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Divergence between hepatic insulin-like growth factor (IGF)-I mRNA expression and IGF-I serum levels in Leishmania (Leishmania) infantum chagasi-infected dogs
Veterinary Immunology and Immunopathology 151 (2013) 163–167
Contents lists available at SciVerse ScienceDirect
Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Short communication
Divergence between hepatic insulin-like growth factor (IGF)-I mRNA expression and IGF-I serum levels in Leishmania (Leishmania) infantum chagasi-infected dogs Flaviane A. Pinho a , Nilton A. Magalhães a , Kleverton R. Silva a , Aline A. Carvalho a , Fernando L.L. Oliveira a , Eduardo M. Ramos-Sanchez b , Hiro Goto b,c , Francisco A.L. Costa a,∗ a
Setor de Patologia Animal, Centro de Ciências Agrárias, Universidade Federal do Piauí, 64049-550, Campus Socopo, S/N, Teresina, PI, Brazil Laboratório de Soroepidemiologia e Imunobiologia, Instituto de Medicina Tropical, USP, 05403-000, Av. Dr. Enéas de Carvalho Aguiar, 470, prédio II, 4◦ andar, São Paulo, SP, Brazil c Departamento de Medicina Preventiva, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brazil b
a r t i c l e
i n f o
Article history: Received 8 August 2012 Received in revised form 20 October 2012 Accepted 23 October 2012 Keywords: Visceral leishmaniasis Dog Insulin-like growth factor-I Liver
a b s t r a c t Visceral leishmaniasis (VL) caused in the New World by Leishmania (Leishmania) infantum chagasi has dog as important peridomestic reservoir in its transmission cycle. Since VL in infected animals is similar to human VL, its study is interesting for human pathology. During Leishmania infection, insulin-like growth factor-I (IGF-I) plays a role in the host-parasite interaction, favoring parasite growth, particularly acting directly on Leishmania. We evaluated IGF-I mRNA expression in different organs/tissues, which was differently modulated in dogs naturally infected by L. (L.) infantum chagasi. We also evaluated the hepatic IGF-I mRNA and serum IGF-I levels in infected dogs. Hepatic mRNA IGF-I expression was higher in the infected dogs than in control animals. However, the serum levels of IGF-I, which are related to the production of this factor in the liver, were reduced in the infected dogs compared with the non-infected controls. Thus, we suggest interference in post-transcriptional processing in IGF-I production in active visceral leishmaniasis. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Visceral leishmaniasis (VL) is a systemic disease caused by Leishmania (Leishmania) infantum chagasi (Shaw, 2006) in the American continent that proliferates within cells of the mononuclear phagocyte system (Hommel, 1999). In humans, symptomatic VL is lethal in some cases (Badaro et al., 1986). Dogs are the most important reservoir in the
∗ Corresponding author at: Universidade Federal do Piauí, Centro de Ciências Agrárias, Departamento de Clínica e Cirurgia Veterinária, Setor de Patologia Animal. Campus da Socopo, S/N, 64049-550, Teresina, Piauí, Brazil. Tel.: +55 86 3215 5760; fax: +55 86 3215 5753. E-mail address: [email protected] (F.A.L. Costa). 0165-2427/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetimm.2012.10.008
peridomestic environment, and they play an important role in the transmission to humans due to their susceptibility to infection, intense cutaneous parasitism and their close contact with humans (Ashford, 1996). Furthermore, the disease in dogs is similar to human VL, making its study interesting for human pathology (Costa et al., 2003). The control or progression by Leishmania infection depends on the specific and non-specific host immune response and the ability of the parasite to evade the host response (Mougneau et al., 2011). Insulin-like growth factor-I (IGF-I), which is present in circulation (Cohick and Clemmons, 1993) and in macrophages (Arkins et al., 1993), is one of the early factors that interact with Leishmania promastigotes in the skin of the host and possibly with amastigotes after internalization by macrophages. Previous
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F.A. Pinho et al. / Veterinary Immunology and Immunopathology 151 (2013) 163–167
studies have shown that IGF-I induces in vitro an increase in the proliferation of promastigotes and axenic amastigotes of Leishmania (Goto et al., 1998). In addition, it was also observed in vivo a significant increase in the lesion size and in the number of viable parasites in the skin of mice infected with Leishmania amazonensis promastigotes pre-incubated with IGF-I (Gomes et al., 2000). Because there are still no studies on the expression of IGF-I in VL, the local expression of IGF-I was initially evaluated in preferential sites for parasitism in the dog, i.e., the skin (ear and periungual region), popliteal lymph node, spleen and liver. Furthermore, we evaluated the serum levels of IGF-I, and because it is primarily produced in the liver (Schimpff et al., 1976; Han et al., 1988), we compared these data with hepatic IGF-I mRNA expression levels. The results showed that the serum levels were not consistent with the hepatic mRNA expression levels in dogs naturally infected with L. (L.) infantum chagasi.
