Clinical and immunological features of patients with atopy and concomitant HTLV-1 infection

Human T-cell lymphotropic virus type 1 (HTLV-1) induces an exacerbated type 1 immune response characterized by high spontaneous IFN-γ and TNF-α production. Allergic rhinitis and asthma are associated with the type 2 immune response, with elevated secretion of IL-4 and IL-5. The aim of this study was to characterize the immune response in atopic HTLV-1 carriers. The cytokine profile of atopic HTLV-1 carriers (N = 10; all females) was compared with that of non-atopic HTLV-1 carriers (N = 14; 9 females and 5 males). Mean patient age of atopic and non-atopic groups was 45 ± 8 and 38 ± 11 years, respectively. All atopic HTLV-1 carriers had rhinitis with or without asthma and a skin prick test positive for Dermatophagoides pteronyssinus antigen 1 (Derp-1). There was no difference in cytokine levels between the two groups in unstimulated peripheral blood mononuclear cell cultures. In cultures stimulated with Derp-1, IFN-γ levels tended to be higher (P = 0.06) and IL-5 levels were higher (P = 0.02) in atopic HTLV-1 patients than in non-atopic subjects. In contrast, IL-10 was lower (P = 0.004) in atopic than in non-atopic HTLV-1-infected subjects. This study shows that HTLV-1 infection with an exaggerated type 1 immune response does not prevent atopy. In this case, the exacerbated type 1 and type 2 immune responses were due to a lack of IL-10 production, a cytokine that plays an important role in down-modulating type 1 and type 2 immune responses and in preventing the development of chronic inflammatory diseases.


Introduction
Human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that predominantly infects CD4 + and CD8 + T cells, inducing high spontaneous production of Th1 cytokines, including tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ) and interleukin 2 (IL-2).A large proportion of HTLV-1-infected subjects are asymptomatic, but morbidity associated with the infection has been well documented.In addition to adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), patients may present an overactive bladder, sicca syndrome, artropathy, uveitis, and periodontal disease (1)(2)(3).Additionally, HTLV-1 infection may influence the immune response to other pathogens, increasing susceptibility and modifying the clinical manifestation of strongyloidiasis (4,5), tuberculosis (6,7), schistosomiasis (8), and scabies (9).The pathology related to HTLV-1 infection is associated with an exaggerated and inappropriately modulated immune response, and a high proviral load (10).The mechanisms related to the ability of HTLV-1 to increase susceptibility or influence the clinical course of other diseases are not fully understood.Regarding

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helminth infections, it is known that the defense mechanism is associated with the type 2 immune response.In patients co-infected with HTLV-1 and strongyloidiasis or HTLV-1 and schistosomiasis there is a reduction in IL-4, IL-5 and IL-13 production in cultures stimulated with parasite antigens (11,12).Moreover, there is an inverse correlation between IFN-γ and IL-4, and between IFN-γ and parasite-specific IgE production (13).Therefore, in such cases, it is clear that the exaggerated type 1 immune response observed in HTLV-1 infection decreases the helminth-driven Th2 response.Asthma and rhinitis are common chronic allergic (atopic) diseases, and, similar to helminth infection, are associated with a strong type 2 immune response, accompanied by the production of high levels of IL-4, IL-5, IL-13, increased numbers of eosinophils, and increased IgE synthesis (14)(15)(16).We have shown previously that the prevalence of atopy and allergic diseases is lower in HTLV-1-infected subjects than in uninfected individuals (17), and that atopy and HTLV-1 infection may co-exist (18).The objective of the present study was to characterize the immune response in patients with HTLV-1 and concomitant atopy, and to determine whether HTLV-1 infection influences the immune response to Dermatophagoides pteronyssinus (Derp) antigen, the most common aeroallergen among atopic patients in Brazil.

Patients
Participants were identified at the HTLV-1 Multidisciplinary Clinic of Hospital Universitário Prof. Edgard Santos (HUPES), Salvador, Bahia, Northwestern Brazil.All patients had positive serology for HTLV-1 determined by an enzymelinked immunosorbent assay (ELISA, Cambridge Biotech Corporation, USA), and confirmed by Western blot (Genelabs, Singapore).Additionally, all sera were negative for HIV, syphilis, hepatitis B, and hepatitis C. A complete neurological exam was performed to determine the presence of mild manifestations of HTLV-1-related myelopathy and the Expanded Disability Status Scale (EDSS) was applied.A survey to evaluate the prevalence of atopy in a sample of asymptomatic HTLV-1 carriers admitted to the clinic was performed previously using a directed questionnaire on the basis of the International Study of Asthma and Allergies in Childhood (ISAAC) (19), and clinical examination (17).Based on this survey, 15 patients with atopy and 15 controls paired for age were selected to participate in the present study.However, at the scheduled appointment to have blood drawn for immunological studies, only 24 HTLV-1-infected subjects showed up for evaluation.All atopic individuals had a history of asthma or chronic rhinitis in the preceding 12 months, and had a positive skin prick test (SPT) to at least one aeroallergen.Each atopic and non-atopic recruited volunteer had at least three stool samples negative for intestinal parasites.

