Amy Li

PPAR-γ is a major driver of the accumulation and phenotype of adipose tissue T reg cells

Obesity and type-2 diabetes have increased markedly over the past few decades, in parallel. One of the major links between these two disorders is chronic, low-grade inflammation. Prolonged nutrient excess promotes the accumulation and activation of leukocytes in visceral adipose tissue (VAT) and ultimately other tissues, leading to metabolic abnormalities such as insulin resistance, type-2 diabetes and fatty-liver disease. Although invasion of VAT by pro-inflammatory macrophages is considered to be a key event driving adipose-tissue inflammation and insulin resistance, little is known about the roles of other immune system cell types in these processes. A unique population of VAT-resident regulatory T (Treg) cells was recently implicated in control of the inflammatory state of adipose tissue and, thereby, insulin sensitivity. Here we identify peroxisome proliferator-activated receptor (PPAR)-γ, the ‘master regulator’ of adipocyte differentiation, as a crucial molecular orchestrator of VAT Treg cell accumulation, phenotype and function. Unexpectedly, PPAR-γ expression by VAT Treg cells was necessary for complete restoration of insulin sensitivity in obese mice by the thiazolidinedione drug pioglitazone. These findings suggest a previously unknown cellular mechanism for this important class of thiazolidinedione drugs, and provide proof-of-principle that discrete populations of Treg cells with unique functions can be precisely targeted to therapeutic ends.

NLR family member NLRC5 is a transcriptional regulator of MHC class I genes

MHC class I plays a critical role in the immune defense against viruses and tumors by presenting antigens to CD8 T cells. An NLR protein, class II transactivator (CIITA), is a key regulator of MHC class II gene expression that associates and cooperates with transcription factors in the MHC class II promoter. Although CIITA also transactivates MHC class I gene promoters, loss of CIITA in humans and mice results in the severe reduction of only MHC class II expression, suggesting that additional mechanisms regulate the expression of MHC class I. Here, we identify another member of the NLR protein family, NLRC5, as a transcriptional regulator of MHC class I genes. Similar to CIITA, NLRC5 is an IFN-γ–inducible nuclear protein, and the expression of NLRC5 resulted in enhanced MHC class I expression in lymphoid as well as epithelial cell lines. Using chromatin immunoprecipitation and reporter gene assays, we show that NLRC5 associates with and activates the promoters of MHC class I genes. Furthermore, we show that the IFN-γ–induced up-regulation of MHC class I requires NLRC5, because knockdown of NLRC5 specifically impaired the expression of MHC class I. In addition to MHC class I genes, NLRC5 also induced the expression of β2-microglobulin, transporter associated with antigen processing, and large multifunctional protease, which are essential for MHC class I antigen presentation. Our results suggest that NLRC5 is a transcriptional regulator, orchestrating the concerted expression of critical components in the MHC class I pathway.

A role for pericytes as microenvironmental regulators of human skin tissue regeneration

The cellular and molecular microenvironment of epithelial stem and progenitor cells is poorly characterized despite well-documented roles in homeostatic tissue renewal, wound healing, and cancer progression. Here, we demonstrate that, in organotypic cocultures, dermal pericytes substantially enhanced the intrinsically low tissue-regenerative capacity of human epidermal cells that have committed to differentiate and that this enhancement was independent of angiogenesis. We used microarray analysis to identify genes expressed by human dermal pericytes that could potentially promote epidermal regeneration. Using this approach, we identified as a candidate the gene LAMA5, which encodes laminin alpha5, a subunit of the ECM component laminin-511/521 (LM-511/521). LAMA5 was of particular interest as we had previously shown that it promotes skin regeneration both in vitro and in vivo. Analysis using immunogold localization revealed that pericytes synthesized and secreted LAMA5 in human skin. Consistent with this observation, coculture with pericytes enhanced LM-511/521 deposition in the dermal-epidermal junction of organotypic cultures. We further showed that skin pericytes could also act as mesenchymal stem cells, exhibiting the capacity to differentiate into bone, fat, and cartilage lineages in vitro. This study suggests that pericytes represent a potent stem cell population in the skin that is capable of modifying the ECM microenvironment and promoting epidermal tissue renewal from non-stem cells, a previously unsuspected role for pericytes.

