extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine

text:
Elevated autocrine EDIL3 protects hepatocellular carcinoma from anoikis through RGD-mediated integrin activation

Background A remolded microenvironment in hepatocellular carcinoma (HCC) caused by abnormally expressed matricellular proteins could promote HCC progression. The cell-matrix interactions mediated by integrins play an important role in tumor microenvironment. Epidermal Growth Factor-like repeats and Discoidin I-Like Domains 3 (EDIL3), an extracellular matrix (ECM) protein with angiogenic and anti-inflammatory effects, is abnormally highly expressed in HCC. Here we aim to analyze its expression in liver and HCC tissues, investigate the underlined mechanisms accounted for HCC progression. Methods EDIL3 expression level is examined in normal liver, cirrhotic liver and HCC at both mRNA and protein level. The association between EDIL3 and clinical outcomes is analyzed. The pattern of EDIL3 expression and location is examined using Immunofluorescence and ELISA. Overexpression or knock-down of EDIL3 in a panel of cell lines are subjected to assays related to proliferation, invasion, and anoikis to investigate the mechanisms of this matrix protein in HCC progression. Recombinant EDIL3 treatment is applied to confirm the results. Results Compared with normal liver and cirrhotic liver, EDIL3 is elevated in HCC. High level of EDIL3 protein is much more commonly in patients with larger tumor or portal vein tumor thrombus (PVTT) formation, associated with poor prognosis. EDIL3 is abundantly expressed in HCC cells and secreted by cancer cells. In vitro and in vivo studies indicate that EDIL3, probably in an autocrine manner, inhibits anoikis and promotes anchorage-independent growth of HCC cells. Further mechanistic studies suggest integrin ligation by EDIL3 and thus that the sustained activation of the FAK-Src-AKT signal is responsible for the anoikis resistance and anchorage independence. Both the administration of cilengitide, a RGD-containing integrin antagonist, and silencing of integrin αV, an important RGD-binding integrin, results in the blockade of anoikis-resistance induced by EDIL3. Conclusion Our study suggests that high levels of autocrine EDIL3 may contribute to a receptive microenvironment for the survival of detached HCC cells and may involve in cancer cell spreading. We also highlight the importance of interaction between EDIL3 and integrin αV and suggest disrupting the ligation of EDIL3 to integrins via RGD-blocking in selected patients may bear potential therapeutic value. Electronic supplementary material The online version of this article (doi:10.1186/1476-4598-13-226) contains supplementary material, which is available to authorized users.

Introduction
Hepatocellular carcinoma (HCC) is a lethal disease with high mortality due to the high rate of postoperative recurrence and metastasis [1]. Both genetic and epigenetic alterations [2] within the HCC and a remolded microenvironment [3,4] surrounding HCC affect the biological behaviors of HCC and thus result in different outcomes of HCC patients. Extracellular matrix (ECM) proteins are the main non-cellular components of the tumor microenvironment; many of them can interact with the tumor cell through direct binding to specific receptors or by modulating the activation of other cytokines [5,6]. Some reports have suggested that microenvironmental changes caused by abnormally expressed matricellular proteins may modulate HCC progression by affecting cell growth, migration, invasion, anoikis and metastasis; thus, targeting ECM proteins may have therapeutic value [3,[7][8][9][10].
Epidermal Growth Factor-like repeats and Discoidin I-Like Domains 3 (EDIL3), also known as DEL-1, is a secreted ECM protein that was firstly characterized in vascular morphogenesis [11]. Structurally, EDIL3 contains 3 EGF-like repeats and 2 discoidin-like repeats, with an RGD motif located in the second EGF-like repeat [11]. Three-dimensional crystal structures reveal a unique RGD finger, which is believed to be advantageous for its ligation with integrins over other RGD-containing ECM proteins [12]. EDIL3 has been intensively studied in vascularization, exhibiting strong angiogenic or vascularizing function through integrin αvβ3 modulation [13,14]. Moreover, this protein plays a role in modulating immunocyte adhesion through interactions with leukocytespecific integrins [15]. The role of EDIL3 in cancer is also revealed in several malignancies, albeit in observational level. For example in pancreatic carcinoma, EDIL3 is one of the SHH-dependent stromal factors that predict poor prognosis [16]; another study focusing on carcinogenesis of ulcerative colitis-associated colorectal cancer suggests EDIL3 may add some power in this process [17]; interestingly, EDIL3 could also be detected in exosomes of bladder cancer cells and facilitate cancer progression trough EGFR signal [18]. In the field of HCC, high-throughput genomic data suggests elevated EDIL3 in HCC compared with adjacent liver [19], and a clinical analysis reveals that EDIL3 might affect the prognosis of HCC [20]. However, no study has addressed how EDIL3 is involved in HCC development and progression.
Anoikis is a special apoptotic process resulting from the loss of or inappropriate cell adhesion. Upon detaching from the primary lesion, the lack of cell-ECM adhesion fails to maintain the pro-survival signals within cancer cells, leading to decreased anti-apoptotic signals, thus activating anoikis [21]. Gaining anoikis resistance is a prerequisite for intra-hepatic spreading and extra-hepatic metastasis of HCC. In addition to adaptive alterations, such as switch in integrin expression patterns or the hyperactivation of receptor tyrosine kinases, the abnormal microenvironment also helps the cancer evading anoikis [22]. Integrins are the key mediators of the cell-ECM interaction, sending pro-survival signals in the presence of ECM ligands, e.g., collagens and laminins, and inducing apoptosis in their unligated status [23]. EDIL3 is an important ligand for αV-coupled integrins via RGD recognition and has been shown to be able to bind these groups of integrins [24] and inhibit anoikis in the endothelium [25].
In the present study, we focus on the role of EDIL3 in HCC and demonstrate that EDIL3 is highly expressed in HCC patients. Moreover, the accumulation of tumorderived EDIL3 in the microenvironment promotes anoikis resistance and anchorage independent growth advantage through the activation of integrin signal pathways.

Results
The EDIL3 level is significantly elevated in HCC tissue and is associated with adverse biological behaviors To observe the expression change in HCC, we first tested the EDIL3 level in 5 normal livers (NL), 10 cirrhotic livers (CL), and 49 HCCs by qRT-PCR and western blot. The result revealed that EDIL3 was mildly expressed in NLs and CLs but was significantly elevated in HCC both at the mRNA and protein level (Figure 1 A, B, Additional file 1: Figure S1A). IHC stain of another array of samples including 3 NLs, 10HCCs and 6 PVTTs confirmed the high expression of EDIL3 in HCC compared with NL and CL ( Figure 1C). Notably, co-stain of EDIL3 and endothelium marker CD31 in HCC samples showed EDIL3 is not only stained with endothelium marker, but also widely and diffusively located in cancer cells clusters ( Figure 1C, D), suggesting that cancer cells becomes an important sources of EDIL3 besides endothelium.
We then explored the clinical value of EDIL3 in HCC by analyzing the protein level in another independent 202 HCC samples gathered on tissue microarrays (TMA). We observed approximately 72% (57/202) of patients were EDIL3-positive, and high levels of EDIL3 positivity was significantly associated with adverse clinicopathological parameters of HCC, including tumor size, PVTT formation, and TNM stage (Table 1). Moreover, Kaplan-Meier survival analysis in the 192 patients with follow-up demonstrated a higher postoperative recurrence risk and poor survival in patients with high EDIL3 expression. Because high EDIL3 expression was much more common in patients with PVTT, we examined EDIL3 protein levels in 6 PVTT samples. The result revealed that EDIL3 stain was very strong in all of the PVTT samples (6/6) as well as microscopic tumor thrombus ( Figure 1C), and this was validated by western blot ( Figure 1B). Taken together, these results reveal the high level of EDIL3 in HCC and suggest its association with adverse clinicopathological parameters and poor prognosis.

EDIL3 is abundantly expressed in HCC cell lines and exhibits unique expression pattern
We examined EDIL3 expression in 7 HCC cell lines and 2 non-HCC cell lines. The mRNA level of EDIL3 varied among all of the cell lines, with the HCC cell lines Huh-7 and CSQT-2 exhibiting much higher mRNA levels of EDIL3 (Figure 2A), while THLE-3 and Lo-2, which derived from normal hepatocyte, also showed mild transcription. Surprisingly, inconsistent with mRNA, EDIL3 protein was detected at almost the same level in the cell lysates of all the cell lines ( Figure 2A) and the Immunofluorescence confirmed the existence of EDIL3 in cytoplasm of all 9 cell lines ( Figure 2C and Additional file 1: Figure S1). Indeed, we also observed this inconsistency in other cancer cell lines (Additional file 2: Figure S2). Because EDIL3 is an ECM protein, we examined the EDIL3 that was secreted in conditioned medium (CM). All cell lines were subjected to standardized condition (see methods for details), and the secreted proteins in the CM was analyzed by Western Blot and ELISA. Not surprisingly, the Huh-7 and CSQT-2 secreted the most EDIL3 into the CM, whereas there was only detectable EDIL3 in the CM of other cell lines (Figure 2A, B). These observations in cell lines reveal a unique autocrine expression pattern of EDIL3, and the high level of EDIL3 in CSQT-2, a PVTT derived cell line, is consistent with the observation in patient's samples above.

