Chikungunya is an acute self‐limiting disease characterized by a number of symptoms, including acute inflammation accompanied mainly by fever, muscle pain, skin rash, myalgia and arthralgia, which usually decrease after 7–10 days.1, 2 In some individuals, this illness can develop into chronic disease.3 It occurs when infected individuals experience an incapacitating arthralgia, which often includes signs of joint inflammation and tenosynovitis, that persists for months or years after the acute phase.4, 5, 6
Acute chikungunya virus (CHIKV) infection generates a strong immune response that is capable of eliminating circulating infectious virus, but the immunobiology of chronic disease is still not well understood.3, 7 T and B lymphocytes and dendritic cells derived from monocytes are not susceptible to CHIKV infection, and their role in killing infected cells or possibly in the pathogenesis of the disease remains to be established.8, 9
CD8 T cells have an important role in virus infection and some authors have shown that CD8+ T cells are involved early in CHIKV infection in humans10 and have been detected in blood samples > 7 weeks after infection.11 However, investigations of CD8+ T cells’ role in mechanisms regarding chikungunya disease (CHIKD) and rheumatic disorders have been limited, mainly by the difficulty of establishing relevant and accessible models to study the disease.
Here, we evaluate the expression of cytotoxic molecules and cytokines in CD8+ T cells in individuals infected by CHIKV with acute and chronic disease. Apoptosis markers were also included to better characterize the expression profile of CD8+ T cells. Our results demonstrate that CD8+ T cells have higher cytotoxic molecule expression, mainly in individuals with acute CHIKD. Analysis of cytokines and apoptotic molecules has also demonstrated that some of these molecules are expressed in an increased manner in both phases of the disease. Our data provide for the first time new insights into the functional competence of CD8+ T cells and suggest that modulation of the expression profile in CD8+ T cells differs between the acute and chronic phases of CHIKD.
MATERIALS AND METHODS
Blood samples from patients infected with CHIKV were obtained from the Lauro Wanderley University Hospital, in the city of João Pessoa‐Brazil. Infected patients were assigned to acute or chronic groups based on the observation of clinical symptoms of CHIKV infection, such as polyarthralgia, and associated symptoms (high fever, rash, swelling), to enable the evaluation of the long‐term and short‐term mechanisms involved in the pathophysiology of CHIKV infection. Clinical and biological examinations and blood sampling were performed in patients with symptoms ranging from 2 to 7 days (acute phase) and from 6 to 12 months (chronic phase). Informed consent was obtained from patients with CHIKV infection and from healthy volunteers (hospital staff) from the same local medical hospital. We recruited acutely and chronically infected individuals who were positive for CHIKV on polymerase chain reaction. Acute group (n = 4), chronic group (n = 6) and healthy individuals (n = 6) were evaluated. Viral RNA extractions were obtained from whole blood or serum samples using the QIAmp Viral Mini Kit (Qiagen Inc., Valencia, Spain) following the manufacturer's predetermined protocol. RNA was analysed by quantitative polymerase chain reaction to detect CHIKV.12 All samples were kept at −80° until the study was started. This study was approved by the National Commission of Ethics in Research (Certificate number CAAE: 59833416.6.0000.5183).
Whole blood was collected from individuals and processed immediately for ex vivo analyses by flow cytometry. Leucocytes obtained from erythrocyte lysis (BD Biosciences, San Jose, CA, USA) were then stained with extracellular anti‐CD8, anti‐CD69, anti‐interleukin‐10 receptor (IL‐10R), anti‐CD107A, anti‐CD95 and anti‐ CD95 ligand (CD95L) antibodies (BD Biosciences, San Jose, CA, USA). For intracellular cell markers, cells were incubated with brefeldin‐A (Sigma Aldrich, St Louis, MO, USA) for 4 hr and then stained with anti‐perforin, anti‐granzyme B, anti‐IL‐17A, anti‐IL‐10, antibodies (BD Biosciences, San Jose, CA, USA). At least 50 000 gated events were acquired for later analysis using a FACS CANTO II (BD Biosciences, San Jose, CA, USA) and analysed using flowjo software version 10.4 (TreeStar, Ashland, OR, USA). Statistical analysis was performed using graphpad prism, Inc. Software (Version 7.0; GraphPad, La Jolla, CA, USA) by Mann–Whitney test, significance was set at P < 0·05.
