Diego Guerrieri

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.

Preconditioning donor with a combination of tacrolimus and rapamacyn to decrease ischaemia–reperfusion injury in a rat syngenic kidney transplantation model

Reperfusion injury remains one of the major problems in transplantation. Repair from ischaemic acute renal failure (ARF) involves stimulation of tubular epithelial cell proliferation. The aim of this exploratory study was to evaluate the effects of preconditioning donor animals with rapamycin and tacrolimus to prevent ischaemia–reperfusion (I/R) injury. Twelve hours before nephrectomy, the donor animals received immunosuppressive drugs. The animals were divided into four groups, as follows: group 1 control: no treatment; group 2: rapamycin (2 mg/kg); group 3 FK506 (0, 3 mg/kg); and group 4: FK506 (0, 3 mg/kg) plus rapamycin (2 mg/kg). The left kidney was removed and after 3 h of cold ischaemia, the graft was transplanted. Twenty‐four hours after transplant, the kidney was recovered for histological analysis and cytokine expression. Preconditioning treatment with rapamycin or tacrolimus significantly reduced blood urea nitrogen and creatinine compared with control [blood urea nitrogen (BUN): P < 0·001 versus control and creatinine: P < 0·001 versus control]. A further decrease was observed when rapamycin was combined with tacrolimus. Acute tubular necrosis was decreased significantly in donors treated with immunosuppressants compared with the control group (P < 0·001 versus control). Moreover, the number of apoptotic nuclei in the control group was higher compared with the treated groups (P < 0·001 versus control). Surprisingly, only rapamycin preconditioning treatment increased anti‐apoptotic Bcl2 levels (P < 0·001). Finally, inflammatory cytokines, such as tumour necrosis factor (TNF)‐α and interleukin (IL)‐6, showed lower levels in the graft of those animals that had been pretreated with rapamycin or tacrolimus. This exploratory study demonstrates that preconditioning donor animals with rapamycin or tacrolimus improves clinical outcomes and reduce necrosis and apoptosis in kidney I/R injury.

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.

Donor preconditioning with rabbit anti-rat thymocyte immunoglobulin ameliorates ischemia reperfusion injury in rat kidney transplantation.

A major concern in transplantation is the preservation of organ function. Ischemia time and microcirculatory disturbance of the organ cannot be avoided and may result in ischemia reperfusion injury (IRI), increasing the risk of delayed graft function (DGF) and acute and chronic rejection. Anti-thymocyte immunoglobulin (rATG) is a polyclonal antibody preparation with multiple effects when administered to recipients. Our objective has been to evaluate whether the administration of rATG to kidney donors instead of recipients, in an experimental model of syngeneic rat transplantation, ameliorates IRI and facilitates immediate graft function recovery. Urea and creatinine levels and necrosis severity scores were significantly lower in kidneys from donors that had received rATG (urea: control: 211±8mg/dl vs. treatment: 110±15mg/dl, p<0.001; creatinine: control: 4.6±0.24mg/dl vs. treatment: 2.6±0.22mg/dl, p<0.001; necrosis severity scores: control: 2.3 vs. treatment: 1.6, p<0.05). TUNEL staining showed 80±13 positive cells in control group and 9±3 (p<0.001) in treatment group. In situ expression of proinflammatory cytokines TNF-α, IL-6, IL-21 and TGF-β1 was reduced in rATG group (p<0.01); the same was observed for KIM-1 and caspase 8 (p<0.001). Cytoprotective genes Bcl2 and HO-1 were upregulated in situ in treatment group (p<0.001). In situ expression of IL-17, caspase 9, IL-23a, CxCl3 and ICAM1 showed no difference between groups (p>0.05). Findings suggest ATG administered to donors may ameliorate the IRI process in kidney transplantation, expressed by lower necrosis and apoptosis scores and the improvement of renal function, which may be explained through the diminished in situ expression of inflammatory mediators.

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.

Inflammatory mediators in exhaled breath condensate of healthy donors and exacerbated COPD patients.

Samples of exhaled breath condensate (EBC) provide a convenient and non-invasive method to study inflammation in lung diseases. The aim of the present study was to evaluate and compare the inflammatory protein mediator levels in EBC from healthy donors (HD) and from patients with exacerbation of chronic obstructive pulmonary disease (COPD) using an EBC collection device with and without a coating of albumin as a carrier. We studied 13 HD and 26 patients with exacerbation of COPD. The concentrations of myeloperoxidase (MPO), IFNγ and secretory leukocyte protease inhibitor (SLPI) in EBC were measured by immunoassays. The EBC samples from HD and COPD patients showed higher concentrations of MPO when samples were recovered with an albumin-coated device. Furthermore, levels of MPO in COPD patients were significantly higher than in HD. An inverse correlation was observed between MPO and spirometric parameters (FVC and FEV1). Almost all samples collected with the albumin-coated device showed higher amounts of IFNγ and SLPI than those collected with the uncoated device. The levels of SLPI in COPD patients were significantly higher than in HD. A direct correlation was observed between FVC% predicted and SLPI. We concluded that coating the collection device with albumin increased the sensitivity of the technique, at least for measurements of MPO, SLPI and IFNγ. Furthermore, the higher levels of MPO and SLPI and lower levels of IFNγ in EBC from COPD patients could reflect the immunological status and the response of lung parenchyma to treatment during the exacerbation of the illness.

