Amy Lai

Inflammatory Effects of Ex Vivo Human Th17 Cells Are Suppressed by Regulatory T Cells

Th17 cells are proinflammatory cells associated with many immune-mediated diseases. Major factors limiting the study of human Th17 cells are the lack of an accepted method for their in vitro differentiation or for isolation of a homogenous population of Th17 cells that do not cosecrete IFN-γ. To overcome these hurdles, we established a novel method to isolate in vivo differentiated Th17 cells from peripheral blood by sorting CD161+CCR4+CCR6+CXCR3−CD4+ T cells. The resulting cells produce high levels of IL-17 but not IFN-γ, express high levels of retinoic acid-related orphan receptor variant 2, and maintain this phenotype upon expansion. Ex vivo Th17 cells exhibit a low cytotoxic potential and are hyporesponsive to polyclonal anti-CD3/anti-CD28 stimulation. Importantly, ex vivo Th17 cells were susceptible to suppression by both naive and memory regulatory T cells (Tregs), which inhibited production of IL-17, IL-22, and CXCL8. Moreover, Tregs suppressed the antifibrotic effects of Th17 cells in a wound-healing model. These findings provide new tools for the study of normal and pathological functions of bona fide Th17 cells in humans. They also provide new insight into the cross-talk between Th17 cells and immune and nonimmune cells, and they establish the paradigm that adoptive Treg-based therapies may effectively limit Th17-mediated inflammation.

Keratinocyte-releasable factors increased the expression of MMP1 and MMP3 in co-cultured fibroblasts under both 2D and 3D culture conditions

Matrix metalloproteinases (MMPs) are key elements in extracellular matrix (ECM) degradation and scar remodeling during the wound-healing process. Our previous data revealed that keratinocyte-releasable factors significantly increased the expression of fibroblast MMPs in monolayer-cultured fibroblasts. In this study, we analyzed the differences in the MMP expressions of fibroblasts in a three-dimensional fibroblast-populated collagen gel (3D FPCG) from that in a two-dimensional monolayer-cultured fibroblasts when both co-cultured with keratinocytes. Differential mRNA and protein expression of fibroblasts were examined by microarray, RT-PCR, and western blot. Our results showed that fibroblasts co-cultured with keratinocytes in a 3D FPCG expressed significantly higher MMP1 and MMP3 at the gene and protein levels. Due to the physiological advantages of a 3D FPCG model to a 2D system, we concluded that the 3D FPCG model may provide a better means of understanding the fibroblast–keratinocyte cross-talk during the wound-healing process.

Dermal fibroblasts influence the expression profile of 14-3-3 proteins in human keratinocytes

We have previously demonstrated that the release of some of the 14-3-3 isoforms from keratinocytes is able to influence the expression of key matrix metalloproteinases (MMPs) in dermal fibroblasts. Conversely, in this study we aimed to investigate whether dermal fibroblasts possess the ability to modulate the expression of 14-3-3 proteins in keratinocytes. In order to address this question, human keratinocytes and dermal fibroblasts were harvested and co-cultured. Intra- and extracellular levels of 14-3-3 proteins (β, η, γ, and σ) were analyzed using western blot analysis, and the gene expression was further assessed by quantitative real-time polymerase chain reaction. Gene analysis revealed an up-regulation of all four 14-3-3 isoforms of interest. In addition, the findings of this study reveal a significant increase in the intracellular levels of 14-3-3 γ and σ in keratinocytes co-cultured with fibroblasts compared to those of the mono-cultured control keratinocytes. Mechanistic investigations also demonstrated the capacity of several mitogen-activated protein kinase-specific inhibitors to markedly reduce induction of 14-3-3 σ in keratinocytes stimulated with fibroblast-conditioned medium. The study concluded that dermal fibroblasts possess the ability to influence the expression of several 14-3-3 isoforms (notably γ and σ) in keratinocytes, suggesting that the two cell types might be capable of bi-directionally influencing the protein expression of one another in vivo.

