Capturing the Finer Points of Gene Expression in Psoriasis: Beaming in on the CCL19/CCR7 Axis

Abstract

Laser capture microdissection–coupled complementary DNA microarray analysis is a powerful tool for studying minor cell populations in tissues. In this issue, Mitsui et al. use this method to characterize the immune infiltrates that localize in the dermis of psoriatic skin. They identify the T-cell activation regulators C–C chemokine ligand 19 and C–C chemokine receptor 7 as potential mediators of immune organization in psoriasis.

Over the past decade, genetic, immunologic, and transcriptome studies have been used to identify key molecular pathways central to the pathogenesis of psoriasis. Together, results from these studies have converged in revealing alterations in antigen presentation, T-cell activation, and bidirectional signaling between inflammatory cells and epidermal keratinocytes (reviewed in Capon et al. (2012)). The clinical success of tumor necrosis factor (TNF) blockers and T-cell-targeted drugs such as cyclosporine emphasizes the functional importance of these two major axes in the pathogenesis of psoriasis. Although their precise mechanism of action is incompletely understood, TNF blockers are thought to act by blocking the activation of a subset of inflammatory dendritic cells (DCs) responsible for T-cell activation (Zaba et al., 2009). The second major signaling axis involving IL-23 and IL-17 has emerged more recently supported by genetic, pharmacologic, and immunologic evidence (Elder et al., 2010). Interestingly, targeted transcriptomic analysis has revealed a strong synergism between these two signaling pathways (Chiricozzi et al., 2011). However, current efforts to understand the interplay among the numerous cell types present in a psoriatic skin lesion are often hampered by the complex tissue architecture of skin.

Although tremendously informative, bulk-tissue arrays present two main limitations: (i) the cellular source of differently expressed genes (DEGs) cannot be directly determined, and (ii) DEGs from relatively minor cell populations can be diluted and missed. The next difficult task is to identify the “prime movers”—that is, the molecular effectors with direct and central roles in the pathogenesis of psoriasis, such as those stimulating the DCs responsible for T-cell activation. Progress toward this goal is presented by Mitsui et al. (this issue, 2012) who used laser capture microdissection (LCM)–coupled complementary DNA microarray analysis to identify mediators of lymphoid organization as potential regulators of T-cell-mediated inflammation in psoriatic skin.

LCM: powered by PCR

LCM is not a new technique: the first report of LCM principles was published in the mid 1970s (Isenberg et al., 1976). However, it took two decades to find the first practical applications in biology (Emmert-Buck et al., 1996) and another decade to improve these applications. Today, various LCM systems are commercially available, and all allow a fairly straightforward isolation of cells from their surrounding tissue. Isolation of single cells (or cell clusters) is performed with the precision of a laser beam directly on histological sections, without the need for enzymatic reactions, growth in culture, or advanced tissue manipulation. Importantly, improvements in nucleic acid extraction, amplification, and analysis methods have made LCM a powerful tool for biologists. Because minuscule amounts of genetic material can be amplified in a sufficient and reliable manner, LCM now allows powerful genetic analyses of small subsets of cells in heterogeneous tissues such as skin. Previous studies have used this method to identify human hair follicle stem cell markers (Ohyama et al., 2006) and to study specific pathways in cell populations associated with skin cancer (Rittié et al., 2007; Koh et al., 2009) and wound healing (Rittié et al., 2011). In this issue, Mitsui et al. (2012) combine LCM and PCR amplification with high-throughput transcriptome analysis to compare normal vs. psoriatic skin, yielding interesting observations that were not previously spotted using bulk tissue arrays.

Psoriatic skin microdissected

Mitsui et al. (2012) have focused on dermal aggregates in psoriatic lesional skin. These dermal aggregates—also referred to as lymphoid or dermal inflammatory cell aggregates—are primarily made up of LAMP3 (formerly known as DC-LAMP)-positive DCs and T cells. Psoriatic dermal aggregates are thought to be important in the pathogenesis of psoriasis, not only because of their elevated numbers in involved compared with non-involved psoriatic and normal skin but also because effective treatments of psoriasis lead to their disappearance (Zaba et al., 2009).

When investigating minute clinical samples such as LCM-isolated psoriatic dermal aggregates, reliable sample preparation and amplification is critical. Mitsui et al. (2012) validated the accuracy of their transcript amplification system by demonstrating a correlation between results obtained using microdissected epidermal and dermal samples and those of bulk skin microarrays made publicly available by other groups. The authors demonstrate a greater contribution of the epidermis compared with the dermis to bulk analysis, as is generally believed but rarely demonstrated. Comparison of psoriatic dermal aggregates, which are typically found in the reticular dermis, with reticular dermis of non-lesional psoriatic skin revealed not only expected transcripts, such as LAMP3 and CD28 (T-cell-specific surface glycoprotein), but also several cytokines and cytokine receptors that were not previously recognized as being selectively expressed in psoriasis. Among them, C–C chemokine ligand (CCL)19 and its receptor C–C chemokine receptor (CCR)7 were the most highly increased; others included CCL22 (chemotactic for immune cells including DCs and activated T cells) and CXCL13 (a B-lymphocyte chemoattractant).

