Original article
Paracoccidioides brasiliensis
GP43-derived peptides are potent modulators
of local and systemic inflammatory response
Fabiana T.C. Konno
a, Juliana Maricato
a, Adriana Y.C. Konno
a, Ma´rcia G. Guereschi
a,
Bruno C. Vivanco
a, Luciano dos Santos Feitosa
b, Ma´rio Mariano
a, Jose´ Daniel Lopes
a,*
aUniversidade Federal de Sa˜o Paulo
eUNIFESP, Department of Microbiology, Immunology and Parasitology, Discipline of Immunology, Sa˜o Paulo, Brazil
bCamilo Castelo Branco University, Biomedical Engineering Center, 08230-030, Rua Carolina Fonseca 584, Sa˜o Paulo, SP, Brazil
Received 22 August 2011; accepted 22 December 2011 Available online 10 January 2012
Abstract
Paracoccidioidomycosis is a systemic granulomatous disease caused by the dimorphic fungusParacoccidioides brasiliensis. Its major antigen is a 43 kDa glycoprotein whose peptides embody different functions: P10 peptide, a T-cell epitope, induces protective response while P4 and P23 peptides inhibit both, macrophage functions and inflammatory reaction, thus facilitating infection. Here we investigated the modulating mechanisms of the immune response exerted by P4 and P23 involved in the latter inhibitory effect on macrophages. Moreover we analyzed the peptides effects in different models in vivo. While evaluating whether P4 and P23 present systemic anti-inflammatory effects in vivo, we showed that their intraperitonial administration decreased footpad swelling in mice infected with eitherP. brasiliensisorMycobacterium bovis. Both, qPCR and ELISA assays suggested that this anti-inflammatory effect depended on alterations in the kinetics of production of innate immunity modulators such as TNF-a, IL6, IL10 and TLR2. IL10 seems to be early produced than TNF-aand IL6, produced later in presence of peptides.
Higher doses or intravenously given P4 and P23 resulted in earlier and more prolonged anti-inflammatory effects. Moreover, continuous treatment with P4 and P23 sustained the anti-inflammatory activity throughout.
Ó2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords:P4 and P23 peptides; GP43; Macrophage; Inflammation;P. brasiliensis
1. Introduction
Paracoccidioidomycosis (PCM) is a systemic granuloma-tous disease caused by the dimorphic fungusParacoccidioides brasiliensis. PCM is prevalent in Brazil and other Latin American countries [1e3]. The acute or subacute form of
PCM affects both genders, predominantly male, and chiefly involves the reticuloendothelial system. The acute form is characterized by impaired cellular immunity, negative delayed-type hypersensitivity reactions, increased systemic proliferation of the fungus, and high mortality rates. The chronic form affects mainly adult males with predominantly
pulmonary and/or mucocutaneous involvement[4]. This form shows exacerbated host cellular immune responses and formation of granulomas containing fungal cells and may develop extensive sequels, including fibrotic lesions and impairment of lung function [3,5]. Cellular immunity is the host most important defense mechanism against both experi-mental and human PCM[6,7].
The glycoprotein gp43 from P. brasiliensis [8,9], is the main PCM diagnostic antigen[10], being recognized by 100% of sera from infected patients[11,12]. This antigen was firstly described as an adhesion protein for its capacity of binding to matrix extracellular components, such as laminin [13,14]. Gp43 plays different roles in the pathogenesisP. brasiliensis. It strongly stimulates in vitro granuloma-like formation by B-1 cells and macrophages [15]. Moreover, a peptide named P10 (QTLIAIHTLAIRYAN), derived from its sequence, mediates specific T-cell activation, leading to protection against PCM. * Corresponding author. Rua Botucatu 862, 4th floor, 04023-901, Sa˜o Paulo,
Brazil. Tel.:þ55 11 55496073; fax:þ55 11 555723328.
