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Duplex real-time reverse transcriptase PCR to determine cytokine mRNA expression in a hamster model of New World cutaneous leishmaniasis.

Espitia CM, Zhao W, Saldarriaga O, Osorio Y, Harrison LM, Cappello M, Travi BL, Melby PC - BMC Immunol. (2010)

Bottom Line: Real-time RT-PCR primers and probes were synthesized for analysis of interleukin (IL)-4, IFN-gamma, TNF-alpha, IL-10, IL-12p40, TGF-beta, IL-13, IL-21, chemokine ligand (CCL) 22, CCL17, Chemokine (C-C motif) receptor 4 and FoxP3 expression.Standard curves and validation experiments were performed for each real-time RT-PCR assay, allowing us to use the comparative Ct (2-DeltaDeltaCt) method to calculate changes in gene expression.Application of the real-time RT PCR assays to a biological model was demonstrated by comparing mRNA expression in skin and lymph node tissues between uninfected and Leishmania panamensis infected hamsters.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Service, Department of Veterans Affairs Medical Center, South Texas Veterans Health Care System, 7400 Merton Minter, San Antonio, Texas, USA.

ABSTRACT

Background: The Syrian hamster, Mesocricetus auratus, has distinct immunological features and is uniquely susceptible to intracellular pathogens. Studies in hamsters are limited by the relative unavailability of tools to conduct immunological studies. To address this limitation we developed duplex real-time reverse transcriptase (RT) PCR assays for the relative quantification of the mRNAs of hamster cytokines, chemokines, and related immune response molecules.

Results: Real-time RT-PCR primers and probes were synthesized for analysis of interleukin (IL)-4, IFN-gamma, TNF-alpha, IL-10, IL-12p40, TGF-beta, IL-13, IL-21, chemokine ligand (CCL) 22, CCL17, Chemokine (C-C motif) receptor 4 and FoxP3 expression. Standard curves and validation experiments were performed for each real-time RT-PCR assay, allowing us to use the comparative Ct (2-DeltaDeltaCt) method to calculate changes in gene expression. Application of the real-time RT PCR assays to a biological model was demonstrated by comparing mRNA expression in skin and lymph node tissues between uninfected and Leishmania panamensis infected hamsters.

Conclusions: The duplex real-time RT PCR assays provide a powerful approach for the quantification of cytokine transcription in hamsters, and their application to a model of cutaneous leishmaniasis suggests that a balanced type 1 and type 2 cytokine response contributes to the chronic, nonprogressive course of disease. These new molecular tools will further facilitate investigation into the mechanisms of disease in the hamster, not only for models of leishmaniasis, but also for other viral, bacterial, fungal, and parasitic infections.

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Relative mRNA expression levels in lymph node from uninfected and 7-day L. panamensis infected hamsters. Experiments were conducted as described for Figure 2. Results are expressed as a relative fold difference between experimental samples and BHK cells to which the value of 1 was arbitrarily assigned. For panels A, B, D, E, F, G, H and L the mean + SD are represented and statistical analysis utilized the unpaired t test. Data presented in panels C, I, J and K were not normally distributed so are shown as the median + interquartile range and the statistical analysis utilized the nonparametric Mann Whitney test. Each uninfected hamster is represented by a filled circle, and each infected hamster is represented by a filled square.
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Figure 4: Relative mRNA expression levels in lymph node from uninfected and 7-day L. panamensis infected hamsters. Experiments were conducted as described for Figure 2. Results are expressed as a relative fold difference between experimental samples and BHK cells to which the value of 1 was arbitrarily assigned. For panels A, B, D, E, F, G, H and L the mean + SD are represented and statistical analysis utilized the unpaired t test. Data presented in panels C, I, J and K were not normally distributed so are shown as the median + interquartile range and the statistical analysis utilized the nonparametric Mann Whitney test. Each uninfected hamster is represented by a filled circle, and each infected hamster is represented by a filled square.

