The IL-1-Like Cytokine IL-33 Is Constitutively Expressed in the Nucleus of Endothelial Cells and Epithelial Cells In Vivo: A Novel ‘Alarmin’?

BACKGROUND: Interleukin-33 (IL-33) is an IL-1-like cytokine ligand for the IL-1 receptor-related protein ST2, that activates mast cells and Th2 lymphocytes, and induces production of Th2-associated cytokines in vivo. We initially discovered IL-33 as a nuclear factor (NF-HEV) abundantly expressed in high endothelial venules from lymphoid organs, that associates with chromatin and exhibits transcriptional regulatory properties. This suggested that, similarly to IL-1alpha and chromatin-associated cytokine HMGB1, IL-33 may act as both a cytokine and a nuclear factor. Although the activity of recombinant IL-33 has been well characterized, little is known yet about the expression pattern of endogenous IL-33 in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that IL-33 is constitutively and abundantly expressed in normal human tissues. Using a combination of human tissue microarrays and IL-33 monoclonal and polyclonal antibodies, we found that IL-33 is a novel nuclear marker of the endothelium widely expressed along the vascular tree. We observed abundant nuclear expression of IL-33 in endothelial cells from both large and small blood vessels in most normal human tissues, as well as in human tumors. In addition to endothelium, we also found constitutive nuclear expression of IL-33 in fibroblastic reticular cells of lymphoid tissues, and epithelial cells of tissues exposed to the environment, including skin keratinocytes and epithelial cells of the stomach, tonsillar crypts and salivary glands. CONCLUSIONS/SIGNIFICANCE: Together, our results indicate that, unlike inducible cytokines, IL-33 is constitutively expressed in normal human tissues. In addition, they reveal that endothelial cells and epithelial cells constitute major sources of IL-33 in vivo. Based on these findings, we speculate that IL-33 may function, similarly to the prototype 'alarmin' HMGB1, as an endogenous 'danger' signal to alert the immune system after endothelial or epithelial cell damage during trauma or infection

A systems biology analysis of adrenergically stimulated adiponectin exocytosis in white adipocytes

Circulating levels of the adipocyte hormone adiponectin are typically reduced in obesity, and this deficiency has been linked to metabolic diseases. It is thus important to understand the mechanisms controlling adiponectin exocytosis. This understanding is hindered by the high complexity of both the available data and the underlying signaling network. To deal with this complexity, we have previously investigated how different intracellular concentrations of Ca2+, cAMP, and ATP affect adiponectin exocytosis, using both patch-clamp recordings and systems biology mathematical modeling. Recent work has shown that adiponectin exocytosis is physiologically triggered via signaling pathways involving adrenergic beta(3) receptors (beta(3)ARs). Therefore, we developed a mathematical model that also includes adiponectin exocytosis stimulated by extracellular epinephrine or the beta(3)AR agonist CL 316243. Our new model is consistent with all previous patch-clamp data as well as new data (collected from stimulations with a combination of the intracellular mediators and extracellular adrenergic stimuli) and can predict independent validation data. We used this model to perform new in silico experiments where corresponding wet lab experiments would be difficult to perform. We simulated adiponectin exocytosis in single cells in response to the reduction of beta(3)ARs that is observed in adipocytes from animals with obesity-induced diabetes. Finally, we used our model to investigate intracellular dynamics and to predict both cAMP levels and adiponectin release by scaling the model from single-cell to a population of cells-predictions corroborated by experimental data. Our work brings us one step closer to understanding the intricate regulation of adiponectin exocytosis.Funding Agencies|Swedish Diabetes Foundation [DIA2017-273, DIA2018-358]; Swedish Research CouncilSwedish Research CouncilEuropean Commission [2019-01239, 2018-05418, 2018-03319, 2019-03767, 2013-7107]; CENIIT [15.09, 20.08]; Swedish Foundation for Strategic ResearchSwedish Foundation for Strategic Research [ITM17-0245]; SciLifeLab; KAW [2020.0182]; Ake Winbergs stiftelse [M19-0449]; H2020 project PRECISE4Q [777107]; Swedish Fund for Research without Animal Experiments [F2019-0010]; ELLIIT; VisualSweden; VINNOVAVinnova [2020-04711]</p

Arrhenius plots presenting the effect of Ca²⁺ on the rate of cAMP-triggered exocytosis.

<p>Absolute values of Δ<i>C</i>/Δ<i>t</i> at t = 2 min plotted against the inverse of temperature (T) in the absence <b>(A)</b> and presence <b>(B)</b> of cytosolic Ca²⁺. The curve represents a linear least-squares fit to the equation lnk = lnA—E_A/RT. E_A is the energy of activation, k is the change in Δ<i>C</i>/Δ<i>t</i> upon a temperature change, A is the pre-exponential factor. R represents the gas constant and T the absolute temperature as usual. The negative slope of the curve gives activation energies of 5.7 kJ mol^-1 (A) and 53 kJ mol^-1 (B).</p

Undifferentiated and mature 3T3-L1 adipocytes as well as demonstration of analysis performance.