Skin prick test
The SPT was performed using a panel with nine relevant local allergens: D. pteronyssinus, D. farinae, Blomia tropicalis, Aspergillus fumigatus, Cladosporum herbarum, Periplaneta americana, Blattella germanica, and dog and cat epithelia (Alk Abello, Denmark), according to a standardized technique (17).Histamine (1:1000) and saline were used as positive and negative controls, respectively.To avoid possible circadian differences in skin reactivity, skin tests were done between 9:00 and 11:00 am.The size of the reaction was determined by measuring the perpendicular diameters of the papule and then halving the sum [(d1 + d2) / 2].The skin reaction was considered to be positive after 20 min when the mean diameter was ≥3 mm in the presence of a positive histamine reaction and in the absence of a reaction to saline control.
Patients taking antiallergic or immunosuppressive drugs, or with a clinical diagnosis of HAM/TSP were excluded.The study was approved by the Hospital Universitário Prof. Edgard Santos Medical Ethics Committee and all participants gave written informed consent to participate.

IFN-γ, IL-5 and IL-10 ELISA
ELISA sandwich techniques were used to measure cytokine levels following instructions described by the manufacturer (PharMingen, USA).Briefly, microtiter plates were coated with purified anti-human cytokines.After blockage with

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bovine serum albumin (BSA), culture supernatants or standards of recombinant cytokines were added.Plates were then incubated again with biotinylated mouse anti-human cytokines.Finally, streptavidin conjugated to horseradish peroxidase (Sigma) was added, followed by the substrate tetramethylbenzidine (Calbiochem, USA).Absorbance was measured at 450 nm using a Labsystem Multiskan ELISA reader.Data are reported as picograms per milliliter based on a standard curve generated using recombinant human cytokines.

Statistical analysis
Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) software, version 9.0 for Windows.The Mann-Whitney U-test was used to compare the distribution of independent numerical variables.The level of significance was set at P < 0.05 for all analyses.

Demographic and clinical data of atopic and non-atopic HTLV-1 patients
The age, gender, clinical status, and the response to aeroallergens of the 10 patients infected with HTLV-1 and with atopy are shown in Table 1.All patients were females, and their mean age was 45 ± 8 years.All atopic patients presented chronic rhinitis, including 3 (30%) cases with concomitant asthma, and 1 (10%) patient who also had eczema.All atopic patients were SPT-positive to D. pteronyssinus antigen (100%), 9 patients responded to D. farinae (90%), and 6 to B. tropicalis (60%).Only 2 patients were positive to B. germanica, 1 patient to A. fumigatus and 1 to cat epithelium (data not shown).No patient presented cutaneous hyperreactivity to C. herbarum, P. americana or dog epithelium antigen.The control group had 14 HTLV-1 carriers (9 females and 5 males) with a mean age of 38 ± 11 years (range: 22 to 55 years).They denied a previous history of atopy, and in all of them the SPT was negative for the allergens tested.

Cytokine profile produced by PBMC of atopic and non-atopic HTLV-1 carriers
The levels of IFN-γ, IL-5 and IL-10 in unstimulated cell cultures of patients with HTLV-1 with (N = 10) and without atopy (N = 14) are shown in Figure 1.High levels of IFN-γ were detected in the supernatants of unstimulated PBMC cultures of HTLV-1-infected patients with or without atopy.There was no statistically significant difference between HTLV-1 patients, and in both groups the production of this cytokine was quite variable, with a median value of 1295 pg/mL, ranging from 34 to 3189 pg/mL for the atopic group, and a median of 450 pg/mL, ranging from 0 to 3454 pg/mL, for the non-atopic group (P = 0.1, Mann-Whitney U-test).There was no significant difference between the levels of IL-5 in unstimulated supernatants of PBMC from atopic patients with HTLV-1 (median: 0 pg/mL; range: 0 to 189 pg/mL) and those from nonatopic patients with HTLV-1 (median: 0 pg/mL; range: 0 to 659 pg/mL), P = 0.7.IL-10 production did not differ between groups, with a median of 9 pg/mL (range: 0 to 273 pg/mL) for atopic HTLV-1, and 30 pg/mL (range: 0 to 357 pg/mL) for non-atopic HTLV-1, P = 0.2.These data show no significant difference in the levels of cytokines in unstimulated cultures between the two groups of patients.
The cytokine levels in supernatants of Derp-1-stimulated PBMC are shown in Figure 2.There was a tendency to higher levels of IFN-γ in atopic HTLV-1-infected subjects compared to non-atopic HTLV-1-infected controls (P = 0.06).The median value of IFN-γ was 1625 pg/mL for atopic patients, with values ranging from 206 to 3840 pg/mL, and 582 pg/mL for non-atopic HTLV-1 carriers, with values ranging from 37 to 3163 pg/mL.

