V. Kumaraswami

Cytokine control of parasite-specific anergy in human lymphatic filariasis. Preferential induction of a regulatory T helper type 2 lymphocyte subset.

The immunological mechanisms involved in maintenance of an asymptomatic microfilaremic state (MF) in patients with lymphatic filariasis remain undefined. MF patients have impaired filarial antigen (Ag)-specific lymphocyte proliferation and decreased frequencies (Fo) of Ag-specific T cells, and yet elevated serum IgE and antifilarial IgG4. To investigate the mechanism of Ag-specific anergy in MF patients in contrast to amicrofilaremic individuals with chronic lymphatic obstruction (CP), the Fo of Ag-specific lymphocytes from peripheral blood mononuclear cells secreting either IL-4 or IFN-gamma were assessed by filter spot enzyme-linked immunosorbent assay, and IL-10 and transforming growth factor-beta (TGF-beta) mRNA transcript levels were assessed by a semiquantitative reverse transcriptase polymerase chain reaction technique. The Fo of filaria-specific IL-4-secreting lymphocytes were equivalent in both MF (geometric mean [GM] = 1:11,700) and CP (GM = 1:29,300 P = 0.08), whereas the Fo of IFN-gamma-secreting lymphocytes were lower in MF (GM = 1:39,300) than in CP (GM = 1:4,200, P < 0.01). When the ratio of IL-4/IFN-gamma (T helper type 2 [Th2]/Th1)-secreting cells was examined, MF subjects showed a predominant Th2 response (8:1) compared with a Th1 response in CP individuals (1:4). mRNA transcript levels of IL-10 were also significantly elevated in MF compared with CP individuals (P < 0.01). Further, IL-10 and TGF-beta were shown to have a role in modulating the Ag-specific anergy among MF subjects, in that neutralizing anti-IL-10 or anti-TGF-beta significantly enhanced lymphocyte proliferation response (by 220-1,300%) to filarial Ags in MF individuals. These findings demonstrate that MF subjects respond to parasite antigen by producing a set of suppressive cytokines that may facilitate persistence of the parasite within humans while producing little clinical disease.

Regulatory Networks Induced by Live Parasites Impair Both Th1 and Th2 Pathways in Patent Lymphatic Filariasis: Implications for Parasite Persistence

Patent lymphatic filariasis is characterized by a profound down-regulation of immune responses with both parasite Ag-specific tolerance and bystander suppression. Although this down-regulation is confined to the Th1 arm of the immune system in response to parasite Ag, we hypothesized a more generalized suppression in response to live parasites. Indeed, when we examined the cytokine profile of a cohort of filaria-infected (n = 10) and uninfected (n = 10) individuals in response to live infective-stage larvae or microfilariae of Brugia malayi, we found significant impairment of both Th1 and Th2 cytokines characterized by diminished production of IFN-γ, TNF-α, IL-4, IL-5, and IL-10 in infected patients. The molecular basis of this impaired Th1/Th2 response was examined, and we identified three major networks of immunoregulation and tolerance. First, impaired induction of T-bet and GATA-3 mRNA underlies the Th1/Th2 deficiency in infected individuals. Second, regulatory networks, as evidenced by significantly increased expression of Foxp3 (natural regulatory T cell marker) and regulatory effectors such as TGF-β, CTLA-4, PD-1, ICOS, and indoleamine 2,3-dioxygenase play an important role in immunosuppression. Third, the compromise of effector T cell function is mediated by the enhanced induction of anergy-inducing factors cbl-b, c-cbl (cbl is abbreviation for Casitas B lymphoma), Itch, and Nedd4. Indeed, blocking CTLA-4 or neutralizing TGF-β restored the ability to mount Th1/Th2 responses to live parasites and reversed the induction of anergy-inducing factors. Hence, we conclude that a profound impairment of live parasite-specific Th1 and Th2 immune responses occurs in lymphatic filariasis that is governed at the transcriptional level by a complex interplay of inhibitory mediators.

Parasite-specific anergy in human filariasis. Insights after analysis of parasite antigen-driven lymphokine production.

The antigen-specific immune unresponsiveness seen in bancroftian filariasis was studied by examining lymphokine production in peripheral blood mononuclear cells (PBMC) or PBMC subpopulations from 10 patients with asymptomatic microfilaremia, 13 patients with elephantiasis and 6 normal North Americans. In each group of patients, the kinetics of the lymphokine response and the response to mitogens and nonparasite antigens did not differ significantly. In marked contrast, when antigen-induced lymphokine production was examined, most patients with microfilaremia were unable to produce either interleukin 2 (IL-2) or gamma-interferon (i.e., were nonresponders), and the few who could (hyporesponders, generally with quite low microfilaremia levels) did so at levels significantly less than those of patients with elephantiasis, all of whom showed strong responses to parasite antigen. Removal of neither adherent cells or T8+ cells affected the parasite-specific anergy seen in those with microfilaremia, suggesting a state of T cell tolerance to the parasite in patients with this most common clinical manifestation of bancroftian filariasis.