2. Materials and methods Adult male and female stray dogs from a Brazilian endemic area (Teresina, state of Piaui) with VL (N = 22) and controls (N = 6), with estimated age range of 2–8 years old and body weight range of 8–30 kg were included in this study. All of the experimental protocols were previously approved by the Research Ethics Committee of Federal University of Piauí (Protocol No. 004/09), and the dogs were anesthetized using 2.5% sodium thiopental (10 mg/kg) before of the procedures. Infection was confirmed through the identification of Leishmania amastigotes in the sternal bone marrow and/or popliteal lymph node aspirates. After the clinical examination, whole blood was collected to obtain serum. Subsequently, the dogs were sacrificed with an overdose of sodium thiopental (33%, 5 ml/kg, IV). Samples of the skin, spleen, liver and popliteal lymph node were obtained for touch imprint cytology and stained with Giemsa for parasite count in 50 random fields under the light microscope using oil immersion at a magnification of 100×. Then we collected 30 mg of each organ to extract total RNA using Trizol reagent (Invitrogen, California, USA). The RNA was reverse transcribed into cDNA using 200 U/ml reverse transcriptase (Invitrogen, California, USA) according to the manufacturer’s protocol (Invitrogen, California, USA). IGF-I mRNA expression was assessed in six infected and six non-infected control dogs with real-time PCR using Sybr Green (2×) (Life Technologies, California, USA) and an Applied Biosystems StepOne Real-time PCR System thermal cycler. The IGF-I primer sequences (NM:L08254) were forward, 5 GGG TTC TAC TTC AAC AAG CCC ACA3 , and reverse, 5 GGG CAC AGT GTG ACA CTA TCT GCC3 . The primers of the housekeeping -actin gene (NM: AF021873) were forward, 5 CGT GAC ATC AAG GAA GAA GCT CTG3 , and reverse, 5 CGG GTT GTA CGT AGC TCT TCT CCA3 . The PCR conditions were as follows: an initial 10 min incubation step at 95 ◦ C and 45 cycles of 15 s at 94 ◦ C, 1 min at 61 ◦ C, and 1 min at 70 ◦ C. The expression levels were analyzed with the relative expression method and presented as 2−ct relative to -actin gene expression (Pfaffl, 2001).
Serum IGF-I concentrations were determined with a chemiluminescent assay (Immulite – Siemens) with an Immulite 2000 automated immunoassay (Diagnostic Products Corporation – DPC, CA, USA) according to the protocol provided by the manufacturer. The assay is based on the detection of the emmited ligh proportional to the amount of the analyte by chemiluminescent dioxetane substrate when it reacts with alkaline phosphatase. In the kit this enzyme is bound to the polystyrene beads coated with either anti-IGF-I or anti-IGFBP3 antibodies. For evaluation, 20 L of the sample is mixed with the provided reagents and processed in the automated system. For the data analysis, nonparametric statistics (Mann–Whitney and Kruskal–Wallis tests) were used. 3. Results and discussion The frequent clinical manifestations in dogs with VL were skin lesions (86.4%), local or generalized lymphadenopathy (77.3%), weight loss (68.2%), onychogryphosis (54.5%), conjunctivitis (36.4%), apathy (31.8%), coryza (27.3%), fever (18.2%), anemia (18.2%), diarrhea (18.2%), subcutaneous edema (4.5%) and dyspnea (4.5%). On cytologic analysis of six infected dogs, the presence of amastigote was observed free and inside the cytoplasm of mononuclear cells in all organs. The frequency was higher in ear (4/6) and lymph nodes (3/6) when compared to liver (1/6) and spleen (1/6). We analyzed the mRNA expression of IGF-I in the skin from various areas and the spleen and we observed a higher tendency of IGF-I mRNA expression in infected dogs compared with the controls. The IGF-I mRNA expression in the liver was significantly higher in the infected dogs compared with the