Yonsei Med J.  2013 May;54(3):707-714. 10.3349/ymj.2013.54.3.707.

Kinetics of IFN-Gamma and TNF-Alpha Gene Expression and Their Relationship with Disease Progression after Infection with Mycobacterium Tuberculosis in Guinea Pigs

• Roh IS
• Cho S
• Eum SY
• Cho SN

• KMID: 1727887
• DOI: http://doi.org/10.3349/ymj.2013.54.3.707

Abstract

PURPOSE
Guinea pig is one of the most suitable animal models for Mycobacterium tuberculosis (M. tb) infection since it shows similarities to pulmonary infection in humans. Although guinea pig shows hematogenous spread of M. tb infection into the whole body, immunological studies have mainly focused on granulomatous tissues in lungs and spleens. In order to investigate the time-course of disease pathogenesis and immunological profiles in each infected organ, we performed the following approaches with guinea pigs experimentally infected with M. tb over a 22-week post-infection period.
MATERIALS AND METHODS
We examined body weight changes, M. tb growth curve, cytokine gene expression (IFN-gamma and TNF-alpha), and histopathology in liver, spleen, lungs and lymph nodes of infected guinea pigs.
RESULTS
The body weights of infected guinea pigs did not increase as much as uninfected ones and the number of M. tb bacilli in their organs increased except bronchotracheal lymph node during the experimental period. The gene expression of IFN-gamma and TNF-alpha was induced between 3 and 6 weeks of infection; however, kinetic profiles of cytokine gene expression showed heterogeneity among organs over the study period. Histophathologically granulomatous lesions were developed in all four organs of infected guinea pigs.
CONCLUSION
Although IFN-gamma and TNF-alpha gene expression profiles showed heterogeneity, the granuloma formation was clearly observed in every organ regardless of whether the number of bacilli increased or decreased. However, this protective immunity was accompanied with severe tissue damage in all four organs, which may lead to the death of guinea pigs.

Keyword

Mycobacterium tuberculosis; pathogenesis; guinea pigs; IFN-gamma and TNF-alpha

MeSH Terms

Animals
Body Weight
*Disease Progression
Female
Gene Expression
Gene Expression Regulation
Guinea Pigs
Interferon-gamma/genetics/*metabolism
Kinetics
Liver/metabolism/pathology
Lung/metabolism/pathology
Lymph Nodes/metabolism/pathology
Mycobacterium tuberculosis
Spleen/metabolism/pathology
Tuberculosis/*genetics/pathology
Tumor Necrosis Factor-alpha/genetics/*metabolism
Tumor Necrosis Factor-alpha
Interferon-gamma

Figure

• 

  Fig. 1 Total body weight of guinea pigs measured after an aerosol infection with Mycobacterium tuberculosis(M. tb). After infection with M. tb, guinea pigs gained less weight compared to the uninfected control. Body weight was calculated as the mean of weights of all infected guinea pigs per time (0 and 1 week: 9, 3 wk: 7, 6 wk: 5, 10 wk: 3, 14 wk: 2 and 22 wk: 1 animal) and only one animal chosen as an uninfected control was used in this graph. The statistical analysis was not performed since the size of experimental group varied at each time point.

• 

  Fig. 2 TNF-α and IFN-γ synthesis in each organ of guinea pigs in response to M. tb infection. Changes in mRNA expression profiles of IFN-γ and TNF-α versus time course of infection with M. tb in each organ (lung, BTLN, spleen and liver) of guinea pigs were determined by RT-PCR analysis at each data point. (A) RT-PCR products were resolved on an agarose gel. (B) The gene expression of IFN-γ (white bar) or TNF-α (grey bar) was normalized with β-actin based on densitometry analysis. Each histogram represents mean±SD IFN-γ or TNF-α gene expression ratio to β-actin from two RT-PCR experiments. BTLN, bronchotracheal lymph node; M. tb, Mycobacterium tuberculosis.

• 

  Fig. 3 Growth curves of M. tb in each organ. The M. tb growth in lungs, BTLNs, spleens and livers of guinea pigs were measured after an aerosol infection with about 106 colony forming units (CFUs) per exposure. The number of mean viable CFUs in each organ at different time intervals following the infection was calculated by plating the serial dilutions of homogenates of each organ on Middlebrook 7H11 agar and counting CFUs after 14-21 days. BTLN, bronchotracheal lymph node; M. tb, Mycobacterium tuberculosis.

• 

  Fig. 4 Histopathological findings of uninfected or M. tb infected guinea pigs. (A-F) Lung (A) normal (40×), (B) at 1 week post-infection; mild interstitial thickening by the accumulation of inflammatory cells (40×), (C) at 3 weeks post-infection; multifocal granulomas composed of macrophages, epithelioid cells and lymphocytes develop (arrows) in the angiocentric area (40×). (D) At 14 weeks post-infection; necrotizing fibrous granulomas with caseous necrosis center of the lesion develops (arrows) (40×), (Ea and Eb) at 22 weeks post-infection (100×); bronchial tuberculosis and diffuse granulomatous inflammation are seen. (F) Acid-fast stain after 22 weeks, multiple red, rod-shaped bacilli are shown (arrows) (1000×). (G and H) BTLN. (G) Normal (40×), (H) severe necrotizing fibrous granuloma after 14 weeks (arrows) (100×). (I and J) Spleen. (I) Normal (40×), (J) the necrotic granuloma around the central arteries after 6 weeks (40×). (K and L) Liver. (K) Normal (40×), (L) the scattered granuloma in the portal area after 6 weeks (arrows) (40×). BTLN, bronchotracheal lymph node; M. tb, Mycobacterium tuberculosis.

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