferon. Curr Rheumatol Rep 2011;13(3):225–32. [DOI] [PubMed] [Google Scholar]
  22. Kiefer K, Oropallo MA, Cancro MP, Marshak-Rothstein A. Role of type I interferons in the activation of autoreactive B cells. Immunol Cell Biol 2012;90(5):498–504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Liao AP, Salajegheh M, Morehouse C, Nazareno R, Jubin RG, Jallal B, et al. Human plasmacytoid dendritic cell accumulation amplifies their type 1 interferon production. Clin Immunol 2010;136(1):130–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Magro CM, Segal JP, Crowson AN, Chadwick P. The phenotypic profile of dermatomyositis and lupus erythematosus: a comparative analysis. J Cutan Pathol 2010;37(6):659–71. [DOI] [PubMed] [Google Scholar]
  25. Mainetti C, Terziroli Beretta-Piccoli B, Selmi C. Cutaneous Manifestations of Dermatomyositis: a Comprehensive Review. Clin Rev Allergy Immunol 2017;53(3):337–56. [DOI] [PubMed] [Google Scholar]
  26. Sontheimer RD. Dermatomyositis: an overview of recent progress with emphasis on dermatologic aspects. Dermatol Clin 2002;20(3):387–408. [DOI] [PubMed] [Google Scholar]
  27. Wong D, Kea B, Pesich R, Higgs BW, Zhu W, Brown P, et al. Interferon and biologic signatures in dermatomyositis skin: specificity and heterogeneity across diseases. PLoS One 2012;7(1):e29161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zaba LC, Fuentes-Duculan J, Eungdamrong NJ, Abello MV, Novitskaya I, Pierson KC, et al. Psoriasis is characterized by accumulation of immunostimulatory and Th1/Th17 cell-polarizing myeloid dendritic cells. J Invest Dermatol 2009;129(1):79–88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zeidi M, Kim HJ, Werth VP. Increased Myeloid Dendritic Cells and TNF-α Expression Predicts Poor Response to Hydroxychloroquine in Cutaneous Lupus Erythematosus. J Invest Dermatol 2019;139(2):324–32. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

1
NIHMS1680028-supplement-1.pdf (16.4KB, pdf)

Data Availability Statement

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