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.

FIGURE 1.

IFN-γ responses to hkH37Rv in TB-IRIS compared with non-IRIS control PBMC during the first 8 wk post-ART initiation. IFN-γ responses of PBMC measured by ELISPOT from 38 HIV-TB–coinfected patients at baseline and for 8 wk post-ART initiation, after stimulation with hkH37Rv. Eleven patients developed TB-IRIS at a median of 14 d. Significantly increased numbers of IFN-γ spot-forming cells (SFC) were identified in TB-IRIS patients at TB-IRIS onset, compared with non-IRIS control patients sampled at 2 wk of ART (p = 0.035). No differences in IFN-γ SFC were identified at other time points. Horizontal lines indicate median values.

FIGURE 2.

Representation of significantly differentially expressed genes. Microarray analysis of RNA from seven matched pairs of IRIS and non-IRIS patients, at 2 wk on ART, showed transcript for perforin and granzyme B genes to be among the four genes most significantly overrepresented in TB-IRIS PBMC after stimulation with H37Rv. The transcripts for Zinc and ring finger 1 and Cbp/p300-interacting transactivator with Glu/Asp-rich C-terminal domain 2 were also significantly raised. The genes significantly upregulated in TB-IRIS (with δ = 0.96 and false discovery rate of 0.000001%) are represented on the volcano plot as labeled. The x-axis shows the log2-fold change, and the y-axis shows a measure of significance (−log10 [p value]).

FIGURE 3.

Secondary validation of microarray analysis by RT-PCR. PBMC from 22 TB-IRIS and 22 non-IRIS control patients were isolated and cultured in the presence or absence of hkH37Rv (MOI 1:1, PBMC:H37Rv) for 6 or 24 h. RNA was extracted and assayed by RT-PCR. β-actin was used to normalize values. Fold induction over unstimulated cultures was calculated by the ΔΔ cycle threshold method, and fold values log10 transformed to normalize. Granzyme B was increased in TB-IRIS compared with controls after both 6 and 24 h (p < 0.0001 and p = 0.002, respectively). Perforin was also significantly increased after 24-h stimulation (p = 0.008). Horizontal lines indicate median values.

FIGURE 4.

Granzyme B and perforin secretion are increased in TB-IRIS. (A and B) Granzyme B secretion measured by ELISA was significantly increased in TB-IRIS patients, in contrast to granzyme A, in hkH37Rv-stimulated PBMC culture supernatants (20 TB-IRIS, 20 controls) compared with unstimulated supernatants (p < 0.0001), and compared with stimulated PBMC culture supernatants from non-IRIS control patients (p = 0.002). (C and D) Granzyme B and perforin measured by ELISPOT tended to be increased in TB-IRIS compared with controls (not statistically significant). Horizontal lines indicate median values.

FIGURE 5.

Effect of prednisone or placebo on granzyme B release in vitro. Granzyme B secretion (pg/ml) from PBMCs, from TB-IRIS patients enrolled in a randomized controlled trial of prednisone versus placebo, measured by ELISA after stimulation with hkH37Rv, with unstimulated culture results subtracted. Prednisone treatment for 2 wk significantly decreased granzyme B secretion from TB-IRIS PBMC compared with pretreatment values. This reduction was not evident in PBMC from placebo-treated TB-IRIS patients.

FIGURE 6.

Increased proportions of iNKT cells in TB-IRIS compared with non-IRIS control patients. Flow cytometric analysis using cell surface staining for CD3⁺Vα24⁺ cells identified an increased proportion of CD3⁺Vα24⁺ cells in TB-IRIS compared with non-IRIS control patients (median, 0.17%; IQR, 0.09–0.22, versus 0.03%; IQR, 0.016–0.106) (A). Using intracellular staining for perforin, CD3⁺Vα24⁺ cells from TB-IRIS patients were identified as a potential source of perforin (B). Horizontal bars indicate median values. Gating strategy was based on selecting CD3⁺ cells from a side scatter/CD3 scatter dot blot, followed by gating on Vα24 and either Vβ11 or perforin-positive cells.