Secretory Leukocyte Protease Inhibitor Secretion Research Articles

Dietary iron modulates gut microbiota and induces SLPI secretion to promote colorectal tumorigenesis

ABSTRACT Dietary iron intake is closely related to the incidence of colorectal cancer. However, the interactions among dietary iron, gut microbiota, and epithelial cells in promoting tumorigenesis have rarely been discussed. Here, we report that gut microbiota plays a crucial role in promoting colorectal tumorigenesis in multiple mice models under excessive dietary iron intake. Gut microbiota modulated by excessive dietary iron are pathogenic, irritating the permeability of the gut barrier and causing leakage of lumen bacteria. Mechanistically, epithelial cells released more secretory leukocyte protease inhibitor (SLPI) to combat the leaked bacteria and limit inflammation. The upregulated SLPI acted as a pro-tumorigenic factor and promoted colorectal tumorigenesis by activating the MAPK signaling pathway. Moreover, excessive dietary iron significantly depleted Akkermansiaceae in the gut microbiota; while supplementation with Akkermansia muciniphila could successfully attenuate the tumorigenic effect from excessive dietary iron. Overall, excessive dietary iron perturbs diet – microbiome–epithelium interactions, which contributes to intestinal tumor initiation.

Secretory leukocyte protease inhibitor (SLPI) as a potential target for inhibiting metastasis of triple-negative breast cancers.

SLPI has been implicated in the progression and metastasis of certain cancers. However, the effects of SLPI seem to be tumor-specific and the mechanisms remain poorly defined. Here, we demonstrate that highly metastatic, triple-negative breast cancer (TNBC) 4T1 cells secreted more SLPI compared to their non-metastatic counterparts. Furthermore, SLPI secretion directly correlated with spontaneous lung metastasis from 4T1 tumors orthotopically implanted in mice. Consistent with our experimental results, we also found that higher SLPI expression levels correlate with worse clinical outcome in basal/TNBC patients. Using high-throughput screening we identified a novel compound, C74, which significantly inhibits SLPI secretion. C74 administration in our mouse model slows the growth of primary 4T1 tumors and inhibits their dissemination to the lung. We also discovered that SLPI physically interacts with the retinoblastoma tumor suppressor protein (Rb) and releases FoxM1 from the Rb-FoxM1 complex, which may activate FoxM1 target genes involved in breast cancer metastasis.

Increased secretory leukocyte protease inhibitor (SLPI) production by highly metastatic mouse breast cancer cells.

The precise molecular mechanisms enabling cancer cells to metastasize from the primary tumor to different tissue locations are still largely unknown. Secretion of some proteins by metastatic cells could facilitate metastasis formation. The comparison of secreted proteins from cancer cells with different metastatic capabilities in vivo might provide insight into proteins involved in the metastatic process. Comparison of the secreted proteins from the mouse breast cancer cell line 4T1 and its highly metastatic 4T1.2 clone revealed a prominent differentially secreted protein which was identified as SLPI (secretory leukocyte protease inhibitor). Western blotting indicated higher levels of the protein in both conditioned media and whole cell lysates of 4T1.2 cells. Additionally higher levels of SLPI were also observed in 4T1.2 breast tumors in vivo following immunohistochemical staining. A comparison of SLPI mRNA levels by gene profiling using microarrays and RT-PCR did not detect major differences in SLPI gene expression between the 4T1 and 4T1.2 cells indicating that SLPI secretion is regulated at the protein level. Our results demonstrate that secretion of SLPI is drastically increased in highly metastatic cells, suggesting a possible role for SLPI in enhancing the metastatic behavior of breast cancer cell line 4T1.

