No Evidence for Circulating Retina Specific Autoreactive T-cells in Latent Tuberculosis-associated Uveitis and Sarcoid Uveitis.

Purpose: To detect circulating retina-specific autoreactive CD4+ T-cells and antiretinal antibodies (ARA) in latent tuberculosis (TB)-associated uveitis or sarcoid uveitis patients.Methods: The presence of crude retinal extract (RE) autoreactive CD4+ T-cells was determined by a highly sensitive flowcytometric-based technique examining co-expression of CD25 and CD134 (OX40) on RE stimulated PBMC. The presence of ARA in available matched serum samples was assessed by indirect immunofluorescence.Results: No autoreactive CD4+ T-cells against RE could be detected in either latent TB-associated uveitis or sarcoid uveitis patients, while ARA were detected in the serum of the majority (5/6) of latent TB-associated uveitis and all (3/3) sarcoid uveitis patients.Conclusion: Even with the use of this highly sensitive flowcytometric technique circulating retina-specific autoreactive CD4+ T-cells could not be detected. In contrast, ARA were detected in the majority of patients indicating an adaptive humoral immune response toward retinal antigens had occurred.

Singapore Ocular Tuberculosis Immunity Study (SPOTIS): Role of T-lymphocyte Profiling in Patients with Presumed Ocular Tuberculosis.

Objective: A prospective clinical study to assess the utility of CD4 + T cell lymphocyte profiling from peripheral blood in patients with ocular tuberculosis (TB).Methods: Thirty-six Asian patients with presumed diagnosis of ocular TB were recruited for T-lymphocyte profiling. MTB antigen specific CD4 assay was set up, and flow cytometric data were analyzed using FlowJo software.Results: There was no significant difference between treatment responders and non-responders for the proportion of CD4 + T cells specific for PPD or ESAT-6+ CFP-10, but treatment responders did have significantly higher frequency of CD38+ (p = .0357) and CD38+ HLA-DR+ (p = .0357) on the PPD-specific CD4 + T cells.Conclusion: This study is one of the first of its kind to look into MTB specific T cell activation marker profiling of peripheral blood in patients with ocular TB. Further studies need to be undertaken to assess the utility of CD4 + T cell phenotypes as a biomarker for ocular TB.

Insights into the molecular pathogenesis of ocular tuberculosis.

Unclear pathogenic mechanisms underlying the ocular tuberculosis (OTB) has resulted in perplexity related to the diagnosis and management of the disease. Developments in experimental research and innovations in molecular diagnostics have recently provided a new understanding of disease pathogenesis and natural history. The current review focuses on the new insights into OTB pathogenesis, derived from in vivo and in vitro studies on Mycobacterium tuberculosis dissemination and localization into the eye, in combination with histopathological studies on chorioretinal tissue and vascular network. Advances in the knowledge of OTB have influenced disease management in the clinical setting and lead to reconsideration of the role of existing treatments and suggesting potential new therapeutic approaches.

Pathogenesis of ocular tuberculosis: New observations and future directions.

Ocular tuberculosis (OTB) encompasses all forms of intra- and extra-ocular inflammation associated with Mycobacterium tuberculosis (Mtb) infection. However, the organism is rarely found in ocular fluid samples of diseased eyes, rendering the pathomechanisms of the disease unclear. This confounds clinical decision-making in diagnosis and treatment of OTB. Here, we critically review existing human and animal data related to ocular inflammation and TB pathogenesis to unravel likely pathomechanisms of OTB. Broadly there appear to be two fundamental mechanisms that may underlie the development of TB-associated ocular inflammation: a. inflammatory response to live/replicating Mtb in the eye, and b. immune mediated ocular inflammation induced by non-viable Mtb or its components in the eye. This distinction is significant as in direct Mtb-driven mechanisms, diagnosis and treatment would be aimed at detection of Mtb-infection and its elimination; while indirect mechanisms would primarily require anti-inflammatory therapy with adjunctive anti-TB therapy. Further, we discuss how that most clinical phenotypes of OTB likely represent a combination of both mechanisms, with one being predominant than the other.

Study of ocular manifestations in tuberculosis and its association with HIV AIDS in a tertiary care hospital.

