Interferon-γ and IL-5 Production Correlate Directly in HIV Patients Co-Infected with Mycobacterium tuberculosis with or without Immune Restoration Disease

Abstract

IL-5 and interferon-γ responses were investigated in mitogen-stimulated whole-blood cultures from HIV patients with and without Mycobacterium tuberculosis disease, to determine whether an imbalance of Th1/Th2 cytokines contributes to susceptibility to M. tuberculosis disease or to immune restoration disease (IRD) associated with M. tuberculosis after starting antiretroviral therapy (ART). Interferon-γ levels were constant on ART, whilst IL-5 levels generally rose over time. We suggest that increased IL-5 production reflects a recovery of CD4⁺ T cell function and that a Th1/Th2 imbalance is not associated with increased susceptibility to M. tuberculosis disease or IRD associated with M. tuberculosis upon starting ART.

T uberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) and antiretroviral therapy (ART)-associated TB (ART-TB) are major causes of illness in HIV patients with Mycobacterium tuberculosis infection who commence ART.¹ TB-IRIS and many cases of ART-TB are forms of immune restoration disease (IRD), which we have argued results from the restoration of a pathogen-specific immune response that causes immunopathology.^2,3 Overall, IRD associated with M. tuberculosis infection is characterized by increased Th1 immune responses, notably interferon-γ (IFN-γ) production.^4

–7

Th1 cytokine responses are important in protection against M. tuberculosis infection through the actions of IL-12 and IFN-γ.⁸ Several studies suggest that infection with HIV leads to a shift from Th1 to Th2-type cytokine expression,^9
–11 which might contribute to increased incidence and severity of TB in HIV patients. If this is true, a reversal of the abnormal Th1/Th2 balance after commencing ART might contribute to the development of IRD. However, few studies report Th1/Th2 cytokine responses in HIV patients after they commence ART.^12,13 Here, we investigated the Th1 and Th2 polarization of circulating T cells by assessing IFN-γ and IL-5 production in whole blood cultures from HIV-1-infected patients with and without disease associated with M. tuberculosis infection after commencing ART.

Plasma was obtained from whole blood IFN-γ release assays [Quantiferon-TB Gold^TM in-tube (QFTGIT); Cellestis, Carnegie, Australia) undertaken before and at 4, 12, and 24 weeks of ART in 306 ART-naïve HIV-1 patients.⁷ Seventy-five patients had treated TB upon starting ART, of whom 15 experienced TB-IRIS. Eleven patients with no history of TB were diagnosed with ART-TB after starting ART. Case definitions of TB-IRIS and ART-TB appear elsewhere.¹ Each TB-IRIS and ART-TB case was matched with 2 controls by sex, pre-ART CD4⁺ T cell count, and TB history. Levels of IFN-γ and IL-5 were assayed in plasma from whole blood cultured for 24 h with phytohemagglutinin (PHA) or purified protein derivative (PPD).

IFN-γ levels were measured by ELISA as per the QFTGIT assay kit instructions. Levels of IL-5 were measured by BD Cytometric Bead Array Flex Sets (BD Biosciences, San Jose, CA). 300 events were collected using a BD FACSArray machine and BD FACSArray System Software v1.0.3 (BD Immunocytometry Systems, San Jose, CA). Analysis was performed using FCAP Array Software v1.0.1 (BD Biosciences). All samples were diluted 1:5 and the lowest limit of detection was 2.7 pg/ml.

Levels of IFN-γ and IL-5 were compared using a Mann–Whitney U test and a Kruskal–Wallis (with Dunn's post-test analysis) nonparametric test. Correlations were performed using the Spearman test. All patients provided written informed consent and the study was approved by the Cambodian National Ethics Committee and the University of New South Wales Human Research Ethics Committee.

Levels of IFN-γ and IL-5 did not differ between TB-IRIS and ART-TB cases and their controls before or during 24 weeks of ART (Fig. 1). However, levels of IL-5 were marginally higher in TB-IRIS controls compared to cases after 12 weeks of ART (p = 0.07, data not shown) and in ART-TB controls compared to cases after 4 weeks of ART (p = 0.10, data not shown).

FIG.1.

Changes in levels of IFN-γ and IL-5 in (a) TB-IRIS cases and controls and (b) ART-TB cases and controls during 24 weeks of ART. Trends are expressed in terms of the median, and asterisks represent statistical significance relative to baseline: * represents p < 0.05 and ** represents p < 0.01. Small numbers of samples (1–3) were missing at later time points in all groups except for ART-TB controls.

IL-5 levels rose significantly in TB-IRIS controls relative to baseline following the commencement of ART (for week 0 vs 4, p < 0.05; for week 0 vs 12, p < 0.05; for week 0 vs 24, p < 0.01; Kruskal–Wallis, Fig. 1a), while levels remained low in TB-IRIS cases before and during 12 weeks of ART. Levels of IFN-γ did not change significantly relative to baseline in TB-IRIS cases or their controls during ART (Fig. 1a). IL-5 levels generally rose in ART-TB cases and controls after the commencement of ART, although the rise was not significant relative to baseline (Fig. 1b). Levels of IFN-γ did not change relative to baseline in ART-TB cases or controls (Fig. 1b).