Sulforaphane induces SLPI secretion in the nasal mucosa

Cells lining the respiratory tract are equipped with mechanisms that dampen the effects of oxidative stress. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a mediator involved in regulating oxidative stress. Recent data indicate Nrf2 also controls expression of secretory leukocyte protease inhibitor (SLPI). Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, enhances Nrf2 activity. Therefore, we hypothesized that SFN supplementation induces SLPI secretion in the nasal mucosa in an Nrf2 dependent manner. Healthy nonsmoking adults ingested SFN-containing broccoli shake homogenate (BSH) for 3 consecutive days. Nasal lavage fluid (NLF) was collected before and after BSH ingestion and analyzed for SLPI protein levels. In follow up invitro experiments, differentiated primary nasal epithelial cells were used to evaluate the relationship between SFN, Nrf2, and SLPI. Epithelial cells were transduced with Nrf2-specific shRNA to examine the regulatory role of Nrf2 on SLPI expression. Supplementation with BSH significantly increased SLPI levels in NLF. SFN supplementation invitro significantly enhanced SLPI secretion and these effects were significantly decreased in cells transduced with Nrf2-specific shRNA. Our data support a relationship between nutritional supplementation, Nrf2 activation, and SLPI secretion. Therefore, ingestion of SFN-containing foods has therapeutic potential to augment SLPI expression in the nasal mucosa.

The negative feedback mechanism between SLPI and IFNγ is impaired in tuberculosis patients (164.7)

Abstract Mycobacterium tuberculosis increases IFNγ secretion which plays an important role in granuloma formation and clearance of it. Also, the exposure of macrophages to M. tuberculosis increases secretory leukocyte protease inhibitor (SLPI), a serine protease inhibitor with antimycobacterial activity. Therefore, the aim of the present study was to determine the role of a putative interaction between IFNγ and SLPI in PBMC derived from healthy donors (HD) and tuberculosis patients (TB). Peripheral blood mononuclear cells (PBMC) from TB and HD were treated with Mtb antigen and IFNγ and SLPI was determined by ELISA. Our results showed that the stimulation with Mtb of PBMC from HD, it decreased SLPI (p<0.01) while increased IFNγ secretion. Furthermore, the blockage of IFNγ reversed the effect of the Mtb antigen on SLPI secretion in PBMC-derived HD. Surprisingly Mtb antigen did not modify the SLPI secretion in PBMC-derived from TB, although it increased IFNγ. Furthermore, the mAb against IFNγ did not affect the SLPI secretion. When plasma levels of SLPI and IFNγ were measured, we found a positive correlation between them in TB but not in HD. Moreover, tuberculosis patients showed more SLPI in plasma, being the levels higher with the severity of the disease. Overall, these results suggest that there is a negative cross talk between IFNγ and SLPI in HD that it is not present in TB patients. Therefore, the higher concentration of IFNγ and SLPI might contribute to control the infection.

Paracrine SLPI secretion upregulates MMP-9 transcription and secretion in ovarian cancer cells

Objectives Secretory leukocyte protease inhibitor (SLPI) is amplified in serous ovarian cancer. We have dissected its function, showing it is a survival factor for ovarian cancer and promotes tumorigenesis and paclitaxel-resistance. We hypothesized that the protease inhibitory function was responsible for modulating SLPI's invasive capacity. Methods Stable HEYA8 ovarian cancer transfectants expressing vector, wild type SLPI, and protease inhibitor null (F-)SLPI were examined in vitro and in xenografts. Invasion, enzyme activity, and MMP production and function assays were applied. SLPI and MMP immunoexpression was graded on tissue microarray and clinical samples. Statistical comparisons used unpaired t test and ANOVA, where appropriate. Results SLPI and F-SLPI cells caused greater parenchymal and peritoneal dissemination over control cells in xenografts and invasion assays (p < 0.001). MMP-9 protease activity was increased in SLPI and F-SLPI cells over control. SLPI, but not F-SLPI, inhibited plasmin activity, necessary for MMP-9 activation and release, and inhibited activation of MMP-9. However, paradoxically, both induced quantitative MMP-9 transcription (p < 0.05) and protein (p < 0.008), yielding an increased net MMP-9 activity in the face of plasmin inhibition. SLPI and MMP-9 expression were strongly correlated in serous ovarian cancers (r 2 = 0.986) and a set of ovarian cancers (p < 0.02). SLPI expression was greater in serous than endometrioid ovarian cancers (p = 0.04). Conclusions SLPI stimulates ovarian cancer invasion, modulated in part by its serine protease inhibitory activity attenuating MMP-9 release. However, SLPI induction of MMP-9, independent of protease inhibition activity, is greater yielding a net pro-invasive behavior. These findings further support SLPI as a molecular target for ovarian cancer.