E-mail address:jdlopes@unifesp.br(J.D. Lopes).
www.elsevier.com/locate/micinf
1286-4579/$ - see front matterÓ2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
In mice it elicits an interferon-gamma (IFN-g) dependent immune protection and immunization with P10 improved the therapeutic effectiveness when combined with antifungal drugs [5,16,17]. Contrarywise, gp43 participates in the installation mechanisms of primary infection by inhibiting both, phagocytosis and fungal intracellular killing[18]. Some endogenous peptides, named Antimicrobial Peptides (AMPs), can act as immune modulators and their production may depend on the host immune response to infections, involving inflammation and vascular effects [19]. Considering that the gp43 molecule induces inhibition of phagocytosis [18,20], Konno et al.[21]demonstrated that two peptides derived from gp43, named P4 and P23, inhibit phagocytosis of zymosan particles and P. brasiliensis yeasts by macrophages. NO is related with the killing capacity of macrophages [22]and its production is also inhibited by P4 and P23. In vivo experi-ments showed that local administration of the peptides in mice infected with P. brasiliensis or BCG promoted decrease of footpad inflammation[21].
Considering the immune modulator function of some AMPs and the inhibitory effect of P4 and P23 on macrophages [21], and assuming that these findings could bear some correlation, we herein investigated mechanisms through which P4 and P23 down-regulate macrophage functions and inflam-matory response. Moreover, we used P4 and P23 with different treatment protocols in vivo to determine the schedule by which they may produce best results in mice.
2. Materials and methods
2.1. Peptide synthesis and purification
Peptides were kindly synthesized by MA Juliano, Federal University of Sao Paulo (UNIFESP), SP, Brazil, by the 9- fluoroenylmethoxycarbonyl technique as described by Atherton and Sheppard [25], with an automated benchtop simultaneous multiple solid-phase peptide synthesizer (PSSM 8 system; Shimadzu, Tokyo, Japan). The amino acid sequences are NLGRDAKRHLSKHWDTFITEDDFKNIAAAGL (P4) and AFEVGAGWYFWTWKTEGAPGWDMQD (P23). Abbrevia-tions for amino acids and nomenclature of peptide structures followed the recommendations of the IUPAC-IUB (Commission on Biochemical Nomenclature) [26].
2.2. Mice
Male Balb/c and C57/Bl6 mice, 6-8 week-old, were obtained from the animal facility of Federal University of Sa˜o Paulo (UNIFESP), SP, Brazil. Male KO-IL10 and CAF mice, 6-8 week-old, were bred and maintained in the facility of the Discipline of Immunology, UNIFESP. Animals were euthanized according to Guide for the Care and Use of Laboratory Animals (NIH guide).
2.3. Bone marrow-derived macrophages (BMM)
BMM were generated as previously described [27] from 6-to 8-week-old Balb/c mice. Cultures were kept at 37 C in
the presence of 5% CO2 in RPMI supplemented with 10% fetal bovine serum and 5% of conditioned medium from cultures of L929 cells, known to be high producers of MCSF. Differentiated macrophages were maintained in culture or were counted and plated at 2106 cells/well onto a 6-well tissue culture plate.
2.4. Phagocytic test
Differentiated BMM were challenged with zymosan parti-cles (Sigma, 2 mg/ml) with or without P4 or P23 (5mg/ml) after incubation at 37C for 2, 4, 8 or 12 h. Afterward, culture supernatants were harvested, kept at 70
C and analyzed for cytokines and NO. Cells were then rinsed with PBS for removal of non-internalized zymosan. RNA was isolated from BMM for PCR quantitative assays.
2.5. Isolation of total RNA and cDNA synthesis
Total RNA was isolated from fresh differentiated BMM disrupted in the presence of Trizol reagent (Invitrogen), following the manufacturer’s instructions. Contaminating DNA was digested with RNAse-free DNAse I (Promega), as described [28]. RNA samples were quantified using a Nano-Drop apparatus (Thermo Scientific, USA), analyzed for integrity in formaldehyde agarose gels and stored at 70C in Dimethyl pyrocarbonate (DEPC)-treated water. cDNA synthesis was made from 3mg of total RNA with SuperScript II reverse transcriptase kit (Invitrogen), as described[29].
