Expression of a novel cytokine, IL-4delta2, in HIV and HIV-tuberculosis co-infection

Background: Correcting the Th2 shift in HIV/AIDS represents a potential intervention strategy. However data on interleukin (IL)-4 expression in HIV or AIDS are un-interpretable because of failure to distinguish between IL-4 and its splice variant and natural antagonist, IL-4δ2.

Objective: To determine Th1 [interferon (IFN)-γ], IL-4δ2 and Th2 (IL-4) expression in whole blood and lung lavage from healthy volunteers and in HIV or HIV-tuberculosis (TB) co-infection.

Design: Cross-sectional with prospective cohort.

Methods: Expression of IL-4δ2, IL-4 and IFN-γ were determined by quantitative real-time PCR, using unstimulated cells from whole blood and lung lavage, in 20 HIV-TB (pulmonary) co-infected patients, 20 matched HIV-positive controls and 20 HIV-negative healthy volunteers. Results were correlated with plasma viral load, CD4 cell counts, radiological scores and response to anti-TB treatment.

Results: Compared to HIV negative donors, stable HIV-positive donors did not have increased levels of mRNA encoding IL-4, IL-4δ2 or IFN-γ in blood or lavage. By contrast, the HIV-TB co-infected donors had increased IL-4 and IFN-γ in both compartments. However the antagonist, IL-4δ2 was increased only in lavage. Consequently the dominant form was IL-4δ2 in lavage, but IL-4 itself in blood. The lung IL-4/IFN-γ ratio correlated with radiological disease extent. With anti-TB treatment, IL-4 levels did not change whilst IL-4δ2 levels increased significantly.

Conclusions: IL-4 and its natural antagonist, IL-4δ2 and are not upregulated in the absence of opportunistic infection. However in HIV-TB co-infection both cytokines increase in lung, but only IL-4 in the periphery. Further studies are required to determine if IL-4 facilitates systemic HIV progression.

Tuberculosis (TB) and HIV are closely interactive pandemics that continue to flourish despite the availability of effective chemotherapy. Accordingly, immunotherapy in the form of widely available vaccines or immune modifiers would be crucial adjuncts for disease containment ^[1]. However the mechanisms underlying HIV related CD4 T cell dysfunction and progression to AIDS remain unclear, and developing a successful HIV or TB vaccine has been more challenging than expected. Consequently, effective design of immunotherapy for either disease will require a thorough understanding of the mechanisms governing disease progression and correlates of protective immunity. In TB, HIV and TB-HIV co-infection there are robust Th1 responses to TB ^[2-4] and HIV ^[5] specific antigens; nevertheless the disease progresses. Therefore it has been suggested that a subversive Th2 component, which undermines the dominant Th1 response, is associated with the pathogenesis of both TB ^[6], HIV ^[7,8] and progression to AIDS ^[9]. The 'Th1 to Th2 switch' hypothesis proposes that immune dysregulation facilitating infection and progression to AIDS is dependent on the balance between Th1 and Th2 responses to HIV ^[9].

However the presence and significance of Th2 cytokines in HIV is controversial ^[10]; moreover Th2 related data in HIV ^[7,11-14] and HIV-TB co-infection ^[15-18] are sparse and discordant. This may be partly explained by technical difficulties in measuring the prototype Th2 cytokine, interleukin (IL)-4, especially in unstimulated human cells and at sites not directly affected by the disease ^[16,19]. More importantly, the available data fail to take into account the presence of a novel cytokine, IL-4delta2 (IL-4δ2), a splice variant and natural antagonist of IL-4 ^[20,21]. This cytokine, recently studied in asthma and pulmonary fibrosis ^[22,23], has Th1 like properties and therefore confounds the interpretation of available Th2 data. Particularly relevant are the recent findings that IL-4δ2, and not IL-4, is upregulated in humans who have non-progressive latent TB infection ^[24,25]. However IL-4δ2 expression has not previously been studied in HIV or HIV-TB co-infection. Understanding the relevant contribution of IL-4/IL-4δ2 in TB and HIV may be important for intervention strategies, which modulate the Th1/Th2 balance in both diseases. Consequently we investigated IL-4δ2 expression in whole blood and lung lavage of donors with TB-HIV co-infection, HIV alone and in non-HIV infected healthy volunteers.