Autocrine EDIL3 promotes anoikis resistance and anchorage-independent growth in HCC cells in vitro
Anoikis resistance and invasion are two important features of the HCC cells that are prone to forming PVTT or metastasis. We continued to investigate the effect of EDIL3 on tumor cell invasion and anoikis. A eukaryotic expression system suitable for secreted protein synthesis was used to generate recombinant EDIL3 with high fidelity (Additional file 3: Figure S3). SMMC-7721 and MHCC-97H, two cell lines with low basal levels of autocrine EDIL3, were chosen to undergo functional assays in the presence of EDIL3. EDIL3 did not affect invasion or proliferation (Additional file 2: Figure S2B). However, using WST-8 and Caspase3/7 activity assay, we found EDIL3 significantly lowered the anoikis rate. EDIL3 treatment reduced the Caspase3/7 activity and increased the viability in cancer suspended in poly-hema coated dishes for long to 72 h ( Figure 3A-D). In soft agar clone formation, EDIL3 also significantly promoted the anchorage-independent growth in SMMC-7721 and MHCC-97H cells ( Figure 3E). These effects above are both dose-dependent, with the 50 nM safely reaching significance.
(See figure on previous page.) Figure 1 EDIL3 is significantly up-regulated in HCC and PVTT compared with normal liver (NL) and cirrhotic livers (CL) and is closely related to the prognosis of HCC. A, The transcriptional level of EDIL3 is measured via qRT-PCR in 5 NLs, 10 CLs and 49 HCCs. The relative mRNA level is normalized to β-actin and is presented as Δ-ΔCq. The mRNA of EDIL3 in HCC is significantly higher than both NL and CL; B, The protein level of EDIL3 in NL, CL, HCC and PVTT was examined by western blot with β-actin as a loading control. The EDIL3/β-actin densitometry is performed and shown as density value below. EDIL3 is mildly expressed In NL and CL while significantly elevated in HCC and PVTT; C, Representative pictures demonstrating EDIL3 staining in different liver samples including NL, CL, HCC, PVTT and microscopic thrombi by IHC. Scale bars, 50 μm or 150 μm; Arrow heads indicate that high EDIL3 expression is largely localized to cancer cells. D, Confocal microscopic observation of immunofluorescence staining of CD31 (green), an endothelium marker, and EDIL3 (red) in HCC samples show EDIL3 is not only localized with endothelium, but also widely and diffusively located in cancer cells clusters. White arrows indicates the endothelium; Grey arrows indicates the cancer cell cluster. E, Kaplan-Meier analysis of overall survival between EDIL3-negative or moderately positive patients and highly positive patients shows a significant survival advantage in EDIL3 low express group. **: P < 0.01. The bold numbers represent the P-values with significant differences.
Furthermore, we transfected SMMC-7721 and MHCC-LM3 with overexpression vectors. Elevated mRNA of EDIL3 did not increase intracellular EDIL3 levels in western blot; however, we did detect an elevation of autocrine EDIL3 by ELISA and western blot of CMs ( Figure 4A). SMMC-7721 overexpressing EDIL3 exhibited a significant advantage of anoikis resistance and anchorageindependence compared with control cells ( Figure 4B-E) without affecting the proliferation and invasion (Additional file 2: Figure S2C). Importantly, the addition of EDIL3 in control cells partly mimicked, albeit to a lower degree, these effect ( Figure 4B-E). We obtained similar results in MHHC-LM3, a cell line that also exhibited a low basal level of autocrine EDIL3 (Additional file 4: Figure S4A-E).
We tested whether knocking down EDIL3 would cause the opposite effects. Using two shRNA sequences, we significantly suppressed the mRNA level of EDIL3, leading to a decrease in secreted EDIL3 in Huh-7 cells but no changes in intracellular EDIL3 in control cells (Additional file 5: Figure S5A). In agreement with the result from overexpression experiments, the decrease in EDIL3 in CM increased the caspase3/7 activity, lowered the WST-8 value and inhibited the colonies formed in soft agar, while adding recombinant EDIL3 partly rescued these inhibitory effects (Additional file 5: Figure S5B-D).
Taken together, these results indicate that EDIL3, especially secreted by cancer cells in our current study, is effective in protecting them from anoikis after suspension, thus supporting anchorage independent growth.

EDIL3 promotes HCC tumorigenesis in vivo
To further elucidate the functional role of anoikis resistance conferred by EDIL3 in vivo, subcutaneous tumor formation assays with EDIL3-overexpressing and control SMMC-7721 were performed in nude mice. The results clearly indicate that the size of tumor formed in SMMC-7721 with EDIL3-overexpression is significantly larger than the control cells in the 2-4 week after subcutaneous implantation ( Figure 5A, C). IHC staining and qRT-PCR confirmed the overexpression of EDIL3 in cancer cells ( Figure 5B, C) and revealed that tumors with high EDIL3 exhibited a much lower apoptosis rate by TUNEL assay, whereas PCNA, a proliferative marker, was dominantly positive in EDIL3-overexpressing tumors but only sporadically positive in control tumors ( Figure 5D). These results demonstrate that EDIL3 supports subcutaneous tumor formation.

Activation of FAK-Src-AKT signaling by EDIL3 ligation is associated with anoikis resistance
Because EDIL3 is a known ligand of integrins, we first examined whether HCC cell lines express receptors for EDIL3, such as integrin αV and α5. All of the cell lines used in this study expressed these integrins (Additional file 3: Figure S3B). We then examined the alteration of integrin-mediated signal pathways that were key to cell survival upon autocrine EDIL3. Indeed, we observed that EDIL3 activates signals downstream of integrins. During the 48 h of suspension, the pro-survival FAK-Src-AKT signal gradually faded away, suggesting the anoikis triggered by detachment. However, the FAK-Src-AKT signal was significantly sustained in EDIL3-overexpressing cells but tapered much more quickly in control cells ( Figure 6A). The administration of recombinant EDIL3 also elevated these signal pathways, although to a lesser degree than overexpression (data not shown). However, pretreating cells with RDG-blocker cilengitide reversed the activation of FAK-Src-AKT ( Figure 6B).

Disrupting integrin ligation restores anoikis susceptibility to HCC
Based on the results above, we continued to examine weather disrupting the EDIL3/ integrins interaction could affect the anti-anoikis effect of EDIL3. In first effort, Cilengitide, an RGD-containing integrinαV antagonist, almost abrogated the anti-anoikis conferred by EDIL3 overexpression by lowering the WST-8 value (and increasing casepase3/7) to the control cells. However, Cilengitide did not result in an obvious pro-apoptotic effect on control cells, which have low EDIL3 expression ( Figure 7A). In another effort, silencing of integrin αV by RNAi also led to a loss of protection of EDIL3. Surprisingly, the relative caspase3/7 intensity declined significantly with the down-regulation of integrin αV ( Figure 7B); indeed, we observed similar results in an integrin α5 knock down assay (data not shown). These results suggest that RGD binding of integrinαV or integrinα5 is crucial for EDIL3-induced anti-anoikis and that integrins alone or when unligated might activate apoptosis in detached cancer cells.
(See figure on previous page.) Figure 2 EDIL3 exhibits a unique expression pattern in cell lines. A, detailed analysis of EDIL3 expression in mRNA level, cell lysates and CMs of 7 HCC and 2 non-HCC cell lines demonstrated an approximate correlation between mRNA and secreted EDIL3 in CMs, whereas EDIL3 in cell lysates exhibited almost same intensity. Notably, Huh-7 and CSQT-2 exhibited a much higher level of secreted EDIL3. B, ELISA assay testing the EDIL3 in CMs of 9 cell lines validates the secreted EDIL3 level is approximately correlated with mRNA level. C, Immunofluorescence staining of EDIL3, F-actin and DAPI in confocal microscope shows EDIL3 is localized within cells and at almost the same intensity in all the 9 cell lines under test, despite their varied mRNA level.

Discussion
This study provides a clear expression pattern of one ECM protein, EDIL3, in clinical samples and highlights the role of autocrine EDIL3 in the integrin-mediated interaction between HCC and ECM, which resulted in an antianoikis and anchorage-independent growth advantage. Our results also suggest a potential therapeutic strategy for personalized medicine by targeting ECM-integrin interactions for cancer cells in a subgroup of HCC patients.
In tissue level, EDIL3 is dominantly expressed and secreted by the endothelium, acting as a regulator of vascularization, immunocyte-endothelium adhesion and platelet microparticle clearance [14,15,26], or by a subset of macrophages to mediate engulfment [27]. Our data reveal that EDIL3 expression is turned on in adult human normal liver (cells) and is amplified during malignant transformation, which is different from mouse data, wherein EDIL3 is undetectable in the liver [15]. The cause of the up-regulation of EDIL3 in HCC is not clear; however, there is evidence suggesting that VEGF induces EDIL3 expression in malignant cells [28] and some inflammatory cytokines in endothelium [25]. Therefore it is possible that EDIL3 is elevated in response to cytokines in HCC. However, other events, such as HBV incorporation or epigenetic modifications, may also affect EDIL3 expression. Meaningfully, by analyzing an adequately sized sample, we demonstrated that when EDIL3 protein is accumulated to a high level, it is associated with larger tumor size and more PVTT formation, as well as greater relapse risk and shorter overall survival, which is consistent with a previous study, although the previous report only reached a positive result in overall survival [20], most likely due to the sample size and difference in patients selection.
In cell lines, however, EDIL3 demonstrates a unique expression. By examining multiple HCC cell lines and normal hepatocyte derived cell line, we find EDIL3 is expressed by both the normal and malignant cell lines, suggesting EDIL3 may not be directly linked to the relative tumorigenicity of HCC cell lines. Interestingly, we reveal that intracellular EDIL3 protein is observed at almost the same level despite the large differences in transcription. This inconsistency in transcription and translation was explained by the difference in secreted EDIL3. Moreover, either overexpressing or knocking-down the transcription level of EDIL3 only affected the secreted EDIL3. Based on these observations in cell lines and human samples, we postulate that EDIL3 is maintained in a standby status by many types of cells in quiescent (or normal) conditions and is secreted upon elevated transcription in physiologic or pathophysiological conditions; notably, the secretion is probably key to its biological functions. However, it will also be interesting to see the effects of EDIL3 knock-out in malignancies, and the potential regulation of EDIL3 secretion.
EDIL3 is well documented as an integrin ligand, modulating a wide range of biological processes that require integrins, such as integrin αV in angiogenesis and integrin αL in the neutrophil adhesion cascade [13,29]. Because integrins such as integrin αV are also abnormally expressed in HCC [30], we postulated that EDIL3 may affect HCC cells through integrin ligation when it is secreted and anchored on the cell membrane, or deposited in the ECM. By overexpressing EDIL3 or recombinant EDIL3 treatment, we observed that secreted EDIL3 reduced the likelihood of anoikis in cancer cells and promoted anchorage-independent growth, both of which are indispensable for HCC spreading and PVTT formation, whereas do not affect proliferation or invasion. Indeed, all of the fresh PVTT samples in our study exhibited a very high level of EDIL3 protein, so it is highly possible that tumor cells bring this protective protein when leaving primary lesion, thus assisting PVTT formation. Similar strategies to overcome anoikis have been reported in other types of cancer, in which another ECM protein, collagen IV, is expressed [31].
The anoikis is a complex apoptosis process resulted from cell detachment from ECM or inappropriate cell-cell adhesion. The lack of ECM contact or the engagement with inappropriate ECM leads to the activation of anoikis from death receptors (extrinsic pathway) or mitochondria (intrinsic pathway) [32]. Integrins on the cell surface are crucial for anoikis modulation by modulating both the extrinsic and intrinsic pathway, passing opposite signals in their ligated or unligated status [21,23,33] or even independent of their ligation status [34]. The 397 FAK-416 Src-473 AKT axis is a well-documented pro-survival pathway in anoikis resistance [32,[35][36][37]. Our results suggest that this axis is sustained when cancer cells are (See figure on previous page.) Figure 3 Treatment of EDIL3 contributes to anoikis resistance and anchorage-independent growth advantage in HCC. A, Different concentration of recombinant EDIL3 is used to treat SMMC-7721 suspended in poly-hema coated dishes. It sustains the viability of SMMC-7721 as demonstrated by caspase3WSt-8 assay in a time and dose dependent manner compared with control protein. B, Recombinant EDIL3 ameliorate the anoikis compared with control protein in SMMC-7721 as demonstrated by caspase3/7 intensity assay in a time and dose dependent manner. C-D, MHCC-97H is subjected to the assays same as SMMC-7721 and obtain consistent result. E, Administration of recombinant EDIL3 increases the anchorage-independent growth of SMMC-7721 and MHCC-97H cells in soft agar compared with control protein in a dose dependent manner. The assays last for 28 days. *: P < 0.05; **: P < 0.01. located in an environment rich with EDIL3, whereas phosphorylation of all three effectors was partly inhibited when RGD binding sites were blocked by cilengitide, suggesting that RGD recognition is critical for this pro-survival signal. Another interesting finding is that the suppression of integrin αV (or α5) expression led to a strong anoikis resistance, suggesting that unligated integrin may trigger anoikis, which is supported by other studies [38,39]. However, intriguingly enough, integrin αV or α5 expression by cancer cells is required for invasion and spreading [40][41][42]. This seemly contradictory role of integrins reflects the importance of a suitable interaction between cancer cells and the microenvironment, or ECM, at the suitable stage of tumor progression; in this study, an EDIL3-integrin alliance may be exploited by detached HCC cells to escape anoikis when cells detach from the primary lesion, which resulted into a higher success rate of metastasis.
Finally, a variety or integrin inhibitors has entered clinical trial of different types of cancers [33,43]. However, the outcomes have fallen short of expectations, revealing the complexity of integrin function as discussed above. Therefore, refined patient selection is required. Cilengitide was initially documented as an angiogenesis and invasion inhibitor [44]. However, our preliminary study suggests a potential role of cilengitide in inhibiting anoikis resistance in EDIL3-overexpressing HCC, thus pointing to a new potential indication of integrin inhibitors in HCC and providing a reference for patient selection.