2.6. Quantitative RT-PCR (qPCR)
For experiments of quantitative RT-PCR SybrGreen based system of detection was used (Applied Biosystems, USA), as described by[30]. Briefly, for detection of IL6, IL10, TNF-a and Toll-like receptor 2 expression levels, the reactions were carried out using 2mM of sense and antisense primers for each gene and GAPDH primers as endogenous control, 10ml of the SybrGreen PCR master mix (Applied Biosystems) and 3ml of cDNA diluted 2.5(synthesized from 3mg of total RNA). As recommended for relative quantification experiments using
(2 min) and 95
C (1 min), followed by 40 cycles at 95 C (15 s) and 60
C (1 min). The melting curve was determined with an additional cycle of 95
C (15 s), 60
C (20 s) and 95
C (15 s). Reactions were performed in triplicates, and the endogenous control (GAPDH) was used as calibrator. Relative expression was measured using∆∆Ct method and calculations were performed as described[34]. Results are representative of 3 individual experiments.
2.7. Cytokine analysis
Cytokine concentrations were measured in culture super-natants using ELISA kits for TNF-a, IL-6 and IL-10 (eBioscience) according to the manufacturer’s protocol. Results are representative of 3 individual experiments, all made in triplicates.
2.8. Analysis of cell phenotype
BMM were stained with monoclonal antibodies and analyzed by flow cytometry using a FACSCalibur System (BD Biosciences). The following primary antibodies were used: phycoerythrin (PE) rat anti-mouse CD11b (BD Biosci-ences), allophycocyanin (APC) rat anti-mouse TLR2. Cell staining was carried out according manufacturer’s protocol. Results are representative of at least 2 individual experiments made using triplicates.
2.9. Influence of peptides on the inflammatory response
The systemic influence of peptides on the inflammatory response was evaluated by inoculation of Mycobacterium bovis(bacillus Calmette Guerin/BCG, Butantan Institute, Sa˜o Paulo, Brazil), at a concentration of 100 mg/ml, orP. brasi-liensiscells in the subcutaneous tissue of the dorsal region of the footpad of male mice (106 CFU of BCG or 2 106P. brasisliensiscells, respectively). Five groups of male Balb/c mice (6e8 weeks old), were used. Treatment by intraperito-neal or intravenous routes with P4 or P23 at concentrations of 100 or 200mg were used in test groups. The kinetics of the inflammatory response was evaluated by dial thickness gauge (Mitutoyo) at different time intervals (30 min, 2, 4, 8, 12, 24 h and every 24 h for 7 days)[35]. Results are representative at least 2 individual experiments with 5 animals per group.
2.10. Histopathology
Groups of 5 Balb/c mice, CAF mice, C57/bl6 mice and KO-IL10 mice were infected in the footpads with P. brasi-liensisor BCG and killed after 8 h. The infected footpads were excised, fixed in 12% buffered formalin, and embedded in paraffin. Sections were stained with hematoxylin-eosin or Grocott method and examined microscopically (Nikon,Tokyo, Japan). Quantification of area withP. brasiliensiswas done in sections stained with Grocott by Image J software (5 slides/30 fields per slide).
2.11. Statistical analysis
Results were expressed as the meanthe standard devia-tions (SD). Data were analyzed by unpaired Student’s t test with Welch’s correction (two-tailed) used for comparison of the two groups when the data met the assumptions of the t tests. Analysis of variance (ANOVA) followed by the TukeyeKramer test was sometimes applied (INSTAT soft-ware, GraphPad, San Diego, CA). P values under 0.05 indi-cated statistical significance.
