Alejandro Palma-Ramos， Gilberto Casillas-Pétriz， Laura Estela Castrillón-Rivera， Jorge Ismael Casta?eda-Sánchez， Roberto Arenas-Guzmán， Maria Elisa Drago-Serrano， Teresita Sainz-Espu?es?

1Department of Biological Systems， Autonomous Metropolitan University Campus Xochimilco， Calzada del Hueso No. 1100， Ciudad de Mexico 04960，Mexico

2Service of Dermatology and Mycology， General Hospital ‘Manuel Gea González'， Calzada de Tlalpan 4800， Tlalpan， Sección XVI， 14080 Ciudad de México， Mexico

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Activation and IL-1β secretion of human peripheral phagocytes infected with Actinomadura madurae， Nocardia asteroides and Candida albicans

Alejandro Palma-Ramos1， Gilberto Casillas-Pétriz1， Laura Estela Castrillón-Rivera1， Jorge Ismael Casta?eda-Sánchez1， Roberto Arenas-Guzmán2， Maria Elisa Drago-Serrano1， Teresita Sainz-Espu?es1?

1Department of Biological Systems， Autonomous Metropolitan University Campus Xochimilco， Calzada del Hueso No. 1100， Ciudad de Mexico 04960，Mexico

2Service of Dermatology and Mycology， General Hospital ‘Manuel Gea González'， Calzada de Tlalpan 4800， Tlalpan， Sección XVI， 14080 Ciudad de México， Mexico

ARTICLE INFO

Article history:

in revised form 22 June 2016

Accepted 14 July 2016

Available online 20 October 2016

Interleukin-1β

Phagocytosis

Actinomycetoma

Formazan blue assay

Objective: To evaluate the ability of Actinomadura madurae （A. madurae） and Nocardia asteroids （N. asteroides）， using Candida albicans （C. albicans） as prototypic control， to elicit the activation and IL-1β secretion of blood phagocytic cells from healthy donors. Methods: Microcopic evaluation of phagocytosis/activation， cell viability and spectrophotometric quantitation of endocytosis/activation， were assessed by using formazan blue test in human blood phagocytes infected with C. albicans， A. madurae or N. asteroides treated with either normal human serum （NHS） or with decomplemented NHS. Interlukin-1β from culture supernatants of infected polymorphonuclear was tested by ELISA kit assay. Results: Microscopic assay showed that phagocytosis and activation of adherent mononuclear phagocytes were greater with C. albicans followed by A. madurae and then by N. asteroides. Spectrophotometric assay in polymornuclear phagocytes infected with NHS-treated pathogens indicated that activation was similarly higher by C. albicans and A. madurae and lower by N. asteroides. Kinetic assays in infected polymorphonuclear cells showed that viability was decreased by C. albicans and N. asteroides or unaffected with A. madurae. Levels of IL-1β at 8 h of incubation were higher with C. albicans followed by A. madurae whereas lower levels were found with N. asteroides. Conclusions: The extent of cell-viability and activation as well IL-1β secretion may be related with the virulence of C. albicans and N. asteroides and other parameters remain to be explored for assesing the virulence of A. madurae.

Document heading doi: 10.1016/j.apjtm.2016.07.026

1. Introduction

Actinomycetoma is a chronic granulomatous subcutaneousdisease， with high prevalence in Mexico， caused by facultative intracellular filamentous bacteria such as actinomycetes， mainly Nocardia and Actinomadura spp.［1，2］. The hallmark triad of actinomycetoma includes tumefaction， fistulization of abscesses and extrusion of grains［3，4］. Tissular lesion induced by the grains is characterized by the presence of phagocytes such as neutrophils and macrophages surrounded by lymphocytes［5］. Analysis of tissue and serum samples from patients with actinomycetoma suggests that persistence may result from a predominant TH2 profile of anti-inflammatory mediators， such as IL-10， which decreases theTH1 response of IFN-γ， essential for the cellular immunity toward intracellular pathogens［6］.

Experimental assays in mice infected with Nocardia brasiliensis （N. brasiliensis） suggested that IFN-γ released at the site of skin lesion by dermal cells stimulates mononuclear phagocytic cells for the secretion of pro-inflammarory cytokines， such as IL-1β. The latter collaborates for early infiltration of neutrophils and on the activation of their bactericidal mechanisms of phagocytosis to clear the infection［7］. The mechanisms of actynomycetoma persistence are not fully known， but in vitro assays in cultures of bone macrophage from mice infected with N. brasiliensis suggests that cell wall lipids of this pathogen enable its ability to evade phagocytosis［8］. The latter entails several key events: attachment， activation， endocytosis， endosome generation and processing， and the intracellular killing［9］.