Conclusion
Our study suggests that high levels of autocrine EDIL3 may contribute to a receptive microenvironment for the survival of HCC cells and therefore assist cancer cell progression, which lead to adverse clinical results. Although other explanations may be concealed in our current effort, the anti-anoikis conferred by high level of EDIL3 is an important mechanism for through which EDIL3 provide protection for HCC cells. Disrupting the ligation of EDIL3 to integrins via RGD-blockers or inhibitors in selected patients with high EDIL3 may bear potential therapeutic value.

Methods and materials
Cell culture The HCC cell line Sk-Hep1 was purchased from the American Type Cell Culture Collection (ATCC), HuH-7 was purchased from RIKEN Resource Centre, Japan, SMMC-7721 was purchased from the Cell Bank of the Chinese Academy of Sciences Cell Bank of Type Culture Collection. The metastatic HCC cell lines MHCC-97 L, MHCC-97H and MHCC-LM3 were obtained from the Liver Cancer Institute, Zhongshan Hospital, Fudan University. The HCC cell line CSQT-2 derived from a portal vein tumor thrombus (PVTT) has been described elsewhere [45]. Human liver epithelial cell line THLE-3 was purchased from ATCC;. All cell lines except THLE-3 were cultivated in DMEM (Dulbecco's modified Eagle medium) supplemented with 10% (v/v) fetal calf serum at 37°C in a humidified incubator under 5% CO 2 condition. THLE-3 was cultivated in BEGM (Lonza) with the addition of BEGM bullet kit according to the instruction on ACTT.

Sample information
All samples were collected in Department of Liver Surgery, Renji Hospital, Shanghai Jiaotong University School of Medicine. Fresh samples, including tumor tissues and PVTTs, were obtained from HCC patients during tumor resection. Normal livers and cirrhotic livers were collected from healthy liver donors and cirrhosis patients, respectively, during liver transplantation. Approximately 202 HCC samples were collected from 2004 to 2010 and were constructed into tissue microarray (TMA). The median age of this cohort of patients was 50 years (range 17-73 years). The majority of patients are HBV-positive (187/202). The follow-up was ended in December 2012, and the median period was 33 months (range 2-90 months). All human samples were obtained with informed consent, and protocols were approved by the ethical review committee of the World Health Organization Collaborating Center for Research in Human Production (authorized by the Shanghai Municipal Government).

Quantitative real-time PCR
Total RNA was extracted using Trizol reagent (Takara) and reverse transcribed using PrimeScript qRT-PCR kit (See figure on previous page.) Figure 4 Overexpression of EDIL3 in SMMC-7721 cells significantly increases the anoikis resistance and anchorage independence. A, Overexpression of EDIL3 is validated by real-time PCR, western blot and ELISA assay, confirming that the increase in transcription leads to a higher autocrine EDIL3. B, EDIL3-overexpressing SMMC-7721 forms more clones in soft agar compared with control cells, which can be partly mimicked by recombinant EDIL3 (50 nM). C, After being suspended for up to 72 h, the overexpressing group demonstrates a high WST-8 value compare to the control cells at 48 h and 72 h, and administration of recombinant EDIL3(50 nM) in control cells partly mimics the effect induced by EDIL3 overexpression. D, Caspase3/7 intensity assay validate that the difference in cell viability between the two groups is caused by lower apoptosis induction. E, Annexin V/PI stain by FACS is performed to validate the apoptosis result. EDIL3 overexpression lowers both the early phase and later phase apoptosis percentage and recombinant protein partly mimics this effect. *: P < 0.05; **: P < 0.01.
(Takara) according to the protocol. Quantitative real-time PCR analyses were performed with SYBR Premix Ex Taq (Takara) on a 7300 Real-time PCR system (Applied Biosystems Inc.), and the primer for EDIL3 was as follows: forward, GTGAACTGTCGGGTTGTTCTGAG; and reverse, 5′-GGTTCCCAAGTGAACATGTCCAT-3′. The primers for ACTB were as follows: forward, 5′-TCACCCACACTGTGCCCATCTACGA-3′; and reverse, 5′-CAGCGGAACCGCTCATTGCCAATGG-3′. The relative expression of EDIL3 was analyzed by the comparative cycle threshold method (ΔΔCt method), which was normalized to ACTB. week and resected after 6 weeks. A, After 6 weeks, EDIL3-overexpressing group shows relatively larger tumors compare with control group. B, qRT-PCR of 5 tumors in each group demonstrates a significant elevation in mRNA level. C, Tumor growth curve reveals a shorter latency in the EDIL3-overexpressing group (2 weeks) vs. the control group (4 weeks) and tumors in the EDIL3-overexpressing group are significantly larger than control group from 2 nd week to 4 th week. D, IHC stain in the same region of the tumor confirms the overexpression of EDIL3 and suggests more active proliferation (PCNA) and lower apoptosis (tunel) upon EDIL3 overexpression. **: P < 0.01.

Protein collection, Western blot and antibodies
The HCC samples were handled with T-Per tissue protein extraction reagent (Thermo Scientific) according to the manufacturer's protocol. Total cell protein was obtained by IP lysis buffer (Beyotime) for further assays. The secreted proteins in conditional media were collected by ethanol precipitation. Briefly, when cells grew to approximately 80% confluence, normal media were replaced by serum-free media. After 24 h, the media were collected, and 95% ethanol was added and kept overnight. The precipitated proteins were collected with SDS loading buffer and underwent standard western blot immediately. Western blot was performed using SDS-PAGE gel for proteins separation and nitrocellulose membrane for proteins blotting. The total volume of protein used in WB is 50 μg for tissue sample assays, and 30-60 μg in cell line assays. The signal was detected by the Odyssey infrared system (LI-COR).

Immunohistochemistry stain and analysis
A total of 202 samples of HCC on 2 TMA slices, 3 NLs, 10 HCCs and 6 PVTTs are subjected to IHC. Paraffin-embedded sections 5 μm thick and TMA were stained according to standard Immunohistochemistry (IHC) protocols. Heat-mediated antigen retrieval in pH 6.0 citric acid was performed. The antibodies used here were EDIL3 (ProteinTech). The scoring of EDIL3 was performed according to the ratio and intensity of positive-staining cells: 0-5% scored 0; 6-35% scored 1; 36-70% scored 2; and more than 70% scored 3. The final score was designated as negative (score 0), positive (score 1 or 2) and high positive (score 3). These scores were determined independently by two experienced pathologists in a blinded manner.

Enzyme-linked immunosorbent assay
HCC cell lines were planted on 10 cm petri dish until 90% confluence, then were incubated in 3 ml serum free media for 48 hour. At the end of the incubation period, the conditional media were harvested and stored at -80C until use. The secreted EDIL3 in media were quantified using ELISA kits according to the manufacturer's instructions (CUSABio).

Immunofluorescence staining
In cell assay, all the HCC cell lines under tests were seeded on cover slides in 24-well plates and incubated overnight. For F-actin staining, cells were incubated with phalloidin-FITC (Sigma-Aldrich) for 75 minutes at room temperature. For EDIL3 staining, cells were incubated with primary antibodies against EDIL3 (Abnova) for 75 minutes at room temperature, followed by an Alexa Fluor 594-conjugated secondary antibody. In tissue staining, two samples embedded in paraffin were subjected to heat-mediated antigen retrieval in pH 6.0 citric acid, and blocked by 10% BSA. The primary antibodies used are EDIL3 (Abnova) and CD31 (Abcam) with Alexa Fluor 594-conjugated secondary antibody against EDIL3 and Fluor 488 against CD31. Immunofluorescence signals were captured using confocal micro-scopy (LSM 510, METALaser Scanning Microscope, Zeiss).

Recombinant EDIL3 expression, purification and characterization
Human EDIL3 (NM_005711 Origene) with the signal peptide truncated were cloned into the episomal expression vector pCEP-Pu-Strep II-tag (N-terminal) in-frame with the sequence of the BM-40 (SPARC/osteonectin) signal peptide downstream of the CMV promoter, which has been described elsewhere [46]. Briefly, the expression vector was transfected into human embryonic kidney 293/Epstein-Barr nuclear antigen cells (EBNA-293) by Xetrem (Roche). The cells were screened with puromycin, and the surviving cells were cultured on a large scale. The culture medium was collected and centrifuged, and the supernatant was subjected to the StrepTactin sepharose column (IBA) for purification. The collected proteins were quantified and validated by Coomassie Brilliant Blue (CBB) staining and western blot. The empty vector was applied as control proteins.

Establishment of stable overexpression or knock-down cell lines
For the overexpression of EDIL3 in cell lines, the full-length cDNA of EDIL3 (NM_005711 Origene) was subcloned into pCDH-CMV-MCS-EF1-Puro vector (System Biosciences). Lenti-virus was packaged in 293 T cells using Lipofecta-mine2000 (Invitrogen), and the virus DNA was transduced into cell lines. For knock-down of EDIL3, short hairpin RNA (shRNA) sequences (Sh1:5′-CCGGCCCAAGTTTGT CGAAGACATTCTCGAGAATGTCTTCGACAAACT TGGGTTTTTG-3′; Sh2: 5′-CCGGGGAGGTTGCA (See figure on previous page.) Figure 7 Disrupting integrin-EDIL3 ligation deprives HCC of anoikis resistance induced by EDIL3 in SMMC-7721 and MHCC-LM3. A, Cilengitide (10 μM) reduces the WST-8 value and increased casepase3/7 of the EDIL3 overexpressing group back to levels of control group, suggesting a blockage of EDIL3's effect. There was not any effect observed in the control group in WST-8 and caspase3/7 assay. Both the two cell lines show consistent results. B, when integrin αV was knocked down to a very low level in both cell lines by two siRNAs (Si2 and Si3), the alteration in WST-8 and casepase3/7 value of EDIL3-overexpressing cells compared with control cells disappear. Interestingly, the WST-8 value and casepase3/7 intensity results suggest the overall anoikis significantly declined upon integrin αV silencing in both siRNA knock-down groups compare with si-control group. **: P < 0.01. TCAGATGAAGACTCGAGTCTTCATCTGATGCAA CCTCCTTTTTG-3′) targeting EDIL3 were cloned into pLKO.1-puro vectors (Roche). ShRNAcontaining plasmids were packaged into lentiviruses and transduced into target cell lines as above. The efficiency of over-expression or knockdown was tested by qRT-PCR and western blot.

