Parasite Immunology, 2014, 36, 207–217
DOI: 10.1111/pim.12105
Bancroftian filariasis: circulating B-1 cells decreased in microfilaria carriers and correlate with immunoglobulin M levels R. MISHRA, P. K. SAHOO, S. MISHRA, K. G. ACHARY, B. DWIBEDI, S. K. KAR & A. K. SATAPATHY Regional Medical Research Centre (ICMR), Chandrasekharpur, Bhubaneswar, Odisha, India
SUMMARY B-1 cells play an important role in the outcome of infection in schistosomiasis, pneumonia and experimental filariasis. However, no information exists regarding status of B-1 cells in clinical manifestations of human filariasis. We investigated the levels of B-1 cells from the total B cells by flow cytometry. Significantly low levels of B-1 cells and IgM antibodies were detected against a wide variety of autoantigens in microfilariae carriers as compared to endemic controls and patients with chronic pathology. A positive correlation was found between IgM antibodies to actin and ss-DNA. Absorption of plasma with soluble actin, myosin and lipopolysaccharides (LPS) resulted in significant removal of antifilarial antibodies. Affinity-purified anti-ssDNA antibodies were found to be reactive to filarial antigens and various autoantigens. Further, a positive correlation was found between polyreactive antibodies and B-1 cells in filarial-infected human subjects. After antifilarial treatment, levels of IgM antibodies to ss-DNA, actin, LPS and filarial antigen increased significantly indicating a role of polyreactive naturally occurring antibodies in filarial infection. Our findings add to the existing evidence that the B-cell defect in BALB.Xid mice account for susceptibility to murine filarial infection and indicate an important role for these antibodies in providing host protection against filarial infection. Keywords B-1 cells, CD5+CD19+ lymphocyte, lymphatic filariasis, polyreactive antibodies
Correspondence: Ashok K. Satapathy, Regional Medical Research Centre (ICMR), Chandrasekharpur, Bhubaneswar, Odisha 751 023, India (e-mails: [email protected], aksatapathy1@ rediffmail.com). Disclosures: The authors declare that they have no conflict of interests. Received: 3 July 2013 Accepted for publication: 28 January 2014 © 2014 John Wiley & Sons Ltd
INTRODUCTION Lymphatic filariasis, a chronic and debilitating disease, is a major public health problem affecting about 120 million cases throughout the tropics and subtropics (1). It is caused by vector-borne nematode parasites such as Wuchereria bancrofti, Brugia malayi and Brugia timori and often leads to clinical manifestations (such as lymphedema, elephantiasis and/or hydrocele). Filarial infection in humans is characterized by polarization of T-cell responses. A Th1 type of hyperimmune response characterized by increased production of IFN-c by antigenspecific lymphocytes is associated with the absence of active filarial infection. In contrast, the hyporesponsive state observed in individuals harbouring worms is associated with higher levels of IL-10 secretion and severely down-regulated IFN-c production by filarial-specific lymphocytes (2–4). This modulation of immune response appears to play an important role in survival of the parasites as well as protection of the host from proinflammatory pathology. The protective potential of T lymphocytes in filarial infection is well documented. However, the role of B cells in host protection against filariasis remains unclear. B lymphocytes exist in two subsets termed as B-1 and B-2 cells. In normal humans and mice, B-1 cells are committed for the production of polyreactive natural antibodies. In contrast, conventional B-cells (B-2) are mainly involved in the production of antigen-driven antibodies (5, 6). Several findings reported in the literature pointed towards a protective role of antibodies in filariasis (7, 8). However, the role of B-1 cells in protection against filarial infection is not yet clearly understood. B-1 cells are considered to be a part of the natural resistance against a variety of diseases. B-1 cells significantly expand in the spleen and peritoneum of Plasmodium chabaudi chabaudi semiresistant BALB/cJ mice, but not in fully susceptible BALB/cByJ mice (9). Mice expressing high levels of B-1 cells (transgenic mice) have been demonstrated to be
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observations pointed towards the possibility of polyreactive antibodies playing a significant role in the parasitological outcome in the exposed population. Although a spectrum of clinical manifestations has been observed in human bancroftian filariasis, the profile of B-1 cells and polyreactive antibodies during filarial infection in human has not been explored. In this communication, we evaluated the profile of B-1 cell populations and its association with polyreactive antibodies in filarial-infected human subjects and report for the first time the presence of polyreactive antibodies to ss-DNA in plasma of individuals with bancroftian filariasis.