control animals (P = 0.0325, Mann–Whitney test). The IGF-I mRNA expression levels in the popliteal lymph node of infected animals tended to be lower than the control animals (Fig. 1). Interestingly, the lymph node is not a preferential organ for parasite growth (Reis et al., 2006). Thus, the data may suggest the association of local IGF-I mRNA expression and parasite growth in the tissue. When we evaluated the IGF-I mRNA expression in the infected animals in relation to the number of clinical signs, we observed that the IGF-I mRNA expression was higher in animals with less clinical signs, except in liver (Table 1). These data may suggest different roles for IGF-I, in situ, in parasite growth during the infectious process, primarily in the earlier phase of infection. Moreover, we also evaluated the serum IGF-I concentration and did not observe any correlation between serum IGF-I concentration and parasite load. In addition, there was a significant decrease in IGF-I mRNA expression levels in the various organs (except for the liver) in animals with a more severe clinical presentation. Analysis the serum IGF-I levels showed a higher proportion of dogs presenting lower levels of serum IGF-I among infected dogs compared with control animals. However, comparing serum IGF-I concentration with clinical manifestation of infected dogs we did not observe any correlation between them (Table 1). In addition, we observed that the infected dogs with lower serum IGF-I levels had an increase of mRNA IGF-I expression in the liver (Fig. 2A
F.A. Pinho et al. / Veterinary Immunology and Immunopathology 151 (2013) 163–167
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Fig. 1. Quantitative analysis of IGF-I mRNA expression (median and 5–95 percentile intervals) in the skin (ear and ungual region), spleen, liver and popliteal lymph node of L. (L.) infantum chagasi-naturally infected and uninfected control dogs. The data are presented as the relative quantification 2−ct against -actin gene expression. # N = number of animals. *P < 0.05 (Mann–Whitney test).
and B). Furthermore, when we compared the ratio of the serum IGF-I concentration with the IGF-I mRNA expression in animals that had both evaluations, we observed a lower ratio in infected dogs compared with the control dogs (P = 0.0159, Mann–Whitney test) (Fig. 2C), confirming the discrepancy between IGF-I mRNA expression and serum levels.
Previously, in human visceral leishmaniasis, the mean concentrations of IGF-I (total and free) and IGF-binding protein 3 were shown to be reduced in children with active VL (Gomes et al., 2007), corroborating our results and showing the similarity between human and canine visceral leishmaniasis. Furthermore, no correlation was observed between the plasma concentrations of IGF-I and
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Table 1 IGF-I mRNA expression in the skin, liver, spleen and popliteal lymph nodes of dogs naturally by L. (L.) infantum chagasi in Teresina (PI). Animals
Number of clinical signs
1 2 3 4 5 6
1 3 4 4 6 8
IGF-I mRNA expressiona Ungueal
Ear
Liver
Spleen
Popliteal lymph nodes
0.14 9.60 0.62 104 6.08 1.42
7.30 3.70 21.0 11.1 1.40 1.20
55.23 121.64 17.22 13.79 761.40 64.10
2.55 3.81 12.63 1.31 1.85 3.14
NT 38.04 28.88 5.14 3.16 0.77
NT: not detectable. a The data are presented as the relative quantification 2−ct in relation to -actin gene expression.
Fig. 2. Serum IGF-I levels in L. (L.) infantum chagasi-naturally infected and uninfected control dogs. The data are presented as the relative frequency distribution according to the IGF-I serum levels in the infected (A) and control dogs (B). Serum IGF-I concentration/IGF-I mRNA expression ratio of the infected (closed columns) and control (open columns) dogs (data from individual animals) (C). Immulite 2000 IGF-I assay.