Mentions: The role of type 1 (IFN-γ and IL-12p40) and type 2 (IL-4, IL-10, IL-13, and IL-21) cytokines in the immunopathogenesis of leishmaniasis is well known [38]. Our studies to dissect the immunopathogenic mechanisms in this model of LCL are ongoing, but several aspects of this preliminary work deserve comment. After relative quantification and normalization, we found that there were tissue-specific differences in the basal expression of several genes (see Figures 3 and 4). There was relatively greater basal expression in the LN compared to the skin for most transcripts (p ≤ 0.05 for IL-4, CCR4, IL-21, TNF-α, TGF-β, IFN-γ, IL-12p40, IL-10, and Foxp3). Conversely, the assays identified that basal expression of CCL22 and CCL17 mRNAs was significantly greater in the normal skin compared to the LN (p≤0.05), consistent with their prominent expression in Langerhans cells (LCs) and immature dendritic cells (DCs) [39]. At an early stage (1 week post-infection) there was concomitant upregulation of the type 1 (IFN-γ and IL-12p40) and type 2 (IL-4, IL-10, IL-13, and IL-21) cytokines at the site of cutaneous infection (Figure 2, p < 0.05 for all). This mixed cytokine response is reminiscent of what is seen in chronic nonhealing, but nonprogressive, L. mexicana infection in some strains of mice [40]. We also found that the levels of CCL22 and CCL17 mRNAs, which are strongly up-regulated upon DC maturation [39,41], were up-regulated at the cutaneous site of infection of hamsters infected with L. panamensis (Figure 3A and 3B; p < 0.05), consistent with the notion DCs are activated by Leishmania [42]. To our knowledge, this is the first demonstration of the induction of CCL17 and CCL22 expression in response to Leishmania infection. These chemokines have been implicated in the recruitment of activated Th2 cells and regulatory T cells, by signaling through the CC chemokine receptor 4 (CCR4) [43,44]. Consistent with this finding, at the site of cutaneous L. panamensis infection, we also found increased expression of Foxp3 (Figure 3L; p < 0.05), a transcription factor that is critical for the development and function of mouse CD4+CD25+ regulatory T cells (Tregs) [45].


Duplex real-time reverse transcriptase PCR to determine cytokine mRNA expression in a hamster model of New World cutaneous leishmaniasis.

Espitia CM, Zhao W, Saldarriaga O, Osorio Y, Harrison LM, Cappello M, Travi BL, Melby PC - BMC Immunol. (2010)

Relative mRNA expression levels in lymph node from uninfected and 7-day L. panamensis infected hamsters. Experiments were conducted as described for Figure 2. Results are expressed as a relative fold difference between experimental samples and BHK cells to which the value of 1 was arbitrarily assigned. For panels A, B, D, E, F, G, H and L the mean + SD are represented and statistical analysis utilized the unpaired t test. Data presented in panels C, I, J and K were not normally distributed so are shown as the median + interquartile range and the statistical analysis utilized the nonparametric Mann Whitney test. Each uninfected hamster is represented by a filled circle, and each infected hamster is represented by a filled square.
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Figure 4: Relative mRNA expression levels in lymph node from uninfected and 7-day L. panamensis infected hamsters. Experiments were conducted as described for Figure 2. Results are expressed as a relative fold difference between experimental samples and BHK cells to which the value of 1 was arbitrarily assigned. For panels A, B, D, E, F, G, H and L the mean + SD are represented and statistical analysis utilized the unpaired t test. Data presented in panels C, I, J and K were not normally distributed so are shown as the median + interquartile range and the statistical analysis utilized the nonparametric Mann Whitney test. Each uninfected hamster is represented by a filled circle, and each infected hamster is represented by a filled square.
Mentions: The role of type 1 (IFN-γ and IL-12p40) and type 2 (IL-4, IL-10, IL-13, and IL-21) cytokines in the immunopathogenesis of leishmaniasis is well known [38]. Our studies to dissect the immunopathogenic mechanisms in this model of LCL are ongoing, but several aspects of this preliminary work deserve comment. After relative quantification and normalization, we found that there were tissue-specific differences in the basal expression of several genes (see Figures 3 and 4). There was relatively greater basal expression in the LN compared to the skin for most transcripts (p ≤ 0.05 for IL-4, CCR4, IL-21, TNF-α, TGF-β, IFN-γ, IL-12p40, IL-10, and Foxp3). Conversely, the assays identified that basal expression of CCL22 and CCL17 mRNAs was significantly greater in the normal skin compared to the LN (p≤0.05), consistent with their prominent expression in Langerhans cells (LCs) and immature dendritic cells (DCs) [39]. At an early stage (1 week post-infection) there was concomitant upregulation of the type 1 (IFN-γ and IL-12p40) and type 2 (IL-4, IL-10, IL-13, and IL-21) cytokines at the site of cutaneous infection (Figure 2, p < 0.05 for all). This mixed cytokine response is reminiscent of what is seen in chronic nonhealing, but nonprogressive, L. mexicana infection in some strains of mice [40]. We also found that the levels of CCL22 and CCL17 mRNAs, which are strongly up-regulated upon DC maturation [39,41], were up-regulated at the cutaneous site of infection of hamsters infected with L. panamensis (Figure 3A and 3B; p < 0.05), consistent with the notion DCs are activated by Leishmania [42]. To our knowledge, this is the first demonstration of the induction of CCL17 and CCL22 expression in response to Leishmania infection. These chemokines have been implicated in the recruitment of activated Th2 cells and regulatory T cells, by signaling through the CC chemokine receptor 4 (CCR4) [43,44]. Consistent with this finding, at the site of cutaneous L. panamensis infection, we also found increased expression of Foxp3 (Figure 3L; p < 0.05), a transcription factor that is critical for the development and function of mouse CD4+CD25+ regulatory T cells (Tregs) [45].

