The Role of NK Cells in the Control of Viral Infection in HTLV-1 Carriers.

The cytotoxic activities of CD8+ T cells have been considered the main defense mechanism against the human T lymphotropic virus type 1 (HTLV-1). As with CD8+ T cells, NK cells can perform cytotoxic degranulation with production of cytotoxic mediators, such as perforins and granzymes. NK cells are also responsible for antibody-dependent cellular cytotoxicity (ADCC) against infected cells, but few studies have evaluated the role of NK cells in HTLV-1 infection. The aim of this study was to characterize the subsets and measure the frequency of NK cells in HTLV-1 carriers (HC) and in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and correlate these findings with the proviral load and development of HAM/TSP. The diagnosis of HTLV-1 infection was performed with a detection antibody against viral antigens by ELISA and confirmed by Western blot. Phenotypic characterization of NK cells was performed by flow cytometry. The frequencies of CD56+, CD56+CD3-, CD56+CD16+, and CD56dim cells were decreased in HAM/TSP patients. The frequency of CD56+CD3- cells was inversely correlated with proviral load in HC but not in HAM/TSP patients. HAM/TSP patients showed decreased frequency of CD56+ and CD56dim cells expressing CD16, the main receptor for ADCC. These data indicate that NK cells may play a key role in the control of HTLV-1 infection by preventing the progression of HC to HAM/TSP.

Impaired TNF, IL-1ß, and IL-17 production and increased susceptibility to Mycobacterium tuberculosis infection in HTLV-1 infected individuals.

IFN-γ and TNF play critical roles in the control of Mycobacterium tuberculosis infection. Despite leading to an exaggerated production of inflammatory cytokines, HTLV-1 infection increases the risk of developing tuberculosis (TB). However, the immune mechanisms accounting for this phenomenon are still unclear. The aim of this study was to evaluate immunological aspects of the HTLV-1/M. tuberculosis co-infection. In this cross-sectional study, the levels of TNF, IL-1ß, and IL-17 were determined by ELISA in the supernatants of either unstimulated or tuberculin purified protein derivative (PPD) stimulated peripheral blood mononuclear cells. Cells from HTLV-1 infected individuals produced lower levels of TNF following PPD stimulation compared to unstimulated cells. IL-1ß and IL-17 production by cells from HTLV-1/M. tuberculosis co-infected individuals was lower than in cells from patients with TB. Impairment in TNF, IL-1ß, and IL-17 production upon stimulation with mycobacterial antigens may contribute to the increased susceptibility to M. tuberculosis infection observed in HTLV-1 infected individuals.

Functional activity of monocytes and macrophages in HTLV-1 infected subjects.

The Human T lymphotropic virus type-1 (HTLV-1) infects predominantly T cells, inducing proliferation and lymphocyte activation. Additionally, HTLV-1 infected subjects are more susceptible to other infections caused by other intracellular agents. Monocytes/macrophages are important cells in the defense against intracellular pathogens. Our aims were to determine the frequency of monocytes subsets, expression of co-stimulatory molecules in these cells and to evaluate microbicidal ability and cytokine and chemokine production by macrophages from HTLV-1 infected subjects. Participants were 23 HTLV-1 carriers (HC), 22 HAM/TSP patients and 22 healthy subjects (HS) not infected with HTLV-1. The frequencies of monocyte subsets and expression of co-stimulatory molecules were determined by flow cytometry. Macrophages were infected with L. braziliensis or stimulated with LPS. Microbicidal activity of macrophages was determined by optic microscopy. Cytokines/chemokines from macrophage supernatants were measured by ELISA. HAM/TSP patients showed an increase frequency of intermediate monocytes, but expression of co-stimulatory molecules was similar between the groups. Macrophages from HTLV-1 infected individuals were infected with L. braziliensis at the same ratio than macrophages from HS, and all the groups had the same ability to kill Leishmania parasites. However, macrophages from HTLV-1 infected subjects produced more CXCL9 and CCL5, and less IL-10 than cells from HS. While there was no correlation between IFN-γ and cytokine/chemokine production by macrophages, there was a correlation between proviral load and TNF and CXCL10. These data showed a dissociation between the inflammatory response and microbicidal ability of macrophages from HTLV-1 infected subjects. While macrophages ability to kill an intracellular pathogen did not differ among HTLV-1 infected subjects, these cells secreted high amount of chemokines even in unstimulated cultures. Moreover the increasing inflammatory activity of macrophages was similar in HAM/TSP patients and HC and it was related to HTLV-1 proviral load rather than the high IFN-γ production observed in these subjects.