Candida albicans （C. albicans） is an opportunistic human pathogen yeast causing chronic mucocutaneous candidiasis that induce a predominant TH2 response［10］. Attachment of C. albicans with phagocytes leads to the expression of pro-inflammatory cytokines with a critical role in the outcome of candidiasis. In vitro assays in cultures of peripheral blood mononuclear cells （PBMC） from healthy donors or human oral epithelia cells infected with C. albicans show that IL-1β contributes to the specific T cell activation and to the recruitment of activated leukocytes and lymphocytes to the site of infection［11］. Other studies in PBMC from healthy volunteers cultured with C. albicans indicate that， the extent of IL-1β stimulation by C. albicans differes in regard other strains［12］. Analysis of C. albicans ability to elicit IL-1β has been proposed for detecting virulence factors as well as differences in virulence of Candida strains as evidenced in PBMC from healthy donors or in human oral epithelial cells［11，13］. Due to the clinical and methodological impact， this study was focused on evaluating the ability of Actinomadura madurae （A. madurae） and N. asteroides，using C. albicans as prototypic control， to elicit the activation and IL-1β secretion of blood phagocytic cells from healthy donors. This assay may contribute to the knowledge of the virulence properties of N. asteroides， and mainly A. madurae causing neglected tropical diseases.

2. Materials and methods

2.1. Microorganisms

This study， included yeast cultures in Sabouraud Dextrose Agar（Cat No 109-02 Difco Lab Michigan USA） of C. albicans ATCC 10231 strains， bacterial cultures in brain heart agar （Cat No. 13825 Merck， Naucalpan， Edo. Mex. México） of A. madurae ATCC 19425，and N. asteroides strain collected from Hospital General ‘Manuel Gea González' isolated from patients. Suspensions were prepared from all strains and were adjusted at 300×106CFU/mL based on the McFarland nephelometer by using RPMI 1640 （Cat No 11875 Gibco， Life Technologies Grand Island New York USA） with 7.5% NaHCO3（Cat No S5761 Sigma） used as a diluent.

For opsonization， a volume of 3 mL of 300×106CFU/mL in RPIM 1640 plus 7.5% NaHCO3was centrifuged at 6 000×g 40 min at room temperature. The pellet was mixed with 2 mL of complemented or decomplemented （by heating 30 min in water bath at 56 ℃） normal human serum （NHS） and incubated for 40 min at 37 ℃. After that， microorganisms were centrifuged as before and suspended in the original volume of the same diluent.

2.2. Microscopic assay of phagocytosis and activation in adherent cells

Samples of adherent cells were prepared as following. A volume of 1 mL of defibrinated blood samples from healthy volunteers were placed onto glass slide surfaces and incubated for 40 min at 37 ℃ in a humid chamber with 5% CO2atmosphere. The resulting adherent cells （in most part， the mononuclear type） were washed twice with sterile saline solution. Adherent cells （3×103approximately） were treated with A. madurae， N. asteroides and C. albicans at 3×104CFU/mL to render a proportion of one cell by 10 microorganisms tested under three different conditions: i） unopsonized or opsonized with: ii）complemented， or iii） decomplemented NHS， as described above.

For this assay， microorganisms were suspended in 1 mL of RPMI 1640/7.5% NaHCO3mixed with 0.5 mL of 0.1% p-nitrotetrazolium blue （NBT， Cat No. N6876 Sigma， dissolved in sterile saline）. Control phagocytes were mixed with RPMI 1640/7.5% NaHCO3containing NBT. Samples were incubated for 45 min at 37 ℃inside a chamber with 7% humidity and 5% CO2. Finally， adherent cells on glass slides were washed twice with ste-rile saline solution and stained with 0.5% safranin as a contrast dye. Samples were fixed with resine and a total of 100 cells were counted under optic microscope by using 40× and 100× objective-lenses. The percentage of relative activation （dependent of NADPH oxi-doreductase enzymes） was computed by dividing the number of activated cells （with intracellular blue formazan insoluble complexes）between the total numbers of phagocytic cells （un-activated （red）plus activated （blue formazan））， and multiplied by 100. Data were expressed as the mean value plus standard deviation （SD） of relative activation （n=3） from one of three repeated assays.

