title: Cytotoxic mediators in paradoxical HIV-tuberculosis immune reconstitution inflammatory syndrome.
abstract: Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) frequently complicates combined antiretroviral therapy and antituberculosis therapy in HIV-1-coinfected tuberculosis patients. The immunopathological mechanisms underlying TB-IRIS are incompletely defined, and improved understanding is required to derive new treatments and to reduce associated morbidity and mortality. We performed longitudinal and cross-sectional analyses of human PBMCs from paradoxical TB-IRIS patients and non-IRIS controls (HIV-TB-coinfected patients commencing antiretroviral therapy who did not develop TB-IRIS). Freshly isolated PBMC stimulated with heat-killed Mycobacterium tuberculosis H37Rv (hkH37Rv) were used for IFN-γ ELISPOT and RNA extraction. Stored RNA was used for microarray and RT-PCR, whereas corresponding stored culture supernatants were used for ELISA. Stored PBMC were used for perforin and granzyme B ELISPOT and flow cytometry. There were significantly increased IFN-γ responses to hkH37Rv in TB-IRIS, compared with non-IRIS PBMC (p = 0.035). Microarray analysis of hkH37Rv-stimulated PBMC indicated that perforin 1 was the most significantly upregulated gene, with granzyme B among the top five (log2 fold difference 3.587 and 2.828, respectively), in TB-IRIS. Downstream experiments using RT-PCR, ELISA, and ELISPOT confirmed the increased expression and secretion of perforin and granzyme B. Moreover, granzyme B secretion reduced in PBMC from TB-IRIS patients during corticosteroid treatment. Invariant NKT cell (CD3(+)Vα24(+)) proportions were higher in TB-IRIS patients (p = 0.004) and were a source of perforin. Our data implicate the granule exocytosis pathway in TB-IRIS pathophysiology. Further understanding of the immunopathogenesis of this condition will facilitate development of specific diagnostic and improved therapeutic options.
authors.affiliation: Clinical Infectious Diseases Research Initiative, University of Cape Town, Cape Town, 7925 South Africa; Department of Medicine, University of Cape Town, Cape Town, 7925 South Africa; Medical Research Council National Institute for Medical Research, London NW7 1AA, United Kingdom; Katalin.Wilkinson@uct.ac.za.
authors.forename: Katalin A
authors.identifier: http://orcid.org/0000-0002-9796-2040
authors.initials: KA
authors.lastname: Wilkinson
authors.affiliation: Clinical Infectious Diseases Research Initiative, University of Cape Town, Cape Town, 7925 South Africa; Division of Medicine, Imperial College London, London W2 1PG, United Kingdom;
authors.forename: Naomi F
authors.initials: NF
authors.lastname: Walker
authors.affiliation: Clinical Infectious Diseases Research Initiative, University of Cape Town, Cape Town, 7925 South Africa; Department of Medicine, University of Cape Town, Cape Town, 7925 South Africa; Division of Medicine, Imperial College London, London W2 1PG, United Kingdom;
authors.forename: Graeme
authors.initials: G
authors.lastname: Meintjes
authors.affiliation: Clinical Infectious Diseases Research Initiative, University of Cape Town, Cape Town, 7925 South Africa;
authors.forename: Armin
authors.initials: A
authors.lastname: Deffur
authors.affiliation: Clinical Infectious Diseases Research Initiative, University of Cape Town, Cape Town, 7925 South Africa; Division of Medical Microbiology, University of Cape Town, Cape Town, 7925 South Africa; National Health Laboratory Service, Cape Town, 7925 South Africa; and.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Nicol
authors.affiliation: Clinical Infectious Diseases Research Initiative, University of Cape Town, Cape Town, 7925 South Africa; Division of Medicine, Imperial College London, London W2 1PG, United Kingdom;
authors.forename: Keira H
authors.initials: KH
authors.lastname: Skolimowska
authors.affiliation: Clinical Infectious Diseases Research Initiative, University of Cape Town, Cape Town, 7925 South Africa;
authors.forename: Kerryn
authors.initials: K
authors.lastname: Matthews
authors.affiliation: Clinical Infectious Diseases Research Initiative, University of Cape Town, Cape Town, 7925 South Africa;
authors.forename: Rebecca
authors.initials: R
authors.lastname: Tadokera
authors.affiliation: Clinical Infectious Diseases Research Initiative, University of Cape Town, Cape Town, 7925 South Africa;
authors.forename: Ronnett
authors.initials: R
authors.lastname: Seldon
authors.affiliation: Department of Medicine, University of Cape Town, Cape Town, 7925 South Africa;
authors.forename: Gary
authors.initials: G
authors.lastname: Maartens
authors.affiliation: Clinical Infectious Diseases Research Initiative, University of Cape Town, Cape Town, 7925 South Africa;
authors.forename: Molebogeng X
authors.initials: MX
authors.lastname: Rangaka
authors.affiliation: School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom.
authors.forename: Gurdyal S
authors.initials: GS
authors.lastname: Besra
authors.affiliation: Clinical Infectious Diseases Research Initiative, University of Cape Town, Cape Town, 7925 South Africa; Department of Medicine, University of Cape Town, Cape Town, 7925 South Africa; Medical Research Council National Institute for Medical Research, London NW7 1AA, United Kingdom; Division of Medicine, Imperial College London, London W2 1PG, United Kingdom;
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Wilkinson
mesh_terms: D000328:Adult; D060085:Coinfection; D003430:Cross-Sectional Studies; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D053804:Granzymes / Q000096:biosynthesis / Q000276:immunology; D015658:HIV Infections / Q000150:complications / Q000276:immunology*; D006801:Humans; D054019:Immune Reconstitution Inflammatory Syndrome / Q000209:etiology / Q000276:immunology*; D007371:Interferon-gamma / Q000096:biosynthesis / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008137:Longitudinal Studies; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis; D054353:Perforin / Q000096:biosynthesis / Q000276:immunology; D060888:Real-Time Polymerase Chain Reaction; D014376:Tuberculosis / Q000150:complications / Q000276:immunology*
chemical_list: D054353:Perforin; D007371:Interferon-gamma; D053804:Granzymes