Percentage of CD8+ T cells was assessed (Fig. 1a) and no alteration was observed in CD8+ levels of patients with either acute or chronic CHIKV infection compared with the control group. CD69 activation marker was evaluated (Fig. 2a), and CD8+ T cells of acute and chronic patients showed an increased expression of this marker in relation to the control individuals, demonstrating activation of these lymphocytes upon CHIKV infection. While evaluating the exocytosis mechanism of CD8+ T‐cell cytotoxic proteins, the CD107A expression was found to be up‐regulated (Fig. 1b) in patients with the acute disease compared with control individuals; this is an important molecule in the cytotoxic synapse that coordinates the exocytosis of granzyme B. Similarly, there was an increase in granzyme B expression (Fig. 1c) in patients with acute and chronic disease and an increase in the expression of perforin (Fig. 1d) in patients with acute disease in relation to healthy individuals.
Percentage of CD8+ T cells and frequencies of CD8+ T cells expressing CD107A, granzyme B (GZMB) and perforin (PRF1) from patients with acute and chronic chikungunya disease (CHIKD). Percentage of CD8+ T cells (a), CD107A (b), granzyme B (c) and perforin (d) from healthy individuals (white bars), acute phase patients (black bars) and chronic phase patients (striped bars) was measured ex vivo. Bars represent the mean ± standard error for each group. Means were compared using Student's t‐test and differences were considered significant when P < 0·05. *P < 0·05; **P < 0·005.
Higher frequencies of CD8+ T cells expressing CD69, Interleukin‐17A (IL‐17A), IL‐10 and IL‐10R are found in patients with acute and chronic chikungunya disease (CHIKD). Percentage of CD69 (a), IL‐17A (b), IL‐10 (c) and IL‐10R (d) from healthy individuals (white bars), acute phase patients (black bars) and chronic phase patients (striped bars) was measured ex vivo. Bars represent the mean ± standard error for each group. Means were compared using Student's t‐test and differences were considered significant when P < 0·05. *P < 0·05, **P < 0·005 and ***P < 0·0001.
The levels of IL‐17A expression (Fig. 2b) were increased in acute CHIKD, and this phenomenon was sustained during the chronic phase, with high expression of this molecule compared with healthy individuals; however, there was a decrease in comparison with the acute phase. Expression of IL‐10 was increased in patients with CHIKV infection, in both acute and chronic phases. Regarding IL‐10R, lower expressions levels were demonstrated in patients with acute CHIKD compared with controls. The cell death induction pathway, CD95/CD95L, on CD8+ T cells was also investigated (Fig. 3); there was increased expression of CD95 in the chronic phase, increased expression of CD95L in both acute and chronic phases, and an increase in the co‐expression of CD95/CD95L in both the acute and chronic phases of CHIKV infection.
Higher frequencies of CD8+ T cells expressing CD95, CD95 ligand (CD95L) and co‐expression of CD95/CD95L are found in patients with acute and chronic chikungunya disease (CHIKD). Percentage of CD95 (a), CD95L (b), CD95/CD95L (c) from healthy individuals (white bars), acute phase patients (black bars) and chronic phase patients (striped bars) was measured ex vivo. Bars represent the mean ± standard error for each group. Means were compared using Student's t‐test and differences were considered significant when P < 0·05. *P < 0·05 and **P < 0·005.
CD8+ T cells in particular are responsible for killing virus‐infected cells by inducing cytolytic mechanisms as well as for producing a variety of antiviral factors;13 however, the characterization of immunological mechanisms involved in CHIKD is not yet well known.7 Activation of naive CD8+ T cells induces a development programme that stimulates effector and memory cell proliferation and differentiation.1, 2 Effector CD8+ T cells exhibit activated functions, such as cytotoxicity and cytokine production, that favour elimination of virus.7 Although no difference was observed in the percentage of CD8+ T cells in peripheral blood of acute and chronic patients in relation to healthy individuals (Fig. 1a), analysis of the CD69 expression, an early activation marker for CD8+ T cells, demonstrated an increased CD8+ T‐cell activation in both acute and chronic disease (Fig. 2a), corroborating previous results observed in humans in the acute phase of infection.10 However, CD8+ T‐cell activation drops considerably in patients with chronic disease when compared with acute disease, even though activation is still higher than in the control group, demonstrating that these cells may still participate in the immune response during the chronic phase of the disease.
There are two major pathways by which CD8+ T lymphocytes mediate cytolytic activity against target cells: release of cytolytic granules through exocytosis and the granule‐independent pathway, which consists of binding to death receptors in target cells.14 Secretory effector lysosomes are characterized by a specific set of membrane and luminal protein markers, including CD107. We demonstrated that there was an increase in CD107A, granzyme and perforin (Fig. 1b,c,d) expression in CD8+ T lymphocytes in patients with acute CHIKD when compared with controls. These results allow us to suggest that CD8+ T cells mediate cytolytic activity against CHIKV‐infected cells in these patients. However, patients with chronic disease did not present increased expression of these proteins compared with healthy controls, demonstrating that these mechanisms are not maintained in later stages of the disease.