resistant to challenge with Streptococcus pneumoniae (10). The involvement of B-1 cells in the growth and development of filarial parasites in experimental animals have also been documented. Unlike immunocompetent mice, which are uniformly resistant to Brugian filariasis, the B cell-deficient mice are permissive to the infection. Reconstitution of immune-deficient recombination-activating gene (Rag)1/2 mice with B-1 cells reportedly confers resistance to filariasis even in the absence of conventional B-2 lymphocytes and most T cells (11). Further, Xid mice, which lack B-1 cells, also support the growth and development of filarial parasites. B cell-deficient mice and mice bearing the Btk mutation were found to be equally susceptible to filarial infection (12). The above results suggested that B-1 cells play a critical role in mediating host resistance to Brugia infection. Further, BALB.Xid mice support higher Litomosoides sigmodontis adult worm and microfilariae (MF) burden compared with wild-type mice (13). Similarly, the role of B-1 cells in the outcome of infections by other helminth parasites, that is, Schistosoma mansoni, was evaluated in Xid mice and found to be highly susceptible (14). In human lymphatic filariasis, a spectrum of clinical manifestations has been observed in filarial endemic areas. Some individuals display acute/chronic clinical manifestations, and others with circulating parasites are found without apparent clinical disease. The third group, that is, endemic controls (EN), is free from demonstrable patent infection and/or disease despite living in the endemic region for several years and is considered to be resistant to filarial infection (15). Although it appears that B-1 cells play an important role in the outcome of infection in experimental filariasis, the status of B-1 cell population in different clinical manifestations of human filariasis is yet to be investigated. B-1 cells are a small subset of B cells that secrete most, if not all, natural antibodies in the apparent absence of antigenic challenge. Natural antibodies are often polyreactive and bind to foreign antigens as well as to self-components (16). B-1 cell-derived natural antibodies are crucial for host survival from infections (17). Defects in their production cause increased mortality after infection with bacteria, such as Salmonella typhimurium and Streptococcus pneumoniae (18), and viruses, such as vesicular stomatitis virus, lymphocytic choriomeningitis virus, vaccinia virus and influenza virus (19). Absence of naturally occurring polyreactive antibodies (produced by B-1 cells) has been associated with susceptibility to filarial infection in Xid mice. Jirds, the most susceptible animal models for a variety of human and animal parasites, are found to be significantly deficient in eliciting antibodies to naturally occurring antigens, viz. single-stranded DNA (ss-DNA), lipopolysaccharides (LPS) and phospholipids (20). These
The study was conducted in the coastal districts of Odisha, India. Initially, a mass survey was undertaken in two villages (Tirumala and Kantia) by collection of 20 lL of nocturnal blood samples. Clinical assessments of subjects were carried out by both taking a detailed clinical history and physical examination. All individuals were negative for malaria. The individuals recruited were previously not treated for filarial infection. Approval for the study was obtained from the institutional ethical committee of the Regional Medical Research Center (Indian Council of Medical Research), Bhubaneswar, Odisha, and informed consent was obtained from the study subjects for the collection of blood samples. Subsequently, venous blood samples (4–5 mL) were collected in sterile heparinized tubes from those subjects who were included in the current investigation. The plasma samples were separated and stored at 70°C until further use. Subjects of both sexes, aged 15–50 years, were included in this study. The status of infection was evaluated by the examination of blood smear for MF, and the plasma samples were also tested by Og4C3 ELISA kit (Trop Bio) for circulating filarial antigen (CFA). The study subjects were categorized as follows: (i) EN – asymptomatic individuals residing in same filarial endemic area without demonstrable circulating Mf and CFA; (ii) MF carriers (AS) – individuals with circulating microfilariae of nocturnal periodicity without clinical symptoms; and (iii) chronic pathology (CH) – patients presenting with persistent lymphedema/elephantiasis or hydrocele for four or more years. Asymptomatic microfilaraemic individuals were treated with a single dose (6 mg/kg) of diethylcarbamazine (DEC). After 6 months, plasma samples were collected and evaluated for levels of IgM antibodies against ss-DNA (Sigma-Aldrich, St. Louis, MO, USA), LPS (Sigma-Aldrich), actin (Sigma-Aldrich) & myosin (Sigma-Aldrich).