Highly Efficient Stable Expression of Indoleamine 2,3 Dioxygenase Gene in Primary Fibroblasts

Indoleamine 2,3 dioxygenase (IDO) is a potent immunomodulatory enzyme that has recently attracted significant attention for its potential application as an inducer of immunotolerance in transplantation. We have previously demonstrated that a collagen matrix populated with IDO-expressing fibroblasts can be applied successfully in suppressing islet allogeneic immune response. Meanwhile, a critical aspect of such immunological intervention relies largely on effective long-term expression of the IDO gene. Moreover, gene manipulation of primary cells is known to be challenging due to unsatisfactory expression of the exogenous gene. In this study, a lentiviral gene delivery system has been employed to transduce primary fibroblasts. We used polybrene to efficiently deliver the IDO gene into primary fibroblasts and showed a significant increase (about tenfold) in the rate of gene transfection. In addition, by the use of fluorescence-activated cell sorting, a 95% pure population of IDO-expressing fibroblasts was successfully obtained. The efficiency of the IDO expression and the activity of the enzyme have been confirmed by Western blotting, fluorescence-activated cell sorting analysis, and Kynurenine assay, respectively. The findings of this study revealed simple and effective strategies through which an efficient and stable expression of IDO can be achieved for primary cells which, in turn, significantly improves its potential as a tool for achieving immunotolerance in different types of transplantation.

SPARC/SFN interaction, suppresses type I collagen in dermal fibroblasts

We previously suggested that keratinocyte releasable factors might modulate the wound healing process by regulating the expression of key extracellular matrix components such as collagenase (matrix metalloproteinase‐1) and type I collagen in fibroblasts. The first one, we called it keratinocyte‐derived anti‐fibrogenic factor (KDAF), identified as stratifin (SFN) also named 14‐3‐3σ, revealing a strong collagenase activity. However, the second factor, which we named keratinocyte‐derived collagen‐inhibiting factor(s) (KD‐CIF) that has shown to control the synthesis of type I collagen, was not known. Upon conducting a series of systematic protein purification methods followed by mass spectroscopy, two proteins: secreted protein acidic rich in cystein (SPARC) and SFN were identified in keratinocyte‐conditioned media. Using co‐immunoprecipitation and 3D modeling, we determined that SFN and SPARC form a complex thereby controlling the type I collagen synthesis and expression in fibroblasts. The levels of these proteins in fibrotic tissues (animal and human) were also evaluated and a differential expression of these proteins between normal and fibrotic tissue confirmed their potential role in development of fibrotic condition. In conclusion, this study describes for the first time an interaction between SPARC and SFN that may have implications for the regulation of matrix deposition and prevention of dermal fibrotic conditions such as hypertrophic scars and keloid. J. Cell. Biochem. 113: 2622–2632, 2012.

Microarray‐based identification of aminopeptidase N target genes in keratinocyte conditioned medium‐stimulated dermal fibroblasts

Of the many processes that affect the outcome of wound repair, epidermal–dermal interactions are essential to extracellular matrix (ECM) remodeling and in particular, soluble factors released by keratinocytes are known to have a direct impact on the production of ECM by dermal fibroblasts. Aminopeptidase N (APN) has recently been proposed as a cell‐surface receptor for stratifin and is responsible for the stratifin‐mediated matrix metalloproteinase‐1 (MMP‐1) upregulation in fibroblasts. The present study examines whether modulation of APN gene expression has any impact on the fibroblast ECM gene expression profile. The result reveals that in the presence of keratinocyte‐derived soluble factors, transient knockdown of APN in dermal fibroblasts affects the expression of key ECM components such as fibronectin, tenascin‐C, MMP‐1, MMP‐3, and MMP‐12. The regulatory effects of APN on fibronectin and selective MMPs appear to be associated with receptor‐mediated signal transduction independently of its peptidase activity. On the contrary, inhibition of the APN enzymatic activity by bestatin significantly reduces the tenascin‐C expression and enhances the contraction of fibroblast‐populated collagen gel, suggesting an activity‐dependent regulation of fibroblast contractility by APN. The overall effects of APN on the expression of fibronectin, tenascin‐C, and MMPs in fibroblasts propose an important role for APN in the regulation of keratinocyte‐mediated ECM remodeling and fibroblast contractile activity. J. Cell. Biochem. 113: 1061–1068, 2012.