Discussion
In the present study, we showed that the exaggerated type 1 immune response observed in HTLV-1 infection does not prevent the occurrence of asthma and rhinitis.Moreover, the characterization of the immune response in individuals with concomitant atopy and HTLV-1 infection highlights the crucial role of IL-10 in down-modulating the immune response.We documented that IFN-γ and IL-5 levels were higher in Derp-1-stimulated cultures of patients with atopy and HTLV-1 infection compared to HTLV-1 carriers with no atopy, and that the concentration of IL-10 was decreased in patients with atopy and HTLV-1 infection.

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We have previously published a case report regarding a patient who had severe asthma and HTLV-1 infection, but we later showed that the prevalence of atopy based on the positivity of SPT and clinical manifestations was lower in HTLV-1-infected subjects compared to uninfected controls (17,18).Although we cannot rule out the possibility that the exacerbated Th1 immune response observed in HTLV-1 infection contributed to the decreased prevalence of atopy determined by the SPT, it is possible that other factors might be involved in reducing the skin test response to aeroallergens in HTLV-1-infected subjects.For instance, the response to the prick test with histamine, used as a positive control, was lower among HTLV-1 carriers (17).Interestingly, other investigators have also found that the test for delayed type hypersensitivity to antigens, a reaction associated with a type 1 immune response, also shows a decreased response by HTLV-1-infected subjects.In such a case, it is more likely that HTLV-1 infection may impair cell migration to the skin, or that the function of the cells responsible for these reactions is down-regulated by unknown mechanisms.The data presented here clearly show that, in patients with HTLV-1 and asthma and rhinitis, Th1 and Th2 responses may not only co-exist, but both of them are enhanced, arguing against the Th1 vs Th2 paradigm in humans.Moreover, all patients studied had positive SPT to at least one allergen, and most of them responded to more than one antigen.
In contrast to the observation made in patients co-infected with HTLV-1 and helminths, in whom HTLV-1 infection decreases type 2 cytokines and IgE in the presence of strongyloidiasis and schistosomiasis (12,13,20), and the observation that helminths decrease IFN-γ production in HTLV-1 infection (21), we did not observe these effects in the present study.Although both helminthic infection and asthma are diseases associated with a type 2 immune response, in helminthic infection there is an increase in regulatory T cells and IL-10 production ( 16).In the present study, IL-10 production was lower in patients with atopy and HTLV-1 infection.
It is known that IL-10 is an essential regulatory cytokine in humans that possibly has an important role in the maintenance of the asymptomatic carrier status in HTLV-1-infected subjects, counterbalancing the pro-inflammatory cytokines IFN-γ and TNF-α (22).Previous studies have shown that the production of IL-10 is reduced in ordinary atopic patients (14)(15)(16).IL-10 is able to inhibit both type 1 and type 2 immune responses through a suppressive effect on macrophages, T-cell proliferation and pro-inflammatory cytokine production (23)(24)(25)(26).Inhibition of T cells by IL-10 has been shown to be related to down-modulation of antigen-presenting cells (27).The down-modulation of the immune response mediated by IL-10 in HTLV-1 carriers suggests that this cytokine may directly modulate T-cell activity because T cells from HTLV-1-infected individuals proliferate and produce cytokines independent of accessory cells (28).It is interesting that IL-10 is usually produced at high levels in HTLV-1 carriers (29) and possibly has an impact on the maintenance of asymptomatic carrier status by counterbalancing the pro-inflammatory cytokines (22).Furthermore, it is known that IL-10 is produced at low levels in asthma patients (14).Thus, the lack of modulation by IL-10 may lead to an increase in both type 1 and type 2 cytokines, allowing the occurrence of atopy in HTLV-1-infected individuals.
The present study shows that atopy may occur in a typical Th1 environment.This is associated with a reduction in IL-10 levels, an important immunoregulatory cytokine that plays a major role in modulating type 1 and type 2 immune responses.