Granuloma transplantation: an approach to study mycobacterium-host interactions

Abstract

The host-pathogen biology during infection with Mycobacterium tuberculosis is incredibly complex and despite accelerating progress in research, remains poorly understood. Our limited understanding hinders the development of new drugs, next generation vaccines, and novel therapies. The granuloma is the site where mycobacteria are both controlled and allowed to persist, but it remains one of the least studied aspects of the host-pathogen relationship. Here, we review the development, application, potential uses, and limitations of a novel model of granuloma transplantation as a tool to study specific host-pathogen interactions that have been difficult to probe. Application of this new model has already contributed to our understanding of granuloma cell traffic, repopulation, and the relationship between systemic immunity and mycobacteria-containing granulomas. The data collected highlight the dynamic interaction between systemic and local immune processes and support a paradigm that defines the granuloma as a highly dynamic structure. Granuloma transplantation also has special potential as a novel latency model that can contribute to our understanding of host protection factors and bacterial mutants, and serve as a platform for drug testing.

Keywords: granuloma; host–pathogen interactions; mycobacteria; transplantation.

Figure 1

Intraperitoneal infection of BCG in mice results in mycobac-teria-containing liver granulomas [(A), left panels]. In this transplant model, approximately 1% of infected donor liver tissue is transplanted underneath the kidney capsule of recipient mice [(A), right panels]. The high oxygen/blood-rich environment maintains the viability of the tissue piece, and BCG-containing granulomas can be identified by microscopy 2 weeks after surgery. With this model, the total number of transplanted granuloma cells and bacterial CFU can be calculated for both acute and chronic transplant pieces (B).

Figure 2

Transplantation of BCG-infected liver from colorless donor mice into GFP-expressing mice provides a simple way to distinguish donor and recipient tissues [(A), left]. When coupled with cell-specific antibodies, the proportion of donor (D) and recipient (R) cells in BCG-containing granulomas can be measured. Colocalization of cell-specific (shown here is CD4+ T-cells) and GFP fluorescent signals identifies recipient cells, while cell-specific signal alone identifies donor cells. In the image shown, donor cells are blue only (DAPI) or blue and red (donor CD4+ T-cell), while recipient cells are green (GFP) only or green and red (recipient CD4+ T-cell). Recipient B-cells repopulate chronic granulomas at a higher rate than acute granulomas (B). Seven days after transplant, 2/3 of B-cells in transplanted granulomas are of recipient origin.

Figure 3

Transplantation of BCG-infected liver using CD11c-EYFP donors or recipients allows tracking of DC migration in and out of acute and chronic granulomas. After transplantation, donor DCs migrate out of granulomas, and recipient DCs also migrate in. The ability of DCs to prime systemic immune responses after surveying granulomas can be measured when coupled with adoptive transfer of antigen-specific T-cells into transplant recipients. Systemic T-cell priming after transplantation of acute granulomas results from CCR7-dependent migration to the lymph nodes of donor DCs that have BCG antigen. The antigen is transferred to recipient DCs and presented to mycobacterial-specific, transferred T-cells. Priming after transplantation of both acute and chronic granulomas was absolutely dependent on recipient MHC II.

Figure 4

Bacillus Calmette–Guerin-infected WT or RAG KO donors are transplanted into WT or RAG KO recipients (A). Four weeks after transplantation, bacterial dissemination is measured by CFU and fluorescent microscopy of recipient spleen and renal lymph nodes (B). Transplantation of infected RAG KO donors into WT recipients, but not WT donors into RAG KO recipients, results in bacterial dissemination (C).

Figure 5

Non-expanding BCG from WT chronic granulomas can be induced to disseminate and expand after transplantation into TNFαKO, but not WT, recipients. Disseminated bacteria are measured by CFU and fluorescent microscopy of recipient target organs such as spleen and renal lymph node. These experiments highlight the system’s potential as a new model of bacterial dissemination to study host protection factors (bottom), bacteria mutants, and new drugs/therapies.