Anoikis assay
Anoikis was induced by culturing cells in poly-HEMA coated plates as described by others [47]. Briefly, poly-HEMA were prepared as a 10 mg/ml solution in ethanol, which covered completely the petri-dish or plates, then dried and repeated once. Cells in serum-free medium were seeded into the coated plates with or without adding the recombinant EDIL3 at the corresponding concentration. To avoid survival effects caused by the clumping of cells, 0.5% methyl cellulose (Sigma-Aldrich) was added into the medium. At the designated time points, the suspended cells were collected and subjected to cell viability assays by WST-8 kit (Dojindo), apoptosis assays by Caspase3/7 Glo kit (Promega), and Annexin V/PI staining on FACS (BD) according to the protocols from the respective manufacturers.

Anchorage-independent growth assay
Colony formation in soft agar was tested to assay anchorage-independent growth. Stable overexpressing EDIL3 cell lines SMMC-7721 and MHCC-LM3 or their control cells were suspended in the upper layer consisting of culture medium with 1% FBS and 0.35% agar, which is above a basal layer of 0.6% agarose in 6-well plates in a triplicate manner. The cell density was 2,500 cells per well. The soft agars were fed twice a week with 0.3 ml of culture medium. In recombinant EDIL3 treatment assay, the soft agars were fed every 2 days with serum-free culture medium containing EDIL3 or empty vector. Colonies were stained with 0.05% crystal violet, and all the visible colonies were counted by microscopy after 21-28 days.

Tumorigenesis in vivo
A total of 1.0 × 10 6 of lenti-vector or lenti-EDIL3 SMMC-7721 cells were implanted subcutaneously into the right flank of 5 BALB/c (nu/nu) mice in each group. Tumor sizes were measured once a week, and mice were sacrificed for the analysis of tumor burden after 6 weeks. All procedures were performed in accordance with The Animal Care and Use Committee of Jiaotong University. The resected mice tumors were fixed in methanol and embedded in paraffin before being subjected to IHC using anti-EDIL3 (ProteinTech), anti-PCNA (Abcam) and anti-TUNEL (Millipore).

RGD-blocking assay and knock-down of ITGAV
Cyclic RGD-containing peptide cilengitide (Selleckchem) or control peptide RGE was used to block the ligation of EDIL3 to integrins on the surface of HCC cells at a final concentration of 10 μM. Briefly, the cells was suspended in Poly-HEMA coated plates and exposed to the peptides about 3 hours after suspension. Then, the cells were subjected to the assays as described above. SiRNA targeting ITGAV (Si2: 5′-GCCAGCCAAUUGAAUUUGATT-3′; Si3: 5′-CCCAGUUGUAUCUCACAAATT -3) was used to knock down the protein level of this integrin and was confirmed by western blot. After 72 h of transfection, the cells were subjected to the assays as above.

Statistical analysis
Statistical analyses were performed with SPSS 16.0 software. The association between EDIL3 expression and clinicopathological parameters was analyzed by Pearson Chi-Square test. Overall survival and relapse risk was calculated by the Kaplan-Meier method and compared by the log-rank test. Statistical significance was determined by two-tailed Student's t test for any differences observed (P < 0.05).

Additional files
Additional file 1: Figure S1. A, The protein level of EDIL3 in NL, CL, HCC and PVTT run on the same gel was quantified by EDIL3/β-actin densitometry analysis respectively. The density of NL or CL samples are significantly lower compare with HCC and PVTT. B, Immunofluorescence staining of EDIL3, F-actin and DAPI in confocal microscope shows EDIL3 is localized within cells and at almost the same intensity in the cell lines.
Additional file 2: Figure S2. The EDIL3 is widely expressed in other cancer cell lines. A, The EDIL3 expression pattern in 6 pancreatic cancer, 5 gastric and 2 endometrial carcinoma cell lines showed a difference mRNA level, while the protein level within cell was almost the same Neither EDIL3 overexpression or administration affects the invasion or proliferation of HCC cells. B,C, Neither administration of recombinant EIDL3 or EDIL3 overexpression led to a change of proliferation or invasion of SMMC-7721, which are examined by WST-8 assay and transwell-matrigel invasion assay respectively. Lenti-EDIL3-E2 stands for monoclonal overexpressing cells.
Additional file 3: Figure S3. A. Validation of purified recombinant EDIL3-StrepII protein by coomassie blue and western blot. Coomassie blue show a single brand, which validate the purity of protein. Both the EDIL3 antibody and StrepII antibody detect a single brand, and the intensity is consistent with concentration, which further validate the accuracy of the result. B, All the normal or HCC cell lines express integrinαV as shown in western blot.
Additional file 4: Figure S4. EDIL3 overexpressed by MHCC-LM3 significantly increases the anoikis resistance and anchorage independence. A, overexpression of EDIL3 is validated by qRT-PCR, western blot and ELISA, confirming the increase in transcription leads to a higher autocrine EDIL3 whereas no change within cell. B, EDIL3 overexpressing MHCC-LM3 formed more clones in soft agar compared with control cells, which can be partly mimicked by recombinant EDIL3 (50nM). C, After being suspended for 72 h, more cells in overexpressing group survived compared with control cells. D, caspase3/7 intensity assay validated the difference in survival between two groups was caused by lower apoptosis. E, Annexin V/PI stain by FACS was performed to validate the apoptosis result. *: P < 0.05; **: P < 0.01.
Additional file 5: Figure S5. Knocking down EDIL3 in Huh-7 increases the anoikis and suppresses the anchorage-independent growth. A, knocking down of EDIL3 by 2 shRNA was validated by real-time PCR, western blot and ELISA, confirming the suppression in transcription leads to a lower autocrine EDIL3. B, after being suspended for 48 h, both two knockdown group showed a less survived cell compared with control group. C, caspase3/7 intensity assay validated the difference in survival is due to more apoptosis in knock-down group. E, Annexin V/PI stain by FACS was performed to validate the apoptosis result. *: P < 0.05; **: P < 0.01.


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Ontogeny of adrenal-like glucocorticoid synthesis pathway and of 20α-hydroxysteroid dehydrogenase in the mouse lung

Background Glucocorticoids exert recognized positive effects on lung development. The genes involved in the classical pathway of glucocorticoid synthesis normally occurring in adrenals were found to be expressed on gestation day (GD) 15.5 in the developing mouse lung. Recently, expression of two of these genes was also detected on GD 17.5 suggesting a more complex temporal regulation than previously expected. Here, we deepen the knowledge on expression of “adrenal” glucocorticoid synthesis genes in the mouse lung during the perinatal period and we also study expression of the gene encoding for the steroid inactivating enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD). Results We performed an ontogenic study of P450scc, 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase 1 (3β-HSD1), 21-hydroxylase, 11β-hydroxylase, 11β-HSD1, and 11β-HSD2 expression up to post natal day (PN) 15. The substrate (progesterone) and the product (deoxycorticosterone) of 21-hydroxylase are substrates of 20α-HSD, thus 20α-HSD (Akr1c18) gene expression was investigated. In lung samples collected between GD 15.5 and PN 15, 11β-hydroxylase was only detected on GD 15.5. In contrast, all the other tested genes were expressed throughout the analyzed period with different temporal expression patterns. P450scc, 21-hydroxylase, 20α-HSD and 11β-HSD2 mRNA levels increased after birth with different patterns including an increase from PN 3 with a possible sex difference for 21-hydroxylase mRNA. Also, the 21-hydroxylase protein was observed by Western blot in perinatal lungs with higher levels after birth. Conclusion Progesterone is present at high levels during gestation and the product of 21-hydroxylase, deoxycorticosterone, can bind the glucocorticoid receptor with an affinity close to that of corticosterone. Detection of 21-hydroxylase at the protein level during antenatal lung development is the first evidence that the adrenal-like glucocorticoid synthesis pathway detected during lung development has the machinery to produce glucocorticoids in the fetal lung. Glucocorticoids from lung 21-hydroxylase appear to modulate lung ontogenesis through paracrine/intracrine actions.

Background
Glucocorticoids (GCs) play an essential role in fetal lung development [1,2]. They exert various effects including thinning of alveolar septa, increase in the number of type I pneumonocytes, decrease in pneumonocyte cell division, and stimulation of secretion of fibroblast paracrine factors that play a role in type II pneumonocyte maturation [3]. Glucocorticoid receptor-deficient mice were reported to die within a few hours after birth because of respiratory failure [4], showing the importance of GCs in the lung development process. The requirement for GCs in normal lung development was also demonstrated with a CRH knockout (KO) mouse model. CRH KO homozygous fetuses from CRH KO homozygous mothers were delivered normally at term, but died on the first day of life with an overall failure in lung development, except when GCs were administered to pregnant dams [2,5]. The overall failure of lung development was characterized by hypercellularity of the mesenchymal compartment and decreased expression of mRNAs associated with mature lung epithelial cells such as those of surfactant protein (SP)-A, SP-B and CCP10.
The endogenous GCs accessible to the developing lung originate from two sources. They can be provided by adrenals through the classical pathway of GC synthesis from cholesterol, or by the lung where cortisone or 11dehydrocorticosterone (11-DHC, rodent) can be reactivated to cortisol or corticosterone, respectively. In the rodent adrenals, cytochrome P450 side-chain cleavage (P450scc), 3βhydroxysteroid dehydrogenase/Δ 5 -Δ 4 isomerase (3β-HSD), 21-hydroxylase, and 11β-hydroxylase are involved in GC synthesis ( Figure 1). All these enzymes play a similar role in the adrenals of the human and many other non-rodent mammals, except that cytochrome P450c17 converts pregnenolone and progesterone into 17OH-pregnenolone and 17OH-progesterone, respectively. This leads to the production of the active GC cortisol instead of corticosterone. In peripheral tissues including the lung, GC synthesis was considered for many years to be limited to cortisol (human) and corticosterone (rodents) reactivation by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) [6] following their prior inactivation by 11β-HSD2. This perception is changing since genes normally involved in the classical adrenal pathway of GC synthesis were found to be expressed in some peripheral sites such as thymus, skin, and brain [7]. We are among the pioneers that have described expression of the adrenal genes outside adrenals [8].
In 2005, we reported that the four genes encoding for the enzymes catalyzing corticosterone synthesis from cholesterol and StAR were expressed in the fetal lung on gestation day (GD) 15.5 in the Balb/c mouse [8]. StAR is essential to this synthesis pathway since it plays a role in the intramitochondrial movement of cholesterol [9]. In fetal lungs studied up to GD 18.5, 11β-hydroxylase mRNA was found only on GD 15.5, which led to the initial conclusion that the adrenal pathway of GC synthesis should be expressed only on GD 15.5 in the developing lung. Further studies showed that 3β-HSD1 and 21hydroxylase were still expressed on GD17.5 [10,11]. This Figure 1 Glucocorticoid synthesis pathway in rodent and non-rodent mammals. Corticosterone (rodent) and cortisol (non-rodent mammals) are the most potent circulating GCs. Their production from cholesterol in adrenals correspond to the classical pathway of GC synthesis. There is no P450c17 enzyme in the rodent adrenal. Cell-specific 11β-HSD-catalyzed reactions occur in peripheral tissues. During the last decade, expression of some or all the enzymes catalyzing GC production from cholesterol has been observed in some non-adrenal tissues including the developing lung.
is important because the product of 21-hydroxylase, deoxycorticosterone (DOC), is known to bind the glucocorticoid receptor [12][13][14][15] with an IC 50 value of 70 nM compared to 60 nM for corticosterone [15]. Therefore, the impact of 21-hydroxylase on lung development may be exerted during a longer period, but the exact developmental time remains to be characterized.
The aim of the present study was to deepen the knowledge on expression of "adrenal" glucocorticoid synthesis genes in the mouse lung during the perinatal period and to study expression of the 20α-HSD gene encoding for a steroid inactivating enzyme active on c21 steroids. An ontogenic study of P450scc, 3β-HSD1, 21-hydroxylase, and 11β-hydroxylase expression in the mouse developing lung was performed up to post-natal day (PN) 15. Genes encoding for 11β-HSD1 and 11β-HSD2 were included for comparison. In addition, the expression of Akr1c18 gene, encoding for 20α-HSD, was included because 20α-HSD is active on the substrate (progesterone) and on the product (DOC) of 21-hydroxylase. This gene has never been studied in the lung. Because a sex difference in the timing of expression of the adrenal-like pathway of GC synthesis was proposed on GD 15.5 in the fetal lung [8], both sexes were studied separately. Finally, an ontogenic study of levels of 21-hydroxylase protein is also included.