OBJECTIVE: To assess and understand the prevalence and clinical presentation of ocular morbidity in patients suffering from tuberculosis and compare it with ocular involvement in patients coinfected with tuberculosis and HIV AIDS. MATERIALS AND METHODS: This was a non-comparative, observational, cross sectional study done on 580 patients, who were diagnosed cases of tuberculosis, pulmonary or extrapulmonary, on or off treatment, visiting the Ophthalmology OPD, Tuberculosis OPD and ART Centre of the institute in the period from March 2015 to March 2018, screened for ocular morbidity. RESULTS: Out of 580, 408 patients had only tuberculosis and 172 had tuberculosis with HIV AIDS. 108 patients were found to have ocular involvement (18.6%) out of which 63 were males and 45 were females. The prevalence of ocular morbidity in patients with only tuberculosis was found to be 16.4% and in those having both tuberculosis and HIV AIDS was found to be 23.8%. CONCLUSION: Our study concludes that posterior uveitis, pan uveitis, periphlebitis and vitritis are the most common ocular manifestations in tuberculosis. In patients with both tuberculosis and HIV most common ocular findings included vitritis and herpes zoster ophthalmicus. Our study also concludes that lower CD4 counts (less than 200) in HIV AIDS patient is significantly associated with ocular involvement.

Immune-mediated tuberculous uveitis - A rare association with papulonecrotic tuberculid.

The tuberculids are a group of distinct clinicopathological form of skin lesions representing hypersensitivity reaction to hematogenous dissemination of Mycobacterium tuberculosis or its antigen from an underlying active or a silent focus of tuberculosis elsewhere in the body in an individual with a strong antituberculous cell-mediated immunity and by definition do not show bacilli on special stains and are culture-negative. Ocular involvement can occur in tuberculosis, both due to direct invasion by the bacilli as well as an immune-mediated reaction; however, immune-mediated tuberculous uveitis occurring as a hypersensitivity response in association with PNT has hardly been reported in the literature. Here we report one such rare case.

Diminished TLR2-TLR9 mediated CD4+ T cell responses are associated with increased inflammation in intraocular tuberculosis.

Intraocular tuberculosis (IOTB) is amongst the leading causes of uveitis in tropical countries. Despite reports on involvement of proinflammatory cytokines, studies on innate immune responses in disease pathogenesis are lacking. Reports from animal models and patients with pulmonary tuberculosis indicate that defects in toll like receptor (TLR)2 and TLR9 signalling predispose them to tuberculosis. In this context, we investigated the role of TLR2, TLR4 and TLR9 in generation of CD4+ T effector (Teff) cell responses during IOTB. Firstly, the cells in vitreous fluids showed lower expression of TLR2 and TLR9 in IOTB as compared to non-uveitis and non-TB uveitis groups. Next, peripheral CD4+ Teff cells of subjects with IOTB showed decreased proliferative responses and lower induction of Tregs following TLR2 and TLR9 stimulation. Further, TLR9 ligation resulted in increased IFN-γ and IL-17a but decreased expression of IL-10 and TGF-ß. Lastly, lower expression of genes involved in TLR9 signalling after direct TLR9 ligation was observed in IOTB. Collectively, our results show that a subdued response to direct TLR2 and TLR9 stimulation in CD4+ T cells is associated with increased proinflammatory responses in IOTB. These findings reveal an important link between innate immune signalling and ensuing adaptive immune responses in IOTB with implications in other forms of extrapulmonary tuberculosis.

Immune Profiling of T Cells Infiltrating Vitreous Humor in Tubercular Uveitis.

PURPOSE: To assess cellular composition and local cytokine response in vitreous humor of tubercular uveitis. METHODS: Cells were collected from vitreous cassettes and peripheral blood of 8 tubercular uveitis and 5 control subjects, undergoing vitrectomy and analyzed by flow cytometry for cellular composition, activation status, proinflammatory cytokine expression, and uptake of TLR9 ligand, CpG ODN 2216. RESULTS: CD3 + T cells with equal proportion of CD4+ and CD8 + T cells formed major fraction of infiltrating cells. The vitreous humor showed higher expression of recent activation marker, CD69, and proinflammatory cytokines, IFN-γ and IL-17A, in CD4 + T cells as compared to peripheral blood. Lastly, intraocular CD4 + T cells showed reduced uptake of ODN 2216 than peripheral blood. CONCLUSIONS: Our results indicate that local antigenic stimuli trigger T cell infiltration and activation of CD4 + T cells that are hyporesponsive to TLR9 stimulation. These infiltrating T cells might be responsible in further aggravating ocular inflammation.