<p><b>A</b> Example of 3T3-L1 adipocytes in the fibroblast-like state before differentiation (left) as well as when differentiated into mature adipocytes (right). <b>B</b> Representative capacitance recording with Δ<i>C</i>_m (delta membrane capacitance) plotted against time showing how analyses were carried out. Δ<i>C</i>/Δ<i>t</i> was measured at the time intervals indicated by the dotted lines by fitting straight functions to the data points (red lines superimposed on the black capacitance trace). Scale bar = 50μm.</p

Ca²⁺-augmented adiponectin secretion is abolished by cooling while secretion stimulated by cAMP alone is unaffected.

<p><b>A</b> Adiponectin secretion as fold-increase compared to control stimulated by 8-Br-cAMP (1 mM) alone or in combination with ionomycin (1 μM) during 30 min incubations at 23°C (blue) or 32°C (red). <b>B</b> As in <b>(A)</b> but using forskolin (10 μM) and IBMX (200 μM; forsk/IBMX) as a stimulator. Data are mean values ± S.E.M. of 11 experiments at each temperature in (A) and 7 (23°C) and 8 (32°C) in (B). <i>*P<0</i>.<i>05; **P<0</i>.<i>01</i>; <i>**P<0</i>.<i>001</i> vs. control at corresponding temperature. †<i>P<0</i>.<i>01</i> vs. 8-Br-cAMP or forsk/IBMX alone at 32°C; ǂ <i>P<0</i>.<i>05</i> vs. 8-Br-cAMP + ionomycin or forsk/IBMX + ionomycin at room temperature. The data in (B) at 32°C are the same as in Fig. 7B in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119530#pone.0119530.ref005" target="_blank">5</a>].</p

Ionomycin, but not 8-Br-cAMP, elevates adipocyte [Ca²⁺]_i.

<p>Example traces of [Ca²⁺]_i responses upon extracellular application of 1 μM ionomycin <b>(A)</b> or 1 mM 8-Br-cAMP <b>(C)</b>. <b>B</b> Average responses to ionomycin at indicated time points between 0 and 15 min. Ionomycin or 8-Br-cAMP was added extracellularly to the dish of cells and remained present throughout the recording as indicated. Note that the peak response to ionomycin shown in (B) was slightly shifted at 23°C (peak at 3.6 min; 5 separate experiments and 122 cells) compared to 32°C (peak at 3.1 min; 4 experiments and 107 cells). The trace in (C) is representative for 101 analysed cells in 4 separate experiments.</p

Proposed model of cooling effects on white adipocyte exocytosis.

<p>White adipocytes release adiponectin containing vesicles belonging to two functionally distinct populations. cAMP stimulates release of vesicles residing in a readily releasable pool in a temperature-independent manner. The Ca²⁺-dependent augmentation of secretion is reduced/abolished by cooling. Intracellular ATP is necessary for the Ca²⁺ effect. See text for more details.</p

Mathematical modeling of white adipocyte exocytosis predicts adiponectin secretion and quantifies the rates of vesicle exo- and endocytosis

Adiponectin is a hormone secreted from white adipocytes and takes part in the regulation of several metabolic processes. Although the pathophysiological importance of adiponectin has been thoroughly investigated, the mechanisms controlling its release are only partly understood. We have recently shown that adiponectin is secreted via regulated exocytosis of adiponectin-containing vesicles, that adiponectin exocytosis is stimulated by cAMP-dependent mechanisms, and that Ca2+ and ATP augment the cAMP-triggered secretion. However, much remains to be discovered regarding the molecular and cellular regulation of adiponectin release. Here, we have used mathematical modeling to extract detailed information contained within our previously obtained high-resolution patch-clamp time-resolved capacitance recordings to produce the first model of adiponectin exocytosis/secretion that combines all mechanistic knowledge deduced from electrophysiological experimental series. This model demonstrates that our previous understanding of the role of intracellular ATP in the control of adiponectin exocytosis needs to be revised to include an additional ATP-dependent step. Validation of the model by introduction of data of secreted adiponectin yielded a very close resemblance between the simulations and experimental results. Moreover, we could show that Ca2+-dependent adiponectin endocytosis contributes to the measured capacitance signal, and we were able to predict the contribution of endocytosis to the measured exocytotic rate under different experimental conditions. In conclusion, using mathematical modeling of published and newly generated data, we have obtained estimates of adiponectin exo- and endocytosis rates, and we have predicted adiponectin secretion. We believe that our model should have multiple applications in the study of metabolic processes and hormonal control thereof