Nicolás O. Amiano

Secretory Leukocyte Protease Inhibitor : A Secreted Pattern Recognition Receptor for Mycobacteria

RATIONALE Human secretory leukocyte protease inhibitor (SLPI) displays bactericidal activity against pathogens such as Escherichia coli and Streptococcus. Furthermore, it has been reported that murine SLPI shows potent antimycobacterial activity. OBJECTIVES The aim of the present study was to investigate whether human recombinant SLPI not only kills mycobacteria but also acts as a pattern recognition receptor for the host immune system. METHODS For the in vivo experiment, BALB/c mice were infected by intranasal instillation with Mycobacterium bovis BCG and viable BCG load in lung homogenates was later determined. For the in vitro experiments, SLPI was incubated overnight with a suspension of M. bovis BCG or the virulent strain Mycobacterium tuberculosis H37Rv, and the percentage survival as well as the binding of SLPI to mycobacteria was determined. Furthermore, bacteria phagocytosis was also determined by flow cytometry. MEASUREMENTS AND MAIN RESULTS Intranasal SLPI treatment decreased the number of colony-forming units recovered from lung homogenates, indicating that SLPI interfered with M. bovis BCG infection. Moreover, SLPI decreased the viability of both M. bovis BCG and H37Rv. We demonstrated that SLPI attached to the surface of the mycobacteria by binding to pathogen-associated molecular pattern mannan-capped lipoarabinomannans and phosphatidylinositol mannoside. Furthermore, we found that in the sputum of patients with tuberculosis, mycobacteria were coated with endogenous SLPI. Finally, we showed that phagocytosis of SLPI-coated mycobacteria was faster than that of uncoated bacteria. CONCLUSIONS The present results demonstrate for the first time that human SLPI kills mycobacteria and is a new pattern recognition receptor for them.

Neutrophil elastase treated dendritic cells promote the generation of CD4(+)FOXP3(+) regulatory T cells in vitro.

We have previously shown that neutrophilic elastase converts human immature dendritic cells (DCs) into TGF-β secreting cells and reduces its allostimulatory ability. Since TGF-β has been involved in regulatory T cells (Tregs) induction we analyzed whether elastase or neutrophil-derived culture supernatant treated DCs induce CD4(+)FOXP3(+) Tregs in a mixed lymphocyte reaction (MLR). We found that elastase or neutrophil-derived culture supernatant treated DCs increased TGF-β and decreased IL-6 production. Together with this pattern of cytokines, we observed a higher number of CD4(+)FOXP3(+) cells in the MLR cultures induced by elastase or neutrophil-derived culture supernatant treated DCs but not with untreated DCs. The higher number of CD4(+)FOXP3(+) T cell population was not observed when the enzymatic activity of elastase was inhibited with an elastase specific inhibitor and also when a TGF-β1 blocking antibody was added during the MLR culture. The increased number of CD4(+) that express FOXP3 was also seen when CD4(+)CD25(-) purified T cells were cocultured with the TGF-β producing DCs. Furthermore, these FOXP3(+) T cells showed suppressive activity in vitro. These results identify a novel mechanism by which the tolerogenic DCs generated by elastase exposure contribute to the immune regulation and may be relevant in the pathogenesis of several lung diseases where the inflammatory infiltrate contains high numbers of neutrophils and high elastase concentrations.

Serine leucocyte proteinase inhibitor-treated monocyte inhibits human CD4+ lymphocyte proliferation

Serine leucocyte proteinase inhibitor (SLPI) is the main serine proteinase inhibitor produced by epithelial cells and has been shown to be a pleiotropic molecule with anti‐inflammatory and microbicidal activities. However, the role of SLPI on the adaptive immune response is not well established. Therefore, we evaluated the effect of SLPI on lymphocyte proliferation and cytokine production. Human peripheral blood mononuclear cells (PBMC) were treated with mitogens plus SLPI and proliferation was assessed by [3H]thymidine uptake. The SLPI decreased the lymphocyte proliferation induced by interleukin‐2 (IL‐2) or OKT3 monoclonal antibodies in a dose‐dependent manner. Inhibition was not observed when depleting monocytes from the PBMC and it was restored by adding monocytes and SLPI. SLPI‐treated monocyte slightly decreased MHC II and increased CD18 expression, and secreted greater amounts of IL‐4, IL‐6 and IL‐10 in the cell culture supernatants. SLPI‐treated monocyte culture supernatant inhibited the CD4+ lymphocyte proliferation but did not affect the proliferation of CD8+ cells. Moreover, IL‐2 increased T‐bet expression and the presence of SLPI significantly decreased it. Finally, SLPI‐treated monocyte culture supernatant dramatically decreased interferon‐γ but increased IL‐4, IL‐6 and IL‐10 in the presence of IL‐2‐treated T cells. Our results demonstrate that SLPI target monocytes, which in turn inhibit CD4 lymphocyte proliferation and T helper type 1 cytokine secretion. Overall, these results suggest that SLPI is an alarm protein that modulates not only the innate immune response but also the adaptive immune response.