3. Results
3.1. Systemic anti-inflammatory effect of P4 and P23
Considering the inhibitory effect of P4 or P23 in inflam-mation observed in vitro and in vivo [21], we investigated whether these peptides had any systemic effect in vivo on the kinetics of inflammation elicited by P. brasiliensiscells inoc-ulated into the subcutaneous tissue of the animals. To show systemic activity, 100 mg of peptides P4 and P23 were given intraperitoneally together with the footpad challenge with yeasts of P. brasiliensis. Results showed decrease in footpad swelling test (FPT) indexes in the first 12 h after inoculation of P. brasiliensiscell (Fig. 1A). In Balb/c mice, decrease in FPT was significant after 8 h of infection with peptides treatment and was maintained until 12 h (Fig. 1A). Histological analysis to determine area with P. brasiliensis (Fig. 1B above) on the footpad of the infected mice and quantification of fungus per field (Fig. 1B below) showed that the treated groups presented higher amounts of fungi. Histological analysis with HE showed no difference (data not shown). These results confirm results of Konno et al. (2009)[21]suggesting that P4 and P23 inhibit the phagocytic ability of macrophages, thereby facilitating fungal growth. Additionally, we did tests of FPT swelling using CAF mice that are assumed as having exacerbated inflammatory reactions. We infected their footpad either withP. brasiliensis cells or BCG and the mice were treated or not with P4 or P23. We observed that P4 and P23 led to significant decrease in FPT swelling indexes after 4 h of infection with treatment. This effect was maintained for 12 h (Fig. 2A,B). Histological anal-ysis of infected footpads showed again areas with higher amounts of yeasts in treated groups (Fig. 2C). Histological analysis with HE showed no difference (data not shown). Thus, P4 and P23 presented systemic anti-inflammatory effect and seemed to inhibit the phagocytic ability of macrophages thereby facilitating fungal growth.
3.2. Influence of peptides on the pro-inflammatory cytokines
the presence or absence of P4 and P23. qPCR analyses were evaluated 2 and 8 h after stimulation and treatment with peptides.
We observed that P4 and P23 promoted increased TNF-a mRNA 2 h after stimulation with zymosan particles; however, after 8 h stimulus, there was no difference in expression of TNF-a between groups (Fig. 3A). To confirm these results, ELISA was performed to quantify TNF-a production by macrophages after 2, 4, 8 and 12 h of treatment with peptides and stimulation. Corroborating with mRNA expression results, groups only stimulated with zymosan particles had increased production of TNF-aas early as 2 h. Groups treated with P4 and P23 showed similar rise only after 4 h of the stimulus (Fig. 3B). Thus, P4 and P23 promoted increase expression of TNF-agene after 2 h of stimulation and this effect seems to result in increased production of TNF-a after 4 h (Fig. 3B). Contra-rywise, macrophages stimulated with zymosan particles increased secretion of TNF-aearlier (2 h). These results suggest that P4 and P23 promoted a noticeable delay in the production of TNF-aby macrophages.
P4 and P23 also had influence in mRNA expression of IL6. In the presence of peptides we observed increase of IL6 mRNA expression 8 h after the stimulation (Fig. 3C). As for P23, 2 h only were needed to promote similar expression (Fig. 3C). This resulted in significant increase of IL6 production after 8 h in groups treated with P4 and P23 while the production of IL-6 by the control group showed no alter-ation in any of the evaluated times (Fig. 3D).
3.3. Influence of P4 and P23 on IL10
IL10 is the most important cytokine with anti-inflammatory properties[36,37]. Analysis by qPCR showed that P4 and P23 promoted increase in expression of IL10 mRNA after 2 h of the stimulation with zymosan particles (Fig. 4A). P23 main-tained this higher level up to 8 h (Fig. 4A). Production of IL10 by macrophages seems more precocious in groups treated with P4 and P23 since macrophages in the control group only promoted a small increase in levels of IL-10 8 h after stimu-lation (Fig. 4B). Moreover, experiments in vivo with infection
Fig. 1. Systemic effect of treatment with P4 and P23 on the evolution ofP. brasiliensiscells-induced edemas in the footpads of mice (n¼5 per group). (A) Footpad
swelling of Balb/c infected with P. brasliensis and treated with P4 or P23 (A). Negative control (Cneg), P4 control (CP4) and P23 control (CP23) groups received
PBS in footpad and intraperitoneal treatment with PBS, P4 or P23 respectively. Positive control (Cpos), P4 and P23 groups was infectedP. brasiliensisand treated
intraperitonealy with PBS, P4 or P23 respectively. (B) Histological analyses ofP. brasiliensiscells stained by Grocott (100). (B) Quantification of area withP.
of KO-IL10 mice footpads withP. brasiliensisshowed that, in this lineage, P4 and P23 had no anti-inflammatory effect since comparison in FTP swelling between control and treated groups was not different (Fig. 4C,D).