Methods

Patients and samples

Twenty patients with clinical and radiological features of pulmonary TB (sputum or lavage fluid positive by nucleic amplification test or culture for Mycobacterium tuberculosis ^[26]) and HIV co-infection, 13 of whom underwent bronchoscopy and bronchoalveolar lavage (BAL), were recruited in London, UK. Control donors were 20 otherwise healthy HIV-positive volunteers who were matched to the index cases for age (within 4 years), sex, and CD4 cell count (< 200, 200-350 or > 350 × 10⁶cells/l); and 20 HIV-negative healthy volunteers. Six volunteers from each control group (HIV positive and negative) underwent BAL. In all HIV-positive donors other concomitant viral, bacterial, fungal and parasitic infections were excluded by PCR, serology and cytology of relevant biological samples. Plasma HIV viral load, total peripheral blood and BAL CD3, CD4 and CD8 cell counts were performed in all HIV-positive participants. Whole blood, obtained from all participants before or within 10 days of initiating anti-TB treatment and also from 10 TB-HIV co-infected patients after anti-TB treatment, was immediately transferred to Paxgene tubes (Qiagen, Germany) to fix the RNA profile.

BAL and radiographic scoring

BAL fluid was obtained from a radiologically affected lung segment as previously described ^[3]. To determine the extent of radiological disease prior to treatment, two radiologists blinded to patient details, scored all patient chest x-rays for air space shadowing, reticular opacities and cavitation.

Enzyme-linked-immunospot and ELISA

Latent TB infection was excluded in HIV-positive and HIV-negative control subjects by overnight T-cell interferon (IFN)-γ responses to TB-specific antigens (T-SPOT TB, Oxford Immunotec, Oxford, UK). In addition ELISA for soluble-CD30, IL-4 and IFN-γ (PelikineCompact, Sanquin reagents, Amsterdam, the Netherlands) was performed on ∼ 10-fold concentrated lavage supernatants.

Reverse transcription and real-time PCR

RNA extraction, reverse transcription (RT) and real-time quantitative RT-PCR for IL-4, IL-4δ2 and IFN-γ were performed on all blood and BAL samples as previously described after quantification and quality control of RNA templates ^[3,27].

Data analysis

Patient and control groups were compared, where appropriate, using the Mann-Whitney U test, Wilcoxon matched pairs test and Spearman rank-sum correlation. Cytokine ratios were expressed as log differences between respective cytokine pairs. Analysis was conducted using GraphPad prism.

Results

HIV-TB co-infected patients and HIV-positive controls had comparable baseline characteristics [mean (SD) age in years, 37.1 (8.6) versus 36.2 (5.7), CD4 cell count 222 (201) versus 279 (198) × 10⁶cells/l, percent of donors with a CD4 count < 200 × 10⁶cells/l: 50 in each group, percent male 50 versus 42, percent black African ethnicity, 79 versus 74, percent using highly active antiretroviral therapy (HAART) 15 versus 40]. Similarly, the HIV-positive and HIV-negative controls were not dissimilar for age, sex and race. However HIV-TB co-infected patients had higher viral load (median copies/ml plasma) than HIV-positive controls (77 760 versus 50, P = 0.0001).

HIV positive and HIV negative healthy controls

In HIV-positive controls versus non-HIV controls IL-4, IL-4δ2 and IFN-γ were similar in whole blood and lung lavage fluid (Fig. 1a, b and c), even when paired samples from both compartments were compared (data not shown). mRNA levels did not differ significantly in those taking HAART compared to those who were not.

Fig. 1

HIV-TB co-infected patients and HIV-positive controls

By contrast mRNAs for IL-4 and IFN-γ were increased in both compartments of TB-HIV co-infected donors, compared to the HIV-positive controls [P < 0.003 for IL-4 (∼ 5-fold increase in blood and ∼ 17-fold increase in lavage) and P < 0.02 for IFN-γ (> 350-fold increase for blood and > 1300-fold increase for lavage), Fig. 1a and c]. However IL-4δ2 mRNA levels were similar in the blood whether the HIV-positive individuals were TB co-infected or not, whereas levels were much higher in lung lavage from co-infected donors (P < 0.005, ∼ 100-fold increase, Fig. 1b). Consequently, in co-infected patients the IL-4/IL-4δ2 ratio was significantly higher in whole blood than in lung lavage (P = 0.0006), where the dominant form of 'IL-4' was in fact the antagonist, IL-4δ2.

CD30 and IL-4 were undetectable by ELISA in BAL from most HIV-positive donors, whether TB-coinfected or not. By ELISA, IFN-γ was increased in BAL from some co-infected patients, but undetectable in 50%. Thus ELISA results were unhelpful.

Relationship to clinical, laboratory and treatment parameters

In HIV-TB co-infected patients the IL-4/IFN-γ mRNA ratio was similar in whole blood and lung lavage and the latter correlated with pre-treatment radiological disease extent (P = 0.02). IL-4 and IL-4δ2 mRNA levels were independent of blood and lavage CD4 and CD8 counts, blood viral load, the presence of disseminated tuberculosis and use of HAART. Following short course anti-TB treatment IL-4 and IFN-γ mRNA levels did not change significantly whereas IL-4δ2 levels increased significantly (P = 0.01, ∼ 85 fold increase, Fig. 2a-c).

