Short Communication: Human Lymphotropic Virus Type 1 Coinfection Modulates the Synthesis of Cytokines by Peripheral Blood Mononuclear Cells from HIV Type 1-Infected Individuals

Abstract

HIV-1 and HTLV-1 infect CD4⁺ T lymphocyte but little is known about the impact of coinfection on patient's immune response. In this study we have evaluated the spontaneous production of interleukin (IL)-2, IL-4, IL-6, and IL-10 and interferon (IFN)-γ by unstimulated peripheral blood mononuclear cell (PBMC) cultures obtained from patients infected by HTLV-1, HIV-1, or both viruses. We have observed that HIV/HTLV-coinfected individuals presented significantly higher production of IL-2 and IFN-γ compared to both HIV single-infected and HTLV single-infected individuals. IL-6 and IL-10 synthesis was similar in all infected groups, but HTLV single-infected individuals presented a lower production of IL-4. These results show that HIV/HTLV-coinfected individuals presented a profile of higher production of Th-1 cytokines, suggesting a predominant stimulus of HTLV-1 in detriment to HIV-1 infection.

T he human lymphotropic virus type 1 (HTLV-1) and the human immunodeficiency virus type 1 (HIV-1) are retroviruses that share some characteristics, including tropism for the CD4⁺ T lymphocyte. Although these viruses infect the same cell, they present a different biological behavior. HIV-1 infection results in a massive destruction of the CD4⁺ T lymphocytes, and if this infection is not treated, it will evolve to acquired immunodeficiency syndrome (AIDS) in the majority of infected individuals.¹ HTLV-1 infection can induce proliferation of CD4⁺ T lymphocyte, and also can cause an aggressive malignant disease, the adult T cell leukemia/lymphoma (ATLL) or a neurologic disorder known as HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP).^2,3 Different from HIV-1 infection, only a small fraction of HTLV-1-infected subjects (1–5%) will develop clinical disease, most of the time some decades after the initial infection.⁴ Both viruses are able to spread in the population by the same routes: sexual, blood transfusion, and mother-to-child transmission, thus, coinfection can occur in areas where both infections are prevalent. In the Northeast of Brazil HIV-1/HTLV-1 coinfection can occur in 10–20% of the HIV-1-infected population.^5,6 Our group has observed that in Bahia, Brazil, HTLV-1 coinfection is associated with a decreased survival time in HIV-1-infected individuals.⁷ As both viruses are able to infect CD4⁺ T cells and are also able to modify the cytokines profile,^8
–10 our purpose was to study the pattern of immune response in coinfected individuals.

In this study we have evaluated 67 patients: 33 HIV-1 singly infected (HIV group), 15 HTLV-1 singly infected (HTLV group), and 19 HIV/HTLV-1 coinfected (HIV/HTLV group), and 13 health volunteers (used as controls), from Complexo Hospitalar Professor Edgard Santos and from Centro Estadual de Diagnóstico, Assistência e Pesquisa, Salvador, Bahia, Brazil. HIV-1 and HTLV-1 infections were confirmed by Western blotting. All the patients included in this study were asymptomatic at the time of blood collection and had no identifiable active clinical condition related to HIV-1 and/or HTLV-1 infections. Informed consent was obtained from all individuals and this study was approved by the institutional ethics review board. HIV-1 RNA plasma viral load was evaluated using the commercial kit VERSANT b DNA HIV-1 RNA 3.0 ASSAY. For T lymphocyte absolute count, cells were stained with BD TriTEST monoclonal antibodies using BD TruCOUNT tubes. Determination of T CD4⁺ and T CD8⁺ cell counts was performed in a FACSCalibur (BD-Biosciences). The spontaneous release of interleukin (IL)-2, IL-4, IL-6, IL-10, and γ-interferon (IFN-γ) was evaluated in cultured peripheral blood mononuclear cells (PBMCs) isolated from patients' and controls' blood, through gradient density centrifugation. Cells were adjusted to 5×10⁵/ml in RPMI-1640 containing 100 U penicillin G and 10 μ/ml of streptomycin supplemented with 10% fetal calf serum. All cultures were kept at 37°C in 5% CO₂ for 72 h, without stimulus, and cytokines were measured from the supernatants using commercial ELISA kits according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). For all cytokines, assays were performed in duplicate.