3.4. Influence of peptides on the molecules involved in inflammation
In addition to genes related to pro- and anti-inflammatory cytokines we also checked the expression of molecules involved in innate immunity as Toll like receptor 2 (TLR2) and NO. Analysis of gene expression demonstrated that mRNA of TLR2 was not significantly different between peptide treated and control groups (data not shown). However, we verified by flow cytometry analysis performed with BMM in 2, 4, 8 and 12 h after incubation with zymosan and peptides that expression of TLR2 was a little delayed in treated groups as compared to the control group (data not shown).
Moreover, as shown by Konno (2009)[21], we observed that only until 2 h both, P4 and P23 significantly down-regulated NO
liberation by BMM stimulated with zymosan particles. After 4, 8 and 12 h NO levels had no significant difference between groups (data not shown).
Altogether these results suggest that P4 and P23 appear to modulate the mRNA expression and production of important inflammatory mediators, hence promoting reduction of the initial effects of the inflammatory process.
3.5. Effect of peptides in vivo using different protocols
Considering the systemic inhibitory effect of P4 and P23 observed in vivo, we investigated whether different adminis-tration protocols could improve the anti-inflammatory effects of P4 and P23. First, we determined duration of anti-inflammatory effects of P4 and P23 after administration. We found that after 24 h of administration of P4 or P23 no effect of peptides was observed (data not shown). We then verified systemic effects of P4 and P23 in longer times. For this, Balb/c mice infected with P. brasiliensis in the footpads received every 8 h P4 or P23 intraperitoneally (100 mg). Footpad
Fig. 2. Systemic effect of treatment with P4 and P23 on the evolution ofP. brasiliensiscells-induced edemas (A) or BCG (B) In the footpad of mice (n¼5 per
group). CAF mice were used in these experiments. Negative control groups (Cneg), received PBS in their footpad and were treated intraperitonealy with PBS.
Positive control (Cpos), P4 and P23 groups were infectedP. brasiliensis(A) or BCG (B) and treated intraperitonealy with PBS, P4 or P23 respectively. (C)
Histological analyses ofP. brasiliensiscells stained by Grocott (100). Quantification of area with P. brasiliensiscells were done with Image J software.
measurement was made every 8 h for 3 days. Significant decrease in FTP swelling indexes was observed along the 3 days tested (Fig. 5A). These results showed that P4 and P23 are able to keep the anti-inflammatory effect when
administered continuously (Fig. 5A). Moreover, histological analyses to detectP. brasiliensisconfirmed again that P4 and P23 promote increased areas with fungi after treatment (Fig. 5B).
Fig. 4. Evaluation of anti-inflammatory IL10 involved in inflammation. Macrophages were stimulated with zymosan particles in presence or not of P4 or P23. mRNA expression was done 2 or 8 h after stimulus with zymosan particles and ELISA was performed after 2, 4, 8 and 12 h after stimulus with zymosan. The methods used were qPCR and ELISA respectively; control group were macrophages stimulated with zymosan only. (A) IL10 mRNA expression and (B) dosage of
IL10 secreted by BMM. Importance of IL10 in vivo was performed with KO-IL10 mice infected in the footpad withP. brasiliensisand treated or not with P4 or
P23. Treatment with P4 and P23 on the evolution ofP. brasiliensiscells-induced edemas in footpad of (C) C57/bl6 mice or (D) in KO-IL10 mice (n¼5 per group).