2.3. Spectrophotometric assay of endocytosis and activation in polymorphonuclear cells

2.3.1. Polymorphonuclear cell culture

Polymorphonuclear cells were isolated from heparinized venous blood from healthy human volunteers by using the PolymorphprepTMgradient method according to the manufacturer's instructions （Cat No. 1114683 Axis-Shield PoC AS， Oslo， Norway）. Polymorphonuclear （PMN） cells recovered from the interphase were washed twice by resuspending with sterile saline solution containing heparin（143 USP/10 mL） and centrifuging at 600×g 20 min at 20 ℃. Cell pellet was suspended in RPMI 1640 with 7.5% NaHCO3， 143 USP/10 mL heparin and supplemented with 10% decomplemented fetal calf serum （FCS， Cat No F2442 Sigma） and incubated in culture plates of 24 wells at 37 ℃ with 7% of humidity and 5% CO2in a final concentration of 3×104cells per well.

2.3.2. Spectrophotometric assay

For this assay， glass tubes （12×75 mm） containing PMN （3×104）were mixed with mi-croorganisms （3×105CFU/mL） opsonized with NHS suspended in RPMI plus NBT/NaHCO3（prepared as described before） to render a proportion of one cell to 10 microorganisms. Control PMN cells were incubated only with RPMI 1640 plus NBT/ NaHCO3without microorganisms. Mixture samples were tested by triplicate and incubated for 45 min at 37 ℃ inside a chamber with 7% humidity and 5% CO2.

After that， each tube was treated with 1 mL of pyridine and immersed for 15 min in a boiling water bath under reflux conditions to avoid the evaporation of pyridine and the oversaturation of formazan complexes. Absorbance of solubilized formazan complexes was evaluated spectrophotometrically at λ=525 nm and the concentration of formazan in μmol/mL was extrapoled from a standard curve with modifications of the original method［14］.

Briefly， standards of ascorbic acid containing 2.34， 4.69， 9.38，18.75， 37.5， 75.00 and 150 μM/mL were prepared by using 0.1% NBT as a solvent. A volume of 0.2 mL of each standard was mixed with 2 mL of 0.1M NaOH and 2 mL of 24 mM NaHCO3. Tubes were incubated for 10 min at room temperature and after the addition of 5 mL of distilled water they were centrifuged for 15 min， at room temperature and 3 000×g. After removing the supernatant， a purple insolubilized pellet from each tube was dissolved in 1 mL of pyridine and immersed for 20 min in a boiling water bath with reflux as described before. Tubes were left at room temperature to read the absorbance at λ= 525 nm using pyridine as a blank sample. Absorbance （Y axis） values compared to their corresponding ascorbic standard （X axis） were plotted for preparing the standard curve. Data of the total endocytosis of PMN were expressed as intracellular formazan in μmol/mL with microorganism or without microorganism （control）. Intracellular formazan in μmol/cell was calculated as the substraction of formazan in PMN with microorganism minus PMN without microorganism （control）divided by the total number of PMN. Finally， the percentage of actual activation was computed as a substration of formazan in PMN with microorganism minus PMN without microorganism （control）divided by the total PMN number and multiplied by 100.

2.4. Kinetic assay of cell viability in infected or uninfected PMN

Suspensions of PMN in RPMI （plus 7.5% NaHCO3， 143 USP/10 mL heparin and 10% decomplemented FCS were infected with A. madurae， N. asteroides and C. albicans （1 cell to 10 microorgnisms）. Uninfected PMN cells in the same diluent were included as controls. Culture samples tested by triplicate were taken each 60 min during 8 h of incubation at 37 ℃ with 5% CO2for monitoring the viability by counting dying cells in a Neubauer chamber by using a 0.4% tripan blue exclusion test. Results were expressed as the mean （n=3）plus SD of % viability from one of three repeated assays. Kynetic viability in cells was assessed in order to determine the time when the cell population is decreased to half and the IL-1β is detected in the culture supernates （see below）.

2.5. Kinetic assays of IL-1β secretion

Purified PMN cells （35×103per well in 24 culture plates in RPMI plus 7.5% NaHCO3， 143 USP/10 mL heparin and 10% decomplemented FCS were infected with A. madurae， N. asteroides and C. albicans in a proportion of 1 to 10 microorganisms by preincubating for 15 min at 37 ℃ with 5% CO2， while uninfected PMN in the same diluent were included as controls. Culture supernatants tested by triplicate were collected each 60 min during 8 h of incubation in order to quantify the IL-1β secretion by using an ELISA kit assay （Quantikine IL-1β Immunoassay Cat No DLB50， R&D Systems Mn， USA）. Levels of IL-1β were expressed as the mean value plus standard deviation of pg/mL. Results were expressed as the mean （n=3） plus SD of pg/mL IL-1β from one of three repeated assays.

