Secretory Leukocyte Protease Inhibitor Expression Research Articles

SLPI overexpression in hMSCs could be implicated in the HSC gene expression profile in AML

Acute myeloid leukaemia (AML) is a severe haematological neoplasm that originates from the transformation of haematopoietic stem cells (HSCs) into leukaemic stem cells (LSCs). The bone marrow (BM) microenvironment, particularly that of mesenchymal stromal cells (hMSCs), plays a crucial role in the maintenance of HSCs. In this context, we explored whether alterations in the secretome of hMSCs derived from AML patients (hMSC-AML) could impact HSC gene expression. Proteomic analysis revealed that the secretome of coculture assays with hMSC-AMLs and HSC from healthy donor is altered, with increased levels of secretory leukocyte protease inhibitor (SLPI), a protein associated with important processes for maintenance of the haematopoietic niche that has already been described to be altered in several tumours. Increased SLPI expression was also observed in the BM plasma of AML patients. Transcriptome analysis of HSCs cocultured with hMSC-AML in comparison with HSCs cocultured with hMSC-HD revealed altered expression of SLPI target genes associated with the cell cycle, proliferation, and apoptosis. Important changes were identified, such as increased expression levels of CCNA2, CCNE2, CCND2, CD133 and CDK1 and decreased levels of CDKN2A and IGFBP3, among others. Overall, these findings suggest that the altered secretome of coculture assays with hMSC-AMLs and HSC from healthy donor, particularly increased SLPI expression, can contribute to gene expression changes in HSCs, potentially influencing important molecular mechanisms related to AML development and progression.

Secretory leukocyte protease inhibitor protects against severe urinary tract infection in mice.

Millions suffer from urinary tract infections (UTIs) worldwide every year with women accounting for the majority of cases. Uropathogenic Escherichia coli (UPEC) causes most of these primary infections and leads to 25% becoming recurrent or chronic. To repel invading pathogens, the urinary tract mounts a vigorous innate immune response that includes the secretion of antimicrobial peptides (AMPs), rapid recruitment of phagocytes, and exfoliation of superficial umbrella cells. Here, we investigate secretory leukocyte protease inhibitor (SLPI), an AMP with antiprotease, antimicrobial, and immunomodulatory functions, known to play protective roles at other mucosal sites, but not well characterized in UTIs. Using a preclinical model of UPEC-caused UTI, we show that urine SLPI increases in infected mice and that SLPI is localized to bladder epithelial cells. UPEC-infected SLPI-deficient (Slpi-/-) mice suffer from higher urine bacterial burdens, prolonged bladder inflammation, and elevated urine neutrophil elastase (NE) levels compared to wild-type (Slpi+/+) controls. Combined with bulk bladder RNA sequencing, our data indicate that Slpi-/- mice have a dysregulated immune and tissue repair response following UTI. We also measure SLPI in urine samples from a small group of female subjects 18-49 years old and find that SLPI tends to be higher in the presence of a uropathogen, except in patients with a history of recent or recurrent UTI, suggesting a dysregulation of SLPI expression in these women. Taken together, our findings show SLPI promotes clearance of UPEC in mice and provides preliminary evidence that SLPI is likewise regulated in response to uropathogen exposure in women.IMPORTANCEAnnually, millions of people suffer from urinary tract infections (UTIs) and more than $3 billion are spent on work absences and treatment of these patients. While the early response to UTI is known to be important in combating urinary pathogens, knowledge of host factors that help curb infection is still limited. Here, we use a preclinical model of UTI to study secretory leukocyte protease inhibitor (SLPI), an antimicrobial protein, to determine how it protects the bladder against infection. We find that SLPI is increased during UTI, accelerates the clearance of bacteriuria, and upregulates genes and pathways needed to fight an infection while preventing prolonged bladder inflammation. In a small clinical study, we show SLPI is readily detectable in human urine and is associated with the presence of a uropathogen in patients without a previous history of UTI, suggesting SLPI may play an important role in protecting from bacterial cystitis.

Afterbirth oral fluid secretory leukocyte protease inhibitor decreased in acute histologic chorioamnionitis in preterm birth.