Chikungunya virus was capable of activating the two CD8+ T‐lymphocyte activation pathways as revealed by investigation of the death receptors. The expression of CD95L and the CD95/CD95L co‐expression was observed in both phases of the disease, demonstrating their importance in the elimination of virus during the acute and chronic phases (Fig. 3). As CD8+ T cells only express CD95L after activation,15 we can propose that these cells remain activated even in the chronic phase of the disease, so they may also be involved in CHIKD's chronicity mechanism. Strong immune cell activation drives the initial antiviral response and may lead to T‐cell exhaustion, and this has been linked subsequently to the viral persistence observed in the chronic phase.16
In an animal model infected with CHIKV, CD4+ T and CD8+ T lymphocytes were found in infiltrate from inflamed joints, whereas other animal studies have proposed that CD4+, but not CD8+, T lymphocytes, perform a significant role in the severity of joint inflammation.3 Others demonstrated that the absence of CD8+ T cells in synovial tissue may contribute to the persistence of CHIKV.16 CD8+ T cells play important roles against viral infection, and our results suggest that these cells have an immunomodulatory function in the systemic circulation of CHIKV.
It is well established that just like CD8+ T cells, other immune cells can synthesize and produce IL‐17A cytokine. The secretion of IL‐17 by CD8+ T cells has been described in several human inflammatory illnesses.17, 18 This cytokine has been a relevant target of many studies involving inflammatory diseases, like rheumatic and joint inflammation.19 In this study, we observed that CD8+ T lymphocytes from patients in the acute phase of disease had a significant increase of IL‐17A cytokine (Fig. 2b) production in relation to the control and chronic groups. Patients in the chronic phase, who are still afflicted with the arthritogenic symptoms, also presented high levels of IL‐17A (Fig. 2b) in relation to the control. These data suggest that CD8+ IL‐17‐producing T cells may be directly involved in joint symptoms and contribute to the establishment of CHIKV‐induced arthritis, as well as in the persistence of this condition in patients who are chronically affected.
Interleukin‐10 is a regulatory cytokine that has anti‐inflammatory properties and develops an important role in controlling host immune responses to pathogens. It acts by preventing the damage caused by an exacerbated inflammatory response and by maintaining tissue homeostasis. Immunopathologies in response to infections and the development of autoimmune diseases are associated with dysregulation of IL‐10.18 Increased IL‐10 expression by CD8+ T cells was observed in the acute and chronic phases of CHIKD (Fig. 2c), which can be explained by a mechanism of compensation for the high activation profile of CD8+ T cells. On the other hand, the expression of IL‐10R (Fig. 2d) was decreased in the acute phase when compared with the control group. Persistence of antigen and viral RNA in synovial tissue was demonstrated in patients with chronic arthralgia, and viral persistence was associated with the expression of interferon‐α, IL‐10 and CCL2.16 However, the viral persistence was not associated with inflammatory cytokines, such as interferon‐γ, tumour necrosis factor‐α and IL‐1β.16 For the establishment of chronic CHIKD, silencing mechanisms of the inflammatory response appear to be involved. Down‐regulation in inflammation during the acute and recovery phases or absence of anti‐inflammatory responses play major roles in the development of chronic arthralgia or arthritis in these patients.3 The decrease of IL‐10R expression in acute CHIKD CD8+ T cells suggests an anti‐inflammation silencing mechanism, which may promote the development of chronic arthralgia, as the immunomodulatory effect of IL‐10 starts with its binding to IL‐10R.20
All these results are important in the field of T‐cell immunity of human CHIKV infection. However, other studies are needed to aid in the elucidation of how gender and aging may influence the immune response in infectious diseases. A recent review showed important points related to several age‐dependent differences that could influence disease severity and host recovery in alphavirus infection.21
In summary, our data demonstrate for the first time the role, and putative mechanisms, of CD8+ T lymphocytes from CHIKV‐infected patients in the acute and chronic phases of infection. Expression of markers demonstrated that the cytolytic exocytosis mechanism is activated in this pathology, indicating the ability of these cells to eliminate CHIKV. The decrease of IL‐10R may allow viral persistence and IL‐17A may be the key cytokine in the development of joint symptoms in the acute phase of infection and in the maintenance of these symptoms in the chronic phase. Our data confirm that acute CHIKV infection generates a strong immune response and suggest the important role of the CD8+ T lymphocyte towards virus clearance in the circulation, mainly by mechanism of activation and cytotoxicity. In addition, these lymphocytes appear to participate in the clinical conditions observed in the disease through the production of IL‐17A, the key cytokine found in arthritis.19
CONFLICT OF INTEREST
All authors declare that there are no conflicts of interest to declare.
We would like to thank to Isabel Sarmento and Anna Stella Pachá from Paraiba`s Health Department.
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