Effect of secretory leukocyte protease inhibitor on migration and invasion of human KB oral carcinoma cells

Secretory leukocyte protease inhibitor (SLPI) plays an important role in promoting the invasion and metastasis of a range of cancer cells. However, there are no reports of the expression and function of SLPI in oral carcinoma cells. In this study, the oral carcinoma cell line KB was used to determine whether SLPI affects the proliferation, migration and invasion of oral carcinoma cells. RT-PCR and Western blotting revealed high levels of endogenous SLPI expression in KB cells as well as a strong increase in SLPI secretion after wounding compared to immortalized normal oral keratinocytes (INOK). The wound healing assay revealed more migration of KB cells than INOK cells, and the SLPI treatment increased the migration of KB cells. KB cell proliferation was increased significantly by the SLPI protein but decreased by SLPI-siRNA. SLPI strongly increased the migration and invasion of KB cells. On the other hand, SLPI-siRNA decreased the migration and invasion of KB cells. This suggests that SLPI plays an important role in the metastasis of oral carcinoma cells.

Estradiol selectively regulates innate immune function by polarized human uterine epithelial cells in culture

The goal of this study was to examine the role of E2 in regulating innate immune protection by human uterine epithelial cells (UECs). Recognizing that UECs produce cytokines and chemokines to recruit and activate immune cells as well as viral and bacterial antimicrobials, we sought to examine the effect of E2 on constitutive and Toll-like receptor (TLR) agonist (lipopolysaccharide (LPS) and poly (I:C))-induced immune responses. The secretion by polarized UECs in culture of interleukin (IL)-6, macrophage inhibitory factor (MIF), and secretory leukocyte protease inhibitor (SLPI) was examined as well as the mRNA expression of human β-defensin-2 (HBD2), tumor necrosis factor (TNF)-α, IL-8, and nuclear factor (NF)-kB. When incubated with E2 for 24–48 h, we found that E2 stimulated UEC secretion of SLPI (fourfold) and mRNA expression of HBD2 (fivefold). Moreover, when antibacterial activity in UEC secretions was measured using Staphylococcus aureus, E2 increased the secretion of soluble factor(s) with antibacterial activity. In contrast, E2 had no effect on constitutive secretion of proinflammatory cytokines and chemokines by UECs but completely inhibited LPS- and poly (I:C)-induced secretion of MIF, IL-6, and IL-8. Estradiol also reversed the stimulatory effects of IL-1β on mRNA expression of TNF-α, IL-8, and NF-kB by 85, 95, and 70%, respectively. As SLPI is known to inhibit NF-kB expression, these findings suggest that E2 inhibition of proinflammatory cytokines may be mediated through SLPI regulation of NF-kB. Overall, these findings indicate that the production of cytokines, chemokines, and antimicrobials by UECs are differentially regulated by E2. Further, it suggests that with E2 regulation, epithelial cells that line the uterine cavity have evolved immunologically to be sensitive to viral and bacterial infections as well as the constraints of procreation.

Murine macrophages produce secretory leukocyte protease inhibitor during clearance of apoptotic cells: implications for resolution of the inflammatory response.