Results
Expression of genes of the "adrenal" pathway of GC synthesis in the antenatal lung after GD 15.5 and in the post-natal developing lung The temporal and sexual expression profiles of P450scc, 3β-HSD1, 21-hydroxylase, and 11β-hydroxylase genes were determined using cDNA prepared from pooled male and female lung tissues (one pool/sex/litter) from GD 15.5 to PN 15. The 11β-hydroxylase transcript was detected at significant levels on GD 15.5, but not between GD 16.5 and PN 15 inclusively (data not shown). These data are in agreement with previously published data obtained between GD 15.5 and 18.5 [8,10], and on GD 17.5 [10]. Conversely, the P450scc, 3β-HSD1 and 21-hydroxylase genes were expressed after GD 15.5 at least up to PN 15 in the alveolar stage ( Figure 2). In fact, mRNAs were detected for these three genes in all analyzed samples, exhibiting specific temporal expression patterns. A two-way ANOVA showed that P450scc mRNA levels varied significantly according to developmental age, but not sex (Table 1). In fact, a significant 18-fold increase was observed from GD 15.5 to GD 19.5, followed by a significant 2.3-fold increase between PN 0 and PN 3, and a marked significant 28-fold decrease from PN 7 to PN 15 (Figure 2A-B). Expression of 3β-HSD1 did not show significant temporal or sexual regulation, although a slight significant trend for decreased expression was observed over developmental time ( Figure 2C-D and Table 1). A statistical analysis showed a significant influence of age but not of sex or sex*age on 21-hydroxylase expression (Table 1). Hence, 21-hydroxylase expression was stable from GD 15.5 to PN 3, presented a 4.3-fold increase between PN 3 and PN 7, and remained stable until PN 15 ( Figure 2E-F). Inter-litter variability was somewhat higher on PN 7 and PN 15 for 21-hydroxylase, which might explain why no statistically significant sex difference was found despite the fact that all the 6 litters of this period showed higher expression levels for females compared to males ( Figure 2E).

Sexual and temporal regulation of 20α-HSD expression in the antenatal and post-natal developing lungs
Progesterone and DOC inactivation can be achieved by 20α-HSD activity, which was observed in cytosolic fractions of adult mouse lungs [16] and in adult human lung slices [17]. However, an ontogenic study of 20α-HSD expression has never been reported in the developing lung. To perform this study, 20α-HSD mRNA levels were measured in mouse developing lung samples collected at various developmental times. Expression of 20α-HSD was observed in all samples analyzed from GD 15.5 to PN 15 ( Figure 3). A statistically significant variation over developmental time was observed (Table 1) including a marked post-natal increase until PN 3 for both sexes. While no significant sex difference was detected by twoway ANOVA, a male-specific decrease of 19-fold before birth was observed ( Figure 3). Expression levels of 20α-HSD were higher than those of 21-hydroxylase during near all the developmental period studied, with a difference of 5.4-fold in favor of 20α-HSD on PN 3.
11β-HSD1 and 11β-HSD2 expression profiles from GD 19.5 to PN 15 A qPCR study of 11β-HSD1 and 11β-HSD2 expression was reported in the fetal lung between GD 15.5 and 18.5 [8]. Here, the study covers the GD 19.5-PN 15 period. Only little changes were measured over developmental time for 11β-HSD1. In contrast, the 11β-HSD2 mRNA levels presented a marked statistically significant increase after birth ( Figure 4 and Table 1).

21-Hydroxylase protein during lung development
The levels of the 21-hydroxylase protein were analyzed by Western blotting of whole lung protein samples and of microsomal fractions. Our data show that not only the 21-hydroxylase mRNA but also the 21-hydroxylase protein are present in the perinatal lung ( Figure 5 and data not shown). Specific signals were higher in lung samples collected after birth compared to those obtained during gestation ( Figure 5). Because 21-hydroxylase is a microsomal protein, microsome preparations were also studied. The upper unspecific signal observed with total protein extracts ( Figure 5A) was undetected or close to the background with microsomal fractions ( Figure 5B). The most intense signals were observed with samples from PN 7 and PN 16. This increase in 21-hydroxylase protein levels correlates with the increase in mRNA levels described above ( Figure 2E-F). Intermediate protein levels were observed in samples from PN 3 (one individual) and PN 2 (pool from one litter). This can be explained by slight variations in mRNA levels from individual to individual or from litter to litter, as those observed in Figure 2E. No sex difference was observed at the specific developmental times tested, except on PN 15, when a slight difference in favor of females was observed (data not shown).

Discussion
This is the first ontogenic study performed in the developing lung up to PN 15 studying expression of the four genes normally involved in the adrenal pathway of glucocorticoid synthesis from cholesterol. 11β-hydroxylase was previously studied in the developing lung after GD 15.5 (from GD 15.5 to GD 18.5 [8] and on GD 17.5 [10]) and no detectable levels of 11β-hydroxylase mRNA were found after GD 15.5. Here, studying the developing lung at several time points from GD 15.5 to PN 15, we conclude that 11β-hydroxylase mRNA can be found only on GD 15.5 in the developing lung. In contrast, P450scc, 3β-HSD1 and 21-hydroxylase mRNAs were found in all the analyzed samples from GD 15.5 to PN 15. Moreover, the 21-hydroxylase protein was observed in all the analyzed samples from GD 15.5 to PN 16. This is important because the substrate of this enzyme, progesterone, is present during gestation while the product of this enzyme, DOC, has the ability to bind the glucocorticoid receptor [12][13][14][15] with an IC 50 value of 70 nM compared to 60 nM for corticosterone [15]. Therefore, the developing lung possesses the machinery to produce active glucocorticoids, at least during gestation, using circulating progesterone as a substrate.
A transient sex difference in 20α-HSD mRNA levels was observed on GD 19.5 in the developing lung, when the female values were 19-fold higher than the male values ( Figure 3). A sex difference in 20α-HSD expression was also reported in the mouse adrenal [18]. In that tissue, 20α-HSD expression was restricted to the transient X-zone located between the cortex and the medulla.
The presence of the 20α-HSD protein in adrenals was shown to correlate with the presence of the X-zone. On PN 10, no sex difference was observed in the lung in our experiments, whereas a sex difference was found in the adrenal where both 20α-HSD and the X-zone were absent specifically in the male tissue. Therefore, no relationship between the sex differences observed in the developing lung and in adrenals can be established. Sex differences in 20α-HSD mRNA levels with higher levels for females were also observed in the skin, liver, and kidney obtained from adult mice [19]. It was suggested that 20α-HSD may play a role in reducing intracellular levels of progesterone originating from the circulation [19]. In the lung, except on GD 19.5, no significant sex difference was observed in 20α-HSD mRNA levels, which is in line with the absence of sex difference in perinatal progesterone levels in rodents [20]. However, progesterone levels are decreasing in late gestation to reach very low levels after birth, which is not the case for 20α-HSD expression.
The expression of 20α-HSD in the developing lung is compatible with a function of this enzyme in modulation of progesterone receptor occupancy, but our data also suggest that another function should exist in the lung. Indeed, we observed a marked increase in 20α-HSD expression after birth in both sexes. Interestingly, this increase just preceded the increase in 21-hydroxylase expression. Therefore, we propose that 20α-HSD could play a role related to the glucocorticoid synthesis pathway. This enzyme can inactivate both the substrate (progesterone) and the product (DOC) of the 21-hydroxylase [18]. Thus, the lung 20α-HSD activity should influence glucocorticoid receptor occupancy.
The coexistence of GC synthesizing and inactivating activities in the developing lung strongly suggests regionalization of GC action. Intracrine as well as paracrine GC  actions in the developing lung are compatible with our data. The concept of intracrine action was first demonstrated for dihydrotestosterone in the prostate [21,22]. It is defined as the action of newly-formed active steroids within the cells that synthesized them. As shown in Figure 6, the 21-hydroxylase activity changes the affinity of the substrate from progesterone receptor to glucocorticoid receptor, while the 20α-HSD activity controls the availability of active steroids (progesterone and DOC). Therefore, 20αHSD may be involved in the fine tuning of both progesterone receptor occupancy, through its impact on progesterone levels, and glucocorticoid receptor occupancy, through the regulation of the levels of the substrate (progesterone) and product (DOC) of 21-hydroxylase. This suggests an optimizing role for 20αHSD in lung development.
11β-HSD2 is expressed in aldosterone-selective target cells and prevents illicit occupation of mineralocorticoid receptor (MR) by GCs [23,24]. A role for mineralocorticoids is proposed in lung development [25] where coexpression of 11β-HSD2 and MR genes were found [26,27]. MR is proposed to be involved in the control of reabsorption of pulmonary fluid by ENaC, which is essential to the transition from uterine to aerial life at birth. However, ENaC is also modulated by glucocorticoids in the developing lung [28,29]. The increase in 11β-HSD2 mRNA observed in our experiment occurred after parturition and correlated well with the beginning of septation. Therefore, we propose that the process of septation may require a greater specificity of MR to mineralocorticoids.

Conclusion
Our data show that expression in the developing lung of most of the genes involved in the adrenal-like glucocorticoid synthesis pathway is maintained beyond GD 15.5. The gene expression results are compatible with corticosterone formation on GD 15.5, and DOC formation after GD 15.5, both steroids having the capability to bind the glucocorticoid receptor. The presence of the 21-hydroxylase protein showed that the fetal lung contains the machinery catalyzing DOC formation from progesterone, which is present in the fetal circulation. Co-expression of 21-hydroxylase and 20α-HSD in the developing lung strongly suggests that this tissue does not act as a classical endocrine gland, but is rather a site of intracrine/paracrine actions.