P*<0.05,P**<0.01 andP***<0.001 when values were compared with control group. Results are representative of at least two individual experiments.
Fig. 3. Evaluation of mRNA expression and production of pro-inflammatory cytokines in macrophages stimulated with zymosan particles in presence or not of P4 or P23. The methods used were qPCR and ELISA respectively. mRNA expression was done 2 or 8 h after stimulus with zymosan particles and ELISA was
performed after 2, 4, 8 and 12 h after stimulus with zymosan. Control group were macrophages only stimulated with zymosan. (A) TNF-amRNA expression and
(B) dosage of TNF-asecreted by BMM. (C) IL6 gene expression and (D) IL6 secreted by BMM.P*<0.05,P**<0.01 andP***<0.001 when values were
In order to verify the effect of peptides in higher doses or different routes of administration, we treated Balb/c mice with P. brasiliensis infected footpads with 200 mg of P4 or P23 intraperitoneally or injected the peptides (100mg) into the tail vein. Both groups showed significant decrease in FTP swelling indexes (Fig. 6A,B,C,D). Further, these smaller FTP swellings were observed earlier (in 2 h after the peptides administration) than the previous experimental conditions. These smaller in footpad swelling indexes were also maintained for longer periods (Fig. 6A,B,C,D).
4. Discussion
Gp43 plays different roles in the pathogenesis P. brasi-liensis. Our group demonstrated that gp43 down regulates macrophage functions in vitro[18]and that two peptides (P4
and P23), derived from gp43, inhibit phagocytosis of zymosan particles andP. brasiliensisyeast by macrophages[21]. These data suggested that gp43, or more specifically P4 and P23 peptides, could facilitate the establishment and dissemination of P. brasiliensisin the initial phase of infection.
Gp43 also has a role in the protection mechanisms against the fungus, since subcutaneous infection with whole yeasts, led to immune protection or exacerbated disease depending on the route of challenge[38]. The protective effect of gp43 was confirmed after immunization with peptide P10, derived from its sequence, which also promoted protection in animal model [16]. This has been one of few examples in which a short sequence of the whole molecule could be correlated with one very specific function. These distinct responses by the same glycoprotein are due to different peptides of gp43 acting at different times of the immune response.
Fig. 5. Effect of long term treatment with P4 and P23 on the evolution ofP. brasiliensis-induced edemas (A) in the footpad of mice (n¼5 per group). Negative
control group (Cneg) received PBS in the footpad and was also treated intraperitonealy with PBS. Positive control (Cpos), P4 and P23 groups was infectedP.
brasiliensisand treated intraperitonealy with PBS, P4 or P23 respectively. Administration of P4, P23 or PBS was performed every 8 h for 3 days. (B) Histological
analyses ofP. brasiliensiscells stained by Grocott (100) and quantification of area withP. brasiliensiscells (8 h after infection and treatment) done with Image J
Production of inflammatory cytokines has been shown to play important roles in the early host response to pathogens [39]. Macrophages are part of the innate immune response constituting the first line of defense against invading patho-gens. They are involved in recognition, phagocytosis and destruction of many organisms.
In addition, macrophages are also involved in antigen presentation and secretion of a wide variety of products, including enzymes, enzyme inhibitors, cytokines, chemokines, among others [40]. Thus, P4 and P23 might interfere in macrophages functions by modifying production and expres-sion of molecules involved in inflammation.
Recently, it has become evident that some endogenous peptides, generally named Antimicrobial Peptides (AMPs), can also act as immune modulators and that their production may depend on the host immune response to infections, involving inflammation and vascular effects [19]. Our laboratory has shown that peptides derived from P. brasiliensis full sequence could interfere with the pathoge-nicity of this mycosis[23]. Other peptide named KP derived Killer toxin (KT) from Pichia anomala exert microbicidal activity over other microorganisms showing KT receptor (KTR)[24].