2.6. Statistical analysis

Data were analyzed with Student's t test or one-way ANOVA by using a Sigma plot for Windows Version 11.0 software （Systat Software Incorporated， San Jose CA， USA） and P＜0.05 were considered to have significant differences.

2.7. Ethic statement and ethical approval

Samples were collected in accordance with relevance guidelines for ethical research design， confidentiality and protection human subjects. Protocol was reviewed and approved by the ethics and biosafety committees of the Universidad Autónoma Metropolitana Unidad Xochimilco. All procedures performed in studies involving human participants were in accordance with the ethical standardsof the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards （World Medical Association Declaration of Helsinki Ethical Principles for Medical Research， 1964）.

Table 1Microscopic evaluation of phagocytosis and activation of adherent cells.

Table 2Spectrophotometric analysis of endocytosis and activation in polymorphonuclear cells.

3. Results

3.1. Microscopic assay of phagocytosis and activation in adherent cells

The effects of pathogens on the phagocytosis and activation were evaluated microscopically in adherent cells purified from blood of healthy donors. By comparision with unopsonized cells included as control （-）， basal phagocytosis in adherent cells was significantly increased （P＜0.05） with C. albicans and A. madurae opsonized with whole and decomplemented NHS （DNHS）， while in the case of N. asteroides， only with whole NHS.

Values of total phagocytosis were: for C. albicans （93±7%）（NHS） and （77±8%） （DNHS）， for A. madurae （87±8%） （NHS）and （63±10%） （DNHS） and for N. asteroides （54±7%） （NHS） and（49±6%） （DNHS） （Table 1）. Values of relative activation were: with C. albicans of cells 100 % （NHS and DNHS）， with A. madurae（52±8%） （NHS） and （44±20%） （DNHS） and with N. asteroides（67±10%） （NHS） and （67±13%） （DNHS） whereas with （DNHS）（Table 1）.

3.2. Photometric assay of endocytosis and activation in polymorphonuclear cells

The effect of microorganisms on endocytosis （total μmol/mL red plus blue formazan complexes） and activation （μmol/cell blue formazan complexes） in PMN cells evaluated by photometric assays are summarized in the Table 2. Porcentage of activation of PMN cells was significantly lower （P＜0.05） by the infection with N. asteroides（6.0%±0.42%） in comparison to C. albicans （43.0%±1.01%） and A. madurae （40.5%± 4.95%）.

3.3. Kinetic assay of cell viability in infected or uninfected PMN

The kinetic of cell viability of PMN cells infected with pathogens was evaluated in order to assess their viability during the infection. From the first 5 h of incubation， viability of PMN cells from the infected and uninfected followed similar kinetic patterns . From the 6 to 8 h of incubation and by comparision with uninfected conditions，viabiality of PMN infected with C. albicans and N. asteroides was significantly decreased （P＜0.05） whereas no differences were found in cells infected with A. madurae （Figure 1）.

3.4. Kinetic assays of IL-1β secretion

The kinetic pattern of IL-1β secretion by PMN cells showed that by comparision with uninfected control cells， secretion of IL-1β was higher in PMN infected: at 1 h by C. albicans and A. madurae； at 2 h by all strains； at 4 and 5 h only by A. madurae； at 6 h by A. madurae and N. asteroides； at 7 h by C. albicans and N. asteroides； and at 8 h by all strains. Comparisons within strains indicated that the levels of IL-1β were significantly higher （P＜0.05） in cells infected by C. albicans （1， 2， 7 and 8 h）， A. madurae （4 and 5 h） and N. asteroides （6 h）. No other differences were found （Figure 2）.

Figure 1. Kinetic of viability monitored during 8 h of PMN cells infected with C. albicans， A. madurae or N. asteroides or uninfected included as controls.

Figure 2. Kinetic of IL-1β secretion in PMN cells infected with C. albicans，A. madurae or N. asteroides or uninfected control cells.

