Microdissection of the cytokine milieu of pulmonary granulomas from tuberculous guinea pigs

Abstract

SummaryLevels of IL-12p40, TNFa, TGFb, IFNg and IL-10 mRNA were assessed by laser capture microdissection followed by quantitative real-time PCR in the pulmonary granulomas of unimmunized and BCG-vaccinated guinea pigs infected by aerosol with virulent Mycobacterium tuberculosis. Lesions microdissected from unimmunized guinea pigs were overwhelmed by the pro-inflammatory TNFa mRNA at both 3 and 6 weeks post infection, indicating the struggle to control the mounting infection. The cytokine profile of granulomas from vaccinated guinea pigs shifted from type 1 cytokine mRNA (IFNg and IL-12p40) at 3 weeks to a predominantly anti-inflammatory environment (TGFb mRNA) at 6 weeks. The relative proportions of cytokine mRNA transcripts in the periphery of the granuloma were different from the centre, reflecting differences in cell composition and architecture. Moreover, analysis of the individual lung lobes at 6 weeks post infection suggests that heterogeneity exists in the cytokine profile between the lobes of the lung.

Latent Tuberculosis Infection: Myths, Models, and Molecular Mechanisms

“…However, like mice, guinea pigs infected with M. tuberculosis develop high-burden disease and succumb to infection (88), indicating that LTBI is not adequately modeled in this species. Also, despite recent advances (87,89,90), the study of TB immune control mechanisms in the guinea pig model has been complicated by the lack of appropriate reagents.…”

Influence ofMycobacterium bovisBCG Vaccination on Cellular Immune Response of Guinea Pigs Challenged withMycobacterium tuberculosis

“…After pulmonary infection with M. tuberculosis, alveolar macrophages and dendritic cells phagocytose bacilli, and it is thought that dendritic cells carry both intact bacteria and their antigens to draining lymph nodes, where recognition by T cells generates cell-mediated immunity (3,10,11,24,29). Macrophages present antigen via both class I and class II major histocompatibility complex (MHC) molecules, resulting in effector CD4 and CD8 T cells which secrete cytokines, including gamma interferon (IFN-␥), giving rise to macrophage activation and intracellular killing of the organism (8,17,22,28,34). The development of a pulmonary granuloma is orchestrated by both chemokines and cytokines, resulting in a continuous recruitment of lymphocytes, granulocytes, macrophages, dendritic cells, and monocytes to the local site of the infection (37,38).…”

Influence of Advanced Age onMycobacterium bovisBCG Vaccination in Guinea Pigs Aerogenically Infected withMycobacterium tuberculosis

“…M. tuberculosis-specific Th1 (IL-12 and IFN-␥) and Th17 responses play roles in the increased expression of cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death-1 (PD-1), while IL-23 induces IFN-␥ and supports the IL-17 response in the lungs. McMurray and colleagues, using a laser capture microdissection (LCM) technique, reported cytokine mRNA responses in situ in the pulmonary granulomas of nonvaccinated and BCG-vaccinated guinea pigs (23,24). TNF-␣ mRNA was dominant in primary lesions microdissected from nonvaccinated guinea pigs at both 3 and 6 weeks postinfection, while the cytokine profiles of granulomas from BCG-vaccinated guinea pigs shifted from type 1 cytokine mRNA (IFN-␥ and IL-12p40) at 3 weeks to a predominantly anti-inflammatory profile dominated by transforming growth factor-␤ (TGF-␤) at 6 weeks (23,24).…”

“…McMurray and colleagues, using a laser capture microdissection (LCM) technique, reported cytokine mRNA responses in situ in the pulmonary granulomas of nonvaccinated and BCG-vaccinated guinea pigs (23,24). TNF-␣ mRNA was dominant in primary lesions microdissected from nonvaccinated guinea pigs at both 3 and 6 weeks postinfection, while the cytokine profiles of granulomas from BCG-vaccinated guinea pigs shifted from type 1 cytokine mRNA (IFN-␥ and IL-12p40) at 3 weeks to a predominantly anti-inflammatory profile dominated by transforming growth factor-␤ (TGF-␤) at 6 weeks (23,24). These results suggest that BCG vaccination modulates cytokine responses in the lungs to promote antimycobacterial functions while controlling the potentially damaging inflammatory response.…”