Animals and tissue preparation
The protocol and procedures were approved by the animal care and use committee and the institutional review board of the Centre de Recherche du CHU de Québec (CPA-CRCHUQ; protocols no. 2005-156, 2008-071 and 2011-053). All animals were kept under a 12 h light/dark cycle and received water and feed ad libitum. Balb/c (Mus musculus) females at the estrus stage and males of the same strain were mated overnight (16 h). The day of copulatory plug was considered as GD 0.5 while the beginning of PN 0 corresponded to parturition. Pregnant females were kept into individual cages until sacrifice. All pregnant females were euthanized by CO 2 inhalation. Pups were sacrificed either by decapitation following hypothermia-induced anesthesia (GD 15.5 to PN 5), or by intra-peritoneal injection of Euthanyl (PN 6 to PN 16; pentobarbital sodium, Bimeda-MTC, Cambridge, ON, Canada). Sex of fetuses/neonates was determined by gonadal morphology. Fetal and neonatal lungs were individually flash-frozen for use in qPCR and protein extraction. Adrenals from dams were also collected and flash-frozen.

Real-time quantitative PCR of target and normalization genes
For each litter, flash-frozen tissues were pooled according to sex. Total RNA was extracted using Tri-reagent (Molecular Research Center, Cincinnati, OH, USA) according to the protocol of the manufacturer. Each RNA sample was purified on a CsCl gradient [30], using a TLA 120.2 rotor in an Optima MAX centrifuge (Beckman, Mississauga, ON, Canada). An aliquot of 4 μg of each total RNA sample was treated with DNase I (0.25 U/μg of RNA), and then reversed transcribed using Superscript II (Life Technologies, Burlington, ON, Canada) and hexameric random primers (pd [N] 6 , Life Technologies) for 50 min at 42°C in a final volume of 20 μl according to the protocol of the manufacturer. Reverse transcriptase was inactivated by heating at 70°C for 15 min. The resulting cDNAs were diluted tenfold before qPCR analyses. The same cDNA preparations were used for all of the analyzed genes. The qPCR reactions were performed on a LightCycler instrument (Roche, Montreal, QC, Canada) using LightCycler-FastStart DNA Master SYBR Green I kits (Roche). After qPCR enzyme activation (10 min, 95°C), PCR cycles were performed as follows: 5 Sec 95°C (denaturation); 5 sec at the annealing temperature; 20 Sec 72°C (elongation); 5 sec at the temperature of fluorescence intensity reading. The specific primer sequences and qPCR conditions are presented in Table 2 Figure 6 Effects of 21-hydroxylase and 20α-HSD on progesterone receptor and glucocorticoid receptor occupancy. PROG, progesterone; PR progesterone receptor; GR, glucocorticoid receptor. for: Cyp11a1 (P450scc), Hsd3b1 (3β-HSD1), Cyp21a1 (21-hydroxylase), Cyp11b1 (11β-hydroxylase), Akr1c18 (20α-HSD), Hsd11b1 (11β-HSD1), Hsd11b2 (11β-HSD2), Gapdh (glyceraldehyde-3-phosphate dehydrogenase), and Hmbs (hydroxymethylbilane synthase, transcript variant 1). For each gene, the relative expression levels were calculated with a standard curve prepared using serial dilutions of an amplicon in 0.1X reverse transcription buffer. The use of this buffer for dilutions ensured comparable qPCR efficiencies between standard curve samples and lung cDNA samples, as reported [31]. Hmbs and Gapdh expression levels were obtained for all the samples and fed in the GeNorm program to generate a normalization factor for each sample [32]. For all the genes, the mean value obtained on GD 19.5 was fixed as onefold. The suitability of Hmbs and Gapdh as reference genes for normalization of developing lung samples had previously been confirmed [33].
For microsomes preparations, samples were homogenized by hand in an all-glass Dounce homogenizer fitted with a B-pestle in ice-cold buffer containing 20% (vol/ vol) glycerol, 1.0 mM EDTA, and 4 mM potassium phosphate, pH 7.0 (KPBS). Homogenates were centrifuged at 12,000 × g for 20 min to remove cell debris. The resulting supernatants were centrifuged at 105,000 × g for 60 min. The pellets were resuspended in KPBS and centrifuged again at 105,000 × g for 60 min. The resulting pellets were resuspended in the Triton X-100-containing extraction buffer described above, and saved as micro somes-enriched extracts. Protein concentrations in microsomal fractions were determined as described above.

Statistics
The effects of age and sex on target gene expression data was assessed by a randomized-block 2-way ANOVA, male and female pools of the same litter being matched. The age of the studied litters was considered as a fixed factor. To ensure that assumptions of normality and homogeneous variances were respected, the dependent variable (expression) was either untransformed (P450scc, 20α-HSD), transformed into its natural log (21-hydroxylase, 11β-HSD1 and 11β-HSD2) or transformed into its reciprocal (3β-HSD1). For each gene, comparisons between the mean expression values (calculated with data from both sexes) at specific age-points were performed using the Bonferroni test on selected pairs. The differences were considered statistically significant when p < 0.05, whereas for 0.1 > p > 0.05, the differences were considered to present a significant trend.


The Emerging Role of the Hematopoietic Stem Cell Niche in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPN) are chronic hematological neoplasms arising from the accumulation of genetic mutations in hematopoietic stem cells (HSCs). This notion is supported by genomic studies on MPN patients, in which recurrent mutations on genes such as JAK2, CALR, and MPL have been identified. Interestingly, recent studies using MPN mouse models have demonstrated that the HSC niche, where HSCs are located and maintained in the bone marrow (BM), plays an important role in MPN initiation and progression. Changes in the BM microenvironment alone can serve as a driver for altered hematopoiesis and hematological malignancies. Additionally, during MPN progression mutant HSCs remodel the HSC niche into a hospitable microenvironment for themselves that accelerates disease progression, creating a positive feedback loop where mutant HSCs thrive, and continue to alter the BM microenvironment to their advantage. In an attempt to provide a fresh perspective in understanding MPN pathogenesis, this review highlights the studies conducted that explore the role of the HSC niche in MPN pathogenesis and progression.

Introduction
Myeloproliferative neoplasms (MPN), previously known as myeloproliferative disorders (MPD), are chronic hematological neoplasms that are characterized by the excessive production of one or more subtypes of myeloid cells [1]. The myeloid cells involved in MPN, including red blood cells, platelets, and myeloid leukocytes such as granulocytes and monocytes, are derived from the hematopoietic stem cells (HSCs) and myeloid progenitors [2]. Thus, most studies on MPN have been concentrated on studying recurrent mutations in HSCs during the disease pathogenesis. Interestingly, HSCs are tightly regulated by both intrinsic gene expression and extrinsic cues from the bone marrow (BM) microenvironment [3], and recent studies on mouse models have shown that the latter may also play a role in MPN pathogenesis. The BM microenvironment refers to the stem cell niche in the BM where HSCs reside and their stemness maintained [4]. The HSC niche is composed of a variety of cells in the BM, including perivascular stromal cells (Nestin+ cells and Cxcl12-abundant reticular cells), sinusoidal endothelial cells, osteoprogenitors, osteoblast, adipocytes, and non-myelinating Schwann cells [5,6]. The vast majority of studies on MPN suggest that MPN development and progression is driven solely by genetic mutations in HSCs, and these mutations are believed to give autonomous proliferative advantage in certain lineages of blood cells [7]. However, several studies have recently suggested that along with HSC mutations, changes in the HSC niche is also an important regulator in MPN development and progression.

MPN as a disease of mutations in HSCs
A number of genomic studies examining MPN patients have identified recurrent somatic mutations that lead to constitutive activation of hematopoietic signaling pathways [8]. For example, Philadelphia chromosome resulting from the translocation of ABL on chromosome 9 and BCR on chromosome 22 has been found in over 95% of chronic myeloid leukemia (CML) patients, being a most important criteria in the diagnosis of CML, a subgroup of MPN [9,10]. In the BCR-ABL negative MPN, JAK2 V617F is detected in approximately 95% of patients with polycythemia vera (PV), and 50-60% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients [11,12]. Thrombopoietin receptor mutation MPL W515L/K has also been identified in 3% of ET and 10% of PMF patients [7]. However, not all recurrent somatic mutations are directly tied to growth signaling pathways. Recently, a set of recurrent mutations on a gene encoding an endoplasmic reticulum chaperone, Calreticulin (CALR), have been identified in 25-27% of patients with ET and 14%-35% of patients with PMF [13][14][15]. Although the overexpression of mutant CALR resulted in elevated STAT5 activation [14], the function of CALR and its connection with JAK/STAT signaling has not been well characterized [7].
The functional validation of these mutations has been demonstrated in mouse models. For example, transplantation of BCR-ABL HSCs leads to CML and serves as a well-established mouse model for this disease [16]. Conversely, transgenic and knock-in mouse models that express JAK2 V617F in HSCs displayed various levels of MPN-like phenotypes [17]. To date, no studies have examined the effect of CALR mutations in the hematopoietic system on HSC physiology and MPN development, which could provide interesting insight into the role of this ER chaperone.

The emerging role of HSC microenvironment in MPN progression
As discussed above, most studies suggest that MPN is a clonal stem cell disease resulting from lesions on HSCs [18]. However, the intrinsic gene expression and the extrinsic microenvironment are two indispensible parts in regulating HSC activity. Therefore, this raises the question of whether the BM microenvironment plays a role in MPN progression.
In fact, an increasing number of studies have suggested that there is indeed an important role of the HSC niche in MPN development and progression. Mouse models have demonstrated that while genetic mutations are still believed to be the driver of hematological cancers, it is possible to induce MPN-like phenotypes or even leukemia by simply altering the BM microenvironment [19][20][21]. For example, retinoic acid receptor γ (RARγ) is a highly conserved nuclear hormone receptor that acts as a ligand-dependent transcriptional regulator and regulates HSC cell fate [19]. RARγ-deficient (RARγ -/-) mice developed a myeloproliferative syndrome with significantly elevated numbers of granulocytes in the BM, spleen, and peripheral blood, and BM transplantation demonstrated that the effect solely resulted from the RARγ -/-BM microenvironment [20]. More direct evidence came in 2010, where a targeted deletion of miRNA-processing endonuclease Dicer1 from osteoprogenitor cells resulted in phenotypes that recapitulated human myelodysplastic syndromes and secondary leukemia [21]. This study suggested for the first time that changes in the BM microenvironment alone can serve as a driver for altered hematopoiesis and even hematological malignancies ( Figure 1). Altering the HSC niche is not only a potential disease-initiating step in the development of hematological disorders, but is also a mechanism to regulate the progression of existing diseases. For instance, BM transplantation recipients expressing constitutively activated parathyroid hormone receptor in osteoblastic cells displayed attenuated MPN phenotypes induced by BCR-ABL positive HSCs when compared to the wild type recipients [22]. Interestingly, during MPN development, normal and mutant HSCs are believed to compete for the available niches, and mutant HSCs has been observed to remodel the function of the BM microenvironment to better suit disease progression. For example, in a BCR-ABL induced MPN model, the endothelial osteoblastic lineage cells isolated from MPN mice displayed an expansion with a concordant loss of supportive activity for wild type HSCs, whereas the MPN HSCs were insensitive to this loss of supportive activity, eventually outcompeting the wild type cells in BM transplantation assays [23]. Similarly, a recent study demonstrated that JAK2 V617F mutant HSCs produced excessive amount of interleukin-1β that triggered BM neural damage in the HSC niche. This neural damage was shown to be an essential process for MPN pathogenesis [24]. Taken together, these studies provided evidence for the presence of a vicious circle in MPN pathogenesis through interactions between mutant HSCs and the BM microenvironment.

