Regulatory T Cells in Human Lymphatic Filariasis: Stronger Functional Activity in Microfilaremics

Infection with filarial parasites is associated with T cell hyporesponsiveness, which is thought to be partly mediated by their ability to induce regulatory T cells (Tregs) during human infections. This study investigates the functional capacity of Tregs from different groups of filarial patients to suppress filaria-specific immune responses during human filariasis. Microfilaremic (MF), chronic pathology (CP) and uninfected endemic normal (EN) individuals were selected in an area endemic for Brugia timori in Flores island, Indonesia. PBMC were isolated, CD4CD25hi cells were magnetically depleted and in vitro cytokine production and proliferation in response to B. malayi adult worm antigen (BmA) were determined in total and Treg-depleted PBMC. In MF subjects BmA-specific T and B lymphocyte proliferation as well as IFN-gamma, IL-13 and IL-17 responses were lower compared to EN and CP groups. Depletion of Tregs restored T cell as well as B cell proliferation in MF-positives, while proliferative responses in the other groups were not enhanced. BmA-induced IL-13 production was increased after Treg removal in MF-positives only. Thus, filaria-associated Tregs were demonstrated to be functional in suppressing proliferation and possibly Th2 cytokine responses to BmA. These suppressive effects were only observed in the MF group and not in EN or CP. These findings may be important when considering strategies for filarial treatment and the targeted prevention of filaria-induced lymphedema

Altered T Cell Memory and Effector Cell Development in Chronic Lymphatic Filarial Infection That Is Independent of Persistent Parasite Antigen

Chronic lymphatic filarial (LF) infection is associated with suppression of parasite-specific T cell responses that persist even following elimination of infection. While several mechanisms have been implicated in mediating this T cell specific downregulation, a role for alterations in the homeostasis of T effector and memory cell populations has not been explored. Using multiparameter flow cytometry, we investigated the role of persistent filarial infection on the maintenance of T cell memory in patients from the filarial-endemic Cook Islands. Compared to filarial-uninfected endemic normals (EN), microfilaria (mf) positive infected patients (Inf) had a reduced CD4 central memory (TCM) compartment. In addition, Inf patients tended to have more effector memory cells (TEM) and fewer effector cells (TEFF) than did ENs giving significantly smaller TEFF ∶ TEM ratios. These contracted TCM and TEFF populations were still evident in patients previously mf+ who had cleared their infection (CLInf). Moreover, the density of IL-7Rα, necessary for T memory cell maintenance (but decreased in T effector cells), was significantly higher on memory cells of Inf and CLInf patients, although there was no evidence for decreased IL-7 or increased soluble IL7-Rα, both possible mechanisms for signaling defects in memory cells. However, effector cells that were present in Inf and CLInf patients had lower percentages of HLA-DR suggesting impaired function. These changes in T cell populations appear to reflect chronicity of infection, as filarial-infected children, despite the presence of active infection, did not show alterations in the frequencies of these T cell phenotypes. These data indicate that filarial-infected patients have contracted TCM compartments and a defect in effector cell development, defects that persist even following clearance of infection. The fact that these global changes in memory and effector cell compartments do not yet occur in infected children makes early treatment of LF even more crucial

Decreased Prevalence of Lymphatic Filariasis among Diabetic Subjects Associated with a Diminished Pro-Inflammatory Cytokine Response (CURES 83)

Epidemiological studies have shown an inverse correlation between the incidence of lymphatic filariasis (LF) and the incidence of allergies and autoimmunity. However, the interrelationship between LF and type-2 diabetes is not known and hence, a cross sectional study to assess the baseline prevalence and the correlates of sero-positivity of LF among diabetic subjects was carried out (n = 1416) as part of the CURES study. There was a significant decrease in the prevalence of LF among diabetic subjects (both newly diagnosed [5.7%] and those under treatment [4.3%]) compared to pre-diabetic subjects [9.1%] (p = 0.0095) and non-diabetic subjects [10.4%] (p = 0.0463). A significant decrease in filarial antigen load (p = 0.04) was also seen among diabetic subjects. Serum cytokine levels of the pro-inflammatory cytokines—IL-6 and GM-CSF—were significantly lower in diabetic subjects who were LF positive, compared to those who were LF negative. There were, however, no significant differences in the levels of anti-inflammatory cytokines—IL-10, IL-13 and TGF-β—between the two groups. Although a direct causal link has yet to be shown, there appears to be a striking inverse relationship between the prevalence of LF and diabetes, which is reflected by a diminished pro-inflammatory cytokine response in Asian Indians with diabetes and concomitant LF

Brugia malayi Microfilariae Induce a Regulatory Monocyte/Macrophage Phenotype That Suppresses Innate and Adaptive Immune Responses