Secretory Leukocyte Protease Inhibitor (SLPI) expression downregulates E-cadherin, induces β-catenin re-localisation and triggers apoptosis-related events in breast cancer cells

Epithelial cadherin (E‐cadherin) is involved in cell–cell adhesion through its extracellular domain, whereas the intracellular domain interacts with adaptor proteins, i.e. β‐catenin, links E‐cadherin to the actin cytoskeleton and participates in signal transduction events. E‐cadherin protects mammary epithelial cells from apoptosis and its loss during tumour progression has been documented. Secretory Leukocyte Protease Inhibitor (SLPI) has anti‐ and pro‐tumourigenic activities but its role in breast cancer has not been fully elucidated. Notwithstanding its relevance, how SLPI affects E‐cadherin in breast cancer is still unknown. This study evaluated the effect of SLPI upon E‐cadherin/β‐catenin expression and apoptosis‐related markers in murine (F3II) and human (MCF‐7) breast tumour cells either treated with exogenous recombinant human SLPI (rhSLPI) or stably transfected with a plasmid encoding its sequence.

Anti-tumor effect of SLPI on mammary but not colon tumor growth†

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor that was related to cancer development and metastasis dissemination on several types of tumors. However, it is not known the effect of SLPI on mammary and colon tumors. The aim of this study was to examine the effect of SLPI on mammary and colon tumor growth. The effect of SLPI was tested on in vitro cell apoptosis and in vivo tumor growth experiments. SLPI over‐expressing human and murine mammary and colon tumor cells were generated by gene transfection. The administration of murine mammary tumor cells over‐expressing high levels of SLPI did not develop tumors in mice. On the contrary, the administration of murine colon tumor cells over‐expressing SLPI, developed faster tumors than control cells. Intratumoral, but not intraperitoneal administration of SLPI, delayed the growth of tumors and increased the survival of mammary but not colon tumor bearing mice. In vitro culture of mammary tumor cell lines treated with SLPI, and SLPI producer clones were more prone to apoptosis than control cells, mainly under serum deprivation culture conditions. Herein we demonstrated that SLPI induces the apoptosis of mammary tumor cells in vitro and decreases the mammary but not colon tumor growth in vivo. Therefore, SLPI may be a new potential therapeutic tool for certain tumors, such as mammary tumors. J. Cell. Physiol. 228: 469–475, 2013.

The IFNG rs1861494 Single Nucleotide Polymorphism Is Associated with Protection against Tuberculosis Disease in Argentina

Interferon gamma (IFNG) plays a key role during Mycobacterium tuberculosis (Mtb) infection, and several polymorphisms located in its gene are associated with risk of tuberculosis in diverse populations. Nevertheless, the genetic resistance/susceptibility to tuberculosis in Argentina is unknown. The IFNG rs1861494 polymorphism (G→A) was reported to alter the binding of transcription factors to this region, influencing IFNG production. Using a case-control study, we found an association between the AA and AG genotypes and tuberculosis resistance (AA vs. GG: odds ratio (OR) = 0.235, p-value = 0.012; AG vs. GG: OR = 0.303, p-value = 0.044; AA vs. AG: OR = 0.776, p-value = 0.427; AA + AG vs. GG: OR = 0.270, p-value = 0.022). Moreover, Mtb-antigen stimulated peripheral blood mononuclear cells (PBMCs) from healthy donors and AA carriers secreted the highest amounts of IFNG in culture supernatants (p-value = 0.034) and presented the greatest percentage of CD4+IFNG+ lymphocytes (p-value = 0.035), in comparison with GG carriers. No association between the polymorphism and clinical parameters of tuberculosis severity was detected. However, our findings indicate that the rs1861494 single nucleotide polymorphism (SNP) could be considered as a biomarker of tuberculosis resistance in the Argentinean population.