anthropometrical variables (Gomes et al., 2007), which suggested no direct association of these low levels to the nutritional status of the patients (Thissen et al., 1994). This same data was observed in our infected dogs. Thus, these data instead suggest a direct association of low serum IGFI levels with the Leishmania infection process. However it is intriguing the progression of the infection in the presence of low serum IGF- levels considering our previous results showing growth promoting role of IGF-I on Leishmania (Goto et al., 1998). It may be that the effect of IGF-I on visceral leishmaniasis strains in their interaction with macrophages is different from other Leishmania species that we had studied before, a point that we are presently considering. Under catabolic conditions, such as septicemia, endotoxemia or the inflammatory process, the concentrations of IGF-I in experimental animals (Fan et al., 1995; Lang et al., 1998; Lelbach et al., 2001) and humans (Lang et al., 1997) are reduced. This reduction may result from the effect of acute inflammatory mediators, such as IL-6, IL-1 and TNF␣, that inhibit the biosynthesis of IGF-I in the liver and other tissues (Lelbach et al., 2001). In VL, malnutrition is present, but cytokines are actively produced; thus, these mediators may contribute to the modulation of the expression of IGF-I in hepatocytes (Thissen et al., 1994; Fan et al., 1995; Lang et al., 1998; Lelbach et al., 2001). Data from the present study showing higher expression levels of IGF-I mRNA in the liver but low serum IGF-I levels in infected animals during an ongoing infection and weight loss instead suggest other factors in the modulation of serum IGF-I production. One possibility is impaired post-transcriptional processing because mRNA expression is increased in infected animals. The translation of mRNA can be controlled by RNA-binding proteins (RBPs) and microRNAs (miRNAs) (Keene, 2007; Lee and Gorospe, 2010), and recent studies have shown that several miRNAs modulate IGF-I post-transcriptional processing in different model systems (Lee and Gorospe, 2010). In this study, we have shown that dogs naturally infected with VL show reduced serum IGF-I levels and high hepatic IGF-I mRNA expression, suggesting posttranscriptional inhibition. We have previously observed that IGF-I is important for the progression of Leishmania amazonensis infection in mice (Goto et al., 1998; Gomes et al., 2000), but the significance of low serum IGF-I levels in VL and its role in L. (L.) infantum chagasi infection may be distinct and requires further studies.
F.A. Pinho et al. / Veterinary Immunology and Immunopathology 151 (2013) 163–167
Acknowledgments This study was supported by CAPES (fellowship to FALC), CNPq (research fellowship to HG), and the Laboratório de Soroepidemiologia LIM/38 (HC-FMUSP). We acknowledge the Laboratório de Hormônios e Genética Molecular, Hospital das Clínicas da Faculdade de Medicina, Universidade de São Paulo, for the serum IGF-I determination. References Arkins, S., Rebeiz, N., Biragyn, A., Reese, D.L., Kelley, K.W., 1993. Murine macrophages express abundant insulin-like growth factor-I class I Ea and Eb transcripts. Endocrinology 133, 2334–2343. Ashford, R.W., 1996. Leishmaniasis reservoirs and their significance in control. Clin. Derm. 14, 523–532. Badaro, R., Jones, T.C., Carvalho, E.M., Sampaio, D., Reed, S.G., Barral, A., Teixeira, R., Johnson Jr., W.D., 1986. New perspectives on a subclinical form of visceral leishmaniasis. J. Infect. Dis. 154, 1003–1011. Cohick, W.S., Clemmons, D.R., 1993. The insulin-like growth factors. Annu. Rev. Physiol. 55, 131–153. Costa, F.A., Goto, H., Saldanha, L.C., Silva, S.M., Sinhorini, I.L., Silva, T.C., Guerra, J.L., 2003. Histopathologic patterns of nephropathy in naturally acquires canine visceral leishmaniasis. Vet. Pathol. 40, 677–684. Fan, J., Char, D., Kolasa, A.J., Pan, W., Maitra, S.R., Patlak, C.S., Spolarics, Z., Gelato, M.C., Lang, C.H., 1995. Alterations in hepatic production and peripheral clearance of IGF-I after endotoxin. Am. J. Physiol. 269, 33–42. Gomes, C.M.C., Giannella-Neto, D., Gama, M.E.A., Pereira, J.C., Campos, M.B., Corbett, C.E.P., 2007. Correlation between the components of the insulin-like growth factor I system, nutritional status and visceral leishmaniasis. Trans. R. Soc. Trop. Med. Hyg. 101, 660–667. Gomes, C.M.C., Goto, H., Matta, V.L.R., Laurenti, M.D., Gidlund, M., Corbett, C.E.P., 2000. Insulin-like growth factor (IGF)-I affects parasite growth and host cell migration in experimental cutaneous leishmaniasis. Int. J. Exp. Pathol. 81, 249–255. Goto, H., Gomes, C.M.C., Corbett, C.E.P., Monteiro, H.P., Gidlund, M., 1998. Insulin-like growth factor I is a growth promoting factor for