The importance of CCL19 and CCR7 expression in psoriatic dermal aggregates

Skin DCs are potent antigen-presenting cells. Immature DCs patrol skin until they capture a (usually) nonself antigen, which triggers differentiation into mature DCs. Mature DCs then migrate to lymph nodes where they present antigens to T and B cells to induce primary immune responses (Luther et al., 2011). Maturation and trafficking of DCs are controlled by multiple chemokines and CCRs. CCL19 is particularly important in DC biology not only because DCs acquire CCL19 and CCL21 responsiveness (via induced expression of CCR7) during the maturation process but also because DCs produce CCL19 and thereby modulate T-cell survival and trafficking (naïve T cells express CCR7). CCL19 maintains the viability of naïve T cells alive in vitro, and mice null for CCL19 show reduced T-cell survival in vivo (Link et al., 2007). Interestingly, double-label immunofluorescence studies by Mitsui et al. (2012) indicate that CCL19 and CCR7 are expressed by both DCs and T cells in psoriatic dermal aggregates, suggestive of a self-reinforcing two-way interaction.

Although bulk tissue analyses indicate that the other CCR7 ligand, CCL21, is increased in psoriatic plaques compared with non-lesional skin (Zhou et al., 2003), Mitsui et al. (2012) report that CCL21 transcript and protein levels are relatively low in psoriatic dermal aggregates. These results suggest that the cellular sources of CCL19 and CCL21 are likely different in psoriatic plaques. This hypothesis is consistent with the current understanding that CCL21 is expressed by the stromal cells of the so-called T-zone of lymph nodes, where it attracts CCR7-positive naïve T cells. Upon contact with CCL19 produced by mature DCs in the lymph node, naïve T cells become activated and subsequently exit the lymph node by at least two mechanisms: first, CCL19 induces CCR7 internalization (more effectively than CCL21); second, CCL19 signaling induces the expression of sphingosine-1-phosphate receptor (or S1PR1, also known as endothelial differentiation gene 1). Acquisition of S1PR1 by activated T cells triggers their egress from the lymph node, mostly owing to the high affinity of S1PR1 for sphingosine-1phosphate (S1P), a phospholipid bound extensively to albumin and other plasma proteins in extracellular fluids including lymph (Shannon et al., 2012).

Concluding remarks and open questions

Overall, this interesting study by Mitsui et al. (2012) sheds light on the nature of the immune cell clusters found in psoriatic skin, called dermal aggregates, which may serve to maintain a localized state of dermal T-cell activation. Although of laudable technical quality, the double-label immunofluorescence experiments presented in the current study only partially identify the cell populations that express CCL19 and CCR7, which could include other immune cells or stromal cells as well (Luther et al., 2011).

Prevention of immune cell aggregation might be a beneficial therapeutic strategy for psoriasis. Using the S1P/S1PR1 axis to prevent activated T cells from egressing lymph nodes is one possible scheme. Indeed, studies in mice have shown that S1PR1-null T cells transferred into blood are unable to leave the low-concentration S1P-containing lymph node for the high-concentration S1P-containing lymph (Rosen and Goetzl, 2005). Conversely, several drugs have been developed to neutralize the S1P gradient existing between the lymph node and extracellular fluids. These drugs cause T-cell retention in lymph nodes: the food colorant 2-acetyl-4-tetrahydroxybutylimidazole (caramel color) is one example (Schwab et al., 2005). In humans, autoantibodies preventing T-cell response to S1P cause chronic lymphopenia (Liao et al., 2009). In light of the work presented in this issue of the JID, it would be interesting to determine the effects of globally decreasing T-cell activation on the formation of psoriatic dermal aggregates, and on the progression of psoriasis itself.

In summary, the observations reported by Mitsui et al. (2012) underscore the importance of detailed spatial analysis of gene expression in a complex autoimmune disease. Skin presents multiple advantages over other organs: it is readily accessible, relevant cell populations can be identified with simple stains, and differences between normal and diseased tissue can be readily mapped by microscopic inspection. All of these properties facilitate the LCM approach. Other dermatological disorders such as atopic eczema, alopecia aerata, vitiligo, and lichen planus are bound to greatly benefit from a similar LCM approach in the future.

Clinical Implications.

• Laser capture microdissection (LCM) allows the characterization of single cells in complex tissues … a new frontier.

• Isolation of single cells (or cell clusters) is performed with the precision of a laser directly on histological sections.

• LCM allows powerful genetic analyses of small subsets of cells in heterogeneous tissues, such as skin.

• The investigators combined LCM and PCR amplification to compare normal skin with psoriatic skin, yielding new mechanistic knowledge about the disease.