Secretory leukocyte protease inhibitor (SLPI) is an innate anti-inflammatory and anti-microbial peptide and produced in amnion of fetal membranes during pregnancy. However, studies on the association between SLPI levels in amniotic fluid and acute chorioamnionitis are limited. Afterbirth oral fluid (AOF) of the baby could be useful for representing the intra-amniotic environment precisely just before delivery. This study aimed to determine the relationship between SLPI levels in AOF and acute histologic chorioamnionitis (HC). AOF of the baby was obtained during delivery from 24(0/7) to 36(6/7) weeks of gestational age (preterm group, n=94) and from 37(0/7) to 41(6/7) weeks of gestational age (term group, n=27) just after birth. SLPI expression levels among five classifications were compared to the intensity of acute HC as follows: no inflammation, acute subchorionitis, acute chorionitis, acute chorioamnionitis, and funisits. The SLPI and matrix metalloproteinase-8 (MMP-8) concentrations of AOF were determined using Enzyme Linked Immunosorbent Assay. Histologic examination of the placenta and membranes was performed after delivery. SLPI concentrations in AOF inversely decreased according to the intensity of acute HC (161.62ng/mL in funisitis, 134.83ng/mL in acute chorioamnionitis, 749.35ng/mL in acute chorionitis, 953.05ng/mL in acute subchorionitis, and 1126.77ng/mL in no inflammation [p=.021]). The MMP-8 concentrations in AOF and maternal serum C-reactive protein were the highest in funisitis. The SLPI/ MMP-8 ratio was low in subgroup with acute chorioamnionitis and funisitis. Along with increased MMP-8 levels, decreased SLPI levels in AOF of the baby could be an additional factor in predicting acute HC immediately after birth.

Spatiotemporal expression and regulation of peptidase inhibitor 3 and secretory leukocyte protease inhibitor at the maternal-fetal interface in pigs.

Two serine protease inhibitors, peptidase inhibitor 3 (PI3) and secretory leukocyte protease inhibitor (SLPI), play important roles in protease inhibition and antimicrobial activity, but their expression, regulation, and function at the maternal-fetal interface in pigs are not fully understood. Therefore, we determined the expression and regulation of PI3 and SLPI in the endometrium throughout the estrous cycle and at the maternal-fetal interface in pigs. Endometrial tissues during the estrous cycle and pregnancy, conceptus tissues during early pregnancy, and chorioallantoic tissues during mid to late pregnancy were obtained, and the expression of PI3 and SLPI was analyzed. The effects of the steroid hormones estradiol-17β (E2) and progesterone (P4) on the expression of PI3 and SLPI were determined in endometrial explant cultures. PI3 and SLPI were expressed in the endometrium during the estrous cycle and pregnancy, with higher levels during mid to late pregnancy than during the estrous cycle and early pregnancy. Early-stage conceptuses and chorioallantoic tissues during mid to late pregnancy also expressed PI3 and SLPI. PI3 protein and SLPI mRNA were primarily localized to endometrial epithelia. In endometrial explant cultures, the expression of PI3 was induced by increasing doses of P4, and the expression of SLPI was induced by increasing doses of E2 and P4. These results suggest that the PI3 and SLPI expressed in the endometrium and conceptus tissues play an important role in antimicrobial activity for fetal protection against potential pathogens and in blocking protease actions to allow epitheliochorial placenta formation.

P064 High intestinal Secretory Leukocyte Protease Inhibitor (SLPI) expression correlates with abundant neutrophilic inflammation and identifies Inflammatory Bowel Disease patients with a strong IL-17 response.