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Leishmania promastigotes and amastigotes. Proc. Natl. Acad. Sci. U.S.A. 95, 13211–13216. Han, V.K., Lund, P.K., Lee, D.C., D’Ercole, A.J., 1988. Expression of somatomedin/insulin-like growth factor messenger ribonucleic acids in the human fetus: identification, characterization, and tissue distribution. J. Clin. Endocr. Metabol. 66, 422–429. Hommel, M., 1999. Visceral leishmaniasis: biology of the parasite. J. Infect. 39, 101–111. Keene, J.D., 2007. RNA regulons: coordination of post-transcriptional events. Nat. Rev. Genet. 8, 533–543. Lang, C.H., Fan, J., Wojnar, M.M., Vary, T.C., Cooney, R., 1998. Role of central IL-1 in regulation peripheral IGF-I during endotoxemia and sepsis. Am. J. Physiol. 274, 956–962. Lang, C.H., Pollard, V., Fan, J., Traber, L.D., Traber, D.L., Frost, R.A., Gelato, M.C., Prough, D.S., 1997. Acute alterations in growth hormone-insulinlike growth factor axis in humans injected with endotoxin. Am. J. Physiol. 273, 371–378. Lee, E.K., Gorospe, M., 2010. Minireview: posttranscriptional regulation of the insulin and insulin growth factor systems. Endocrinology 151, 1403–1408. Lelbach, A., Scharf, J.G., Ramadori, G., 2001. Regulation of insulin-like growth factor-I and insulin-like growth factor binding protein-1, -3 and -4 in cocultures of rat hepatocytes and kupffer cells by interleukin6. J. Hepatol. 35, 558–567. Mougneau, E., Bihl, F., Glaichenhaus, N., 2011. Cell biology and immunology of Leishmania. Immunol. Rev. 240, 286–296. Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, 2002–2007. Reis, A.B., Martins-Filho, O.A., Teixeira-Carvalho, A., Carvalho, M.G., Mayrink, W., Franc¸a-Silva, J.C., Giunchetti, R.C., Genaro, O., Corrêa-Oliveira, R., 2006. Parasite density and impaired biochemical/hematological status are associated with severe clinical aspects of canine visceral leishmaniasis. Res. Vet. Sci. 81, 68–75. Schimpff, R.M., Donnadieu, M., Glasnovic, J.C., 1976. The liver as a source of somatomedin. Acta Endocrinol. 83, 365–372. Shaw, J.J., 2006. Further thoughts on the use of the name Leishmania (Leishmania) infantum chagasi for the aetiological agente of American visceral leishmaniasis. Mem. Inst. Oswaldo Cruz 101, 577–579. Thissen, J.P., Ketelslegers, J.M., Underwood, L.E., 1994. Nutritional regulation of the insulin-like growth factor. Endocrinology 15, 80–101.
Contents lists available at SciVerse ScienceDirect
Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Short communication
Divergence between hepatic insulin-like growth factor (IGF)-I mRNA expression and IGF-I serum levels in Leishmania (Leishmania) infantum chagasi-infected dogs Flaviane A. Pinho a , Nilton A. Magalhães a , Kleverton R. Silva a , Aline A. Carvalho a , Fernando L.L. Oliveira a , Eduardo M. Ramos-Sanchez b , Hiro Goto b,c , Francisco A.L. Costa a,∗ a
Setor de Patologia Animal, Centro de Ciências Agrárias, Universidade Federal do Piauí, 64049-550, Campus Socopo, S/N, Teresina, PI, Brazil Laboratório de Soroepidemiologia e Imunobiologia, Instituto de Medicina Tropical, USP, 05403-000, Av. Dr. Enéas de Carvalho Aguiar, 470, prédio II, 4◦ andar, São Paulo, SP, Brazil c Departamento de Medicina Preventiva, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brazil b
a r t i c l e
i n f o
Article history: Received 8 August 2012 Received in revised form 20 October 2012 Accepted 23 October 2012 Keywords: Visceral leishmaniasis Dog Insulin-like growth factor-I Liver
a b s t r a c t Visceral leishmaniasis (VL) caused in the New World by Leishmania (Leishmania) infantum chagasi has dog as important peridomestic reservoir in its transmission cycle. Since VL in infected animals is similar to human VL, its study is interesting for human pathology. During Leishmania infection, insulin-like growth factor-I (IGF-I) plays a role in the host-parasite interaction, favoring parasite growth, particularly acting directly on Leishmania. We evaluated IGF-I mRNA expression in different organs/tissues, which was differently modulated in dogs naturally infected by L. (L.) infantum chagasi. We also evaluated the hepatic IGF-I mRNA and serum IGF-I levels in infected dogs. Hepatic mRNA IGF-I expression was higher in the infected dogs than in control animals. However, the serum levels of IGF-I, which are related to the production of this factor in the liver, were reduced in the infected dogs compared with the non-infected controls. Thus, we suggest interference in post-transcriptional processing in IGF-I production in active visceral leishmaniasis. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Visceral leishmaniasis (VL) is a systemic disease caused by Leishmania (Leishmania) infantum chagasi (Shaw, 2006) in the American continent that proliferates within cells of the mononuclear phagocyte system (Hommel, 1999). In humans, symptomatic VL is lethal in some cases (Badaro et al., 1986). Dogs are the most important reservoir in the