Peptides P4 and P23 can be regarded as AMPs, assuming that their inhibitory effect on macrophages[21]might be due to the fact that they can be acting as immune modulators. We then investigated some mechanisms by which P4 and P23 are
involved in reducing the inflammatory response, attempting to further understand their roles on macrophage activities.
To understand the immunoregulatory phenomena involved in the action of P4 and P23, we analysed anti- and pro-inflammatory molecules produced by macrophages, such as mRNA expression and production of TNF-a. TNF-a is a pleiotropic cytokine produced by different types of cells. However, cells of the monocytic lineage, such as macro-phages, are its primary synthesizers[41]. TNF-ais a powerful pro-inflammatory agent, rapidly released after infection, that regulates many facets of macrophage functions [42]. Results suggested that P4 and P23 delayed the production of TNF-a by macrophages, which could be related with inhibitory effects on macrophages early in inflammation[21].
IL6 is an important pleiotropic pro-inflammatory cytokine that regulates immunological reactions, participating in host defense, inflammation, haematopoiesis and oncogenesis[43]. P4 and P23 also had influenced in mRNA expression of IL6. In their presence we observed increase of IL6 mRNA expression 8 h after stimulation with zymosan. Furthermore, P4 and P23 seem to induce increased production of IL6 as compared with control group in all evaluated times. Such IL6 increase could be a compensation mechanism for to the delay in the production of TNF-a, also a pro-inflammatory cytokine secreted in the early inflammatory process.
IL10 is the most important anti-inflammatory cytokine whose most important sources in vivo seem to be monocytes
Fig. 6. Effect of different administration protocols of P4 and P23 on the evolution ofP. brasiliensis-induced edemas in the mice footpads. Mice infected in the
footpad withP. brasiliensiswere treated with higher dose of P4 and P23 (200mg) or with peptides (100mg) administrated in tail vein (n¼5 per group). (A)
Treatment with increased dose of P4 (B) or P23. Administration of P4 (C) or (D) P23 in tail vein. Negative control (Cneg), P4 control (CP4) and P23 control
(CP23) groups received PBS both in the footpads and as treatment, P4 or P23 respectively. Positive control (Cpos), P4 and P23 groups were infectedP. brasiliensis
and treated intraperitonealy with PBS, P4 or P23 respectively. Results are representative of 2 independent experiments.P*<0.05,P**<0.01 andP***<0.001
and macrophages[36,37], wherein it diminishes production of inflammatory mediators and inhibits antigen presentation[44]. As P4 and P23 seem to be anti-inflammatory peptides, we studied their effect on mRNA gene expression and production of IL10. In macrophages they seem more precocious in groups treated with P4 and P23 since the control group only presented a small increase in the levels of IL-10 8 h after stimulation. IL10 blocks activation of cytokine synthesis and several accessory cell functions, rendering this cytokine a potent suppressor of effector functions of macrophages and other immune cells [42]. This fact could explain exacerbated increase of IL6 while there is decreased production of IL10.
We also checked molecules involved in innate immunity, such as Toll like receptor 2 (TLR2) and NO. TLR are trans-membrane proteins that interact with invariant molecular structures from pathogens (PAMPs)[45]. Expression of TLRs is modulated during inflammation mainly by molecules such as those cytokines previously evaluated. Flow cytometry analysis showed that expression of TLR2 was delayed in treated groups as compared to control group (data not shown). Delayed expression of TLR2 in BMM treated with peptides seems to be related to the inhibitory effect of peptides since TLRs are directly involved with activation of innate immunity [43]. Moreover, these receptors appear to be used byP. bra-siliensis yeast cells to gain access into macrophages and to escape from other fungicidal or fungistatic mechanisms of innate immunity [46]. Thus, delayed expression of TLR2 in BMM could be associated with inhibitory effect in phagocytic potential of macrophages in the presence of our peptides[21]. Activated macrophages migrate to sites of inflammation where they encounter and lyse pathogens. This is accom-plished by an increased production of toxic oxygen species and via induction of nitric oxide synthase (iNOS) to produce nitric oxide (NO)[47]. Konno et al. (2009)[21]demonstrated that P4 and P23 down-regulated NO liberation by bone marrow macrophages stimulated with zymosan particles after incubation for 1 h 30 min. We here observed that after 2 h both, P4 and P23, significantly down-regulated NO liberation by BMM stimulated with zymosan particles. After 2 h no difference was observed (data not shown).
