Regulators of thymic stromal lymphopoietin production by human adipocytes

Highlights

• TSLP is produced by subcutaneous human adipocytes in response to TSH, IL-1β, or IFN-γ.

• TSLP responses to TSH were dependent on PKA and ERK1/2 signaling.

• TSLP responses to IL-1β were dependent on IKKβ signalling.

• Intra-abdominal omental adipocytes also displayed TSLP responses.

Abstract

Thymic stromal lymphopoietin (TSLP) is a cytokine that is known to play a role in inflammatory conditions, especially asthma and atopic dermatitis. It is also recognized to be expressed in human adipose tissue. TSLP production from human adipocytes is stimulated by thyroid-stimulating hormone (TSH). This study aimed to identify TSH-dependent signaling routes that regulate TSLP, to determine if TSLP production is stimulated by other cytokines (IL-1β and TNF-α), and to examine if TSLP production depends on the adipose depot. Human abdominal differentiated adipocytes were stimulated with TSH, IL-1β, or TNF-α. Activation of cell signaling kinases was measured by phospho-immunoblot analysis, and TSLP in medium was assessed by ELISA. TSLP responses from abdominal subcutaneous and omental adipocytes were compared. TSH-stimulated TSLP secretion from subcutaneous adipocytes was enhanced by IBMX (raises cAMP levels) and was blocked by UO126 (inhibitor of MEK1/2-ERK1/2). TSLP secretion was stimulated by IL-1β and by TNF-α. SC-514 (inhibitor of IKKβ/NF-κB) only reduced the former. There was no effect of SB203580 (p38 MAPK inhibitor) or SP600125 (JNK inhibitor) on the stimulation by TSH, IL-1β or TNF-α. Interferon-γ inhibited TSLP responses to TSH, IL-1β, and TNF-α; IL-4 only blocked the response to TNFα. Intra-abdominal omental adipocytes also release TSLP in response to TSH, IL-1β, and TNF-α. We conclude TSLP is produced by human differentiated adipocytes derived from subcutaneous or omental depots in response to a variety of agonists. Further studies will be needed to understand what role it may play in adipose biology.

Introduction

Thymic stromal lymphopoietin (TSLP) is a member of the interleukin (IL)-7 cytokine family involved in type 2 immune responses. It has been implicated in allergic and inflammatory conditions such as atopic dermatitis, asthma, and inflammatory bowel disease, as well as having a role in cancer growth and metastasis [1], [2]. Other investigations have pointed to a role for TSLP in platelet activation and cardiovascular disease [3], [4], [5], [6], [7], [8]. TSLP is expressed by a variety of cells, including epithelial cells, keratinocytes, and immune cells [9].

In 2012, expression of TSLP was first reported in visceral (intra-abdominal) human adipose tissue [10]. Its level of mRNA expression was lower in severely obese men and women with the metabolic syndrome (state of insulin resistance associated with central obesity, hypertension and low-grade inflammation) compared to those without the metabolic syndrome. The authors of that study did not examine the precise cellular source of TSLP within the adipose tissue (e.g, adipocytes, immune cells, etc), and their analysis of TSLP expression was restricted to the mRNA level.

More recently, we reported that TSLP protein is expressed and released by human subcutaneous abdominal differentiated adipocytes in culture in response to TSH [11]. Adipocytes are an established extra-thyroidal target of TSH. This may account, in part, for the pro-inflammatory and pro-atherogenic state observed in patients with elevated circulating levels of TSH, a condition termed subclinical hypothyroidism [12], [13].

TSH is known to activate cAMP-dependent protein kinase (PKA) in differentiated human adipocytes, measured by an increase in cAMP-responsive binding protein (CREB) phosphorylation [14], [15]. PKA inhibitor H89 blocks the TSH-stimulated increase in TSLP mRNA expression in differentiated adipocytes [11]. TSH activates the inhibitor of κB kinase (IKK)β/nuclear factor (NF)-κB pathway in human adipocytes [14], but inhibitors of this pathway had no effect on the release of TSLP protein from human adipocytes in response to TSH [11]. Dexamethasone, an established transcriptional repressor of TSLP in many cell types, [16], blocked the TSH-stimulated TSLP response in the adipocytes [11].

Adipocytes are known to produce a variety of cytokines, and recent literature reviews highlight the importance of IL-1β and TNF-α in adipose inflammation [17], [18]. Furthermore, these same cytokines have been studied as regulators of TSLP in cell models of asthma, atopic dermatitis, and other epithelial inflammatory conditions [19]. Studying the effect of the immune modulators such as IFN-γ, IL-4, and dexamethasone on TSLP production in this context is an established framework of evaluation [20]. It is not yet known if IL-1β and TNF-α influence TSLP levels in adipocytes, and whether this might be susceptible to these immune modulators.

Adipocyte behaviour can be a function of anatomic site of origin [21]. Given the association of abdominal central obesity with insulin resistance and type 2 diabetes, comparison between adipocytes from subcutaneous versus omental depots are often studied [22]. The expression of some adipocyte cytokines differs according to anatomic adipose site e.g. abdominal subcutaneous versus intra-abdominal (visceral or omental) adipose depot. For example, omental adipocytes produce more IL-6 than do their subcutaneous counterparts [23]. There is no information on whether TSLP production by human adipocytes varies according to anatomic-depot.

We now provide new data on the regulation of TSLP by TSH in human abdominal subcutaneous adipocytes. We also identify IL-1β and TNF-α as inducers of TSLP production in these cells and delineate the signal pathways that are involved. Finally, we show that subcutaneous and intra-abdominal omental adipocytes both express TSLP in response to these agonists.