Macrophage-derived secretory leukocyte protease inhibitor (SLPI) can be induced locally as well as systemically in response to microbial products such as LPS and lipotechoic acid. It is not known whether phagocytosis of apoptotic cells, an essential function of macrophages, can regulate expression and secretion of SLPI. In this study, we report that exposure of peritoneal macrophages of BALB/c mice or murine macrophage cell lines RAW264.7 and J774.1 to apoptotic target cells induced an elevation in SLPI secretion. Secreted SLPI retained its antichymotrypsin activity. SLPI expression in thymuses from BALB/c mice that had been injected with anti-CD3 Ab to induce apoptosis of thymocytes was also elevated both at the mRNA and protein levels. Colchicine, a microtubular inhibitor, blocked the internalization of apoptotic cells by macrophages but not SLPI secretion, suggesting that surface recognition of apoptotic cells is sufficient for the induction of SLPI. Exposure of RAW264.7 cells to apoptotic CTLL-2 cells induced both SLPI and TNF-alpha, and addition of IFN-gamma inhibited SLPI but augmented TNF-alpha production. Transfection of either the secreted or a nonsecreted form of SLPI into RAW264.7 cells led to suppression of TNF-alpha production in response to apoptotic cells. Thus, macrophages secrete an increased amount of SLPI when encountering apoptotic cells, which may help to attenuate potential inflammation during clearance of these cells.

Secretion and gene expression of secretory leukocyte protease inhibitor by human airway submucosal glands.

Submucosal glands were isolated within 4 h of death from tracheae and bronchi obtained from autopsied lungs, and the secretory response of secretory leukocyte protease inhibitor (SLPI) was examined with ELISA and a secretory index. Although human neutrophil elastase (HNE) at low concentrations increased SLPI secretion above the control level (i.e., 149% of control level at 10(-11) M), HNE at high concentrations significantly decreased it below the control level (i.e., 16% of control level at 10(-7) M). The decrease in SLPI concentration was shown to result from the degradation of SLPI by excessive HNE. Methacholine induced significant secretion (i.e., 363% of control level at 10(-5) M) that was abolished by both M(1) and M(3) receptor antagonists. A semiquantitative analysis of SLPI mRNA by RT-PCR and Southern blot showed that compared with the superficial epithelium, submucosal glands had a 30-fold or higher level of SLPI mRNA. Both HNE and methacholine significantly increased the level of SLPI mRNA in submucosal glands in a dose-dependent manner (i.e., 357% of control level at 10(-7) M and 175% of control level at 10(-5) M, respectively). These findings indicate that human airway submucosal glands can transcribe 30-fold or more SLPI mRNA than the superficial epithelium and that SLPI mRNA transcription and secretion are regulated by both HNE and muscarinic receptors.

Secretory leukocyte protease inhibitor in punch biopsies from human colonic mucosa

Secretory leukocyte protease inhibitor (SLPI) is a well-known protease inhibitor. Its function is thought to be protease/protease-inhibitor balance. Free proteolytic activity, mainly pancreatic elastase, anionic trypsin and granulocytic elastase, has been demonstrated in faecal extracts from patients with ulcerative colitis. We wanted to verify that SLPI is actually secreted from normal human colonic mucosa. Also, we wanted to ascertain whether studies of SLPI secretion based on punch biopsies were dependent on biopsy area or on biopsy circumference. Normal colonic mucosa was sampled during surgery for colonic cancer. A total of 36 samples from four patients were used. Mucosa preparation was carried out using a punch biopsy technique, and samples of 3, 4 and 6 mm diameter were used. All media contained SLPI at varying concentrations. When expressed in terms of the sample area, the secretion per millimetre-squared seemed to decrease with increasing area. When calculated as secretion per circumference, secretion seemed to be constant. In conclusion, SLPI was secreted from normal human colonic mucosa. The SLPI secretion seemed dependent on the circumference of the biopsy rather than on the area of the biopsy.

Identification of Genes Involved in Innate Responsiveness to Bacterial Products by Differential Display