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MATERIALS AND METHODS
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Collection of Setaria digitata Peritoneal dwelling bovine filarial parasites were collected in phosphate-buffered saline (PBS) supplemented with 1% glucose, from a local abattoir. Freshly collected adult male and female parasites were transported to the laboratory. Parasites were washed extensively in PBS for the preparation of filarial antigens.
B-1 cells in human lymphatic filariasis
commercially available ELISA kits (e-Bioscience). Briefly ELISA plates (NUNC, Maxisorp) were coated with capture antibodies. Subsequently, plasma samples of different clinical categories and recombinant human IL-10 (standard) were incubated for 2 h at 37°C. After washing, IL-10 levels were detected with biotinylated anti-human IL-10 antibodies followed by avidin-peroxidase. The plates were developed by TMB (tetramethylbenzidine, GeNei), and O.D. was measured at 450 nm.
Preparation of filarial antigens Filarial antigens were prepared from adult female S. digitata parasite by the method described elsewhere (7). The parasites were washed extensively in PBS, ground in a glass homogenizer and ultrasonicated (Artek sonic dismembrator; BioLogics, Inc., Manassas, VA, USA) for 15 min. The soluble antigens were collected by centrifuging at 2500 9 g for 10 min and kept frozen at 20°C for further use.
Circulating filarial antigen assay Plasma samples were examined for CFA. The commercially available Trop Bio ELISA kit was used for the quantification of antigens according to the manufacturers instructions (Tropical Biotechnology Pty. Ltd, Townsville, Australia) and expressed as antigen units.
ELISA Specific antibody responses were measured by solid-phase immunoassay using ss-DNA, LPS, actin, myosin and filarial antigens. Standard ELISA procedure was used to detect IgM levels against the above antigens. Briefly NUNC Maxisorp (Sigma-Aldrich, St. Louis, MO, USA) plates were coated with 1 lg/well antigens diluted in PBS and incubated at 37°C for 4 h followed by overnight incubation at 4°C. After blocking with 1% skimmed milk-PBS, human plasma samples were incubated for 2 h at 37°C. The plates were thoroughly washed and probed with anti-human IgM peroxidase (1 : 1000). Enzyme activity was measured using orthophenylenediamine (OPD) (Sigma-Aldrich), and absorbance was read at 492 nm using an ELISA reader. The results were expressed as arbitrary ELISA units using an internal standard. Total IgM in plasma samples of different clinical categories was measured by sandwich ELISA according to manufacturers instructions using commercially available ELISA kits (e-Bioscience, San Diego, CA, USA).
Flow cytometry Staining of cells with antibodies was conducted according to standard protocols. Fifty microlitre of freshly collected whole blood was incubated with 20 lL of specific monoclonal antibodies conjugated with FITC or PE along with appropriate isotype controls for 30 min at 4°C, lysed with 2 mL lysing solution (BD FACS lysing solution) followed by washing with sheath fluid. Cells were analysed by a flow cytometer (FACS Calibur; Becton & Dickinson, San Jose, CA, USA). Data were analysed using CELL QUEST PRO software (Becton & Dickinson).
Absorption of plasma Plasma samples were heat inactivated at 56°C for 30 min. Heat-inactivated (1/200) plasma was incubated with autoantigens (actin, myosin, ss-DNA and LPS) and filarial antigens for 2 h at 37°C. The aggregates were sedimented in a microfuge, and the supernatants were collected. Pre-and postabsorbed diluted plasma samples were then tested for levels of IgM antibodies to the above antigens by ELISA. The cross-reactivity of antibodies to ss-DNA was further assessed by inhibition with soluble DNA.
