Pathogenesis and Pathology of Intraocular Tuberculosis

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1 Ocular Immunology & Inflammation, 2015; 23(4): Copyright! Taylor & Francis Group, LLC ISSN: print / online DOI: / REVIEW ARTICLE Pathogenesis and Pathology of Intraocular Tuberculosis Soumyava Basu, MD 1, Denis Wakefield, MD 2, Jyotirmay Biswas, MD 3, and Narsing A. Rao, MD 4 Downloaded by [Soumyava Basu] at 21:19 14 August LV Prasad Eye Institute, Bhubaneswar, India, 2 Department of Pathology, School of Medicine Sciences, Faculty of Medicine, University of New South Wales, Inflammation and Infectious Research Center, Sydney, Australia, 3 Department of Uveitis and Ocular Pathology, Sankara Nethralaya, Chennai, Tamil Nadu, India, and 4 Deparment of Ophthalmology and Pathology, USC Eye Institute, USC Keck School of Medicine, University of Southern California, Los Angeles, California, USA ABSTRACT Intraocular tuberculosis (TB) is an extremely paucibacillary form of extrapulmonary TB. It likely results from bacterial dissemination to the eye from lungs, localization in ocular tissues, followed later by reactivation and appearance of clinical signs. These have been partly demonstrated in the guinea pig model of ocular TB. Alternative hypotheses have been suggested but are not supported by adequate evidence. Mycobacterial recognition by macrophages and dendritic cells probably leads to activation of several immune pathways, primarily the Th1 and Th17 pathways, as in other TB infections. Histopathology of bacteriologically proven ocular TB tissues reveals granulomatous inflammation with central caseous necrosis containing occasional acidfast organisms. Recent reports have also demonstrated intraretinal granuloma in the vicinity of retinal vessels and T-cell infiltration of epiretinal membranes, in cases of TB retinal vasculitis. Keywords: Animal model, immunology, itraocular tuberculosis, ocular, pathogenesis, pathology Intraocular tuberculosis (TB) is unique among all forms of extrapulmonary TB for 2 reasons: first, it has several clinical manifestations within a single organ, and second, live Mycobacterium tuberculosis is rarely isolated from clinical samples accessible to the ophthalmologist. This has led to a protracted debate on respective roles of immune-mediated inflammation and direct bacteria-mediated inflammation in the pathogenesis of intraocular TB. 1 2 The key differentiating factor between these two mechanisms is their association with latent and active TB, respectively. However, latent TB infection is characterized by the presence of a reactive tuberculin skin test or T-cell response to M. tuberculosis antigens in the absence of clinical or radiological findings of TB. 3 During latent TB, M. tuberculosis may exist in several different organs and cell types, but there cannot be any clinical (pulmonary or extrapulmonary) disease. 4 Thus the presence of clinical signs of active intraocular inflammation rules out any role of latent TB in such eyes. Recent developments support a more direct role of M. tuberculosis in the pathogenesis of intraocular TB. Polymerase chain reaction (PCR) has been used to amplify the M. tuberculosis genome from up to 70% of clinically suspected cases of intraocular TB. 5 7 Anti- TB therapy has been shown to significantly reduce recurrent inflammation in presumed ocular TB as compared to corticosteroid therapy alone We can therefore speculate that intraocular TB is likely to have pathogenic mechanisms similar to other forms of extrapulmonary TB. Thus if we extrapolate the pathogenesis of extrapulmonary TB to intraocular TB, these mechanisms can be divided into 3 different stages: dissemination of bacteria from the site of primary infection, i.e. lungs, to the eye; localization of bacteria in different ocular tissues, and bacterial reactivation and initiation of inflammation in those tissues Received 12 February 2015; revised 16 April 2015; accepted 12 May 2015; published online 4 August 2015 Correspondence: Narsing A. Rao, Department of Ophthalmology and Pathology, USC Eye Institute, USC Keck School of Medicine, University of Southern California, Los Angeles, CA, USA. narsing.rao@med.usc.edu 353