Abstract Background Inflammatory bowel disease, comprising Crohn’s disease (CD) and ulcerative colitis (UC), is driven by aberrant host-microbial immune interactions. Disease heterogeneity and severity hamper effective treatment, thus new therapeutic strategies tailored to the patient’s underlying immune defect are required. In mice, upon epithelial barrier breach and microbiota driven inflammation, colonic intestinal epithelial cells increase Secretory Leukocyte Protease Inhibitor (SLPI) expression. SLPI can exert anti-microbial activity and inhibit NF-kB activation. Therefore, we hypothesized that SLPI expression is increased in the intestine of IBD patients and may differentiate a subgroup of patients with a distinctive underlying immune disease. Methods Intestinal biopsies and plasma from therapy-naive pediatric patients (age 6-18) with CD (n=41), UC (n=22) or IBD-negative controls (n=15) were collected. Intestinal SLPI protein was detected by immunohistochemistry (IHC), scored semi-quantitatively, and patients were dichotomized into ‘SLPI-high’ and ‘SLPI-low’ groups. RNA sequencing and quantitative PCR (qPCR) were performed. Plasma protein concentrations were assessed with Olink technology®. Results We observed increased SLPI mRNA and protein expression in intestinal tissue of CD and UC patients, compared to IBD-negative controls. Expression was the strongest in macroscopically inflamed tissue and more dominant in colon than in small intestinal ileum. Colonic SLPI expression was associated with endoscopic severity of inflammation, with histologic disease activity, and with clinical disease activity in CD and UC patients. Furthermore, high epithelial SLPI expression associated with abundant neutrophil infiltration and calprotectin expression. RNA-seq revealed that biopsies of SLPI-high patients had a signature of inflammatory neutrophilic inflammation including the genes OSM, IL-1B, and CXCL8, which have been associated with severe disease progression and resistance to anti-TNF treatment. Most notably, biopsies of SLPI-high patients had a signature of increased IL-17 signaling, which was supported by IHC detection of high frequencies of IL-17 producing cells in the paired biopsies and higher plasma concentrations of CCL20, MMP10 and IL-17A. Conclusion Together, these results demonstrate that intestinal SLPI expression identifies severe IBD patients with high intestinal SLPI expression and immune features of therapy resistance, who have increased IL-17A responses and subsequent neutrophilic inflammation compared to SLPI-low patients. As such, epithelial SLPI expression is a histological parameter that could support tailored treatment strategies in pediatric IBD patients at the time of diagnosis.

Up-regulation of secretory leukocyte protease inhibitor in human samples might have a potential role of predicting prostate cancer recurrence and progression after surgery and hormonal therapy.

Using new castration-resistant prostate cancer (CRPC) cell lines developed from LNCaP cells as a model for CRPC, we searched for novel biomarkers by analyzing the proteins secreted in culture supernatants. The results showed that the levels of secretory leukocyte protease inhibitor (SLPI) in these cell lines were 4.7-6.7 times higher than those secreted in parental LNCaP. Patients with localized prostate cancer (PC) and who expressed SLPI had a significantly lower prostate-specific antigen (PSA) progression-free survival rate than those who did not. Multivariate analysis revealed that SLPI expression was an independent risk factor for PSA recurrence. By contrast, when immunostaining of SLPI was performed on consecutive prostate tissue samples obtained from 11 patients, both in hormone naive (HN) and castration resistant (CR) conditions, only one patient expressed SLPI in the HNPC state; however, four of the 11 patients expressed SLPI in the CRPC state. In addition, two of these four patients were resistant to enzalutamide, and there was a discrepancy between their serum PSA levels and radiographic progression of the disease. These results suggest that SLPI can be a predictor of prognosis in patients with localized PC and disease progression in CRPC patients.

High expression of secretory leukocyte protease inhibitor (SLPI) in stage III micro-satellite stable colorectal cancer is associated with reduced disease recurrence

Secretory leukocyte protease inhibitor (SLPI) is a pleiotropic protein produced by healthy intestinal epithelial cells. SLPI regulates NF-κB activation, inhibits neutrophil proteases and has broad antimicrobial activity. Recently, increased SLPI expression was found in various types of carcinomas and was suggested to increase their metastatic potential. Indeed, we demonstrated that SLPI protein expression in colorectal cancer (CRC) liver metastases and matched primary tumors is associated with worse outcome, suggesting that SLPI promotes metastasis in human CRC. However, whether SLPI plays a role in CRC before distant metastases have formed is unclear. Therefore, we examined whether SLPI expression is associated with prognosis in CRC patients with localized disease. Using a cohort of 226 stage II and 160 stage III CRC patients we demonstrate that high SLPI protein expression is associated with reduced disease recurrence in patients with stage III micro-satellite stable tumors treated with adjuvant chemotherapy, independently of established clinical risk factors (hazard rate ratio 0.54, P-value 0.03). SLPI protein expression was not associated with disease-free survival in stage II CRC patients. Our data suggest that the role of SLPI in CRC may be different depending on the stage of disease. In stage III CRC, SLPI expression may be unfavorable for tumors, whereas SLPI expression may be beneficial for tumors once distant metastases have established.

FAPhigh α-SMAlow cancer-associated fibroblast-derived SLPI protein encapsulated in extracellular vesicles promotes ovarian cancer development via activation of PI3K/AKT and downstream signaling pathways.