Background Monocytes and macrophages contribute to the dysfunction of immune responses in human filariasis. During patent infection monocytes encounter microfilariae in the blood, an event that occurs in asymptomatically infected filariasis patients that are immunologically hyporeactive. Aim To determine whether blood microfilariae directly act on blood monocytes and in vitro generated macrophages to induce a regulatory phenotype that interferes with innate and adaptive responses. Methodology and principal findings Monocytes and in vitro generated macrophages from filaria non-endemic normal donors were stimulated in vitro with Brugia malayi microfilarial (Mf) lysate. We could show that monocytes stimulated with Mf lysate develop a defined regulatory phenotype, characterised by expression of the immunoregulatory markers IL-10 and PD-L1. Significantly, this regulatory phenotype was recapitulated in monocytes from Wuchereria bancrofti asymptomatically infected patients but not patients with pathology or endemic normals. Monocytes from non-endemic donors stimulated with Mf lysate directly inhibited CD4+ T cell proliferation and cytokine production (IFN-γ, IL-13 and IL-10). IFN-γ responses were restored by neutralising IL-10 or PD-1. Furthermore, macrophages stimulated with Mf lysate expressed high levels of IL-10 and had suppressed phagocytic abilities. Finally Mf lysate applied during the differentiation of macrophages in vitro interfered with macrophage abilities to respond to subsequent LPS stimulation in a selective manner. Conclusions and significance Conclusively, our study demonstrates that Mf lysate stimulation of monocytes from healthy donors in vitro induces a regulatory phenotype, characterized by expression of PD-L1 and IL-10. This phenotype is directly reflected in monocytes from filarial patients with asymptomatic infection but not patients with pathology or endemic normals. We suggest that suppression of T cell functions typically seen in lymphatic filariasis is caused by microfilaria-modulated monocytes in an IL-10-dependent manner. Together with suppression of macrophage innate responses, this may contribute to the overall down-regulation of immune responses observed in asymptomatically infected patients

The development of TH2 responses from infancy to 4 years of age and atopic sensitization in areas endemic for helminth infections

BACKGROUND: Helminth infections and allergies are associated with TH(2) responses. Whereas the development of TH(2) responses and allergic disorders in pediatric populations has been examined in affluent countries, no or little data exist from low income regions of the world. The aim of this study is to examine factors influencing the development of TH(2) responses of children born in areas endemic for helminth infections and to relate these factors to atopic sensitization at 4 years of age. METHODS: Data were collected from pregnant mothers on helminth infections, education and socioeconomic status (SES). Total IgE, IL-5 in response to mitogen, and helminth antigens were measured in children at 2, 5, 12, 24 and 48 months of age. Skin prick testing (SPT) and allergen-specific IgE were determined at 4 years of age. RESULTS: Strong TH(2) responses were seen at 5 months of age and increased with time. Although maternal filarial infection was associated with helminth-antigen specific TH(2) responses, it was low maternal education or SES but not helminth infection, which was associated with the development of high total IgE and PHA-induced IL-5. At 4 years of age when allergen reactivity was assessed by SPT, the high general TH(2) responses did not translate into higher prevalence of SPT. The risk factor for SPT reactivity was low maternal education which decreased the risk of SPT positivity to allergens (adjusted OR, 0.32; 95% CI, 0.12 – 0.87) independently of maternal filarial infection which tended to reduce the child’s risk for being SPT positive (adjusted OR, 0.35; 95% CI, 0.07 – 1.70). CONCLUSIONS: In areas endemic for helminths, potent TH(2) responses were seen early in life, but did not translate into a higher SPT reactivity to allergens. Therefore, in many parts of the world TH(2) responses in general and IgE in particular cannot be used for diagnosis of allergic diseases

Effects of chronic ascariasis and trichuriasis on cytokine production and gene expression in human blood: a cross-sectional study.

Background Chronic soil-transmitted helminth (STH) infections are associated with effects on systemic immune responses that could be caused by alterations in immune homeostasis. To investigate this, we measured the impact in children of STH infections on cytokine responses and gene expression in unstimulated blood. Methodology/Principal Findings Sixty children were classified as having chronic, light, or no STH infections. Peripheral blood mononuclear cells were cultured in medium for 5 days to measure cytokine accumulation. RNA was isolated from peripheral blood and gene expression analysed using microarrays. Different infection groups were compared for the purpose of analysis: STH infection (combined chronic and light vs. uninfected groups) and chronic STH infection (chronic vs. combined light and uninfected groups). The chronic STH infection effect was associated with elevated production of GM-CSF (P = 0.007), IL-2 (P = 0.03), IL-5 (P = 0.01), and IL-10 (P = 0.01). Data reduction suggested that chronic infections were primarily associated with an immune phenotype characterized by elevated IL-5 and IL-10, typical of a modified Th2-like response. Chronic STH infections were associated with the up-regulation of genes associated with immune homeostasis (IDO, P = 0.03; CCL23, P = 0.008, HRK, P = 0.005), down-regulation of microRNA hsa-let-7d (P = 0.01) and differential regulation of several genes associated with granulocyte-mediated inflammation (IL-8, down-regulated, P = 0.0002; RNASE2, up-regulated, P = 0.009; RNASE3, up-regulated, p = 0.03). Conclusions/Significance Chronic STH infections were associated with a cytokine response indicative of a modified Th2 response. There was evidence that STH infections were associated with a pattern of gene expression suggestive of the induction of homeostatic mechanisms, the differential expression of several inflammatory genes and the down-regulation of microRNA has-let-7d. Effects on immune homeostasis and the development of a modified Th2 immune response during chronic STH infections could explain the systemic immunologic effects that have been associated with these infections such as impaired immune responses to vaccines and the suppression of inflammatory diseases