Extensive tissue-regenerative capacity of neonatal human keratinocyte stem cells and their progeny

Given our recent discovery that it is possible to separate human epidermal stem cells of the skin from their more committed progeny (i.e., transit-amplifying cells and early differentiating cells) using FACS techniques, we sought to determine the comparative tissue regeneration ability of these keratinocyte progenitors. We demonstrate that the ability to regenerate a fully stratified epidermis with appropriate spatial and temporal expression of differentiation markers in a short-term in vitro organotypic culture system is an intrinsic characteristic of both epidermal stem and transit-amplifying cells, although the stem cell fraction is most capable of achieving homeostasis. Early differentiating keratinocytes exhibited limited short-term tissue regeneration under specific experimental conditions in this assay, although significant improvement was obtained by manipulating microenvironmental factors, that is, coculture with minimally passaged dermal cells or exogenous supply of the ECM protein laminin-10/11. Importantly, transplantation of all classes of keratinocyte progenitors into an in vivo setting demonstrated that tissue regeneration can be elicited from stem, transit-amplifying, and early differentiating keratinocytes for up to 10 weeks. These data illustrate that significant proliferative and tissue-regenerative capacity resides not only in keratinocyte stem cells as expected, but also in their more committed progeny, including early differentiating cells.

Establishment of 3D organotypic cultures using human neonatal epidermal cells

This protocol describes an ex vivo three-dimensional coculture system optimized to study the skin regenerative ability of primary human keratinocytes grown at the air–liquid interface on collagen matrices embedded with human dermal fibroblasts. An option for enrichment of keratinocyte stem cells and their progeny using fluorescence-activated cell sorting is also provided. Initially, dermal equivalents, comprising human passaged fibroblasts seeded in a collagen matrix, are grown on porous filters (3 μm) placed in transwells. After 1 week, primary human keratinocytes are seeded on this base. One week later, an air-lift transition is performed, leading to the differentiation of the keratinocytes, which are macroscopically visible as artificial skin after a couple of days. The cultures can be harvested 1 week after the air-lift and processed for immunohistochemistry or gene expression analysis. The overall procedure can be completed in 3 weeks, including the preparation of the dermal equivalent and the seeding of the primary keratinocytes.

Side population in adult murine epidermis exhibits phenotypic and functional characteristics of keratinocyte stem cells.

Based on functional studies in the bone marrow, it has been suggested that the ability to efflux Hoechst 33342 may represent a universal stem cell trait. In this phenotypic and functional characterization of the Hoechst side population (SP) in adult murine epidermis, we demonstrate that these cells are a rare subset of the keratinocyte stem cell-enriched α6briCD71dim fraction comprising SSClow/K14+/CD34−/Oil red O−/c-kit−/CD45− keratinocytes. Epidermal SPs have the smallest cell and nuclear size but exhibit the highest nuclear-to-cytoplasmic ratio of any fraction examined, consistent with a primitive cell type. Although SPs demonstrated poor cumulative in vitro proliferative output, they exhibited sustained epidermal tissue-regenerative activity in vivo compared with unfractionated and non-SP cells. Collectively, these results indicate that the epidermal SP contains the most potent keratinocyte stem cell population in skin epithelium.