Ovarian cancer is the most lethal gynecological malignancy worldwide with high metastasis and poor prognosis rates. Cancer‐associated fibroblasts (CAFs), a heterogeneous population of cells that constitutes a major component of the tumor microenvironment, secrete extracellular vesicles (EVs) loading with proteins, lipids, and RNAs to promote tumorigenesis. However, the specific roles of CAF‐derived proteins contained in EVs in ovarian cancer remain poorly understood at present. Using the gene expression microarray analysis, we identified a list of dysregulated genes between the α‐SMA+CAF and FAP+CAF subpopulations, from which secretory leukocyte protease inhibitor (SLPI) was chosen for further validation. Quantitative PCR, western blot, immunohistochemistry, and enzyme‐linked immunosorbent assays were used to assess SLPI expression in ovarian cancer cells, tissues, CAFs, and EVs. Additionally, we evaluated the effects of exogenous SLPI on proliferation, migration, invasion, and adhesion of ovarian cancer cells in vitro. Our results showed SLPI protein was upregulated in CAFs, particularly in the FAPhighα‐SMAlowCAF subpopulation, and associated with increased tumor grade and decreased overall survival (OS). Importantly, CAF‐derived SLPI protein could be encapsulated in EVs for delivery to ovarian cancer cells, thus facilitating cell proliferation, migration, invasion, and adhesion via activating the PI3K/AKT and downstream signaling pathways. Moreover, high plasma expression of SLPI encapsulated in EVs was closely correlated with tumor stage in ovarian cancer patients. Our collective results highlight an oncogenic role of plasma EV‐encapsulated SLPI secreted by CAFs in tumor progression for the first time, supporting its potential utility as a prognostic biomarker of ovarian cancer.

Secretory leukocyte peptidase inhibitor inhibits the inflammatory response and apoptosis of lipopolysaccharide-induced human proximal renal tubular cells

To screen out the potential key genes of sepsis-associated acute kidney injury (AKI), and provide theoretical and experimental evidence for the treatment of sepsis-associated AKI. (1) Bioinformatics analysis: two gene expression datasets (GSE30718 and GSE53773) were downloaded for bioinformatics analysis from the Gene Expression Omnibus (GEO). These two datasets recorded mRNA microarray data from kidney biopsies before and after kidney transplantation, and a subset of patients developed AKI after kidney transplantation. Differential analysis was conducted, and the genes with the same differential expression and a higher area under the receiver operator characteristic curve (AUC) in both databases were used as the target gene for subsequent cell experiments. (2) Cell validation experiment: human proximal renal tubular cells HK2 were cultured in vitro, and lipopolysaccharide (LPS) was used for establishing LPS-HK2 cell model (LPS 10 mg/L for 6 hours, LPS model group), and the blank control group was set. Then, small interfering RNA (siRNA) technology was used to knock down the target gene obtained by bioinformatics analysis in LPS-HK2 cells (gene knockdown group), and a gene negative control group was set. The real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-qPCR) technique was used to detect the expression of the target gene in HK2 cells. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors in the cell supernatants. Western blotting was used to detect the expressions of key apoptosis proteins. (1) Results of bioinformatics analysis: 325 genes in the two datasets showed the same expression trend, of which 144 were significantly down-regulated and 181 were significantly up-regulated, while the expression difference of secretory leukocyte protease inhibitor (SLPI) in the two datasets was both statistically significant. Further receiver operator characteristic curve (ROC curve) analysis confirmed that the SLPI expression in GSE30718 and GSE53773 datasets had a high diagnostic efficiency for AKI, with AUC of 0.83 and 0.92, respectively. Therefore, SLPI was selected as the target gene for subsequent cell validation experiment. (2) Cell validation experiment: the RT-qPCR analysis showed that the expression of SLPI in LPS-HK2 cells of the LPS model group was significantly higher than that of the blank control group (2-ΔΔCT: 1.80±0.14 vs. 1.00±0.11, P < 0.01), and the change trend was the same with the results of bioinformatics analysis. Furthermore, knockdown SLPI gene analysis showed that the levels of inflammatory factors in LPS-HK2 cells supernatants in the gene knockdown group were significantly higher than those in the negative control group [Interleukin-6 (IL-6, ng/L): 509.58±27.08 vs. 253.87±75.83, IL-1β (ng/L): 490.99±49.52 vs. 239.67±26.97, tumor necrosis factor-α (TNF-α, ng/L): 755.22±48.66 vs. 502.06±10.92, all P < 0.01]. The above results indicated that SLPI could inhibit the inflammatory response of HK2 cells induced by LPS. The expressions of key apoptosis proteins Bax and caspase-3 in LPS-HK2 cells in the gene knockdown group were significantly higher than those in the negative control group [Bax protein (Bax/GAPDH): 1.38±0.12 vs. 1.00±0.10, caspase-3 protein (caspase-3/GAPDH): 1.44±0.15 vs. 1.00±0.11, both P < 0.05], and Bcl-2 expression was significantly decreased (Bcl-2/GAPDH: 0.83±0.08 vs. 1.00±0.05, P < 0.05), the above results indicated that SLPI could inhibit the apoptosis of cells in the inflammatory response. SLPI can inhibit the inflammatory response and apoptosis of HK2 cells induced by LPS, which may be involved in the protective mechanism of renal tubular cells in the response to sepsis, and is a potential target for the treatment of sepsis-associated AKI.