Multivariable regression analysis in Schistosoma mansoni-infected individuals in the Sudan reveals unique immunoepidemiological profiles in uninfected, egg+ and non-egg+ infected individuals

Background: In the Sudan, Schistosoma mansoni infections are a major cause of morbidity in schoolaged children and infection rates are associated with available clean water sources. During infection, immune responses pass through a Th1 followed by Th2 and Treg phases and patterns can relate to different stages of infection or immunity. Methodology: This retrospective study evaluated immunoepidemiological aspects in 234 individuals(range 4–85 years old) from Kassala and Khartoum states in 2011. Systemic immune profiles(cytokines and immunoglobulins) and epidemiological parameters were surveyed in n = 110 persons presenting patent S. mansoni infections (egg+), n = 63 individuals positive for S. mansoni via PCR in sera but egg negative (SmPCR+) and n = 61 people who were infection-free (Sm uninf). Immunoepidemiological findings were further investigated using two binary multivariable regression analysis. Principal Findings: Nearly all egg+ individuals had no access to latrines and over 90% obtained water via the canal stemming from the Atbara River. With regards to age, infection and an egg+ status was linked to young and adolescent groups. In terms of immunology, S. mansoni infection per se was strongly associated with increased SEA-specific IgG4 but not IgE levels. IL-6, IL-13 and IL-10 were significantly elevated in patently-infected individuals and positively correlated with egg load. In contrast, IL-2 and IL-1β were significantly lower in SmPCR+ individuals when compared to Sm uninf and egg+ groups which was further confirmed during multivariate regression analysis. Conclusions/Significance: Schistosomiasis remains an important public health problem in the Sudan with a high number of patent individuals. In addition, SmPCR diagnostics revealed another cohort of infected individuals with a unique immunological profile and provides an avenue for future studies on non-patent infection states. Future studies should investigate the downstream signalling pathways/mechanisms of IL-2 and IL-1β as potential diagnostic markers in order to distinguish patent from non-patent individuals

Characterising the Mucosal and Systemic Immune Responses to Experimental Human Hookworm Infection

The mucosal cytokine response of healthy humans to parasitic helminths has never been reported. We investigated the systemic and mucosal cytokine responses to hookworm infection in experimentally infected, previously hookworm naive individuals from non-endemic areas. We collected both peripheral blood and duodenal biopsies to assess the systemic immune response, as well as the response at the site of adult worm establishment. Our results show that experimental hookworm infection leads to a strong systemic and mucosal Th2 (IL-4, IL-5, IL-9 and IL-13) and regulatory (IL-10 and TGF-β) response, with some evidence of a Th1 (IFN-γ and IL-2) response. Despite upregulation after patency of both IL-15 and ALDH1A2, a known Th17-inducing combination in inflammatory diseases, we saw no evidence of a Th17 (IL-17) response. Moreover, we observed strong suppression of mucosal IL-23 and upregulation of IL-22 during established hookworm infection, suggesting a potential mechanism by which Th17 responses are suppressed, and highlighting the potential that hookworms and their secreted proteins offer as therapeutics for human inflammatory diseases

Mixed Th1 and Th2 Mycobacterium tuberculosis-specific CD4 T cell responses in patients with active pulmonary tuberculosis from Tanzania.

Mycobacterium tuberculosis (Mtb) and helminth infections elicit antagonistic immune effector functions and are co-endemic in several regions of the world. We therefore hypothesized that helminth infection may influence Mtb-specific T-cell immune responses. We evaluated the cytokine profile of Mtb-specific T cells in 72 individuals with pulmonary TB disease recruited from two Sub-Saharan regions with high and moderate helminth burden i.e. 55 from Tanzania (TZ) and 17 from South Africa (SA), respectively. We showed that Mtb-specific CD4 T-cell functional profile of TB patients from Tanzania are primarily composed of polyfunctional Th1 and Th2 cells, associated with increased expression of Gata-3 and reduced expression of T-bet in memory CD4 T cells. In contrast, the cytokine profile of Mtb-specific CD4 T cells of TB patients from SA was dominated by single IFN-γ and dual IFN-γ/TNF-α and associated with TB-induced systemic inflammation and elevated serum levels of type I IFNs. Of note, the proportion of patients with Mtb-specific CD8 T cells was significantly reduced in Mtb/helminth co-infected patients from TZ. It is likely that the underlying helminth infection and possibly genetic and other unknown environmental factors may have caused the induction of mixed Th1/Th2 Mtb-specific CD4 T cell responses in patients from TZ. Taken together, these results indicate that the generation of Mtb-specific CD4 and CD8 T cell responses may be substantially influenced by environmental factors in vivo. These observations may have major impact in the identification of immune biomarkers of disease status and correlates of protection