Retinal Pigment Epithelial Cells Control Early Mycobacterium tuberculosis Infection via Interferon Signaling.

Purpose: Mycobacterium tuberculosis (Mtb) bacilli have been found in retinal pigment epithelial (RPE) cells from uveitis patients without signs of systemic tuberculosis (TB) infection. RPE cells are important for ocular immune privilege and uveitis development. Methods: To address a potential role for Mtb-infected RPE cells in the development of uveitis, we delineated the response to Mtb infection in human RPE cells and primary human macrophages, the main target cell of Mtb. Primary human RPE cells, the human RPE cell line ARPE-19, and monocyte-derived proinflammatory M1 and anti-inflammatory M2 macrophages were infected with DsRed-expressing Mtb strain H37Rv. Infection rates and clearance were addressed along with RNA sequencing analysis, a confirmation analysis by dual-color reverse-transcriptase multiplex ligation-dependent probe amplification (dcRT-MLPA) and cytokine secretion. Results: RPE cells robustly controlled intracellular outgrowth of Mtb early after infection. The response in RPE cells to control Mtb survival was dominated by interferon (IFN) signaling and further characterized by prominent regulation of cell death/survival-associated genes and low-level production of Th1-associated cytokines. In contrast, macrophages engaged a plethora of responses including IFN signaling and communication between innate and adaptive immune cells to induce granuloma formation. Conclusions: Together, our data demonstrate that RPE cells display a strong response to Mtb infection that appears, however, incomplete in comparison to the macrophage response to Mtb. The RPE response might reflect a balance between mechanisms aimed at Mtb eradication and mechanisms that limit retinal inflammation.

NLRP3 inflammasome activation by mycobacterial ESAT-6 and dsRNA in intraocular tuberculosis.

The molecular basis of intraocular tuberculosis (TB) is not well understood. In this study, we investigated the role of two constituents of viable Mycobacterium tuberculosis - Early Secreted Antigenic Target-6 (ESAT-6), and mycobacterial RNA- in inflammasome activation in the retinal pigment epithelium (RPE), a key site of inflammation in intraocular TB. We found that ESAT-6 induced caspase-1 activation and inflammasome priming in mouse RPE cells, substantially more in wild-type than in Tlr2/3/4/7/9-/-, Myd88-/- or Nlrp3-/- RPE cells. Sub-retinal ESAT-6 injection resulted in greater RPE degeneration in wild-type than in Nlrp3-/- mice. In human ocular TB tissue sections, NLRP3 staining was noted in retina as well as RPE. Mycobacterial RNA, specifically its double stranded component, also induced caspase-1 activation, and the double stranded RNA was immunolocalized to human ocular TB sections. Our observations suggest that inflammasome activation in RPE by viable M. tuberculosis could potentially contribute to human intraocular TB.

Role of Regulatory T Cells in Tubercular Uveitis.

PURPOSE: To study the role of regulatory T cells (Tregs) in patients with tubercular uveitis. METHODS: Frequencies of peripheral Tregs, Th1, Th17 cells, and intracellular cytokines were determined in 17 tubercular uveitis patients and 18 disease controls. Function of Tregs, Th1, and Th17 cells was assessed in vitro. Simultaneously, ocular levels of IFN-γ, IL-17A, IL-4, and IL-10 were also measured. RESULTS: Frequencies of peripheral Tregs in tubercular uveitis subjects were significantly lower compared with disease controls. Furthermore, expression of TGF-ß and IL-2Rα, but not CTLA4, was reduced in Tregs of the tubercular uveitis group. The tubercular uveitis group demonstrated heightened Th1, Th17 responses following in vitro stimulation with phorbol myristate acetate (PMA)/ionomycin. Interestingly, Treg suppression assay did not show a significant difference between the two groups. Ocular levels of IFN-γ, IL-17A, and IL-10 were also elevated in tubercular uveitis group. CONCLUSIONS: Low Treg frequency and hyporesponsive function contribute to proinflammatory responses manifesting at ocular level in tubercular uveitis.

Autoreactive T Cells in Immunopathogenesis of TB-Associated Uveitis.