Levels of secretory leukocyte protease inhibitor expression in acute wounds.

Even with our best practices, we are frequently unable to prevent slow and stalled wound healing-particularly in people with impaired circulation and conditions such as diabetes. As a result, greater insight into the nature of wound healing and alternative treatment approaches is needed. An avenue that may be of particular promise is increasing understanding of the role of secretory leukocyte protease inhibitor (SLPI) as there is evidence that it enhances wound healing, its expression increases in response to inflammation and infection, and it exhibits anti-protease, anti-inflammatory, antiviral antibacterial and antifungal activities. The response of SLPI levels to wounding and skin injury was assessed by taking punch skin biopsies from healthy volunteers and assessing the levels of SLPI at the site of injury at the time of wounding (baseline) as well as one, two, three, four, seven, nine and 12 weeks later. A total of 35 volunteers took part in the study. Significant elevations were found: levels of SLPI were greatly increased, 12 times that at baseline, and remained elevated at three weeks despite re-epithelialisation having occurred. These findings not only suggest that levels of SLPI rise rapidly following wounding, but that these elevations are sustained, and continue to increase even when re-epithelialisation has occurred. These results suggest that the role and potential benefits of this protease inhibitor deserve further exploration.

Secretory leukocyte protease inhibitor regulates nerve reflex-mediated skin barrier function in psoriasis.

BackgroundSecretory leukocyte protease inhibitor (SLPI), a ~12 kDa protein is an important regulator of innate and adaptive immunity and a component of tissue regenerative programmes. SLPI expression is markedly elevated in chronically inflamed skin, including that of individuals suffering from psoriasis. However, the role of SLPI in these diseases remains elusive.ObjectivesThe poor understanding of the early stages of the development of psoriasis is a major obstacle to successful intervention in the skin pathology. We hypothesized that SLPI and peripheral nerves that might be activated early in the progression of the disease likely form a functional relationship to maintain skin barrier homeostasis and respond to a variety of threats.MethodsWe used skin biopsies of healthy donors and individuals with psoriasis to show expression pattern of SLPI. A role of SLPI in psoriasis was mechanistically assessed using SLPI‐deficient mice and an imiquimod (IMQ)‐induced experimental model of psoriasis.ResultsWe show that mice lacking SLPI had exaggerated skin alterations that extended beyond the treatment site in an imiquimod‐induced psoriasis. The spatiotemporally distinct skin responses in SLPI‐deficient mice, compared to their wild‐type littermates, resulted from a compromised skin barrier function that manifested itself in heightened transepidermal water loss through the larger skin area surrounding the IMQ‐challenged skin. The increased pathogenic skin changes in the absence of SLPI were reversible through pharmacological treatment that blocks a nerve‐reflex arc.ConclusionsTogether, these data indicate that SLPI plays a protective role in psoriasis through preventing skin dryness, inherent in the pathogenesis of psoriasis and that this SLPI action depends on neuronal input operating in a reflex manner. These findings reveal a previously unrecognized mechanism that maintains cutaneous homeostasis, which involves a crosstalk between the nervous system and a protein anatomically poised to fortify the epidermal permeability barrier.