Cutting Edge: Diminished T Cell TLR Expression and Function Modulates the Immune Response in Human Filarial Infection

Patent lymphatic filariasis is characterized by profound Ag-specific T cell hyporesponsiveness with impaired IFN-γ and IL-2 production. Because T cells have been shown to express a number of TLR and to respond to TLR ligands, we hypothesized that diminished T cell TLR function could partially account for the T cell hyporesponsiveness in filariasis. T cells expressed TLR1, TLR2, TLR4, and TLR9, and the baseline expression of TLR1, TLR2, and TLR4, but not TLR9 was significantly lower in T cells of the filarial-infected individuals compared with the uninfected individuals (both endemic and nonendemic). TLR function was significantly diminished in the T cells of filarial-infected individuals based on decreased T cell activation/cytokine production in response to TLR ligands. Thus, diminished expression and function of T cell TLR is a novel mechanism underlying T cell immune tolerance in lymphatic filariasis.

Regulation of the immune response in lymphatic filariasis: perspectives on acute and chronic infection with Wuchereria bancrofti in South India.

Delineating the immune responses in lymphatic filariasis has been complicated not only by the rapidly expanding knowledge of new immunological mediators and effortors, but also by new methodologies (in particular, circulating filarial antigen detection) for defining and categorizing filarial‐infected individuals. By using assays for circulating antigen in the sera collected as part of the many immunological studies performed on individuals in a Wuchereria bancrofti‐endemic region of South India, we have attempted to explore the influence of patency on the antigen‐driven proliferative and cytokine responses seen in peripheral blood mononuclear cells of individuals with varying clinical manifestations of lymphatic filarial infection. Moreover, we have provided perspectives on the differences between acute and chronic infection with W. bancrofti and suggested mechanisms that may underly the modulation of the immune response as patency occurs.

A Controlled Trial of Ivermectin and Diethylcarbamazine in Lymphatic Filariasis

Ivermectin is a new antifilarial drug that can be given in a single oral dose. To compare the efficacy and side effects of ivermectin with those of diethylcarbamazine, the standard antifilarial treatment, we conducted a randomized, double-blind trial in 40 South Indian men with lymphatic filariasis caused by Wuchereria bancrofti. Patients were randomly assigned to one of three treatments: a single low dose of ivermectin (mean [+/- SE], 21.3 +/- 0.7 micrograms per kilogram of body weight; n = 13) followed by placebo for 12 days; a single high dose of ivermectin (mean, 126.2 +/- 3.7 micrograms per kilogram; n = 13) followed by placebo for 12 days; or diethylcarbamazine for 13 days (6 mg per kilogram per day for 12 days preceded by 3 mg per kilogram for 1 day; n = 14). Eleven patients were initially assigned to receive placebo and after five days were reassigned to one of the three treatment groups. At day 12 there was complete clearance of microfilariae from the blood in all 26 men who took ivermectin and in 11 of the 14 men who took diethylcarbamazine. At six months the numbers of detectable microfilariae (as a percentage of the pretreatment values) were 18.3 percent after low-dose ivermectin and 19.5 percent after high-dose ivermectin, as compared with 6.0 percent after diethylcarbamazine (P less than 0.05). The side effects were confined to the first five days and were similar in the three treatment groups. We conclude that in lymphatic filariasis, the clinical response to a single dose of ivermectin compares favorably with that after the standard 12-day course of diethylcarbamazine. Given the practical advantages of single-dose administration, ivermectin should become a useful medication for the control of bancroftian filariasis.

Diminished Expression and Function of TLR in Lymphatic Filariasis: A Novel Mechanism of Immune Dysregulation

Lymphatic filariasis is a disease characterized by immune dysregulation involving APC and T cell populations. To assess the contribution of TLR in mediating this dysregulation, we examined the expression of TLR1, TLR2, TLR4, and TLR9 on B cells and monocytes of filaria-infected and uninfected individuals. Baseline expression of TLR was significantly lower in B cells but not in monocytes of the filaria-infected group compared with the uninfected group. Upon stimulation with filarial Ag, a diminished up-regulation of TLR was observed in both B cells and monocytes of infected individuals. Finally, stimulation of B cells and monocytes with TLR ligands resulted in decreased B cell and monocyte activation/cytokine production, indicating a state of immune tolerance. This dysregulation is associated with diminished CD4+ T cell production of IFN-γ and IL-5. The diminished expression and function of TLR is thus a likely consequence of chronic Ag stimulation and could serve as a novel mechanism underlying the dysfunctional immune response in filariasis.