To explore gene regulation by bacterial lipopolysaccharide (LPS), we compared mRNA profiles of macrophage cell lines from two strains of mice congenic for a locus markedly affecting their ability to respond to LPS. Differential display detected four differentially expressed transcripts. One transcript encoded the mouse homolog of human secretory leukocyte protease inhibitor (SLPI), which was expressed by LPS-hyporesponsive macrophage cells (Lpsd) but not by LPS-normoresponsive cells (Lpsn). Among five macrophage cell lines, secretion of SLPI was inversely correlated with ability to produce nitric oxide (NO) and tumor necrosis factor α in response to LPS. Stable transfection of LPS-responsive macrophages with SLPI suppressed LPS-induced responses. Interferon-γ (IFN-γ), which corrects the defective LPS response inLpsdmacrophages, suppressed the LPS-induced expression of SLPI and restored LPS response to SLPI-overexpressing macrophages. Besides its role as a LPS response inhibitor, mouse SLPI is also a lipoteichoic acid response inhibitor. The expression of SLPI was strongly enhanced by interleukin-10 and -6. SLPI may be an important antiinflammatory molecule in host defense against gram-negative and gram-positive bacteria.

The elastase-induced expression of secretory leukocyte protease inhibitor is decreased in remodelled airway epithelium

During airway inflammation, proteinases such as human leukocyte elastase are actively secreted. Secretory leukocyte protease inhibitor is a major serine proteinase inhibitor, secreted by bronchial, bronchiolar and lung epithelial cells. We recently identified secretory leukocyte protease inhibitor in human nasal epithelium, exclusively in remodelled areas of the surface epithelium. We now investigated the influence of remodelling and inflammation of the nasal tissue on the in vitro capacity of these cells to respond to human leukocyte elastase. Primary cultures of surface epithelial cells were established from various nasal polyp samples. At confluency, cell cultures were exposed to different human leukocyte elastase concentrations. The secretory leukocyte protease inhibitor immunocytolocalisation, expression and secretion were then investigated. Immunocytochemistry, showed a human leukocyte elastase dose-dependent increase of secretory leukocyte protease inhibitor containing cells and a basal extracellular localization of secretory leukocyte protease inhibitor after incubation with 100 μg/ml human leukocyte elastase. The relative amount of secretory leukocyte protease inhibitor mRNA transcripts increased with respect to the human leukocyte elastase concentration. Nevertheless, the potential stimulation of secretory leukocyte protease inhibitor secretion by human leukocyte elastase was lower in the more remodelled and inflamed tissue. Our results suggest that the contribution of the surface epithelial cells of poorly remodelled tissues to the protection against the deleterious effect of neutrophil proteinases is severely decreased in highly remodelled and inflamed tissues.

Regulation of the secretory phenotype of human airway epithelium by retinoic acid, triiodothyronine, and extracellular matrix.

The purpose of our studies was to identify factors which regulate the composition of airway secretions produced by normal human tracheobronchial epithelial (NHTBE) cells. Individual factors were removed from the culture media of NHTBE cells grown in air-liquid interface (ALI) cultures (which support mucociliary differentiation) and the effects on mucin, lysozyme (LZ), and secretory leukocyte protease inhibitor (SLPI) secretion and gene expression were examined. Deletion of hydrocortisone, epinephrine, transferrin, or gentamycin-amphotericin from the media had no reproducible effects; deletion of insulin was incompatible with culture growth. We identified 3 factors, namely retinoic acid (RA), triiodothyronine (T3) and collagen gel substratum, which had a major impact on the profile of NHTBE secretions. Removal of RA from the media caused a drastic decrease in mucin secretion and a decrease in expression of the mucin genes MUC2 and MUC5AC.LZ and SLPI secretions were increased in these cultures. Paradoxically LZ mRNA was decreased, while SLPI mRNA levels were increased. Removal of T3 selectively increased mucin secretion, MUC2 gene expression was not affected, but MUC5AC mRNA levels reproducibly increased, suggesting that the expression of these two mucin genes is differentially regulated. LZ and SLPI secretion levels were not significantly affected by deletion of T3 from the culture media; however, LZ mRNA levels were increased in the absence of T3 while SLPI transcript levels were not affected. Omission of the attachment substratum, type I collagen gel, resulted in significant increases in all 3 secretory products. MUC2 and MUC5AC steady state mRNA levels were not consistently affected. In contrast LZ and SLPI gene expression were reproducibly increased. Our studies show that individual factors in the epithelial environment can regulate expression of specific secretory cell gene products in a highly selective manner.