Paracrine regulation of fibroblast aminopeptidase N/CD13 expression by keratinocyte-releasable stratifin†

As wound healing proceeds into the tissue remodeling phase, cellular interactions become dominated by the interplay of keratinocytes with fibroblasts in the skin, which is largely mediated through paracrine signaling and greatly affects the molecular constitution of the extracellular matrix. We have recently identified aminopeptidase N (APN)/CD13 as a potential fibroblast receptor for 14‐3‐3 sigma (also known as stratifin), a keratinocyte‐releasable protein with potent matrix metalloproteinase 1 (MMP1) stimulatory activity. The present study demonstrates that the expression of APN on dermal fibroblasts is regulated through paracrine signaling by keratinocyte‐derived soluble factors. By using an in vitro keratinocyte‐fibroblast co‐culture system, we showed that APN expression in dermal fibroblasts is induced in the presence of keratinocytes or in response to keratinocyte‐conditioned medium. Conditioned medium collected from differentiated keratinocytes further increases APN protein production, suggesting an amplified stimulatory effect by keratinocyte differentiation. Recombinant stratifin potently induces APN synthesis in a dose‐dependent manner. A consistent correlation between the protein expression levels of APN and MMP1 was also observed. These results confirm paracrine regulation of APN expression in dermal fibroblasts by keratinocyte‐derived stimuli, in particular stratifin, and provide evidence that APN may serve as a target in the regulation of MMP1 expression in epidermal–mesenchymal communication. J. Cell. Physiol. 226: 3114–3120, 2011.

Topical application of aminopeptidase N-neutralizing antibody accelerates wound closure

Upon release from keratinocytes, 14-3-3 sigma (also known as stratifin) acts on the dermal fibroblast and modulates its production of extracellular matrix proteins. Subsequent to the recent identification as a receptor responsible for stratifin-mediated matrix turnover in dermal fibroblasts, aminopeptidase N has been implicated in the regulation of epidermal–dermal communication and expression of key matrix proteases and adhesion molecules. In light of the growing importance of aminopeptidase N in modulation of the fibroblast phenotype, the present study evaluates the potential of targeting the ectoenzyme in cutaneous repair, and demonstrates that neutralization of aminopeptidase N led to acceleration of wound closure. This was attributed to at least in part an increase of collagen deposition and fibroblast contractility in the granulation tissue. These findings confirmed the important role of aminopeptidase N in post-injury tissue remodeling and wound contraction.

From affirmative action hiring to the globalisation of legal academia

Scholars have long agreed upon the interrelated rationales for a diverse law faculty, which include the recognition of the value of multi-perspective and multicultural education and scholarship, the promotion of non-discrimination and prevention of discrimination in the legal academy and legal community at large, and the benefits of minority mentors and role models for minority students. This article will make use of the United States Supreme Court’s 2003 landmark decision in Grutter v Bollinger to illuminate how its diversity rationale in the admission of law students should extend to the hiring of minority law faculty members. Further, it will argue that “racial minorities” should include not only African Americans, Hispanics, and Native Americans, but also Asian Americans. Finally, law schools should include foreign professors in their affirmative action hiring efforts in this era of globalisation. Although this article focuses more upon faculty hiring than student admission, to the extent that it appropriates the Grutter rationale to discuss affirmative action hiring, affirmative action admission will also be a significant part of the discussion. After all, a diverse law faculty and a diverse student body are inseparable components of a supportive and friendly law school environment.