Immunologic tolerance in lymphatic filariasis. Diminished parasite-specific T and B lymphocyte precursor frequency in the microfilaremic state.

To explore the mechanisms of antigen-specific immune unresponsiveness seen in microfilaremic patients with bancroftian filariasis, T and B cell precursor frequency analysis was performed using PBMC from individuals with either asymptomatic microfilaremia (MF, n = 7) or chronic lymphatic obstruction (CP, n = 20). Highly purified CD3+ cells were partially reconstituted with adherent cells and their proliferative response to parasite antigens determined in cultures of T cells by limiting dilution analysis. A filter immunoplaque assay also assessed the frequency of both total and parasite-specific Ig-producing B cells. While the lymphocyte proliferation to mitogens and to a nonparasite antigen (Streptolysin-O, [SLO]) were similar in all groups of patients, the frequency of parasite-specific CD3+ T cells was significantly lower (geometric mean [GM], 1/3,757) in MF patients when compared to that in CP patients (GM 1/1,513; P less than 0.001). Similarly, the proportion of lymphocytes producing parasite-specific IgE or IgG was significantly lower in MF patients (IgE mean, 0.2%; IgG mean, 0.33%) compared with CP patients (IgE mean, 3.2%; IgG mean, 1.76%; P less than 0.05 for both comparisons). These observations imply that low numbers of parasite-specific T and B lymphocytes may be partially responsible for the severely diminished capacity of lymphocytes from patients with MF to produce parasite-specific antibody and to proliferate to parasite antigen in vitro. Such differences in parasite-specific lymphocyte responses suggest that tolerance by clonal anergy may be a critical mechanism for maintaining the microfilaremic state.

Filarial lymphedema is characterized by antigen-specific Th1 and th17 proinflammatory responses and a lack of regulatory T cells.

Background Lymphatic filariasis can be associated with development of serious pathology in the form of lymphedema, hydrocele, and elephantiasis in a subset of infected patients. Methods and Findings To elucidate the role of CD4+ T cell subsets in the development of lymphatic pathology, we examined specific sets of cytokines in individuals with filarial lymphedema in response to parasite antigen (BmA) and compared them with responses from asymptomatic infected individuals. We also examined expression patterns of Toll-like receptors (TLR1–10) and Nod-like receptors (Nod1, Nod2, and NALP3) in response to BmA. BmA induced significantly higher production of Th1-type cytokines—IFN-γ and TNF-α—in patients with lymphedema compared with asymptomatic individuals. Notably, expression of the Th17 family of cytokines—IL-17A, IL-17F, IL-21, and IL-23—was also significantly upregulated by BmA stimulation in lymphedema patients. In contrast, expression of Foxp3, GITR, TGFβ, and CTLA-4, known to be expressed by regulatory T cells, was significantly impaired in patients with lymphedema. BmA also induced significantly higher expression of TLR2, 4, 7, and 9 as well Nod1 and 2 mRNA in patients with lymphedema compared with asymptomatic controls. Conclusion Our findings implicate increased Th1/Th17 responses and decreased regulatory T cells as well as regulation of Toll- and Nod-like receptors in pathogenesis of filarial lymphedema.

Human type 1 and 17 responses in latent tuberculosis are modulated by coincident filarial infection through cytotoxic T lymphocyte antigen-4 and programmed death-1.

Mycobacterium tuberculosis and filarial coinfection is highly prevalent, and the presence of a tissue-invasive helminth may modulate the predominant type 1 T helper (Th1; interferon [IFN]-gamma-mediated) response needed to control M. tuberculosis infection. By analyzing the cellular responses to mycobacterial antigens in patients who had latent tuberculosis with or without filarial infection, we were able to demonstrate that filarial infection coincident with M. tuberculosis infection significantly diminishes M. tuberculosis-specific Th1 (interleukin [IL]-12 and IFN-gamma) and type 17 T helper (Th17; IL-23 and IL-17) responses related to increased expression of cytotoxic T lymphocyte antigen (CTLA)-4 and programmed death (PD)-1. Blockade of CTLA-4 restored production of both IFN-gamma and IL-17, whereas PD-1 blockade restored IFN-gamma production only. Thus, coincident filarial infection exerted a profound inhibitory effect on protective mycobacteria-specific Th1 and Th17 responses in latent tuberculosis, suggesting a mechanism by which concomitant filarial (and other systemic helminth) infections predispose to the development of active tuberculosis in humans.