Influence of HTLV-1 on the clinical, microbiologic and immunologic presentation of tuberculosis.

BACKGROUND: HTLV-1 is associated with increased susceptibility to Mycobacterium tuberculosis infection and severity of tuberculosis. Although previous studies have shown that HTLV-1 infected individuals have a low frequency of positive tuberculin skin test (TST) and decreasing in lymphoproliferative responses compared to HTLV-1 uninfected persons, these studies were not performed in individuals with history of tuberculosis or evidence of M. tuberculosis infection. Therefore the reasons why HTLV-1 infection increases susceptibility to infection and severity of tuberculosis are not understood.The aim of this study was to evaluate how HTLV-1 may influence the clinical, bacteriologic and immunologic presentation of tuberculosis. METHODS: The study prospectively enrolled and followed 13 new cases of tuberculosis associated with HTLV-1 (cases) and 25 patients with tuberculosis without HTLV-1 infection (controls). Clinical findings, bacterial load in the sputum, x-rays, immunological response and death were compared in the two groups. RESULTS: There were no differences in the demographic, clinical and TST response between the two study groups. IFN-γ and TNF-α production was higher in unstimulated cultures of mononuclear cells of case than in control patients (p < 0.01). While there was no difference in IFN-γ production in PPD stimulated cultures, TNF-α levels were lower in cases than in controls (p = 0.01). There was no difference in the bacterial load among the groups but sputum smear microscopy results became negative faster in cases than in controls. Death only occurred in two co-infected patients. CONCLUSION: While the increased susceptibility for tuberculosis infection in HTLV-1 infected subjects may be related to impairment in TNF-α production, the severity of tuberculosis in co-infected patients may be due to the enhancement of the Th1 inflammatory response, rather than in their decreased ability to control bacterial growth.

Evaluation of the microbicidal activity and cytokines/chemokines profile released by neutrophils from HTLV-1-infected individuals.

Human T cell lymphotropic virus type-1 (HTLV-1) induces activation and spontaneous proliferation of T cells with production of type-1 pro-inflammatory cytokines. It modifies the immune response to other antigens and increases susceptibility to infectious diseases. However, little is known about innate immunity in HTLV-1 infection. HTLV-1-infected individuals have higher spontaneous neutrophil activation than HTLV-1-seronegative individuals, as shown by the nitroblue tetrazolium (NBT) assay. This study was conducted to evaluate neutrophil function in HTLV-1-infected individuals. Participants in the study included 18 HTLV-1-infected individuals and 14 HTLV-1-seronegative controls. We evaluated the ability of neutrophils (PMNs) to control a parasite infection, to produce peroxynitrite, cytokines and chemokines and to express activation markers in cultures when stimulated with LPS or infected with Leishmania. When compared with the control group, there was no difference in the percentage of PMNs infected with Leishmania or in the number of amastigotes/100 PMNs in HTLV-1-infected individuals. The microbicidal activity of the PMNs and the levels of CXCL8 and CCL4 released by these cells did not show a difference between HTLV-1-infected individuals and the control group. In both the HTLV-1 group and the control group, infection with Leishmania or stimulation of PMNs led to cellular activation. These observations suggest that neutrophils from HTLV-1-infected individuals have preserved their ability to become activated and to produce chemokines and peroxynitrite after stimulation and that the susceptibility to infection by intracellular Leishmania amazonensis in HTLV-1-infected individuals does not depend on impairment of neutrophil function.

Modulation of T cell responses in HTLV-1 carriers and in patients with myelopathy associated with HTLV-1.