IFN-γ levels correlated directly with IL-5 in all groups studied, but the correlations were weak in the ART-TB cases (Table 1). It was notable that no group displayed an inverse relationship between IFN-γ and IL-5 before or during ART. Rather the capacity to secrete IL-5 rose during ART, while IFN-γ production remained similar. This may reflect a recovery of T cell function on ART. These findings are similar to a study of Australian HIV patients where IFN-γ responses to PHA remained steady while IL-5 responses increased for the first 2 years on treatment.¹²

Table 1.

Correlations Between IL-5 and IFN-γ Levels within TB-IRIS and ART-TB Cases and Controls over 24 Weeks of ART

	Pre-ART		4 Weeks		12 Weeks		24 Weeks
	r	p	r	p	r	p	r	p
TB-IRIS cases	0.14	0.61	0.75	<0.01	0.69	<0.01	0.87	<0.001
TB-IRIS controls	0.31	0.09	0.26	0.2	0.35	0.07	0.51	<0.01
ART-TB cases	0.11	0.74	0.14	0.71	0.21	0.55	0.26	0.46
ART-TB controls	0.34	0.12	0.44	0.04	0.41	0.06	0.47	0.03

These findings also support a previous study showing no inverse relationship between levels of IFN-γ and IL-5 in Australian HIV patients after commencing ART.¹³ This included measurement of mRNA for IL-4 and its inhibitor IL4δ2, as well as cells stimulated with anti-CD3.¹³ In the present study, IL-4 could not be detected in PHA-stimulated cultures, possibly because many HIV patients were severely immunodeficient upon commencing ART (data not shown).

We also assayed IL-5 in plasma from PPD-stimulated whole blood cultures in a subset of patients (4 TB-IRIS cases, 4 ART-TB cases, and 16 matched controls) but levels were below the cut-off of the assay (<2.7 pg/ml, data not shown) in all samples. IFN-γ levels increased over 24 weeks of ART in the same samples in TB-IRIS cases and controls but remained stable in ART-TB cases and controls (data not shown). IFN-γ responses to PPD assessed in the entire cohort are reported elsewhere.⁷

These data provide an insight into the recovery of Th1- and Th2-polarised T cells in HIV-infected patients. The rise in IL-5 production during ART appears to reflect a recovery of T cell function. We find no evidence that a Th1/Th2 imbalance contributes to the susceptibility of HIV patients to M. tuberculosis disease pre-ART, TB-IRIS, or ART-TB.

Footnotes

Acknowledgments

This study was supported by AusAID and by the National Health and Medical Research Council (Australia). We thank all participants for providing samples for the study, as well as Anna Romeo for her expertise and help with the BD FACSArray machine. We also thank Cellestis for their contribution to the study.

Author Disclosure Statement

The authors have no conflicts of interest to declare.

References

Meintjes

, Lawn

, Scano

et al. Tuberculosis-associated immune reconstitution inflammatory syndrome: Case definitions for use in resource-limited settings. Lancet Infect Dis, 2008; 8:516–523.

French

, Lenzo

, John

et al. Immune restoration disease after the treatment of immunodeficient HIV-infected patients with highly active antiretroviral therapy. HIV Med, 2000; 1:107–115.

French

, Price

, Stone

. Immune restoration disease after antiretroviral therapy. AIDS, 2004; 18:1615–1627.

Bourgarit

, Carcelain

, Martinez

et al. Explosion of tuberculin-specific Th1-responses induces immune restoration syndrome in tuberculosis and HIV co-infected patients. AIDS, 2006; 20:F1–7.

Meintjes

, Wilkinson

, Rangaka

et al. Type 1 helper T cells and FoxP3-positive T cells in HIV-tuberculosis-associated immune reconstitution inflammatory syndrome. Am J Respir Crit Care Med, 2008; 178:1083–1089.

Bourgarit

, Carcelain

, Samri

et al. Tuberculosis-associated immune restoration syndrome in HIV-1-infected patients involves tuberculin-specific CD4 Th1 cells and KIR-negative gammadelta T cells. J Immunol, 2009; 183:3915–3923.

Elliott

, Vohith

, Saramony

et al. Immunopathogenesis and diagnosis of tuberculosis and tuberculosis-associated immune reconstitution inflammatory syndrome during early antiretroviral therapy. J Infect Dis, 2009; 200:1736–1745.

Bhatt

, Salgame

. Host innate immune response to Mycobacterium tuberculosis. J Clin Immunol, 2007; 27:347–362.

Clerici

, Shearer

. A TH1– >TH2 switch is a critical step in the etiology of HIV infection. Immunol Today, 1993; 14:107–111.

10.

Klein

, Dobmeyer

et al. Demonstration of the Th1 to Th2 cytokine shift during the course of HIV-1 infection using cytoplasmic cytokine detection on single cell level by flow cytometry. AIDS, 1997; 11:1111–1118.

11.

Lienhardt

, Azzurri

, Amedei

et al. Active tuberculosis in Africa is associated with reduced Th1 and increased Th2 activity in vivo. Eur J Immunol, 2002; 32:1605–1613.

12.

Keane

, Price

, Lee

et al. Restoration of CD4 T-cell responses to cytomegalovirus is short-lived in severely immunodeficient HIV-infected patients responding to highly active antiretroviral therapy. HIV Med, 2004; 5:407–414.

13.

Price

, Keane

, Lee

, Lim

, McKinnon

, French

. A T2 cytokine environment may not limit T1 responses in human immunodeficiency virus patients with a favourable response to antiretroviral therapy. Immunology, 2006; 119:74–82.