Purpose: Intraocular inflammation in tuberculosis-associated uveitis (TBU) is usually widespread, and responds unpredictably to treatment. Herein, we analyze the intraocular T-cell response in TBU for its surface phenotype, antigenic specificity, and functional characteristics to explain the above observations. Methods: We isolated T cells from vitreous humor samples of patients with TBU and non-TB uveitis (controls). These were directly stained for surface markers CD4, CD8, CD45RO, CD45RA, CCR7, as well as intracellular cytokines IFN-γ, TNF-α, and IL-17 and analyzed on flow cytometry. Antigenic specificity was determined by activating with Mycobacterium tuberculosis-specific antigen Early Secreted Antigenic Target-6 (ESAT-6) or retinal crude extract (RCE). Activation-induced cell death (AICD) characteristics of each T-cell population were analyzed by staining for PI-Annexin V, Fas-FasL, phospho-Akt, and phospho-Erk1/2. Results: Immunophenotyping of vitreous humor samples demonstrated polyfunctional effector and central memory CD4+ T helper cells coexpressing IFN-γ, TNF-α, and IL-17. Both ESAT-6 and RCE (autoreactive) specificity was found in T cells extracted from TBU samples; however, the mycobacterial and autoreactive T-cell populations differed in their sensitivity to AICD. Autoreactive T cells appeared to resist AICD through decreased expression of apoptotic markers, FasL and caspase-3, sustained phosphorylation of Akt, and lowered Erk1/2 activity. Conclusions: Autoreactive T cells are present in TBU eyes and are relatively resistant to AICD. An understanding of this epiphenomenon could be crucial in planning treatment of TBU patients, and interpreting response to anti-TB therapy.

Infectious chorioretinitis in an immunocompetent patient: A diagnostic dilemma.

A 44-year-old male presented with a history of defective vision in the right eye for the past 5 months with the previous history of tubercular cervical lymphadenitis. On examination, right eye revealed panuveitis with dense vitritis and chorioretinitis in the superotemporal quadrant. His Mantoux test was positive (25 mm × 25 mm induration), QuantiFERON-TB Gold was test positive, aqueous aspirate was positive for Mycobacterium tuberculosis genome, negative for viruses and toxoplasma, and hence he was initiated on four-drug antitubercular therapy (ATT) with oral steroids. On follow-up, he had worsening of vitritis and intravenous methylprednisolone was given suspecting paradoxical reaction to ATT; however, a repeat AC tap was positive for toxoplasma B1 genome, IgG antitoxoplasma antibody was also positive in serum and aqueous; hence, we switched to systemic antitoxoplasma therapy. He underwent a therapeutic vitrectomy along with intravitreal clindamycin and dexamethasone for persistent vitreous membranes and vitritis. The patient responded well to the treatment with a reduction in vitritis and scarring of the lesion.

Disseminated tuberculosis presenting as hemophagocytic lymphohistiocytosis in an immunocompetent adult patient: a case report.

BACKGROUND: Hemophagocytic lymphohistiocytosis is a frequently fatal and likely underdiagnosed disease. It is a rare occurrence in adults and usually secondary to an insult such as viral infections, bacterial infections, autoimmune connective tissue disorders, malignancies and immunocompromised states, in contrast to its childhood counterpart, which is due to a genetic defect but may share some of same genetic etiologies. It is characterized by multisystem inflammation due to unregulated proliferation and infiltration of macrophages and CD8 T cells in the bone marrow, which leads to phagocytosis of red blood cells, platelets, lymphocytes and their precursors. CASE PRESENTATION: A 40-year-old Sri Lankan woman presented with a high-grade fever of 2 weeks' duration and the initial workup, including a thorough clinical examination, and all the investigations, including a septic screen, were normal. On the 18th day of hospital admission, she was found to have yellowish retinal lesions, which were confirmed as choroid tubercles by the consultant eye surgeon. Two days later she became pancytopenic and a bone marrow biopsy confirmed the diagnosis of hemophagocytic lymphohistiocytosis. She was treated with conventional category-1 antituberculous drugs and an initial 2 weeks with high-dose oral dexamethasone. All the choroid tubercles gradually disappeared and she recovered completely without any complications. CONCLUSIONS: In an adult patient with hemophagocytic lymphohistiocytosis, it is pivotal to understand the underlying etiology, as it needs extensive immunosuppression. If this patient had been treated with immunosuppressants without antituberculous medications, it would have been lethal with disseminated or central nervous system tuberculosis. So, in areas where tuberculosis is endemic, if no underlying cause is found, it may be worth considering antituberculous treatment for these patients. Re-evaluation with thorough clinical examination is of utmost importance in any patient with pyrexia of unknown origin as well as in any disease with unusual manifestations.