Affinity purification of anti-ss-DNA antibodies
Plasma samples were analysed for levels of IL-10 by sandwich ELISA as per manufacturers instructions using
Soluble ss-DNA was conjugated to cyanogen bromide-activated Sepharose 4B (Sigma-Aldrich) using poly-L-lysine (PLL, Sigma-Aldrich) by the method described by Gilliam, et al. (21) with minor modification. Anti-ss-DNA antibodies were purified by the procedure as described elsewhere (22). Anti-ss-DNA antibodies were isolated from a pool of 50% ammonium sulphate precipitated filarial plasma using a column of ss-DNA-Sepharose. About 3 mL of precipitated plasma samples was passed through a column of ss-DNA-Sepharose and washed extensively with PBS to remove the nonbound proteins. The bound antibodies were eluted with elution buffer [20 mM sodium carbonate, pH (105), containing 5% dimethyl sulfoxide] and adjusted to pH 70. The reactivity of the purified
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Quantification of IL-10
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antibodies to a panel of autoantigens (actin, myosin, ssDNA and LPS) and filarial antigens was tested by ELISA.
distribution, median age, Mf status, Mf density, CFA status and CFA density for the different categories.
Frequency of B-1 cell population in human lymphatic filariasis
Affinity determination Relative affinity of antibodies to ss-DNA, actin, myosin, LPS and filarial antigens was determined as described elsewhere (23). Briefly, the ELISA plates were coated with actin, myosin, LPS and filarial antigens in PBS followed by incubation with affinity-purified anti-ss-DNA antibodies from human filarial plasma. The plates were washed with PBS-T and incubated with 2, 4 and 6 M of potassium thiocyanate for 1 h at 37°C. Plates were then washed extensively, and peroxidase-conjugated anti-human IgM was added to each well, and the enzyme activity was quantified using OPD.
Statistical analysis Data analyses were performed using GRAPH PAD PRISM v-3 (Graph Pad Software, Inc., San Diego, CA, USA).Twotailed unpaired Students t-test was used for comparison of two experimental groups. Coefficients of correlation were determined by the Pearson correlation test. Differences were considered as statistically significant when P < 005.
The profile of B-1 cells was evaluated by immunophenotyping. B-1 lymphocytes are a subset of B cells that express the surface marker CD5 together with CD19. Freshly collected blood samples from uninfected and filaria-infected subjects were stained with anti-CD5-FITC and anti-CD19PE and were analysed by flow cytometer. Lymphocytes gated on forward scatter (FSC) and side scatter (SSC) plot were used for quantification of B-1 cells (Figure 1a). Cells that appeared in the upper right quadrant, expressing both CD5 and CD19, are B-1 cells (Figure 1b). The frequency of B-1 cells was quantified from the total B cells. The frequencies of total B cells were not significantly different among the clinical groups (data not shown). Quantitative analysis of B-1 cells in the clinical spectrum of lymphatic filariasis revealed a significant difference in profile of B-1 cells between MF carriers and other clinical groups (Figure 1c). The profile of the B-1 cell population was significantly lower in MF carriers in comparison with EN and people displaying chronic pathology.
IgM antibodies to filarial antigens and autoantigens in clinical manifestations of filariasis
RESULTS Based on the status of microfilaraemia, antigenaemia and chronic disease manifestations, the study subjects were classified into three categories, as described in Materials and Methods. Table 1 shows the number of subjects, sex
Villages in Odisha included Tirumala and Kantia. Samples from Tirumala and Kantia were used for the data shown in this table; AS, asymptomatic microfilariae carriers; CH, subjects with chronic disease; EN, endemic controls (putatively immune subjects from areas of endemicity); CFA, circulating filarial antigen; GMI, geometric mean index for positive subjects; Mf, microfilariae.