SLPI suppresses hepatocellular carcinoma progression via endoplasmic reticulum stress induced apoptosis.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Secretory leukocyte protease inhibitor (SLPI) has been reported to function as a regulatory factor in several cancers. However, its biological functions and underlying mechanisms in HCC remain to be uncovered. Here, we aimed to explore the effect of SLPI in HCC. In our study, we found that the mRNA and protein expression levels of SLPI were significantly down-regulated in HCC tissues and hepatoma cell lines and low level of SLPI predicted worse survival in our HCC cohorts. In term of function, silencing of SLPI markedly promoted whereas overexpression SLPI suppressed proliferation, migration and invasion capabilities of HCC cells in vitro, and ectopic expression of SLPI inhibited the tumorigenicity of HCC cells in vivo. Mechanistic studies demonstrated that SLPI played a protective role in HCC progression via activating endoplasmic reticulum stress (ER stress)-mediated apoptosis of hepatoma cells, which could be regulated by MAPK signaling pathways. In summary, our findings highlight that SLPI could serve as a potential prognostic biomarker and putative tumor suppressor by enhancing ER stress-induced apoptosis in HCC cells mediated by MAPK signaling pathways, which provides new insights into promising therapeutic targets for HCC treatment.

Oral secretory leukocyte protease inhibitor (SLPI): Associations with oropharyngeal cancer and treatment outcome.

BackgroundRates of oropharyngeal cancer (OPC) associated with alcohol & tobacco use have decreased, while human papillomavirus (HPV) associated OPC has increased among men in the US. Secretory leukocyte protease inhibitor (SLPI), detectable in a variety of secretions, has been implicated in cancers of the head and neck, associated with tumor progression and anti-viral activity. Using the recently verified oral gargle specimen, this study aimed to assess the association of salivary SLPI expression with risk of OPC and response to treatment.MethodsA case-control study design compared levels of salivary SLPI among OPC cases to age and tobacco smoking matched healthy controls. Oral HPV DNA and SLPI was quantified from oral gargle specimens. Logistic regression estimated odds ratios (OR) and 95% confidence intervals (CI) for associations of oral SLPI and risk of OPC and treatment outcomes.ResultsIn crude and adjusted analyses of 96 OPC cases and 97 age- and smoking-matched controls, OPC was not significantly associated with oral gargle SLPI levels. Among cases, oral SLPI was associated with tonsillectomy (p = 0.018) and among controls oral SLPI was associated with HPV in the oral gargle (p = 0.008). Higher concentrations of SLPI was significantly associated with increased odds of incomplete treatment response (T2: OR: 12.39; 95% CI: 1.44–106.72; T3: OR: 9.86; 95% CI: 1.13–85.90) among all cases, but not among P16+ cases.ConclusionsSalivary SLPI was not associated with OPC risk but was associated with higher odds of an incomplete treatment response.

Secretory leukocyte protease inhibitor and progranulin as possible regulators of cervical remodeling in pregnancy

Secretory leukocyte protease inhibitor (SLPI) and progranulin (PGRN) are secretory proteins with an anti-inflammatory property. Their involvement in cervical remodeling in pregnant uterus is not yet elucidated. Thus, this study aimed to explore the significance of SLPI and PGRN in the maintenance of pregnancy by investigating the factors associated with their expression levels at the cervix. Concentrations of SLPI and PGRN proteins were measured in cervical mucus samples collected from asymptomatic pregnant women at 24–26 weeks of gestation (n = 166). The concentrations of those molecules were analyzed with clinical parameters related to risk for preterm delivery (PD). In pregnant mice, we evaluated the effect of lipopolysaccharide-induced inflammation and progesterone effect modulation on cervical mRNA expression of SLPI and PGRN. The cervical PGRN level was significantly lower in women with short cervix (<35 mm) and with a history of threatened PD. In women with short cervix, cervical SLPI concentrations were positively correlated with inflammatory cytokines, interleukin-6 (R2 = 0.75) and interleukin-8 (R2 = 0.71). In pregnant mice, cervical mRNA expressions of PGRN and SLPI were increased in response to progesterone supplementation and were suppressed by a progesterone antagonist, mifepristone. Lipopolysaccharide-induced inflammation caused remarkable upregulation in cervical SLPI mRNA level but not in PGRN. Progesterone and local inflammation are the factors controlling expression levels of PGRN and SLPI at the cervix. The observed relationship of PGRN and SLPI levels in the cervical mucus with PD-related clinical parameters supports that those anti-inflammatory molecules possibly play a significant role in appropriate regulation of cervical remodeling.