2 354 S. Basu et al. PATHOGENETIC MECHANISMS Bacterial Dissemination On inhalation of infected aerosol, M. tuberculosis is engulfed by alveolar macrophages and transported to the hilar lymph nodes. This leads to priming of T-cells followed by initiation of adaptive immunity, a process that takes 5 7 days. However, during this period, macrophages or dendritic cells carrying M. tuberculosis or even free bacteria may disseminate to different parts of the body, including eyes. 12 This was demonstrated in an animal model in which uveal granulomatous lesions containing acid-fast organisms were noted following infection of guinea pigs with aerosol containing M. tuberculosis. 13 These choroidal granulomas were shown to be hypoxic and were accompanied by vascular endothelial growth factor expression in the retina and retinal pigment epithelium (RPE).14 Interestingly, when complete Freund s adjuvant that contains heat-killed M. tuberculosis, was injected subcutaneously in mice, mycobacterial DNA could be detected in the retina, besides brain, spleen, and liver. 15 There was upregulation of TLR2, TLR4, and various pro-inflammatory cytokines in retinal microglia but no inflammatory cell infiltration in the retina or uvea. Localization in Ocular Tissues The RPE appears to be most suited, among different ocular cell types, to harbor M. tuberculosis within the eye. It has several alveolar macrophage-like properties such as phagocytosis and expression of various tolllike receptors (TLRs). 16 Rao et al. have demonstrated the presence of M. tuberculosis (histologic examination and PCR) within the RPE in a case of panuveitis. 17 More recently, M. tuberculosis H37Ra was shown to be phagocytized in human RPE cell cultures. 18 The degree of phagocytosis was similar to monocytes leukemia macrophage (THP-1) cells, but infected RPE cells had greater survival rates than THP-1 cells, suggesting that they are equipped to survive for a long time and provide sanctuary for M. tuberculosis as dormant organisms. Bacterial Reactivation and Initiation of Inflammation M. tuberculosis localized in ocular tissues may remain latent for long periods without any apparent clinical disease. This can be extrapolated from earlier studies in which mycobacterial DNA was demonstrated in multiple organs and cell types of patients who died of non-tuberculous causes. 4 While HIV disease, immunosuppressive therapy, and various chronic systemic FIGURE 1. Ziehl-Neelsen acid-fast staining of granuloma shows presence of acid-fast bacteria (arrow). diseases are known risk factors for reactivation of latent TB, it is still unknown how mycobacteria get reactivated in extrapulmonary sites, to cause disease in apparently healthy individuals. Of note, mycobacterial secreted proteins called resuscitation-promoting factors have been associated with reactivation of chronic infection. 19 There is also limited data on various patterns of immune response to M. tuberculosis, once it reaches the eye. Recent work points to the role of a 6 kd secreted mycobacterial protein, early secreted antigenic target-6 (ESAT-6), in activating innate immune responses in the eye (unpublished data). However, it is not clear how a paucibacillary infection by few organisms give rise to such a wide range of clinical manifestations, as seen in intraocular TB. While focal choroiditis lesions have been demonstrated in the guinea pig model, it is not known how some eyes develop retinal vasculitis or multifocal serpiginoid choroiditis lesions. Alternative Hypothesis The absence of definitive evidence of M. tuberculosis in the majority of clinically diagnosed cases has led to development of alternative hypotheses for pathogenesis of intraocular TB. Garip et al. described a case of bilateral granulomatous anterior uveitis, following intravesical injection of Bacille-Calmette-Guérin (BCG) for treatment of bladder carcinoma. 2 They demonstrated T-cell proliferation and cytokine secretion in vitro in response to purified protein derivative (PPD), and several retinal antigens such as interphotoreceptor retinoid-binding protein, retinal soluble antigen, and cellular retinal binding protein. Furthermore, amino acid sequence alignments revealed significant homologies between proteins Ocular Immunology & Inflammation

3 from M. tuberculosis, BCG, and retinal antigens. The authors concluded that antigenic mimicry between tubercular and retinal antigens could be a potential cause of uveitis in patients with latent TB. This hypothesis is supported by cytokine analysis of TB-associated uveitis that showed significantly increased interleukin-6 (IL-6) and other chemokines, but not IL-12, tumor necrosis factor a (TNF-a) and interferon gamma (IFN gamma) that characterize active TB. 20 However, this hypothesis does not explain the lack of retinal inflammation in the reported case of post-bcg uveitis, 2 or the predominant involvement of retinal vasculature or RPE/ choroid in clinical presentations of ocular TB. DIFFERENCES IN DISEASE PATHOGENESIS BETWEEN ENDEMIC AND NONENDEMIC REGIONS Intraocular TB accounts for a much greater proportion of uveitis cases in TB-endemic regions as compared to nonendemic regions. In endemic regions, all age groups, including children, could be affected, while in nonendemic regions the disease is usually limited to high-risk populations such as immigrants, HIV-infected individuals, and the elderly. 