∗ Corresponding author at: Universidade Federal do Piauí, Centro de Ciências Agrárias, Departamento de Clínica e Cirurgia Veterinária, Setor de Patologia Animal. Campus da Socopo, S/N, 64049-550, Teresina, Piauí, Brazil. Tel.: +55 86 3215 5760; fax: +55 86 3215 5753. E-mail address: [email protected] (F.A.L. Costa). 0165-2427/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetimm.2012.10.008
peridomestic environment, and they play an important role in the transmission to humans due to their susceptibility to infection, intense cutaneous parasitism and their close contact with humans (Ashford, 1996). Furthermore, the disease in dogs is similar to human VL, making its study interesting for human pathology (Costa et al., 2003). The control or progression by Leishmania infection depends on the specific and non-specific host immune response and the ability of the parasite to evade the host response (Mougneau et al., 2011). Insulin-like growth factor-I (IGF-I), which is present in circulation (Cohick and Clemmons, 1993) and in macrophages (Arkins et al., 1993), is one of the early factors that interact with Leishmania promastigotes in the skin of the host and possibly with amastigotes after internalization by macrophages. Previous
164
F.A. Pinho et al. / Veterinary Immunology and Immunopathology 151 (2013) 163–167
studies have shown that IGF-I induces in vitro an increase in the proliferation of promastigotes and axenic amastigotes of Leishmania (Goto et al., 1998). In addition, it was also observed in vivo a significant increase in the lesion size and in the number of viable parasites in the skin of mice infected with Leishmania amazonensis promastigotes pre-incubated with IGF-I (Gomes et al., 2000). Because there are still no studies on the expression of IGF-I in VL, the local expression of IGF-I was initially evaluated in preferential sites for parasitism in the dog, i.e., the skin (ear and periungual region), popliteal lymph node, spleen and liver. Furthermore, we evaluated the serum levels of IGF-I, and because it is primarily produced in the liver (Schimpff et al., 1976; Han et al., 1988), we compared these data with hepatic IGF-I mRNA expression levels. The results showed that the serum levels were not consistent with the hepatic mRNA expression levels in dogs naturally infected with L. (L.) infantum chagasi.
2. Materials and methods Adult male and female stray dogs from a Brazilian endemic area (Teresina, state of Piaui) with VL (N = 22) and controls (N = 6), with estimated age range of 2–8 years old and body weight range of 8–30 kg were included in this study. All of the experimental protocols were previously approved by the Research Ethics Committee of Federal University of Piauí (Protocol No. 004/09), and the dogs were anesthetized using 2.5% sodium thiopental (10 mg/kg) before of the procedures. Infection was confirmed through the identification of Leishmania amastigotes in the sternal bone marrow and/or popliteal lymph node aspirates. After the clinical examination, whole blood was collected to obtain serum. Subsequently, the dogs were sacrificed with an overdose of sodium thiopental (33%, 5 ml/kg, IV). Samples of the skin, spleen, liver and popliteal lymph node were obtained for touch imprint cytology and stained with Giemsa for parasite count in 50 random fields under the light microscope using oil immersion at a magnification of 100×. Then we collected 30 mg of each organ to extract total RNA using Trizol reagent (Invitrogen, California, USA). The RNA was reverse transcribed into cDNA using 200 U/ml reverse transcriptase (Invitrogen, California, USA) according to the manufacturer’s protocol (Invitrogen, California, USA). IGF-I mRNA expression was assessed in six infected and six non-infected control dogs with real-time PCR using Sybr Green (2×) (Life Technologies, California, USA) and an Applied Biosystems StepOne Real-time PCR System thermal cycler. The IGF-I primer sequences (NM:L08254) were forward, 5 GGG TTC TAC TTC AAC AAG CCC ACA3 , and reverse, 5 GGG CAC AGT GTG ACA CTA TCT GCC3 . The primers of the housekeeping -actin gene (NM: AF021873) were forward, 5 CGT GAC ATC AAG GAA GAA GCT CTG3 , and reverse, 5 CGG GTT GTA CGT AGC TCT TCT CCA3 . The PCR conditions were as follows: an initial 10 min incubation step at 95 ◦ C and 45 cycles of 15 s at 94 ◦ C, 1 min at 61 ◦ C, and 1 min at 70 ◦ C. The expression levels were analyzed with the relative expression method and presented as 2−ct relative to -actin gene expression (Pfaffl, 2001).
