J. Muse Davis

The Role of the Granuloma in Expansion and Dissemination of Early Tuberculous Infection

Granulomas, organized aggregates of immune cells, form in response to persistent stimuli and are hallmarks of tuberculosis. Tuberculous granulomas have long been considered host-protective structures formed to contain infection. However, work in zebrafish infected with Mycobacterium marinum suggests that granulomas contribute to early bacterial growth. Here we use quantitative intravital microscopy to reveal distinct steps of granuloma formation and assess their consequence for infection. Intracellular mycobacteria use the ESX-1/RD1 virulence locus to induce recruitment of new macrophages to, and their rapid movement within, nascent granulomas. This motility enables multiple arriving macrophages to efficiently find and phagocytose infected macrophages undergoing apoptosis, leading to rapid, iterative expansion of infected macrophages and thereby bacterial numbers. The primary granuloma then seeds secondary granulomas via egress of infected macrophages. Our direct observations provide insight into how pathogenic mycobacteria exploit the granuloma during the innate immune phase for local expansion and systemic dissemination.

Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos.

Infection of vertebrate hosts with pathogenic Mycobacteria, the agents of tuberculosis, produces granulomas, highly organized structures containing differentiated macrophages and lymphocytes, that sequester the pathogen. Adult zebrafish are naturally susceptible to tuberculosis caused by Mycobacterium marinum. Here, we exploit the optical transparency of zebrafish embryos to image the events of M. marinum infection in vivo. Despite the fact that the embryos do not yet have lymphocytes, infection leads to the formation of macrophage aggregates with pathological hallmarks of granulomas and activation of previously identified granuloma-specific Mycobacterium genes. Thus, Mycobacterium-macrophage interactions can initiate granuloma formation solely in the context of innate immunity. Strikingly, infection can redirect normal embryonic macrophage migration, even recruiting macrophages seemingly committed to their developmentally dictated tissue sites.

Tuberculous Granuloma Induction via Interaction of a Bacterial Secreted Protein with Host Epithelium

Garnering Information on Granulomas In tuberculosis, the tuberculous granuloma has been viewed traditionally as a host-protective structure that serves to “wall off” mycobacteria. However, recent work in the zebrafish embryo showed that mycobacteria convert the nascent granuloma into a vehicle for bacterial expansion and dissemination. Thus, intercepting granuloma formation could provide a strategy for treating tuberculosis, an urgent public health goal in light of the epidemic of extensively drug-resistant tuberculosis. Now Volkman et al. (p. 466, published online 10 December; see the Perspective by Agarwal and Bishai) present the molecular pathway by which mycobacteria induce granulomas in zebrafish. Inhibition of this pathway attenuates infection by reducing granuloma formation, suggesting a therapeutic target for tuberculosis treatment. Epithelial cells play a role in tubercular granuloma formation and mycobacterial virulence. Granulomas, organized aggregates of immune cells, are a hallmark of tuberculosis and have traditionally been thought to restrict mycobacterial growth. However, analysis of Mycobacterium marinum in zebrafish has shown that the early granuloma facilitates mycobacterial growth; uninfected macrophages are recruited to the granuloma where they are productively infected by M. marinum. Here, we identified the molecular mechanism by which mycobacteria induce granulomas: The bacterial secreted protein 6-kD early secreted antigenic target (ESAT-6), which has long been implicated in virulence, induced matrix metalloproteinase–9 (MMP9) in epithelial cells neighboring infected macrophages. MMP9 enhanced recruitment of macrophages, which contributed to nascent granuloma maturation and bacterial growth. Disruption of MMP9 function attenuated granuloma formation and bacterial growth. Thus, interception of epithelial MMP9 production could hold promise as a host-targeting tuberculosis therapy.

Neutrophils Exert Protection in the Early Tuberculous Granuloma by Oxidative Killing of Mycobacteria Phagocytosed from Infected Macrophages

Neutrophils are typically the first responders in host defense against invading pathogens, which they destroy by both oxidative and nonoxidative mechanisms. However, despite a longstanding recognition of neutrophil presence at disease sites in tuberculosis, their role in defense against mycobacteria is unclear. Here we exploit the genetic tractability and optical transparency of zebrafish to monitor neutrophil behavior and its consequences during infection with Mycobacterium marinum, a natural fish pathogen. In contrast to macrophages, neutrophils do not interact with mycobacteria at initial infection sites. Neutrophils are subsequently recruited to the nascent granuloma in response to signals from dying infected macrophages within the granuloma, which they phagocytose. Some neutrophils then rapidly kill the internalized mycobacteria through NADPH oxidase-dependent mechanisms. Our results provide a mechanistic link to the observed patterns of neutrophils in human tuberculous granulomas and the susceptibility of humans with chronic granulomatous disease to mycobacterial infection.

Pseudomonas aeruginosa Type III secretion system interacts with phagocytes to modulate systemic infection of zebrafish embryos

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause serious infection in those with deficient or impaired phagocytes. We have developed the optically transparent and genetically tractable zebrafish embryo as a model for systemic P. aeruginosa infection. Despite lacking adaptive immunity at this developmental stage, zebrafish embryos were highly resistant to P. aeruginosa infection, but as in humans, phagocyte depletion dramatically increased their susceptibility. The virulence of an attenuated P. aeruginosa strain lacking a functional Type III secretion system was restored upon phagocyte depletion, suggesting that this system influences virulence through its effects on phagocytes. Intravital imaging revealed bacterial interactions with multiple blood cell types. Neutrophils and macrophages rapidly phagocytosed and killed P. aeruginosa, suggesting that both cell types play a role in protection against infection. Intravascular aggregation of erythrocytes and other blood cells with resultant circulatory blockage was observed immediately upon infection, which may be relevant to the pathogenesis of thrombotic complications of human P. aeruginosa infections. The real‐time visualization capabilities and genetic tractability of the zebrafish infection model should enable elucidation of molecular and cellular details of P. aeruginosa pathogenesis in conditions associated with neutropenia or impaired phagocyte function.