Statistical analysis was performed using SPSS version 17. For normally distributed variables, Student's t test or one-way ANOVA was used. For no normally distributed variables, Kruskal–Wallis or Mann–Whitney tests were applied. Statistical significance was considered when p≤0.05.

Viral load was similar in the HIV group and HIV/HTLV group (mean±SD, 2.3±1.8 log and 1.6±0.0 log, respectively; p=0.9). Patients using antiretroviral therapy were 48.5% (n=16) in the HIV group, and 63.2% (n=12) in the HIV/HTLV group. Viral load was below 50 copies of HIV-1 RNA/ml of plasma in 33.3% (n=11) of the HIV group and in 52.6% (n=10) of the HIV/HTLV group (p=0.08). The HIV/HTLV group showed an inversion in the T CD4⁺/T CD8⁺ ratio as observed for the HIV group (0.5±0.3 and 0.7±0.6). The mean CD4⁺ T cells counts were 410±237 (HIV group), 878±464 (HTLV group), 514±312 (HIV/HTLV group), and 944±233 (control) and the CD8⁺ T cell count was 773±355 (HIV group), 447±291 (HTLV group), 1003±390 (HIV/HTLV group), and 482±149 (control); the differences among the groups were statistically significant (p=0.01 for CD4⁺ and for CD8⁺ T cell counts).

We observed that IL-2 production was enhanced in the HTLV (median and interquartile range=14.3; 3.7–15.6 pg/ml) and HIV/HTLV groups (18.0; 8.9–46.5 pg/ml) when compared to the control (7.8; 7.2–8.8 pg/ml) or HIV group (5.5; 2.6–7.8 pg/ml) and differences were statistically significant (p=0.009 for the HIV×HTLV groups and p<0.001 for the HIV×HIV/HTLV groups). IL-2 levels in the HIV group (5.5; 2.6–7.8 pg/ml) were lower than in the control group (7.8; 7.2–8.8 pg/ml; p=0.004). We could verify that IL-2 production was significantly higher in the HIV/HTLV (38.7±67.1 pg/ml) group compared to the HTLV group (14.3; 3.7–15.6 pg/ml; p=0.007). For IFN-γ we observed similar results, with a significantly higher production of that cytokine in the HIV/HTLV group (11.0; 8.4–23.0 pg/ml) compared to the HIV (5.6; 4.9–8.0 pg/ml, p<0.001) and control groups (5.8; 1.8–8.2 pg/ml, p<0.001). The pattern of IL-4 and IL-6 production was similar for the HIV and HIV/HTLV groups (23.3; 11.9–31.2×20.3; 12.2–30.9 pg/ml, for IL-4 and 25.5; 21.9–36.3×23.4; 14.3–35.9 pg/ml for IL-6), but significantly lower for the HTLV singly infected group (8.8; 7.8–18.2 pg/ml for IL-4 and 15.3; 14.2–28.4 for IL-6) in comparison to the HIV/HTLV group (p=0.03 for both cytokines). We also evaluated IL-10 release by PBMCs and all infected groups presented a higher IL-10 production compared to controls (p=0.006). Table 1 summarizes these findings.

Table 1.

Spontaneous Cytokines Release by Cultured Peripheral Blood Mononuclear Cells from Patients and Controls, After 72 h

	Groups
Cytokines (pg/ml)	HIV-1 (n=33)	HTLV-1 (n=15)	HIV-1/HTLV-1 (n=19)	Control (n=13)	p value ^a
IL-2	5.5 (2.6–7.8)^b	14.3 (3.7–15.6)	18.0 (8.9–46.5)	7.8 (7.2–8.8)	<0.001
IFN-γ	5.6 (4.9–8.0)	10.2 (8.2–16.0)	11.0 (8.4–23.0)	5.8 (1.8–8.2)	<0.001
IL-4	23.3 (11.9–31.2)	8.8 (7.8–18.2)	20.3 (12.2–30.9)	22.2 (6.5–27.3)	0.160
IL-6	25.5 (21.9–36.3)	15.3 (14.2–28.4)	23.4 (14.3–35.9)	36.4 (19.6–48.8)	0.213
IL-10	2000.0 (719.3–2000.0)	2000.0 (646.0–2000.0)	2000.0 (300.2–2000.0)	40.4 (31.2–763.4)	<0.001

p values refer to HIV group compared to HIV/HTLV group.