Serum IGF-I concentrations were determined with a chemiluminescent assay (Immulite – Siemens) with an Immulite 2000 automated immunoassay (Diagnostic Products Corporation – DPC, CA, USA) according to the protocol provided by the manufacturer. The assay is based on the detection of the emmited ligh proportional to the amount of the analyte by chemiluminescent dioxetane substrate when it reacts with alkaline phosphatase. In the kit this enzyme is bound to the polystyrene beads coated with either anti-IGF-I or anti-IGFBP3 antibodies. For evaluation, 20 L of the sample is mixed with the provided reagents and processed in the automated system. For the data analysis, nonparametric statistics (Mann–Whitney and Kruskal–Wallis tests) were used. 3. Results and discussion The frequent clinical manifestations in dogs with VL were skin lesions (86.4%), local or generalized lymphadenopathy (77.3%), weight loss (68.2%), onychogryphosis (54.5%), conjunctivitis (36.4%), apathy (31.8%), coryza (27.3%), fever (18.2%), anemia (18.2%), diarrhea (18.2%), subcutaneous edema (4.5%) and dyspnea (4.5%). On cytologic analysis of six infected dogs, the presence of amastigote was observed free and inside the cytoplasm of mononuclear cells in all organs. The frequency was higher in ear (4/6) and lymph nodes (3/6) when compared to liver (1/6) and spleen (1/6). We analyzed the mRNA expression of IGF-I in the skin from various areas and the spleen and we observed a higher tendency of IGF-I mRNA expression in infected dogs compared with the controls. The IGF-I mRNA expression in the liver was significantly higher in the infected dogs compared with the control animals (P = 0.0325, Mann–Whitney test). The IGF-I mRNA expression levels in the popliteal lymph node of infected animals tended to be lower than the control animals (Fig. 1). Interestingly, the lymph node is not a preferential organ for parasite growth (Reis et al., 2006). Thus, the data may suggest the association of local IGF-I mRNA expression and parasite growth in the tissue. When we evaluated the IGF-I mRNA expression in the infected animals in relation to the number of clinical signs, we observed that the IGF-I mRNA expression was higher in animals with less clinical signs, except in liver (Table 1). These data may suggest different roles for IGF-I, in situ, in parasite growth during the infectious process, primarily in the earlier phase of infection. Moreover, we also evaluated the serum IGF-I concentration and did not observe any correlation between serum IGF-I concentration and parasite load. In addition, there was a significant decrease in IGF-I mRNA expression levels in the various organs (except for the liver) in animals with a more severe clinical presentation. Analysis the serum IGF-I levels showed a higher proportion of dogs presenting lower levels of serum IGF-I among infected dogs compared with control animals. However, comparing serum IGF-I concentration with clinical manifestation of infected dogs we did not observe any correlation between them (Table 1). In addition, we observed that the infected dogs with lower serum IGF-I levels had an increase of mRNA IGF-I expression in the liver (Fig. 2A
F.A. Pinho et al. / Veterinary Immunology and Immunopathology 151 (2013) 163–167
165
Fig. 1. Quantitative analysis of IGF-I mRNA expression (median and 5–95 percentile intervals) in the skin (ear and ungual region), spleen, liver and popliteal lymph node of L. (L.) infantum chagasi-naturally infected and uninfected control dogs. The data are presented as the relative quantification 2−ct against -actin gene expression. # N = number of animals. *P < 0.05 (Mann–Whitney test).
and B). Furthermore, when we compared the ratio of the serum IGF-I concentration with the IGF-I mRNA expression in animals that had both evaluations, we observed a lower ratio in infected dogs compared with the control dogs (P = 0.0159, Mann–Whitney test) (Fig. 2C), confirming the discrepancy between IGF-I mRNA expression and serum levels.