As B-1 cells are committed to the production of polyreactive natural antibodies mostly of IgM, the levels of such antibodies that react with a wide variety of autoantigens such as actin, myosin, LPS, ss-DNA and filarial antigens were evaluated in human filariasis. Initially, the total IgM levels in plasma samples of different clinical groups of filariasis were measured. Plasma levels of total IgM in different clinical categories were comparable (Figure 2a). We quantified IgM antibodies reacting to ss-DNA in the spectrum of clinical manifestations. Levels of IgM antibodies to ss-DNA, actin, myosin, LPS and filarial antigens are shown in Figure 2(b). Significantly lower levels of IgM antibodies to ss-DNA were found in microfilaraemic subjects as compared to EN and patients with chronic manifestations. Similarly, levels of IgM antibodies to actin, myosin, LPS and filarial antigens were significantly lower in MF carriers compared to patients with chronic manifestations and EN (Figure 2b). We also assessed IgM antibody levels after antifilarial treatment. IgM antibody levels to ss-DNA, actin, myosin, LPS and filarial antigens in asymptomatic microfilaria positive individuals were increased significantly after treatment compared with untreated plasma (Figure 2c).
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Table 1 Details of the study population in different areas of Odisha endemic for bancroftian filariasis Characteristic
EN (n = 50)
CH (n = 50)
AS (n = 50)
Sex, no. male/no. female Age, median (range), years Mf status, % Mf density, GMI CFA status, % CFA density, GMI Clinical symptoms
41/9
46/4
47/3
26 (8–75)
335 (15–72)
285 (6–65)
0 0 0 0 No
0 0 2 160 Yes
100 153 100 138642 No
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Plasma levels of IL-10 in human filariasis (a)
Regulatory cytokines like IL-10 is known to be secreted by B-1 cells (24). To demonstrate the effect of B-1 cells on IL-10 production, plasma levels of IL-10 of different clinical categories of filariasis were measured. Increased levels of IL-10 were observed in microfilaraemic individuals as well as patients with chronic as compared to endemic normals (Figure 3). IL-10 production was not affected by reduced levels of B-1 cells in microfilaraemic individuals.
Relationship between antibodies to ss-DNA and frequency of B-1 cells
(b)
We demonstrated that during human filariasis, microfilaraemic individuals exhibited a significant decrease in the frequency of CD5+ B cells and developed low levels of IgM antibodies to various autoantigens as well as filarial antigens. These interesting findings led us to study the relationship between profile of B-1 cells and IgM antibodies to ss-DNA in different clinical manifestations. A positive correlation was found between levels of B-1 cells and IgM antibodies to ss-DNA (Figure 4), indicating that CD5+ B cells appear to be involved in the synthesis of polyreactive naturally occurring antibodies in human bancroftian filariasis.
Relationship between levels of IgM antibodies to ss-DNA and autoantigens (c)
Figure 1 Quantitative analysis of B-1 cells in the clinical spectrum of lymphatic filariasis. Freshly collected blood cells from individuals of filarial endemic areas were stained with anti-CD5FITC and anti-CD19-PE antibodies. (a) Lymphocytes gated (R1) on forward scatter (FSC) and side scatter (SSC) plot were examined for CD5 and CD19 co-expression. (b) The gated double-positive (CD5+CD19+) cell population of upper right quadrant were B-1 cells. The cell populations of both upper right and lower right quadrant denote the total B-cell population. (c) Percentage of B-1 cell population was quantified from the total B cells in three different clinical groups of filariasis endemic controls (EN; n = 35, CH; n = 35 and AS; n = 29). Subjects in the AS group had significantly lower levels of B-1 cells than subjects in the other two study populations, that is, EN and CH. (EN vs. AS, P < 001; CH vs. AS, P < 004). © 2014 John Wiley & Sons Ltd, Parasite Immunology, 36, 207–217
Plasma levels of IgM antibodies were found to be significantly lower in MF carriers compared with EN and patients with chronic manifestations. As reactivity to ss-DNA is the hallmark of polyreactive antibodies, plasma of different clinical manifestations was tested for levels of IgM antibodies to ss-DNA and filarial antigens by ELISA. A significant positive correlation was found between levels of IgM antibodies to filarial antigens and ss-DNA (n = 85, r = 0599, P < 00001; Figure 5a). The relationship between anti-ssDNA antibodies and antibodies to actin, myosin and LPS is shown in Figure 5(b–d), respectively. A positive correlation was also found between anti-ss-DNA and anti-actin IgM antibody levels (n = 85, r = 0558, P < 00001; Figure 5b). A similar significant positive correlation was found between levels of anti-ss-DNA antibodies and anti-myosin antibodies (n = 127, r = 0674, P < 00001 (Figure 5c), anti-ssDNA antibody levels and anti-LPS antibody levels (n = 141, r = 0605, P < 00001; Figure 5d).