Results are shown as median and interquartile range (IQR).

HTLV-1, human lymphotropic virus type 1; IL, interleukin; IFN, interferon.

HIV-1/HTLV-1 coinfection can be observed in some areas where these viruses are prevalent,^5,6 but little is known about the impact of coinfection on the natural history of HIV-1 infection. Some authors have observed that HIV-1 viral load was not affected by coinfection with HTLV-1, but they could verify that coinfection resulted in an increased CD4⁺ T cell count among coinfected patients in the context of AIDS-defining diseases.^11,12 The expected consequence of such an “artificial” increase in CD4⁺ T cells count is a delay in introducing antiretroviral therapy for coinfected subjets.¹³ In addition, indirect evidence of the functional disturbance of such cell population is the presence of opportunistic infection in patients with a high CD4⁺ T cell count.¹⁴ In this study we have observed no differences in viral load among the HIV and HIV/HTLV groups, in agreement with previous observations.^11,12 However, as the patients enrolled in our study were asymptomatic at the time of sample collection, no differences in CD4⁺ T cell counts were observed among HIV-1-infected individuals and HIV-1/HTLV-1-coinfected ones.

We also have evaluated the spontaneous release of IL-2, IL-4, IL-6, IL-10, and IFN-γ by cultured PBMCs from individuals infected by HIV-1, or HTLV-1, or by both viruses. We have observed a decrease in the synthesis of IL-2, and IFN-γ, and an increase in IL-10 production in the HIV-1 singly infected group. These results are in accordance with previous observations reporting that HIV-1 infection alters the synthesis of many cytokines, leading to a predominant Th-2 immune response.¹⁵ On the other hand, HTLV-1 infection is associated with an increase in type 1 cytokines production (as IL-2, and IFN-γ), which can be observed even in asymptomatic HTLV-1-infected subjects.^9,10 Our results show that the HTLV-1 singly infected group presented an increase in IL-2, IL-10, and IFN-γ levels as previously described.^9,10 We observed that the coinfected group presented an increased release of IL-2 and IFN-γ, showing a pattern of immune response closer to the HTLV-1 singly infected group than to that observed for the HIV-1 singly infected group, with a predominant type 1 (Th1) response.

In a previous study we observed that coinfected individuals presented a shorter survival life time compared to HIV-1 singly infected subjects.⁷ This suggests that despite the predominance of a type 1 immune response, in vitro, immune cells from coinfected individuals are dysfunctional. In fact, it was shown that CD4⁺ T cells from HIV-1/HTLV-1-coinfected individuals present higher levels of activation markers.¹⁶ Studies on the functionality of the T CD4⁺ and T CD8⁺ cells from coinfected subjects are warranted to clarify the complex factors involved in the immune response of HIV-1/HTLV-1-coinfected patients.

The real impact of coinfection by HIV-1 and HTLV-1 is still controversial. While some authors detected a negative effect of HTLV-1 on AIDS progression,⁷ several points remain without a clear response. Recent studies published by our group suggest that coinfected children present similar findings to those observed in adults, in terms of survival, CD4⁺ T cells increase, and presence of clinical symptoms.¹⁷ This evidence reinforces the fact that coinfection can modify the natural course of AIDS, due to a differential immunomodulation, to a delay in introducing antiretroviral therapy (as a consequence of higher CD4⁺ cells count), or another unidentified cause. The present work provides additional support for the hypothesis that coinfection can modify AIDS progression. We believe screening for HTLV-1 infection in HIV-1-infected individuals is mandatory in areas where these agents are prevalent. Careful follow-up of coinfected individuals is highly recommended to identify potential changes in the natural course of both infections.

Footnotes

Acknowledgments

This work was supported by Conselho Nacional de Pesquisa [Edital Universal 476504/2006-6]. We thank Alex Torres for performing the T lymphocytes count and André Ramos for performing viral load determinations.

Author Disclosure Statement

No competing financial interests exist.

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