OBJECTIVE: Human T lymphotropic virus-type 1 (HTLV-1) activates the immune system leading to a persistent and exacerbated T-cell response with increased production of IFN-gamma and TNF-alpha. Overproduction of pro-inflammatory cytokines is correlated with the development of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), although some HTLV-1 carriers also show high levels of these cytokines. In this study, the ability of regulatory cytokines and cytokine antagonists to inhibit spontaneous IFN-gamma production was investigated. METHOD: IFN-gamma levels were measured by ELISA before and after addition of cytokines or anti-cytokines. RESULTS: Addition of IL-10 significantly reduced spontaneous IFN-gamma synthesis in cell cultures from HTLV-1 carriers, while no differences were observed in HAM/TSP patients. There was also a tendency to decreased IFN-gamma levels in cell cultures from HTLV-1 carriers with exogenous addition of TGF-beta. In paired analysis, neutralization of IL-2 significantly decreased IFN-gamma production in HTLV-1 carriers but not in HAM/TSP patients. Neutralization of IL-15 was less effective than neutralization of IL-2 in modulating IFN-gamma production. In HTLV-1 carriers, anti-IL-2 and simultaneous addition of anti-IL-2 and anti-IL-15 decreased IFN-gamma synthesis by 46 and 64%, respectively, whereas in patients with HAM/TSP simultaneous neutralization of both anti-cytokines only decrease IFN-gamma levels by 27%. CONCLUSIONS: Although a large proportion of HTLV-1 carriers produced high levels of pro-inflammatory cytokines similar to those observed in HAM/TSP patients, immune response can be downregulated by cytokines or cytokine antagonists in most HTLV-1 carriers. This modulation can be an important step in the prevention of tissue damage and progression from the HTLV-1 carrier state to HAM/TSP.

Association of cytokines, neurological disability, and disease duration in HAM/TSP patients.

OBJECTIVE: To identify clinical and immunological markers associated with HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP). METHOD: 237 HTLV-I infected individuals were clinically assessed. They were classified according to the Expanded Disability Status Scale (EDSS) and Osames Motor Disability Score (OMDS). Cytokine levels were determined in HTLV-I seropositive individuals. RESULTS: 37 patients had HAM/TSP. There was a correlation between the degrees of disability assessed by both scales. There was also a correlation between the duration of HAM/TSP and the severity of disability assessed by either EDSS or OMDS. Higher levels of IFN-gamma were detected in unstimulated peripheral blood mononuclear cells (PBMC) from HAM/TSP patients as compared with HTLV-I carriers. CONCLUSION: This study shows the validity of the neurological scales to classify the degree of neurological disability in HTLV-I carriers and suggests a progressive behavior of HAM/TSP. This study also shows that IFN-gamma in PBMC supernatants are markers of HAM/TSP.

Association of cytokines, neurological disability, and disease duration in HAM/TSP patients

OBEJETIVO: Identificar marcadores clínicos e imunológicos associados com a mielopatia associada ao HTLV-I/paraparesia espástica tropical (MAH/PET). MÉTODO: 237 indivíduos infectados pelo HTLV-I foram clinicamente avaliados. Eles foram classificados de acordo com a escala expandida do estado de incapacidade de Kurtzke (EDSS) e escala de incapacidade motora de Osame (OMDS). Níveis de citocinas foram determinados nos indivíduos. RESULTADOS: 37 pacientes tinham MAH/PET. Houve correlação entre os graus de incapacidade pelas escalas. Houve também correlação entre a duração da MAH/PET e o grau da incapacidade pelas escalas. Níveis elevados de IFN-g foram detectados em células mononucleares de sangue periférico (CMSP) não estimuladas de pacientes com MAH/PET quando comparados com indivíduos HTLV-I positivos assintomáticos. CONCLUSÃO: Os dados demonstram a validade das escalas neurológicas para classificar o grau de incapacidade neurológica em portadores do HTLV-I e sugerem o comportamento progressivo da MAH/PET. Este estudo também demonstra que os níveis de IFN-g em sobrenadante de CMSP são marcadores da MAH/PET.

