Chronic Psoriatic Skin Inflammation Leads to Increased Monocyte Adhesion and Aggregation

Abstract

Psoriasis patients exhibit an increased risk of death by cardiovascular disease (CVD) and have elevated levels of circulating intermediate (CD14(++)CD16(+)) monocytes. This elevation could represent evidence of monocyte dysfunction in psoriasis patients at risk for CVD, as increases in circulating CD14(++)CD16(+) monocytes are predictive of myocardial infarction and death. An elevation in the CD14(++)CD16(+) cell population has been previously reported in patients with psoriatic disease, which has been confirmed in the cohort of our human psoriasis patients. CD16 expression was induced in CD14(++)CD16(-) classical monocytes following plastic adhesion, which also elicited enhanced β2 but not β1 integrin surface expression, suggesting increased adhesive capacity. Indeed, we found that psoriasis patients have increased monocyte aggregation among circulating PBMCs, which is recapitulated in the KC-Tie2 murine model of psoriasis. Visualization of human monocyte aggregates using imaging cytometry revealed that classical (CD14(++)CD16(-)) monocytes are the predominant cell type participating in these aggregate pairs. Many of these pairs also included CD16(+) monocytes, which could account for apparent elevations of intermediate monocytes. Additionally, intermediate monocytes and monocyte aggregates were the predominant cell type to adhere to TNF-α- and IL-17A-stimulated dermal endothelium. Ingenuity Pathway Analysis demonstrated that monocyte aggregates have a distinct transcriptional profile from singlet monocytes and monocytes following plastic adhesion, suggesting that circulating monocyte responses to aggregation are not fully accounted for by homotypic adhesion, and that further factors influence their functionality.

Copyright © 2015 by The American Association of Immunologists, Inc.

Figures

Figure 1. CD14++CD16+ (intermediate) cells are increased in psoriasis patients compared to healthy controls

(A) The percentage of intermediate monocytes (CD14++CD16+) is increased in psoriasis patients (n=19, open squares) when compared to healthy controls (n=23, solid squares; p=.056) while (B) the percentage of classical (circles) and non-classical monocytes (triangles) do not differ between psoriasis patients and healthy controls. (C) Representative flow plots showing gates and population distribution. (D) Classical monocytes (red, open circles) negatively correlate with disease severity measured by PASI (r=−0.541, p=0.017), while intermediate monocytes (blue, open square) positively correlate with disease severity (r=0.638, p=0.003).

Figure 2. Psoriasis patients have increased total doublets within PBMCs

(A) The prominent intermediate monocyte population in psoriasis patients was selected and in (B), cells were overlaid electronically into a monocyte scatter gate as shown in the FSC-A vs. SSC-A plot. (C) The majority of the cells were doublets, as shown in representative plots of FSC-W vs. FSC-H, demonstrating singlet and doublet populations in PBMCs (n=42) and whole blood (n=8). (D) Analysis of all patients demonstrated that the total doublet percentage is significantly increased in psoriasis patients when compared to controls (open diamonds versus solid diamonds, n=19 and n=23; respectively; p=.006).

Figure 3. Amnis Imagestream visualization of cell surface expression of CD14 (APC) and CD16 (FITC) and monocyte-monocyte doublets

(A) Representative images of each type of monocyte (classical, intermediate, and non-classical) as imaged by the Amnis imagestream cytometer. CD14 (red) is expressed on the surface of the classical cells, CD16 (green) is expressed on the surface of the non-classical cells, while both CD14 and CD16 (orange) co-localize to the surface of the intermediate monocyte. (B) Psoriasis patients (n=3) have increased monocyte doublets when compared to controls (n=3) (black bar versus white bar, respectively; as expressed by a ratio of (% monocyte doublets/% monocyte singlets)). (C) Monocytes form aggregate pairs in the form of monocyte:monocyte or monocyte:lymphocyte. The classical CD14+ cells participate in the most doublet formation as a homogenous pair (CD14+ binding to another CD14+) or as a heterogeneous pair (CD14+ binding to a lymphocyte). The CD14+ cells within the intermediate doublet gate also participate in homo- and heterogeneous pairs, but not to the same extent. Additionally, double positive CD14+CD16+ monocytes participate in binding to CD3+ lymphocytes (as shown in D). Non-classical CD16+ cells rarely participate in homogenous doublet pairs. (D) Representative images of the doublet pairs described in C. Platelets (labeled with CD42b (pink)) participate in doublet formation but are not necessary for monocyte:monocyte aggregation.

Figure 4. CD14++CD16+ cells are present and detectable in psoriatic plaques

Frozen involved human psoriatic plaque stained for CD14 (red) and CD16 (green) with nuclei stained by DAPI (blue). Yellow arrows represent CD14++CD16+ (intermediate) monocytes. Representative image from a psoriasis patient with a PASI score of 33.4 (n=7).

Figure 5. Upregulation of CD16 expression and enhanced adhesion

(A) Representative images: PBMCs at baseline (left panel) were adhered for 4 hours (middle panel) or overnight (right panel) to plastic and then stained for CD14 and CD16 (n=3). After 4 hours, expression of CD16 begins to increase; after overnight culture almost the entire classical population has upregulated CD16 and transitioned into the intermediate gate. (B) Representative images of monocyte gating of negatively selected control monocytes at baseline (left panels) and after 1 hour culture with unstimulated endothelial HMVEC-D cells (middle panels) and HMVEC-D cells stimulated with TNF-α (1ng/ml) and IL-17A (100ng/ml) (right panels); (n=4).

Figure 6. Increased β2 integrin expression on psoriasis classical monocytes

At baseline, psoriasis patients (solid red lines; n=3) demonstrate increased surface expression of (A) CD11b and (B) CD11c with a slight upregulation of (C) CD18 on the classical population when compared to controls (solid blue lines; n=3). After a 30 minute plastic adhesion, control (dashed blue lines) and psoriasis (dashed red lines) classical monocytes upregulate (A) CD11b, (B) CD11c, and (C) CD18, while there is no change in (D) ICAM-1, (E) VCAM-1, or (F) VLA-4. Isotypes are shown on all panels in grey.

Figure 7. Gene networks in doublets and adhered classical monocytes

(A) Heat map of different gene expression patterns of CD14+-doublets (Dblt) and tissue culture adhered classical singlet monocytes (Adh) in control (C; n=5) and psoriasis (P; n=5) individuals. Each individual patient’s singlet classical monocytes were used as the comparator. (B) Heat map of gene expression patterns of psoriasis singlet classical monocytes. An average of the classical singlet monocytes was used as the comparator.

Figure 8. Gene network of psoriasis doublets

(A) Psoriasis doublet gene network generated using the average of all psoriasis doublets (n=5); an average of all control doublets (n=5) is the comparator. (B) Leukocyte extravasation signaling pathway network of psoriasis doublets generated by IPA.

Figure 9. Monocyte doublets are increased in KC-Tie2 mice

(A) Representative figures of doublet gating strategy. Percentages represent doublets within the CD11b+Ly6Chi gate. (B) Splenic doublets are significantly increased in KC-Tie2 mice (n=10) when compared to littermate WT controls (n=5) (p=.008).