20 The clinical presentation in immigrants is similar to endemic areas though the disease appears to be less severe. In general, the diagnostic approach in endemic areas is to rule out intraocular TB, while in nonendemic regions, one needs to rule out other infections to diagnose intraocular TB. Studies done in pulmonary TB patients show that there could also be differences in the respective roles of M. tuberculosis reactivation and reinfection in endemic and nonendemic regions. This is studied by DNA fingerprinting of mycobacterial isolates from recurrent TB cases. If the fingerprinting patterns are the same in consecutive episodes of TB, it suggests reactivation, while if they are different, it indicates reinfection. Initial reports suggested that reactivation alone accounted for recurrent TB in nonendemic regions, while it could be both reactivation and reinfection in endemic regions. 21 However, conflicting reports have appeared in subsequent years from both endemic and nonendemic regions IMMUNOPATHOGENESIS The initial immune response to M. tuberculosis in naïve immunocompetent subjects leads to the development of cell-mediated immunity, which helps confer immunity to the bacteria and also results in hypersensitivity to mycobacterial antigens. The immune response to active M. tuberculosis infection is initiated by the recognition of the bacteria, mainly by macrophage! 2015 Taylor & Francis Group, LLC Pathogenesis and Pathology of Intraocular TB 355 and dendritic cells through TLRs, which stimulate the production of cytokines such as IL-12 and TNF-a. IL-12 subsequently activates a Th1-cell-dominated adaptive immune response, which is largely responsible for containing the infection. 24 M. tuberculosis demonstrates a remarkable array of defense mechanisms that facilitate their survival. 24 Inhaled mycobacteria are phagocytized in the alveolar space by macrophages, dendritic cells, and possibly epithelial cells. One of the crucial steps in the pathogenesis of TB is the observation that mycobacteria have a unique ability to delay the initiation of an adaptive immune response. Studies in murine models of TB have shown that mycobacteria are able to delay the onset of a T-cell-mediated response by 2 3 weeks. This is believed to be due to a failure of dendritic cells to migrate to the regional lymph nodes and initiate a substantial immune response Mycobacteria also have adapted a number of other impressive strategies to allow them to continue to survive and multiply locally within the host. These strategies include inhibiting maturation and fusion of the phagosome-lysosome complex, inhibiting autophagy, and decreasing the interferon-gamma receptormediated signaling, which is a major antimicrobial defense mechanism. 27 The mycobacterial granulomata are the basic lesions of tuberculous infection and said to be the battleground of immunity in this disease. Recent studies in the zebra fish indicate that mycobacterial granulomata are dynamic and demonstrate rapidly trafficking immune cells, into and out of the granulomata. 28 Granulomata consist of a central area of macrophages with M. tuberculosis surrounded by T-cells with a surrounding fibrotic reaction. Granulomata help to localize and sequester the infecting microorganisms and limit dissemination of the mycobacterium. 25 Recent studies in man and animal models of TB have considerably expanded our understanding of the role of cell-mediated immunity in this disease. The best-characterized cell involved in the pathogenesis of tuberculosis is the CD4 Th-1 cell. These cells secrete IFN-gamma and TNF-a, which play a pivotal role in the protective immunity against M. tuberculosis. 24,29 30 Gamma interferon induction is regulated by IL-12, which is also secreted by activated macrophages and phagocytic cells. The widespread use of biological agent, anti-tnf therapy in diseases such as rheumatoid arthritis has highlighted the important role of this cytokine in the pathogenesis of TB. The use of this biological agent leads to an increased incidence of TB. Macrophages and dendritic cells present mycobacterial antigens via the MHC class II molecule on their surface to CD4 Th-1 cells. These cells are crucial to granulomata formation. Recently it has been realized that mycobacterial infection is also associated with the activation of a CD4 Th subset, the Th 17