NLRC5 Cooperates with the RFX Transcription Factor Complex To Induce MHC Class I Gene Expression

Tight regulation of MHC class I gene expression is critical for CD8 T cell activation and host adaptive-immune responses. The promoters of MHC class I genes contain a well-conserved core module, the W/S-X-Y motif, which assembles a nucleoprotein complex termed MHC enhanceosome. A member of the nucleotide-binding domain, leucine-rich repeat (NLR) protein family, NLRC5, is a newly identified transcriptional regulator of MHC class I genes. NLRC5 associates with and transactivates the proximal promoters of MHC class I genes, although the molecular mechanism of transactivation has not been understood. In this article, we show that NLRC5-mediated MHC class I gene induction requires the W/S and X1, X2 cis-regulatory elements. The transcription factors RFX5, RFXAP, and RFXANK/B, which compose the RFX protein complex and associate with the X1 box, cooperate with NLRC5 for MHC class I expression. Coimmunoprecipitation experiments revealed that NLRC5 specifically interacts with the RFX subunit RFXANK/B via its ankyrin repeats. In addition, we show that NLRC5 can cooperate with ATF1 and the transcriptional coactivators CBP/p300 and general control nonderepressible 5, which display histone acetyltransferase activity. Taken together, our data suggest that NLRC5 participates in an MHC class I-specific enhanceosome, which assembles on the conserved W/S-X-Y core module of the MHC class I proximal promoters, including the RFX factor components and CREB/ATF1 family transcription factors, to promote MHC class I gene expression.

NLRC5: a newly discovered MHC class I transactivator (CITA)

Major histocompatibility complex (MHC) class I and class II are crucial for the function of the human adaptive immune system. An NLR protein, CIITA (MHC class II transactivator), is a master regulator of MHC class II gene expression as well as of some of the genes involved in MHC class II antigen presentation. It has recently been discovered that another member of the NLR protein family, NLRC5, transcriptionally activates MHC class I genes, and thus acts as CITA (MHC class I transactivator), a counterpart to CIITA. In addition to MHC class I genes, NLRC5 can induce the expression of β2M, TAP1 and LMP2, essential components of MHC class I antigen presentation. These findings indicate that NLRC5 and CIITA are transcriptional regulators that orchestrate the concerted expression of critical components in the MHC class I and MHC class II pathways, respectively.

The nucleotide-binding domain of NLRC5 is critical for nuclear import and transactivation activity.

Major histocompatibility complex (MHC) class I and class II are crucial for the function of the human adaptive immune system. A member of the NLR (nucleotide-binding domain, leucine-rich repeat) protein family, NLRC5, has recently been identified as a transcriptional regulator of MHC class I and related genes. While a master regulator of MHC class II genes, CIITA, has long been known, NLRC5 specifically associates with and transactivates the proximal promoters of MHC class I genes. In this study, we analyzed the molecular requirements of NLRC5 nuclear import and transactivation activity. We show that NLRC5-mediated MHC class I gene induction requires an intact nuclear localization signal and nuclear distribution of NLRC5. In addition, we find that the nucleotide-binding domain (NBD) of NLRC5 is critical not only for nuclear translocation but also for the transactivation of MHC class I genes. Changing the cellular localization of NLRC5 is likely to immediately impact MHC class I expression as well as MHC class I-mediated antigen presentation. NLRC5 may thus provide a promising target for the modulation of MHC class I antigen presentation, especially in the setting of transplant medicine.

FACS Enrichment of Human Keratinocyte Stem Cells

Recent work from our laboratory has led to the development and validation of fluorescence-activated cell sorting (FACS)-based techniques to prospectively isolate viable keratinocyte stem cells from both human and murine skin. Here we describe a step-by-step method to apply our technique to isolate epidermal keratinocytes from skin tissue, process them for immunofluorescent staining for cell surface markers, and subject them to fluorescence-activated cell sorting to obtain the stem, transient amplifying, and early differentiating keratinocyte fractions. These viable cells can then be placed into culture for further analysis or directly into keratinocyte assays, such as organotypic cultures or in vivo transplantation. This method will be useful for the complete biological characterization of keratinocyte progenitors with respect to wound healing, carcinogenesis, and therapeutic manipulation.