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

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.

Figure 1

Real-time RT PCR standardization and validation. Standard curves (A), validation experiment (B) and amplification plots (C) for the hamster CCR4 duplex real-time Reverse Transcription (RT) PCR using TaqMan One-Step RT-PCR Master Mix, FAM-labeled probe for the target gene (hamster CCR4) and VIC-labeled probe for the endogenous control (hamster γ-actin).

Figure 2

Course of disease and parasite burden in L. panamensis infected hamsters. 6-8 week old female hamsters (n = 6) with 3 × 10⁶ luciferase (luc)-transfected L. panamensis promastigotes in the dermis of the snout. Lesion size was measured weekly and is shown as the lesion area (mm²) in panel A. The intralesional parasite burden was determined by in vivo imaging and is shown as the total intralesional amastigotes in panel B. Images of the luc-L. panamensis burden in two representative hamsters are shown in panel C.

Figure 3

Relative mRNA expression levels in skin from uninfected and 7-day L. panamensis infected hamsters. Skin was harvested from the uninfected or infected snout at 7 days p.i. and the expression of multiple mRNAs determined by duplex real-time RT PCR. 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, D, G, H, J and L the mean + SD are represented in the bar graph and statistical analysis utilized the unpaired t test. Data presented in panels B, C, E, F, I 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.

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.