4. Discussion

Due to the critical role of phagocytosis in the outcome of infections caused by A. madurae［3］， C. albicans［15］ and N. asteroides［16］， the ability of these pathogens to modulate the function of phagocytic cells from healthy donors was explored. According to the findings，phagocytosis of adherent cells （mostly of mononuclear type）was higher when C. albicans， A. madurae and N. asteroides were opsonized with NHS （containing antibodies and complement），although lesser extent was seen with DNHS （depleted of complement）. Previous assays to assess the effect of opsonization of pathogens showed that phagocytsis and the killing of C. albicans by human monuclear cells appear to be optimal when the yeast is opsonized with NHS［17］. Opsonization with antibodies triggers the killing activity of macrophages only with N. asteroides in logphase cells but not in stationary-phase cells［16］. Phagocytosis of mononuclear cells of opsonized A. madurae is unknown， although in some actinomycetes opsonization with complement fragments and specific antibodies is correlated with the reduction in viability of Actinomyces viscosus by PMN cells［18］. Under host conditions， the substantive impact of opsonization to N. asteroides and C. albicans for enabling the phagocytosis by mononuclear cells is not fully clear［15，16］ or unknown for A. madurae. Thus， under our experimental conditions， in vitro assays suggest that opsonization with antibodies and complement may contribute to phagocytosis of the pathogens by mononuclear cells.

In this study C. albicans， A. madurae and N. asteroides elicited differential patterns of activation of adherent and PMN cells. These effects may result， in part， from the expression of surface molecules of attachment with phagocyte receptors able to elicit signal pathways leading to engulfment and activation［9］. The presumable role of surface molecules on the interaction with phagocytic cells in C. albicans may include mannoproteins and/or β-glucans［19，20］. Surface components of interaction with phagocytes in N. asteroides and A. madurae are not yet described， but may include cell wall lipids reported from N. brasiliensis［8］ or type-2 fimbriae lectin described in actinomycetes like Actinomyces［21］.

Kinetic assays showed that viability was decreased in PMN cells infected with C. albicans and N. asteroides. Decreased PMN cell viability may result from components associated with the virulence including hydrolytic enzymes from C. albicans［19，20］ or mycolic compounds described in N. brasiliensis［8，22］. During the kinetic assay， cell viability was unaffected by the infection with A. madurae， however， it contains cell wall components with potentential deleteriuos effects on cell viability［4］.

Interaction of surface components of pathogens with phagocytes via pattern recognition receptors elicits signal pathways leading to pro-inflammatory cytokines［9］. In this regard， elicitation of pro-inflammatory cytokines like IL-1β has been related with mannoproteins and β-glucans from C. albicans in human phagocityc cells［13，23］ and mycolyl glycolipids as described in Nymphaea rubra in mouse peritoneal macrophages［24］.

Accordingly， kinetic assays of IL-1β secretion were evaluated inPMN cells infected with pathogens. An overall analysis showed that decreased viability occurred along with increased IL-1β levels at（7-8） h of infection with C. albicans or at （6-8） h with N. asteroides. These findings might result from variations in the extent of the accumulative virulence effects from each pathogen that accounted for the increased IL-1β levels and decreased cell viability. Analysis of the ability to elicit IL-1β is useful for detecting virulences factors as well as differences in the virulence of Candida strains as described in human mononuclear cells and oral human epithelia［11，13］ In this assay conducted in PMN cells， findings suggest then that higher levels of IL-1β and lower viability may be associated with the virulence of C. albicans and N. asteroides. Levels of IL-1β and PMN cell viability were in general terms unaffected by the infection with A. madurae what reflect reduced virulence of this pathogen in regard C. albicans and N. asteroides.

Pathogen viability after the infection of phagocytes was not evaluated. However， in spite of this limitation， findings of this assay may contribute to designing methological strategies with clinical impact to evaluate the virulence based on the ability of pathogens associated with subcutaneous or mucocutaneous chronic infections to elicit the response of phagocityc cells and the concomitant generation of pro-inflammatory cytokines. This assay may also contribute to the knowledge about the virulence properties of N. asteroides and A. madurae causing neglected tropical diseases.

Conflict of interest statement

We declare that we have no conflict of interest

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21 May 2016

Alejandro Palma-Ramos， Department of Biological Systems，Autonomous Metropolitan University Campus Xochimilco， Calzada del Hueso No. 1100， Ciudad de Mexico 04960， Mexico.

Teresita Sainz-Espu?es， Department of Biological Systems，Autonomous Metropolitan University Campus Xochimilco， Calzada del Hueso No. 1100， Ciudad de Mexico 04960， Mexico.

E-mail: trsainz@correo.xoc.uam.mx

Tel: （+52） 55 5483 7000 （ext 3624）

Fax: （+52） 55 5483 7237