Fig. 2

Discussion

IL-4δ2 is a novel cytokine that has Th1 properties as it is a competitive antagonist of IL-4 ^[20,21]. Published studies of HIV have used assays that do not distinguish between the agonist, IL-4, and antagonist, IL-4δ2 ^[7,11-14]. Moreover, due to the insensitivity of existing immmunoassays for IL-4 ^[19] investigators have invariably used unphysiological stimulation protocols which have a Th1 bias ^[28]. Interpretation is further complicated by heterogeneity in the stage of HIV studied (primary infection, stable HIV or AIDS), the site of sample acquisition (peripheral blood, lymph nodes or lung) and the type of assay used to measure IL-4. Therefore published IL-4 results are conflicting with some studies supporting ^[7,12,13] and some opposing ^[11,14] the 'Th1 to Th2 shift' hypothesis. Consequently the issue of whether a switch to Th2 responses facilitates the progression of HIV ^[8,9] remains controversial ^[10,29]. We have measured both IL-4 and the antagonist, IL-4δ2, in unstimulated cells from both blood and lung lavage using a validated probe-based real-time quantitative assay ^[30]. We found that Th2 profiles were similar in HIV infected and non-infected healthy volunteers and this was independent of CD4 cell count, viral load or use of HAART at the time of sample acquisition. Our data argue against the implication of Th2 cytokines in the pathogenesis of HIV in the absence of other opportunistic infections. It remains possible that cells from HIV-infected donors release more IL-4 (or IL-4δ2; the assays used would have measured both together) when stimulated in vitro with mitogens ^[12,13]. It is also noteworthy that we did not study subjects with primary HIV infection; however Th2 findings even in this group are discordant ^[11,13].

By contrast, in HIV-TB co-infected patients both IL-4 and IFN-γ are upregulated whilst IL-4δ2 increased in the lungs but not in the blood. Consequently, co-infected patients had a significantly higher IL-4/IL-4δ2 ratio in whole blood compared to lung lavage. That these observations are biologically meaningful is corroborated by the correlation of the lung Th1/Th2 (IL-4/IFN-γ) ratio with pre-treatment radiological scores. The mechanisms that underpin increased viral replication during TB co-infection are poorly understood. We confirm here that in HIV-positive hosts, TB induces IL-4 as it does in HIV seronegative TB ^[6], and the profile of IL-4 and IL-4δ2 in HIV-TB co-infection is similar to that found in seronegative TB ^[3]. In the latter group the IL-4 induced by TB can subvert mycobactericidal host responses by facilitating dysfunction of Toll-like receptor and nitric ozide pathways ^[31-33]. In HIV-positive patients, IL-4 may facilitate HIV progression by up-regulating HIV co-receptors, encouraging syncytium-inducing strains ^[34], inhibiting anti-viral α-defensins ^[35,36] and activating viral transcription ^[34]. The high IFN-γ levels found in co-infected patients may also facilitate HIV progression by up-regulating HIV co-receptors ^[37] though we found no correlation between plasma viral load and IFN-γ mRNA levels. However, plasma viral load is only a surrogate marker of total viral burden and we did not measure viral load at the main site of disease, the lung.

Immunoassays have poor sensitivity in detecting protein in BAL due to the considerable dilutional effect in lavage fluid ^[38]. Although this study did not examine the cell types making IL-4, HIV-negative subjects with TB produced significantly more IL-4 than controls in whom IL-4 was produced pre-dominantly (∼ 95%) by non-T cells ^[3].

Completion of chemotherapy was accompanied by a significant increase in IL-4δ2 and a reduced IL-4/IL-4δ2 ratio. Further studies are warranted to evaluate the utility of this ratio as a marker of disease activity or successful treatment. If this is borne out it may be of value for future trials of new therapeutic interventions.

In conclusion our data suggest a role for Th2 cytokines in the pathogenesis of HIV-TB co-infection but not in otherwise stable HIV infection. Further studies are required to determine if this applies to other HIV related opportunistic infections, and whether the selective increase in IL-4 expression in the periphery of dually infected patients contributes to an increase in replication of HIV.

Acknowledgements

The authors thank I.A. Cropley, S. Bhagani, M. Tyrer, M. Beckles and staff at the Royal Free Hospital HIV and TB clinics/ward for assisting with patient recruitment; Luciene Lopes and Ian Gerrard for their technical advice and F.C. Lampe for her statistical input.

Sponsorship: Supported by the British Lung Foundation.