Human immunodeficiency virus type-1 Tat protein induces secretory leukocyte protease inhibitor expression in African green monkey but not human cells.

African monkeys are resistant to HIV-1 infection due to intrinsic restriction mechanisms found in their cells. However, although they can be infected by monkey-adapted modified HIV-1 particles that are designed to overcome known restriction factors, virus numbers drop to undetectable levels in immunocompetent animals. These results indicate the possibility of the presence of yet unidentified factor(s) that restrict HIV-1 in old-world monkey (OWM) cells after integration of the viral genome into the host cell chromosome. In the light of these findings, we hypothesized that OWMs might have evolved resistance mechanism(s) against HIV-1 by switching specific gene(s) on in response to the synthesis of viral proteins in infected cells. In an attempt to mimic post-infection status, we expressed HIV-1 Tat gene in African green monkey cells and compared the whole proteome with normal cells and identified secretory leukocyte protease inhibitor (SLPI), a protein with known extracellular anti-HIV-1 activity, as an over-expressed protein in the presence of HIV-1 Tat protein by 2D-PAGE and mass spectrometry analysis. We also showed that overexpression of SLPI in the presence of HIV-1 Tat was specific to monkey cells. Our results also suggest that SLPI had a previously undiscovered intracellular anti-HIV activity in addition to its extracellular activity.

Expression of the immune modulator secretory leukocyte protease inhibitor (SLPI) in colorectal cancer liver metastases and matched primary tumors is associated with a poorer prognosis

ABSTRACT Secretory leukocyte protease inhibitor (SLPI), a pleiotropic protein expressed by healthy intestinal epithelial cells, functions as an inhibitor of NF-κB and neutrophil proteases and exerts antimicrobial activity. We previously showed SLPI suppresses intestinal epithelial chemokine production in response to microbial contact. Increased SLPI expression was recently detected in various types of carcinoma. In addition, accumulating evidence indicates SLPI expression is favorable for tumor cells. In view of these findings and the abundance of SLPI in the colonic epithelium, we hypothesized SLPI promotes colorectal cancer (CRC) growth and metastasis. Here, we aimed to establish whether SLPI expression in CRC is related to clinical outcome. Using a cohort of 507 patients with CRC who underwent resection of liver metastases, we show that high SLPI protein expression in both liver metastases and primary CRC is associated with significantly shorter overall survival after resection of liver metastases. The prognostic value of SLPI in CRC patients with liver metastases implies a role for SLPI in the formation of metastasis of human CRC. Based on the immune regulatory functions of SLPI, we anticipate that expression of SLPI provides tumors with a mechanism to evade infiltration by immune cells.

TGF-β1 Impairs Vitamin D-Induced and Constitutive Airway Epithelial Host Defense Mechanisms

Airway epithelium is an important site for local vitamin D (VD) metabolism; this can be negatively affected by inflammatory mediators. VD is an important regulator of respiratory host defense, for example, by increasing the expression of hCAP18/LL-37. TGF-β1 is increased in chronic obstructive pulmonary disease (COPD), and known to decrease the expression of constitutive host defense mediators such as secretory leukocyte protease inhibitor (SLPI) and polymeric immunoglobulin receptor (pIgR). VD has been shown to affect TGF-β1-signaling by inhibiting TGF-β1-induced epithelial-to-mesenchymal transition. However, interactions between VD and TGF-β1, relevant for the understanding host defense in COPD, are incompletely understood. Therefore, the aim of the present study was to investigate the combined effects of VD and TGF-β1 on airway epithelial cell host defense mechanisms. Exposure to TGF-β1 reduced both baseline and VD-induced expression of hCAP18/LL-37, partly by increasing the expression of the VD-degrading enzyme CYP24A1. TGF-β1 alone decreased the number of secretory club and goblet cells and reduced the expression of constitutive host defense mediators SLPI, s/lPLUNC and pIgR, effects that were not modulated by VD. These results suggest that TGF-β1 may decrease the respiratory host defense both directly by reducing the expression of host defense mediators, and indirectly by affecting VD-mediated effects such as expression of hCAP18/LL-37.