Bottom Line: Real-time RT-PCR primers and probes were synthesized for analysis of interleukin (IL)-4, IFN-gamma, TNF-alpha, IL-10, IL-12p40, TGF-beta, IL-13, IL-21, chemokine ligand (CCL) 22, CCL17, Chemokine (C-C motif) receptor 4 and FoxP3 expression.Standard curves and validation experiments were performed for each real-time RT-PCR assay, allowing us to use the comparative Ct (2-DeltaDeltaCt) method to calculate changes in gene expression.Application of the real-time RT PCR assays to a biological model was demonstrated by comparing mRNA expression in skin and lymph node tissues between uninfected and Leishmania panamensis infected hamsters.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Service, Department of Veterans Affairs Medical Center, South Texas Veterans Health Care System, 7400 Merton Minter, San Antonio, Texas, USA.

ABSTRACT

Background: The Syrian hamster, Mesocricetus auratus, has distinct immunological features and is uniquely susceptible to intracellular pathogens. Studies in hamsters are limited by the relative unavailability of tools to conduct immunological studies. To address this limitation we developed duplex real-time reverse transcriptase (RT) PCR assays for the relative quantification of the mRNAs of hamster cytokines, chemokines, and related immune response molecules.

Results: Real-time RT-PCR primers and probes were synthesized for analysis of interleukin (IL)-4, IFN-gamma, TNF-alpha, IL-10, IL-12p40, TGF-beta, IL-13, IL-21, chemokine ligand (CCL) 22, CCL17, Chemokine (C-C motif) receptor 4 and FoxP3 expression. Standard curves and validation experiments were performed for each real-time RT-PCR assay, allowing us to use the comparative Ct (2-DeltaDeltaCt) method to calculate changes in gene expression. Application of the real-time RT PCR assays to a biological model was demonstrated by comparing mRNA expression in skin and lymph node tissues between uninfected and Leishmania panamensis infected hamsters.

Conclusions: The duplex real-time RT PCR assays provide a powerful approach for the quantification of cytokine transcription in hamsters, and their application to a model of cutaneous leishmaniasis suggests that a balanced type 1 and type 2 cytokine response contributes to the chronic, nonprogressive course of disease. These new molecular tools will further facilitate investigation into the mechanisms of disease in the hamster, not only for models of leishmaniasis, but also for other viral, bacterial, fungal, and parasitic infections.

Show MeSH
Related in: MedlinePlus