α-Lipoic Acid Protects Against Ischemia-Reperfusion Injury in Simultaneous Kidney-Pancreas Transplantation.

Background Multiple factors have been implicated in the process of ischemia-reperfusion injury (IRI) in organ transplantation. Among these factors, oxidative damage seems to initiate the injury. &agr;-lipoic acid (ALA) is a potent antioxidant that is used in patients with diabetic polyneuropathy. The aim of the present study was to determine the effect of ALA in patients undergoing simultaneous kidney-pancreas transplant by evaluating the functional recovery of the graft and biochemical markers of IRI. Methods Twenty-six patients were included in the following groups: (i) untreated control; (ii) donor and recipient (DR) ALA-treated, in which ALA was administered both to the deceased donor and to the recipients; and (iii) recipient ALA-treated group. The expression of inflammatory genes, as observed in biopsies taken at the end of surgery, as well as the serum cytokines, secretory leukocyte protease inhibitor, regenerating islet-derived protein 3&bgr;/pancreatitis-associated protein, amylase, lipase, glucose, and creatinine levels were quantified as markers of organ function. Results The DR group showed high levels of TGF&bgr; and low levels of C3 and TNF&agr; in the kidneys, whereas high levels of C3 and heme oxygenase were identified in pancreas biopsies. Decreases in serum IL-8, IL-6, secretory leukocyte protease inhibitor, and regenerating islet-derived protein 3 &bgr;/pancreatitis-associated protein were observed after surgery in the DR group. Serum lipase and amylase were lower in the DR group than in the control and recipient groups. Early kidney dysfunction and clinical pancreatitis were higher in the control group than in either treatment group. Conclusions These results show that ALA preconditioning is capable of reducing inflammatory markers while decreasing early kidney dysfunction and clinical posttransplant pancreatitis.

Protective effect of immunosuppressive treatment before orthotopic kidney autotransplantation

BACKGROUND Ischemia reperfusion injury (IRI) is one of the risk factors for delayed graft function, acute rejection and long term allograft survival after kidney transplantation. IRI is an independent antigen inflammatory process that produces tissue damage. Our objective was to study the impact of immunosuppressive treatment (IS) on IRI applying only one dose of IS before orthotopic kidney autotransplantation. METHODS Twenty-four rats allocated in four groups were studied. One group served as control (G1: autotransplanted rats without IS) and the rest received IS 12 h before kidney autotransplantation (G2: Rapamycin, G3: Mycophenolate mofetil and G4: Tacrolimus). RESULTS Improved renal function and systemic inflammatory response were found among IS groups compared to the control group (Delta Urea p<0.0001; Delta Creatinine p<0.0001; Delta C3 p<0.001). The number of apoptotic nuclei in renal medulla in G1 was higher than in IS groups (p<0.0001). Tubular damage was less severe in IS groups respecting G1 (p<0.001). C3, TNF-α and IL-6 expression in kidney samples was reduced when IS was used compared to the control group. No differences were observed among the different immunosuppressive drugs tested. However, Heme oxygenase-1(HO-1) was increased only in Rapamycin treatment. CONCLUSIONS These data suggest that the use of IS administered before transplant attenuates the IRI process after kidney transplantation in an animal model.

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.

Amelioration of renal damage by administration of anti-thymocyte globulin to potential donors in a brain death rat model

Brain death (BD), a non‐immunological factor of renal injury, triggers an inflammatory process causing pathological signs of cell death in the kidney, such as necrosis and apoptosis. Kidneys from brain dead donors show lower success rates than kidneys from living donors and one strategy to improve transplantation outcome is to precondition the donors. For the first time, anti‐rat thymoglobulin (rATG) was administered in an experimental brain death animal model to evaluate if it could ameliorate histopathological damage and improve organ function. Animals were divided into three groups: V (n = 5) ventilated for 2 h; BD (n = 5) brain death and ventilated for 2 h; and BD+rATG (n = 5) brain death, ventilated for 2 h, rATG was administered during brain death (10 mg/kg). We observed lower creatinine levels in treatment groups (means): V, 0·88 ± 0·22 mg/dl; BD, 1·37 ± 0·07 mg/dl; and BD+rATG, 0·64 ± 0·02 mg/dl (BD versus BD+rATG, P < 0·001). In the BD group there appeared to be a marked increase of ATN, whereas ATN was decreased significantly in the rATG group (V, 2·25 ± 0·5 versus BD, 4·75 ± 0·5, P < 0·01; BD+rATG, 2·75 ± 0·5 versus BD 4·75 ± 0·5 P < 0·01). Gene expression was evaluated with reverse transcription–polymerase chain reaction; tumour necrosis factor (TNF)‐α, interleukin (IL)‐6, C3, CD86 showed no significant difference between groups. Increased IL‐10 and decreased CCL2 in BD+rATG compared to BD (both cases P < 0·01). Myeloperoxidase was increased significantly after the brain death setting (V: 32 ± 7·5 versus BD: 129 ± 18). Findings suggest that rATG administered to potential donors may ameliorate renal damage caused by BD. These findings could contribute in the search for specific cytoprotective interventions to improve the quality and viability of transplanted organs.