Conclusions and Perspective
While most studies on MPN have been concentrated on genetic mutations in HSCs, emerging studies now present evidence that the HSC niche also has an important role in MPN pathogenesis. Under physiological conditions, the HSC niche maintains HSCs quiescence and prevents differentiation. However, this function of the HSC niche is compromised during MPN pathogenesis. The change of the BM microenvironment plays an essential role in advancing MPN disease progression, and the disease progression in return reinforces the microenvironment remodeling and thereby further tilting the already imbalanced hematopoiesis towards generating more dysregulated myeloid cells. With the active involvement of the HSC niche, the progression of MPN is no longer merely the accumulation of genetic mutations, but a positive feedback loop directly involving mutant HSCs and multiple BM components.
Despite multiple studies suggest that MPN progression can be driven through the HSC and niche feedback loop, there are many unanswered questions surround the specific role of HSC niche dysregulation in MPN. For instance, is the altered niche a cause or a consequence of the disease? Also, is the HSC niche dysregulation a stand-alone driver of the disease, an essential step in its progression, or a facilitating accelerator? It is also unknown which niche components participate in the remodeling process, and what their individual roles are. Studying these questions along with many others will begin to provide insight into this complicated disease process. The answers may vary depending on the biological context and more investigation of these queries is needed.
Moreover, a bigger question is whether the results on HSC niche in MPN obtained in mice can be translated to humans. While the genetic mutations identified in MPN patients can be feasibly validated for causal effects in mouse models, genetically modifying the HSC niche in healthy human or MPN patients is impractical. Targeting the HSC niche through pharmacological intervention may represent a viable option for studying the role of BM microenvironment in MPN pathogenesis, and ultimately uncover a new therapeutic option for MPN treatments. However, this needs to be based on a thorough understanding of the individual compartments in the HSC niche, their contributions to MPN development, and the signaling pathways that mediate interactions between the MPN HSC niche and stem cells. In summary, recent studies suggest an important role exists for the HSC niche in the progression of MPN. Learning how HSCs and the niche are altered to promote MPN progression will be an important step to understanding and treating this disease.


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Protective host defense against disseminated candidiasis is impaired in mice expressing human interleukin-37

The effect of the anti-inflammatory cytokine interleukin-37 (IL-37) on host defense against Candida infections remains unknown. We assessed the role of IL-37 in a murine model of disseminated candidiasis using mice transgenic for human IL-37 (hIL-37Tg). Upon exposure to Candida albicans pseudohyphae, macrophages from hIL-37Tg mice release 39% less TNFα compared to cells from wild-type (WT) mice (p = 0.01). In vivo, hIL-37Tg mice displayed a decreased capacity to recruit neutrophils to the site of infection. These defects were associated with increased mortality and organ fungal growth in hIL-37Tg compared to WT mice. We conclude that IL-37 interferes with the innate protective anti-Candida host response by reducing the production of proinflammatory cytokines and suppressing neutrophil recruitment in response to Candida, resulting in an increased susceptibility to disseminated candidiasis.

INTRODUCTION
The interleukin-1 (IL-1) family includes inflammatory cytokines (such as IL-1α, IL-1β, IL-18, and IL-33) that activate target cells through a receptor-mediated mechanism; anti-inflammatory cytokine antagonists (such as IL-1Ra) block IL-1-dependent activation by competing for binding to the IL-1 receptors (Dinarello, 2011). Twelve years ago, using in silico searches, additional members of the IL-1 family namely IL-36, IL-37, and IL-38 were discovered, but only recently have their properties been studied (Dinarello, 2013). In the case of IL-37, this cytokine has emerged as fundamental inhibitor of innate inflammation using mice transgenic for human IL-37 as well as reducing endogenous IL-37 in human blood monocytes (Nold et al., 2010). For example, mice transgenic for human IL-37 are protected against systemic endotoxemia, chemical induced colitis, ConA hepatitis, and ischemic reperfusion injury, as reviewed in Dinarello and Bufler (2013). The mechanism by which IL-37 affords anti-inflammatory protection can be via a caspase-1-dependent nuclear translocation or by an extracellular mechanism engaging the IL-18 receptor (Bulau et al., 2014). It is important however, to recognize that antiinflammatory strategies based upon IL-37 could have a negative impact on susceptibility to infections by inhibiting host defense. Since in models of live infection blocking inflammation mediated by cytokines such as IL-1 or TNFα can be either detrimental or beneficial for the outcome of the host, we therefore carried-out studies of disseminated candidiasis in mice expressing human IL-37.

ANIMALS
Transgenic mice expressing human IL-37 (hIL-37Tg) have been previously described Nold et al. (2010). Control C57BL/6J (WT) mice were purchased from the Jackson Laboratory and maintained under specific-pathogen free conditions. The mice were fed sterilized laboratory chow (Hope Farms, Woerden, The Netherlands) and water ad libitum. The experiments were approved by the Ethics Committee on Animal Experiments of Radboud University Nijmegen.

Candida albicans AND GROWTH CONDITIONS
Candida albicans ATCC MYA-3573 (UC 820), a strain well described elsewhere (Lehrer and Cline, 1969), was used in all experiments. Candida was grown overnight in Sabouraud broth at 37 • C, cells were harvested by centrifugation, washed twice, and resuspended in culture medium (RPMI-1640 Dutch modification, ICN Biomedicals, Aurora, OH, USA; van der Graaf et al., 2005). To generate pseudohyphae, C. albicans blastoconidia were grown at 37 • C in culture medium, adjusted to pH 6.4 by using hydrochloric acid. Pseudohyphae were killed at 100 • C for 1 h and resuspended in culture medium to a hyphal inoculum size that originated from 10 6 blastoconidia per ml (referred to as 10 6 pseudohyphae per ml).

CYTOKINE RESPONSES OF C. albicans-STIMULATED MACROPHAGES AND SPLENOCYTES
Resident peritoneal macrophages were harvested from groups of five hIL-37Tg and wild-type (WT) mice by injecting 4 ml of sterile phosphate-buffered saline (PBS) intrapertitoneal containing 0.38% sodium citrate (Kullberg et al., 1993). After washing, the cells were resuspended in culture medium in 96-well microtiter plates (Greiner, Alphen, The Netherlands) at 10 5 cells/well, in a final volume of 200 μl. The cells were stimulated with either control medium or heat killed C. albicans at 1 × 10 7 microorganisms/ml. After 24 h of incubation at 37 • C, www.frontiersin.org the plates were centrifuged (500 g, 10 min), and the supernatants were removed. The cells were lysed with three freeze-thaw cycles. Samples were stored at −80 • C until cytokine assays were performed.
To assess cytokine production in splenocytes, spleen were gently squeezed into a sterile 200 mm filter chamber. The cells were washed and resuspended in RPMI 1640, counted in a Bürker chamber and adjusted to 5 × 10 6 /ml. 200 μL of the cell suspension was stimulated with 1 × 10 7 heat-killed C. albicans/ml. Measurement of TNFα and IL-6 concentrations was performed in supernatants collected after 48 h of incubation at 37 • C in 5% CO 2 in 48-well plate.

CYTOKINE ASSAYS
TNFα was determined by specific radioimmunoassay (detection limit 20 pg/ml), as previously described Netea et al. (1996). IL-6 concentrations were determined by a commercial ELISA (Biosource, Camarillo, CA, USA, detection limit 16 pg/ml) according to the instructions of the manufacturer.

C. albicans INFECTION MODEL
hIL-37Tg mice and WT mice were injected intravenously with C. albicans blastoconidia (1 × 10 6 CFU/mouse for survival and 5 × 10 5 CFU/mouse for testing fungal burden) in a 100 μl volume of sterile pyrogen-free PBS. Survival was assessed daily for 2 weeks. Subgroups of five animals were killed on days 3 and 7 of infection. To assess the tissue outgrowth of the microorganisms, the kidneys of the sacrificed animals were removed aseptically, weighed, and homogenized in sterile saline in a tissue grinder. The number of viable Candida cells in the tissues was determined by plating serial dilutions on Sabouraud dextrose agar plates. The CFU were counted after 24 h of incubation at 37 • C, and expressed as log CFU/g tissue.

Candida OUTGROWTH IN PMN DEPLETED MICE
Sexually mature female C57bl/6 mice (8-12 weeks old, weights 20-25 g) were used. The animals were kept under routine laboratory conditions (21-22 • C, relative humidity 60% and a 12 h light-dark cycle), fed a standard commercial pellet diet (RHM, Hope Farms, The Netherlands), and given acidified tap water ad libitum. The mice were injected intraperitoneal with rat anti-mouse GR1 or isotype control (100 μg in 200 μl PBS; eBioscience) 4 days prior to the experiment to deplete the mice of PMNs. On day zero, the mice were injected intraperitoneal with 1 × 10e5 live C. albicans conidia. After 2 h the mice were anesthetized with isoflurane and blood samples were obtained for measurement of blood cells counts. Subsequently, the mice were sacrificed by cervical dislocation. The number of viable Candida cells in the peritoneal lavage was determined by plating serial dilutions on Sabouraud dextrose agar plates and the CFU were counted after 24 h of incubation at 37 • C and expressed as log CFU.

IN VITRO CYTOKINE PRODUCTION BY PRIMED SPLENOCYTES
To assess cytokine production during infection, primed spleen cells from mice on day 7 of infection with 2 × 10 5 CFU of C. albicans per mouse were stimulated in vitro with heat-killed Candida conidia or pseudohyphae (1 × 10 6 microorganisms/ml). Spleen cells were washed and resuspended in RPMI1640, counted in a Bürker chamber and the number was adjusted to 5 × 10 6 /ml. 200 μL of the cell suspension was stimulated with 1 × 10 6 heat killed C. albicans/ml. Measurement of IFNγ, IL-10, and IL-17 concentrations was performed in supernatants collected after 48 h of incubation at 37 • C in 5% CO 2 in 48-well plate.