SLPI Expression on Kidney Biopsies Correlates with Better Clinical Outcomes

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor produced mainly by epithelial cells. It has been described that SLPI could be a biomarker for acute kidney injury. However, we have described that SLPI has a protective role on epithelial kidney cells and favors the immunossupresive activity by decreasing lymphocyte cell proliferation. Moreover, we found that plasma levels of SLPI is high in transplant patient and there is an indirect correlation with renal function. We hypothetized that local levels of SLPI but not plasma levels of SLPI has a nephrotective activity. Therefore, the aim of the present study was to assess the expression of SLPI on kidney biopsies at the time of transplantation and to correlate the expression of it with other inflammatory markers and clinical outcomes. The study included 18 kidney biopsies obtained at the end of the surgery of transplant patients. We evaluated the transcripts levels of SLPI and others factors that are usually associated with tissue injury (such as C3 and TNF-α) or protection (as TGF-β and HMOX-1) by RT-qPCR. Figure 1A shows that there is a direct correlation between the levels of the SLPI transcript and HMOX-1 or TGF-β but not with C3 or TNF-α, suggesting that SLPI present in the graft, could be related to genes linked to cytoprotection and anti-inflammatory mediators.To determine if these results had a clinical impact, we next evaluated the relationship between SLPI transcript levels at the time of biopsy and creatinine levels during the first 10 days post-transplant. Table 1 shows that tissue SLPI transcript levels correlated indirectly with plasma creatinine values, in particular at day 10 post-transplant, suggesting the existence of an association between local SLPI transcript levels and a better short-term renal function.Remarkably, there was not a correlation between tissue SLPI mRNA and plasma levels of SLPI measured at day 1 post-transplant. However, a direct correlation was observed between plasma level of SLPI (meassured at day 1) and the creatinine levels of the first days post-transplant (Table 1). Overall, these results suggest that local SLPI could have a nephrotective activity while plasma levels of SLPI may reflect a kidney injury. ANPCYT PICT2014-2504. UBACYT2014-2017. Fundación del 3er Milenio. Fundación GADOR.

PO-314 Loss of host secretory leukocyte protease inhibitor reduces lung adenocarcinoma burden

IntroductionLung cancer accounts for approximately 11.8% of all cancer diagnoses within the European Union.1 One area of interest in lung cancer is the role of the secretory leukocyte protease inhibitor (SLPI) in lung tumorigenesis. SLPI has been identified to possess anti-bacterial, anti-protease and anti-inflammatory activity. However, SLPI levels are substantially increased in lung adenocarcinoma patient tumour tissue, with SLPI-/- mice presenting with significantly reduced pulmonary nodules in a urethane model.2 Zelvyte et al showed a significant increase in plasma SLPI levels in lung cancer patients compared to healthy controls, and that metastatic patients exhibited increased SLPI plasma levels compared to local disease however this was not significant.3Material and methodsHuman tissue microarrays were stained for SLPI. 5 × 105 3 LL murine lung adenocarcinoma cells were subcutaneously (S.C.) injected into C57BL/6 WT and SLPI-/- mice. Evan’s blue dye (45 mg/kg) was intravenously injected for vessel functionality assessment. S.C. tumours were excised and stained for H and E, CD31, Ki67, TUNEL and CD45. In an experimental metastasis (I.V.) model 1 × 106 3 LL cells were intravenously injected into C57BL/6 WT and SLPI-/- mice. Flow cytometry and qPCR analysis was performed on S.C. tumours and experimental metastasis lung tissue.Results and discussionsHuman lung adenocarcinoma tissue exhibited substantially increased SLPI expression. In a subcutaneous model SLPI-/- mice exhibited significantly reduced tumour growth and cellular proliferation compared to WT mice. Tumours from SLPI-/- mice also displayed significantly reduced functional vessel number and vessel leakiness compared to WT mice. Host SLPI loss altered the immune microenvironment in both the subcutaneous and experimental metastasis model upon qPCR and flow cytometry analysis.ConclusionThe findings to date highlight that SLPI may play a notable role in lung tumorigenesis. This may be due to altered neo-angiogenesis or tumour immune cell infiltration and requires further scientific investigation. These findings propose a role for SLPI as a biomarker or potential treatment target in lung cancer.ReferencesWorld Health Organisation, issuing body. 2012 Available at: http://globocan.iarc.fr/Pages/fact_sheets_cancer.aspx. Accessed 02–13, 2018.Nukiwa T, et al. Cancer Sci 2008 May;99(5):849–855.Zelvyte I, et al. Anticancer Res 2004 Jan-Feb;24(1):241–247.

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.