Summing up, P4 and P23 modulate production of important inflammatory mediators promoting reduction of the beginning inflammatory process.
Considering the inhibitory effect of P4 or P23 in inflam-mation observed in vitro and in vivo[21], we then investigated whether P4 and P23 showed any systemic effect in vivo on the kinetics of inflammation. Results of footpad swelling test (FPT) showed decreased indexes in the first 12 h after inoculation ofP. brasiliensiscells in different strains of mice. Only in KO-IL10 mice, no effect of P4 and P23 was observed. This finding suggests again the importance of IL10 in the participation of P4 and P23 in inflammation. Furthermore, histological analysis to quantify P. brasiliensis yeasts in the footpad of the infected mice showed that the treated groups presented higher amounts of fungi. These results combined with results of Konno et al. (2009) [21] suggest that P4 and P23 are able to inhibit the phagocytic ability of macrophages thereby facilitating fungal
growth. In conclusion, P4 and P23 showed systemic anti-inflammatory effect and seem to inhibit the phagocytic func-tion of macrophages thereby facilitating fungal growth in different models here studied. Similar results were seen when using infection with BCG, suggesting that the anti-inflammatory effect of peptides is not restricted to the PCM model. Additionally, Konno et al., 2009[21]demonstrated by Jameson Wolff (Protean software) analyses that P23 has low antigenic indexes whereas P4 show antigenic indexes slightly higher in its N-terminal region. P4 and P23 have no epitopes that could be presented to T cells. So, according with these char-acteristics of P4 and P23 observed in silico analyses and bio-logical effects, we assumed that the peptides do not elicit specific immune response. If I was so, an immune response to the fungus in presence of P4 or P23 would be more similar to control group. Moreover, when we evaluated production of cytokines in vivo in sera of mouse infected in footpad withP. brasiliensisand treated or not intraperitoneally with P4 or P23, we found no difference in production of systemic IL6, TNF-a and IL10 between groups in any of the times evaluated (2, 4, 8, 12 h) (data not shown). These results suggest that P4 and P23 can act only in the inflammatory focus and that they are not able to alter cytokine production systemically. We then evaluated whether continuous treatment with peptides could maintain the reduced inflammation. Our findings demonstrated that treat-ment with P4 and P23 every 8 h induced continuous anti-inflammatory effects during the evaluated period. Moreover, P4 and P23 administered in higher doses or intravenously had improved effects since their anti-inflammatory action was observed earlier and lasted longer. Because P4 and P23 are exposed at the gp43 molecule surface[21], it can be assumed that they could be responsible for the role as immune modulator. Moreover, findings here presented demonstrated that P4 and P23 have a potent effect on the inhibition of inflammation through modulation of inflammatory molecules. Additionally, analyses of peptides by BLAST-R [http://blast.ncbi.nlm.nih. gov/Blast] showed that P4 have no homology with any puta-tive conserved domain of human proteins (Homo sapiensTaxid 9606). P23 have low homology with HCG1779566 protein CRA isoform whose product and function are unknown (hypothetical proteins) (score of 28.2 bits, identity of 9/13 and e-value of 0.001) and with VDJ regions of the immunoglobulin heavy chain (score of 26.9 bits, 6/7 of identity and e-value of 0.002). Our findings open the possibility, after better under-standing and characterization of P4 and P23, that these mole-cules may have application in the general practice. This will depend on better understanding of their pharmacological actions, on increasing their stability in the circulation and on better evaluation of their possible toxicity in parallel with the time and dosing for ideal anti-inflammatory results.
Acknowledgements
Appendix. Supplementary material
Supplementary material associated with this article can be found, in the online version, atdoi:10.1016/j.micinf.2011.12. 012.
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