PHAGOCYTOSIS AND KILLING OF C. albicans BY MACROPHAGES
Phagocytosis and killing of C. albicans blastoconidia were assessed as described elsewhere (Vonk et al., 2006). Exudate peritoneal phagocytes from groups of five hIL-37Tg mice and WT mice were elicited by an i.p. injection of 10% proteose peptone. After 72 h, cells were collected in separate sterile tubes by washing the peritoneal cavity with 4 ml of ice-cold PBS that contained 50 U/ml heparin. Phagocytes were centrifuged (for 10 min at 2250 g), counted in a Bürker chamber, and resuspended in culture medium. 5 × 10 5 cells were dispensed into the wells of a 96-well flat bottom plate (Costar), allowed to adhere for 2 h, and washed to remove non-adherent cells. Subsequently, the cells were incubated with 1 × 10 4 CFU C. albicans, which were opsonized for 45 min at 24 • C in modified Eagle's medium (MEM; Gibco Life Technologies) containing 2.5% fresh mouse serum (effector:target ratio, 40:1). After 15 min, supernatants were aspirated, and monolayers were gently washed with MEM to remove non-ingested microorganisms. The supernatant and well washings that contained the noningested Candida blastoconidia were plated in serial dilutions on Sabouraud agar plates. The percentage of phagocytosed microorganisms was defined as [1-(number of uningested CFU/CFU at the start of incubation)] × 100. Killing of C. albicans by phagocytes was assessed in the same monolayers. After removal of the nonphagocytized Candida blastoconidia, 200μl of culture medium, consisting of Sabouraud in MEM (50% vol/vol), was added to the monolayers. After 3 h of incubation at 37 • C in air and 5% CO 2 , the wells were scraped gently with a plastic paddle and washed with 200μl distilled H 2 O to achieve lysis of phagocytes in three cycles, and 10-fold dilutions of each sample were spread on Sabouraud agar plates and incubated at 37 • C for 24 h. The percentage of yeast killed by the phagocytes was determined as follows: [1-(CFU after incubation/number of phagocytized CFU)] (Nold et al., 2010) × 100. Phagocyte-free incubations of blastoconidia were included as a control for yeast viability. In earlier experiments we have shown that 90-97% of attached/internalized C. albicans are intracellular (Vonk et al., 2006).

STATISTICAL ANALYSIS
The differences between groups were analyzed by the Mann-Whitney U -test. The level of significance between groups was set at p < 0.05. The data are presented as cumulative results of all experiments performed.

hIL-37Tg MICE ARE MORE SUSCEPTIBLE TO DISSEMINATED C. albicans INFECTION
To investigate the role of IL-37 in the host defense against invasive C. albicans infection, hIL-37Tg mice, and WT mice were infected with C. albicans blastospores. The survival during disseminated candidiasis was 20% for WT-type mice after 2 weeks, whereas all Frontiers in Microbiology | Infectious Diseases mice had died by day 10 in the hIL-37Tg group (Figure 1A). The fungal burden in the kidneys, the target organ of disseminated candidiasis in mice (Spellberg et al., 2005), was not significantly different at day 3 (mean ± SD; WT: 3.6 ± 0.83, IL-37Tg 3.8 ± 0.44 log CFU/g kidney), and 1-log higher in the hIL-37Tg mice than in WT mice on day 7 of infection (p < 0.05; Figure 1B). Histology revealed an accumulation of Candida in the pyelum of the kidneys of hIL-37Tg mice which was not observed in the WT mice ( Figure 1C).

NEUTROPHIL RECRUITMENT AND PHAGOCYTOSIS AND KILLING OF C. albicans
In order to understand the enhanced lethality and greater fungal burden of the hIL-37Tg mice, we investigated the dynamics of the influx and function of neutrophils. Peritoneal cells were harvested and counted 4 h after intraperitoneal injection of heat-killed Candida. A significant lower influx of neutrophils was apparent 4 h after challenge with heat killed Candida in the hIL37Tg mice compared to WT mice (Figure 2A). Phagocytosis of C. albicans by hIL-37Tg cells was similar to that by cells of WT mice (37 vs. 39% phagocytized in 15 min; p > 0.05). hIL-37Tg cells killed 92% of phagocytized Candida blastoconidia after 3 h, which was not different from the killing activity of cells from WT cells (88%, p > 0.05, Figure 2B).
To assess whether the decrease of neutrophils within the first hours has a significant biological effect, we investigated the difference of fungal burden between neutrophil depleted mice and non-neutrophil depleted mice in a short in vivo Candida infection model. The lack of neutrophils at the site of infection resulted in a log increase in fungal growth within 2 h compared to mice in which neutrophil influx was normal ( Figure 2C). These differences support the importance of early influx of neutrophils in restricting Candida growth in vivo.

IL-37 INHIBITS PROINFLAMMATORY CYTOKINE PRODUCTION BY MACROPHAGES AND SPLENOCYTES STIMULATED WITH C. albicans
To investigate the inhibitory effect of IL-37 at the level of cytokine production, resident peritoneal macrophages, or naïve splenocytes of hIL-37Tg and WT mice were exposed to heat-killed C. albicans blastoconidia and pseudohyphae in vitro. Cytokine production by unstimulated macrophages of each mouse strain was below the detection limit for TNFα and IL-6. However, after stimulation with C. albicans pseudohyphae, the production of TNFα was significantly lower in macrophages from the hIL-37Tg mice compared to cells from WT mice. There were no differences in the production of IL-6 in the same cultures (Figure 3).

hIL-37Tg MICE DISPLAY INCREASED Th17 AND IL-10 RESPONSES
Proinflammatory T helper responses such as Th1 and Th17 responses play an important role in fungal host defense (Gow et al., 2012). The Th17 response during disseminated fungal infection can be both protective and detrimental (Huang et al., 2004;Zelante et al., 2007;Lin et al., 2009). To study the impact of IL-37 on proinflammatory adaptive immune responses, we investigated the Th1 and Th17 characteristic cytokines IFNγ and IL-17. Splenocytes isolated from hIL-37Tg mice with a higher fungal burden at day 7 produced significant more IL-17 in response to C. albicans pseudohyphae (Figure 4). IFNγ production in response to C. albicans pseudohyphae was undetectable in WT and hIL-37Tg splenocytes at day 7 (Figure 4). Since IL-37 mainly has anti-inflammatory effects (Nold et al., 2010;Boraschi et al., 2011), we investigated levels of IL-10 in cells from IL-37Tg There was no significant difference in IL-10 production in splenocytes from hIL-37Tg compared to WT cells in response to C. albicans, although hIL-37Tg mice showed a trend toward an increased IL-10 production (Figure 4).

DISCUSSION
In the present study, we demonstrate that overexpression of IL-37 is detrimental for the early host defense against C. albicans in a murine model of disseminated candidiasis. This conclusion is based on the increased fungal load in the kidney, the target organ of disseminated candidiasis, in hIL-37Tg mice compared to WT mice. The greater outgrowth of the fungus in the tissues is most probably due to impaired early influx of neutrophils into the infected tissues, which in turn may be due to lower production of neutrophil-inducing cytokines, such as TNF. Therefore, the Percentage of Candida blastoconidia of the initial inoculum that was ingested by macrophages of hIL-37Tg and WT mice after 15 min of phagocytosis and percentage of phagocytized Candida blastoconidia that was killed after incubation at 37 • C for 3 h is shown. The results are pooled data with a total of five hIL-37Tg mice and five WT mice (mean ± SEM, *p < 0.05, Students t -test). (C) Fungal outgrowth of peritoneal wash-outs in neutrophil (PMN) depleted and non-PMN depleted WT mice 2 h after intraperitoneal infection with live Candida. The results are pooled data with a total of four hIL-37Tg mice and four WT mice (mean ± SEM, *p < 0.05, Students t -test).
presence of IL-37 in the early stages of infection results in an impaired innate immune response that is essential to limit fungal dissemination.
Recently, several biological functions of the cytokine IL-37 have been described. IL-37 has a protective role in a murine model

FIGURE 3 | IL-37 inhibits proinflammatory cytokine production by macrophages and splenocytes stimulated with C. albicans. (A)
Resident peritoneal macrophages from hIL-37Tg mice and WT mice were stimulated with either 1 × 10e7 microorganisms/ml heat-killed Candida blastoconidia or pseudohyphae. (B) Splenocytes from hIL-37Tg mice and WT mice were stimulated with either 1 × 10e7 microorganisms/ml Candida blastoconidia or pseudohyphae. Production of TNFα and IL-6 in the supernatants was measured after 24 h of stimulation at 37 • C for macrophages and 48 h of stimulation at 37 • C for splenocytes. The results are pooled data with a total of seven hIL-37Tg mice and six WT mice (mean ± SEM, *p < 0.05, Mann-Whitney U-test).
of lethal endotoxemia (Nold et al., 2010;Bulau et al., 2011), and it has been reported that mice overexpressing IL-37 are protected from DSS colitis (McNamee et al., 2011). These reports point to significant anti-inflammatory properties of IL-37. In the present study we extend these findings to a live infection FIGURE 4 | hIL-37Tg mice display increased Th17 and IL-10 responses. Splenocytes from WT and hIL-37Tg mice were re-stimulated with RPMI (gray bars) or heat-killed 1 × 10 6 C. albicans pseudohyphae cells/ml (black bars) 7 days after intravenous infection with C. albicans. IL-17, IFNγ, and IL-10 were measured 48 h after stimulation with ELISA. *p < 0.05; n = 5 mice per group. Bars indicate mean ± SEM. model, the murine model of disseminated candidiasis, in which we assessed whether IL-37 hampers the innate immune defense against a fungal infection. We observed that mice overexpressing IL-37 were more susceptible to invasive candidiasis, due to the inability to control fungal growth in the kidney during infection. This effect is at least partly due to a defective neutrophil recruitment, a cell population central for the local antifungal host defense (Kullberg et al., 1990), and this supports the previously reported finding that leukocyte recruitment to the site of inflammation is decreased in hIL-37Tg mice (McNamee et al., 2011). The importance of neutrophils in the control of Candida outgrowth in vivo is further supported in this study by the observation that neutrophil depleted mice have a log increase within 2 h in fungal burden compared to mice that have a normal neutrophil influx. In addition, macrophages and splenocytes overexpressing IL-37 produced significantly less TNFα than WT macrophages when exposed to C. albicans. This is in line with other studies, showing that macrophages from hIL-37Tg mice produce less TNF and that IL-37 overexpression reduces TNF-dependent inflammation in a murine model of colitis (McNamee et al., 2011). An involvement of defective TNF production in the increased susceptibility of hIL-37Tg mice to disseminated candidiasis is also consistent with earlier studies showing that mice lacking TNF or the TNF receptor are highly susceptible to disseminated candidiasis (Netea et al., 1999).
Another observation of this study is that neutrophils from hIL-37Tg mice do not display an intrinsic defect in phagocytosis and killing, suggesting that IL-37 does not directly impair neutrophil function. The increased Th17 response in hIL-37Tg mice present on day 7 most likely reflects an increased cellular immune response due to higher fungal burden (antigen) exposure. Whether this increased Th17 response has directly contributed to the higher susceptibility of hIL-37Tg mice to disseminated candidiasis remains to be determined. Interestingly, hIL-37Tg splenocytes showed a consistent trend at day 7 toward an increased production of the anti-inflammatory cytokine IL-10 when exposed to C. albicans. This observation is consistent with the increased IL-10 production found in hIL-37Tg mice with experimental colitis, and supports the concept that IL-37 can contribute to systemic anti-inflammatory effects (McNamee et al., 2011).
In conclusion, IL-37 reduces proinflammatory cytokine production induced by Candida in macrophages, and decreases neutrophil recruitment in vivo in response to C. albicans. These data highlight the potent anti-inflammatory effects of IL-37, and underline that the timing of IL-37 expression in the tissue at the site of inflammation is critical for the outcome of the host during inflammatory processes.

ACKNOWLEDGMENTS
This study was supported by the Niels Stensen Foundation and a Veni Grant of the Netherlands Foundation for Scientific Research to Frank L. van de Veerdonk. Charles A. Dinarello was supported by NIH grant AI-15614.