4 356 S. Basu et al. cells IL-17 is necessary for the formation of pulmonary granulomata in animal models of this disease. Interestingly, CD8 T-cells have also been implicated in the pathogenesis of TB. These cells are able to process antigen on cells such as epithelial cells via their class I MHC molecule. This may potentially increase the breadth of immune response to mycobacterial antigens. CD8 cells also had a number of antimicrobial effector mechanisms that may play a role in helping to kill mycobacteria. 30 Other cells of the immune system have also been implicated in the pathogenesis of TB. These include innate immune cells such as natural killer or NK cells and neutrophils. A human gene-wide gene expression study found an association with neutrophils type I INF signaling in patients with TB. 32 There has also been considerable interest in recent times in the role of T reg cells in modulating and controlling the immune response, including to mycobacterial infection. 30 T reg cells suppress CD4 Th-1 cell activity via secretion of IL-10 and transforming growth factor beta (TGF-B) amongst other cytokines. They may also play a role in regulatory immune response to mycobacteria. Thus the immune response to mycobacteria involves both innate and adaptive immunity and most of the regulatory and effector cells of the immune response appear to play a role in the pathogenesis of this disease. Pathology of Ocular TB Enucleated eyes of ocular TB grossly reveal granulomatous lesions which may involve sclera, cornea, conjunctiva, iris and ciliary body, vitreous adjacent to pars plana ciliaris, retina choroid. 33 However, posterior segment involvement is common and may include features of endophthalmitis, panophthalmitis, and may simulate intraocular tumors. The histopathology of such ocular involvements characteristically reveals granulomatous inflammation with central necrosis, recognized as caseous necrosis, and shows occasional acid-fast organisms. The granulomatous response consists of abundant epithelioid histiocytes, occasional giant cells of Langerhans type, and peripheral mononuclear cells, primarily made up of lymphocytes. Such granulomatous inflammation shows the presence of occasional acid-fast bacteria in the majority of specimens; however, immunocompromised individuals show poorly defined granulomas with numerous acid-fast bacteria. The latter are demonstrated by Ziehl-Neelsen staining (Figure 1). Since the granulomas in immunocompetent individuals contain occasional bacteria, the staining may not reveal the presence of organisms. In such cases, the bacterial DNA could be detected by polymerase chain reaction (PCR). 34 Necrosis of ocular tissue is a common feature in intraocular TB, and this process involves RPE and adjacent uvea. The necrotic RPE displays numerous acid-fast organisms, which are confirmed to be M. tuberculosis by quantitative PCR. 17 The necrotic process can involve iris and ciliary epithelium and such necrotic changes clinically present as pigmented hypopyon. 35 In older literature, histological evidence of intraocular TB has been associated with a wide range of clinical signs including retinal vasculitis/periphlebitis, retinitis, choroiditis, and iritis. 36 Recently, retinal biopsy from a patient with PCR-proven tubercular retinal vasculitis revealed the presence of discrete intraretinal granuloma in the vicinity of blood vessels. 37 These granuloma stained for HLA-DR that could be seen in both retinal microglia and bloodderived macrophages, but the scanty lymphocytic infiltration accompanying the granuloma suggested a primarily microglial origin. Such intraretinal granuloma possibly represent the healed or active chorioretinitis patches overlying blood vessels, which characterize tubercular retinal vasculitis in TB-endemic countries. 38 Immunohistochemical analysis of epiretinal membranes from suspected Eales disease patients have also revealed predominant T-lymphocytes, again suggesting a role for cellmediated immunity. 39 Nearly half of these patients were positive for M. tuberculosis by nested PCR. CONCLUSION In summary, intraocular TB represents a unique form of extrapulmonary TB due to its extreme paucibacillary nature and multiple clinical manifestations. However, the pathogenic mechanisms appear to be similar to other forms of extrapulmonary TB. DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. REFERENCES 1. Tabbara KF. Tuberculosis. Curr Opin Ophthalmol. 2007;18: Garip A, Diedrichs-Möhring M, Thurau SR, Deeg CA, Wildner G. 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