Previously, in human visceral leishmaniasis, the mean concentrations of IGF-I (total and free) and IGF-binding protein 3 were shown to be reduced in children with active VL (Gomes et al., 2007), corroborating our results and showing the similarity between human and canine visceral leishmaniasis. Furthermore, no correlation was observed between the plasma concentrations of IGF-I and
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Table 1 IGF-I mRNA expression in the skin, liver, spleen and popliteal lymph nodes of dogs naturally by L. (L.) infantum chagasi in Teresina (PI). Animals
Number of clinical signs
1 2 3 4 5 6
1 3 4 4 6 8
IGF-I mRNA expressiona Ungueal
Ear
Liver
Spleen
Popliteal lymph nodes
0.14 9.60 0.62 104 6.08 1.42
7.30 3.70 21.0 11.1 1.40 1.20
55.23 121.64 17.22 13.79 761.40 64.10
2.55 3.81 12.63 1.31 1.85 3.14
NT 38.04 28.88 5.14 3.16 0.77
NT: not detectable. a The data are presented as the relative quantification 2−ct in relation to -actin gene expression.
Fig. 2. Serum IGF-I levels in L. (L.) infantum chagasi-naturally infected and uninfected control dogs. The data are presented as the relative frequency distribution according to the IGF-I serum levels in the infected (A) and control dogs (B). Serum IGF-I concentration/IGF-I mRNA expression ratio of the infected (closed columns) and control (open columns) dogs (data from individual animals) (C). Immulite 2000 IGF-I assay.
anthropometrical variables (Gomes et al., 2007), which suggested no direct association of these low levels to the nutritional status of the patients (Thissen et al., 1994). This same data was observed in our infected dogs. Thus, these data instead suggest a direct association of low serum IGFI levels with the Leishmania infection process. However it is intriguing the progression of the infection in the presence of low serum IGF- levels considering our previous results showing growth promoting role of IGF-I on Leishmania (Goto et al., 1998). It may be that the effect of IGF-I on visceral leishmaniasis strains in their interaction with macrophages is different from other Leishmania species that we had studied before, a point that we are presently considering. Under catabolic conditions, such as septicemia, endotoxemia or the inflammatory process, the concentrations of IGF-I in experimental animals (Fan et al., 1995; Lang et al., 1998; Lelbach et al., 2001) and humans (Lang et al., 1997) are reduced. This reduction may result from the effect of acute inflammatory mediators, such as IL-6, IL-1 and TNF␣, that inhibit the biosynthesis of IGF-I in the liver and other tissues (Lelbach et al., 2001). In VL, malnutrition is present, but cytokines are actively produced; thus, these mediators may contribute to the modulation of the expression of IGF-I in hepatocytes (Thissen et al., 1994; Fan et al., 1995; Lang et al., 1998; Lelbach et al., 2001). Data from the present study showing higher expression levels of IGF-I mRNA in the liver but low serum IGF-I levels in infected animals during an ongoing infection and weight loss instead suggest other factors in the modulation of serum IGF-I production. One possibility is impaired post-transcriptional processing because mRNA expression is increased in infected animals. The translation of mRNA can be controlled by RNA-binding proteins (RBPs) and microRNAs (miRNAs) (Keene, 2007; Lee and Gorospe, 2010), and recent studies have shown that several miRNAs modulate IGF-I post-transcriptional processing in different model systems (Lee and Gorospe, 2010). In this study, we have shown that dogs naturally infected with VL show reduced serum IGF-I levels and high hepatic IGF-I mRNA expression, suggesting posttranscriptional inhibition. We have previously observed that IGF-I is important for the progression of Leishmania amazonensis infection in mice (Goto et al., 1998; Gomes et al., 2000), but the significance of low serum IGF-I levels in VL and its role in L. (L.) infantum chagasi infection may be distinct and requires further studies.
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