Clinical application of an in-house ELISPOT assay in patients with suspicious tuberculous uveitis and no signs of active tuberculosis.

PURPOSE: The purpose of this study is to evaluate the rate of Mycobacterium tuberculosis infection in uveitis patients using an ELISPOT-IFN-gamma (ELISPOT-MTP) assay and a tuberculin skin test (TST). METHODS: Fifty-three patients with suspicious tuberculous uveitis, seen at the Ocular Immunology and Uveitis Service, Scientific Institute San Raffaele, Milan, Italy, were compared with 233 healthy control subjects. All uveitis patients, together with healthy control subjects, underwent in-house ELISPOT-MTP assay and then the TST. RESULTS: None of the patients had signs of active tuberculosis. A total of 75.5% of uveitis patients showed positive TST reaction while 58.5% responded positively to ELISPOT-MTP. In healthy individuals, these responses were 30.5% and 25.3%, respectively (p<0.0001). In a different diagnosis subset, TST and ELISPOT positivity were, respectively, 80% and 50% in anterior uveitis; 75% and 50% in intermediate uveitis; 100% and 87.5% in serpiginous-like choroiditis; 90% and 80% in posterior uveitis; and 57.1% and 42.9% in panuveitis. Serpiginous-like choroiditis and posterior uveitis patients had a higher number of ELISPOT-MTP positive results and a higher grade of intensity of ELISPOT-MTP responses compared to healthy control subjects (p=0.0098). CONCLUSIONS: Our uveitis patients had higher M tuberculosis infection rate and grade of intensity response than healthy control subjects detected by ELISPOT-MTP. This response is statistically significant and higher in patients with serpiginous-like choroiditis and posterior uveitis.

The value of an immune response to Mycobacterium tuberculosis in patients with chronic posterior uveitis revisited: utility of the new IGRAs.

PURPOSE: To explore the utility of a specific immune response to Mycobacterium tuberculosis in a population of immunosuppressed idiopathic chronic posterior uveitis patients, by means of a tuberculosis-specific interferon-gamma release assay. DESIGN: Prospective, interventional case series. METHODS: A total of 31 referred patients with severe idiopathic chronic uveitis or panuveitis and 52 controls were screened for a specific immune response to tuberculosis. After ruling-out specific uveitis entities, presumed tuberculosis-related uveitis was initially considered when ophthalmologic findings were consistent with tubercular uveitis, and a specific immune response to M. tuberculosis confirmed by QuantiFERON, despite inability to detect M. tuberculosis. Clinical responses to antitubercular treatment were recorded. RESULTS: The prevalence of an immune response to M. tuberculosis was 15.38% in controls and 32.25% in uveitis patients (OR=2.619, P=0.07). Two patients were QuantiFERON indeterminate (6.4%). After excluding seven specific uveitis entities (OR=3.66, P=0.03), eight QuantiFERON-positive and one QuanTIFERON-negative uveitis patients were initially treated for presumed tuberculosis-related uveitis. All but one had no evidence of active systemic involvement. None had been previously diagnosed with tuberculosis, but unsuccessfully treated with immunosuppressors. After a 9-month tuberculostatic treatment, seven QuantiFERON -positive and one QuantiFERON-negative patients exhibited decreased intraocular inflammation, visual acuity improvement, and no relapses. Estimated QuantiFERON sensitivity and specificity were 82 and 100%, respectively, with a PPV=100% and an NPV=86%. CONCLUSIONS: QuantiFERON was useful for antituberculous treatment decision-making in chronic posterior uveitis immunosuppressed patients from areas with an intermediate-high prevalence of tuberculosis.

Presumed ocular tuberculosis in an immunocompetent eight-year-old boy.

In recent years, tuberculosis has re-emerged as a serious public health problem, raising the possibility that tuberculous eye disease may also have become more prevalent. Ocular tuberculosis usually occurs in apparently healthy individuals. It is rarely observed in patients with active pulmonary disease. An eight-year-old boy was admitted to our department because of chronic granulomatous anterior uveitis on his left eye. His medical history was unremarkable. There were no systemic symptoms of tuberculosis. He had a positive purified protein derivative test reaction. In our case, the diagnosis of ocular tuberculosis was presumptive and depended upon indirect evidence. The patient was started on anti-tuberculosis therapy with three drugs, which were continued for 12 months, with complete healing of the ocular lesions, including a marked improvement in the gait of the patient. Tuberculosis remains one of the most important causes of morbidity and mortality in developing countries.