Zebrafish and Frog Models of Mycobacterium marinum Infection

Mycobacterium marinum infection of poikilothermic animals, such as fish and frogs, results in chronic granulomatous diseases that bear many similarities to mycobacterioses in mammals, including tuberculosis. This unit describes three animal models of M. marinum infection that can be used to study basic aspects of Mycobacterium‐host interactions and granuloma development, as well as trafficking of immune cells in host tissues. Protocols are included that describe intraperitoneal infection of adult leopard frogs (Rana pipiens) and zebrafish (Danio rerio). Protocols also describe subsequent monitoring of the infection by enumeration of bacterial cfu, mean time to death, or visual examination of infected tissue using both conventional histological stains and fluorescence microscopy of fluorescently marked bacteria. Furthermore, protocols are included that describe the infection of embryonic zebrafish and the subsequent analysis of the infection in real time using DIC and fluorescence microscopy.

Leptospira interrogans stably infects zebrafish embryos, altering phagocyte behavior and homing to specific tissues

Leptospirosis is an extremely widespread zoonotic infection with outcomes ranging from subclinical infection to fatal Weils syndrome. Despite the global impact of the disease, key aspects of its pathogenesis remain unclear. To examine in detail the earliest steps in the host response to leptospires, we used fluorescently labelled Leptospira interrogans serovar Copenhageni to infect 30 hour post fertilization zebrafish embryos by either the caudal vein or hindbrain ventricle. These embryos have functional innate immunity but have not yet developed an adaptive immune system. Furthermore, they are optically transparent, allowing direct visualization of host–pathogen interactions from the moment of infection. We observed rapid uptake of leptospires by phagocytes, followed by persistent, intracellular infection over the first 48 hours. Phagocytosis of leptospires occasionally resulted in formation of large cellular vesicles consistent with apoptotic bodies. By 24 hours, clusters of infected phagocytes were accumulating lateral to the dorsal artery, presumably in early hematopoietic tissue. Our observations suggest that phagocytosis may be a key defense mechanism in the early stages of leptospirosis, and that phagocytic cells play roles in immunopathogenesis and likely in the dissemination of leptospires to specific target tissues.

Evaluation of the pathogenesis and treatment of Mycobacterium marinum infection in zebrafish

Mycobacterium marinum–infected zebrafish are used to study tuberculosis pathogenesis, as well as for antitubercular drug discovery. The small size of zebrafish larvae coupled with their optical transparency allows for rapid analysis of bacterial burdens and host survival in response to genetic and pharmacological manipulations of both mycobacteria and host. Automated fluorescence microscopy and automated plate fluorimetry (APF) are coupled with facile husbandry to facilitate large-scale, repeated analysis of individual infected fish. Both methods allow for in vivo screening of chemical libraries, requiring only 0.1 μmol of drug per fish to assess efficacy; they also permit a more detailed evaluation of the individual stages of tuberculosis pathogenesis. Here we describe a 16-h protocol spanning 22 d, in which zebrafish larvae are infected via the two primary injection sites, the hindbrain ventricle and caudal vein; this is followed by the high-throughput evaluation of pathogenesis and antimicrobial efficacy.

“The Very Pulse of the Machine”: The Tuberculous Granuloma in Motion

What is happening inside the tuberculous granuloma? In this issue of Immunity, Egen et al. (2008) present live images of tuberculous granulomas of the mouse, demonstrating the influx and incessant wandering of T lymphocytes.

Brachyury expression in tailless Molgulid ascidian embryos.

SUMMARY The T‐box transcription factor gene Brachyury is important for the differentiation of notochord in all chordates, including the ascidians Halocynthia roretzi and Ciona intestinalis. We isolated Brachyury from molgulid ascidians, which have evolved tailless larvae multiple times independently, and found the genes appear functional by cDNA sequence analyses. We then compared the expression of Mocu‐Bra in tailed Molgula oculata embryos to two tailless species, Molgula occulta (Mocc‐Bra) and Molgula tectiformis (Mt‐Bra). Here we show that both tailless species express Brachyury in the notochord lineage during embryogenesis. Initial expression of Mocu‐Bra is normal in tailed M. oculata embryos; 10 precursor notochord cells divide twice to result in 40 notochord cells that converge and extend to make a notochord down the center of the tail. In contrast, in tailless Molgula occulta, Mocc‐Bra expression disappears prematurely, and there is only one round of division, resulting in 20 cells in the final notochord lineage that never converge or extend. In M. occulta×M. oculata hybrid embryos, expression of Mocu‐Bra is prolonged, and the embryos form a tail with 20 notochord cells that converge and extend normally. However, in Molgula tectiformis, a different tailless ascidian, Mt‐Bra was expressed only in the 10 notochord precursor cells, which never divide, converge, or extend. In summary, neither Brachyury function nor the early establishment of the notochord lineage appears to be impaired in tailless embryos. In light of these results, we are continuing to investigate how and why notochord development is lost in tailless molgulid ascidian embryos.