Spontaneous neutrophil activation in HTLV-1 infected patients

Human T cell lymphotropic Virus type-1 (HTLV-1) induces lymphocyte activation and proliferation, but little is known about the innate immune response due to HTLV-1 infection. We evaluated the percentage of neutrophils that metabolize Nitroblue tetrazolium (NBT) to formazan in HTLV-1 infected subjects and the association between neutrophil activation and IFN-gamma and TNF-alpha levels. Blood was collected from 35 HTLV-1 carriers, from 8 patients with HAM/TSP (HTLV-1- associated myelopathy); 22 healthy individuals were evaluated for spontaneous and lipopolysaccharide (LPS)-stimulated neutrophil activity (reduction of NBT to formazan). The production of IFN-gamma and TNF-alpha by unstimulated mononuclear cells was determined by ELISA. Spontaneous NBT levels, as well as spontaneous IFN-gamma and TNF-alpha production, were significantly higher (p<0.001) in HTLV-1 infected subjects than in healthy individuals. A trend towards a positive correlation was noted, with increasing percentage of NBT positive neutrophils and levels of IFN-gamma. The high IFN-gamma producing HTLV-1 patient group had significantly greater NBT than healthy controls, 43±24 percent and 17±4.8 percent respectively (p< 0.001), while no significant difference was observed between healthy controls and the low IFN-gamma-producing HTLV-1 patient group (30±20 percent). Spontaneous neutrophil activation is another marker of immune perturbation resulting from HTLV-1 infection. In vivo activation of neutrophils observed in HTLV-1 infected subjects is likely to be the same process that causes spontaneous IFN-gamma production, or it may partially result from direct IFN-gamma stimulation.

Helminthic infection down-regulates type 1 immune responses in human T cell lymphotropic virus type 1 (HTLV-1) carriers and is more prevalent in HTLV-1 carriers than in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis.

Human T cell lymphotropic virus type 1 (HTLV-1) infection is associated with an exacerbated type 1 immune response and secretion of high levels of proinflammatory cytokines. In contrast, helminthic infection induces a type 2 immune response. In the present study, the cytokine profile in HTLV-1 carriers coinfected with helminths (Strongyloides stercoralis and/or Schistosoma mansoni) was compared with that in HTLV-1 carriers not coinfected with helminths. Levels of interferon (IFN)- gamma were higher in HTLV-1 carriers not coinfected with helminths than in HTLV-1 carriers coinfected with helminths (P<.05). The overall frequency of IFN- gamma -expressing CD8+ and CD4+ cells was decreased in HTLV-1 carriers coinfected with helminths (P<.05). The percentage of interleukin (IL)-5- and IL-10-expressing T cells in HTLV-1 carriers coinfected with helminths was higher than that in HTLV-1 carriers not coinfected with helminths (P<.05). Moreover, we found that the prevalence of helminthic infection was 7-fold higher in HTLV-1 carriers than in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (P<.05). These data show that helminthic infection decreases activation of type 1 cells, which may influence the clinical outcome of HTLV-1 infection.

Spontaneous neutrophil activation in HTLV-1 infected patients.

Human T cell lymphotropic Virus type-1 (HTLV-1) induces lymphocyte activation and proliferation, but little is known about the innate immune response due to HTLV-1 infection. We evaluated the percentage of neutrophils that metabolize Nitroblue tetrazolium (NBT) to formazan in HTLV-1 infected subjects and the association between neutrophil activation and IFN-gamma and TNF-alpha levels. Blood was collected from 35 HTLV-1 carriers, from 8 patients with HAM/TSP (HTLV-1- associated myelopathy); 22 healthy individuals were evaluated for spontaneous and lipopolysaccharide (LPS)-stimulated neutrophil activity (reduction of NBT to formazan). The production of IFN-gamma and TNF-alpha by unstimulated mononuclear cells was determined by ELISA. Spontaneous NBT levels, as well as spontaneous IFN-gamma and TNF-alpha production, were significantly higher (p<0.001) in HTLV-1 infected subjects than in healthy individuals. A trend towards a positive correlation was noted, with increasing percentage of NBT positive neutrophils and levels of IFN-gamma. The high IFN-gamma producing HTLV-1 patient group had significantly greater NBT than healthy controls, 43+/-24% and 17+/-4.8% respectively (p< 0.001), while no significant difference was observed between healthy controls and the low IFN-gamma-producing HTLV-1 patient group (30+/-20%). Spontaneous neutrophil activation is another marker of immune perturbation resulting from HTLV-1 infection. In vivo activation of neutrophils observed in HTLV-1 infected subjects is likely to be the same process that causes spontaneous IFN-gamma production, or it may partially result from direct IFN-gamma stimulation.