Cross-reactivity of anti-ss-DNA antibodies The cross-reactivity of anti-ss-DNA antibodies was assessed by inhibition with soluble autoantigens.
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(a)
(b)
(i)
(ii)
(iii)
(iv)
(v)
Figure 2 Plasma levels of total IgM antibodies and IgM antibodies to filarial antigens and various auto antigens [ss-DNA, actin, myosin, lipopolysaccharides (LPS)] of different clinical categories of filariasis. (a) Plasma levels of total IgM in endemic controls (EN), CH and AS groups were quantified by commercially available ELISA kit. Plasma levels of total IgM were comparable between different clinical groups of filariasis (n = 20). (b) IgM antibodies to filarial antigen and various autoantigens in EN, CH and AS groups were measured by ELISA. IgM antibodies to ss-DNA, actin, myosin, LPS and filarial antigens were significantly low in plasma of AS individuals as compared to EN and CH. (i) In case of IgM antibodies to ss-DNA, P < 0001 in EN vs. AS and P < 0003 in CH vs. AS; (ii) IgM antibodies to actin, P < 001 in EN vs. AS and P < 00001 in CH vs. AS; (iii) IgM antibodies to myosin, P < 0007 in EN vs. AS and P < 00001 in CH vs. AS; (iv) IgM antibodies to LPS, P < 0001 in EN vs. AS and P < 00001 in CH vs. AS. (v) IgM antibodies to filarial antigen, P < 00001 in EN vs. AS and P < 00004 in CH vs. AS (c) Plasma levels of IgM antibodies to filarial antigen and various auto antigens such as actin, myosin, LPS and ss-DNA of AS individuals (n = 20) were measured before and after antifilarial treatment by ELISA. A significant increase in levels of IgM antibodies to the above antigens was observed after antifilarial treatment.
(c)
Figure 3 Plasma levels of IL-10 of different clinical categories of filariasis. Plasma concentrations of IL-10 in endemic controls (EN), AS and CH groups were quantified by commercially available ELISA kit. Levels of IL-10 were significantly high in plasma of AS and CH groups compared with EN (P < 00001 in EN vs. AS and P < 00001 in EN vs. CH; n = 20).
Single-step absorption of plasma with soluble actin, myosin and LPS reduced anti-ss-DNA IgM antibodies (Figure 6a). Similarly, the reactivity to myosin, LPS and actin was effectively decreased by absorption with ss-DNA (Figure 6b), indicating that most of the ss-DNA-binding antibodies found in human plasma are cross-reactive. Further, the reactivity to ss-DNA, actin, myosin and LPS was effectively decreased by absorption with filarial antigens (Figure 6c), which also indicated that most of the soluble filarial antigen-binding antibodies cross-react with ss-DNA.
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Reactivity of affinity-purified anti-ss-DNA antibodies As naturally occurring polyreactive antibodies have been demonstrated to play a role in innate immunity as well as in immune regulation, the polyreactive property of DNAbinding antibodies was evaluated. Antibodies to ss-DNA were affinity purified from plasma of filarial patients using a column of ss-DNA-Sepharose. Affinity-purified anti-ssDNA antibodies were tested against a panel of antigens to evaluate their polyreactive nature. Affinity-purified © 2014 John Wiley & Sons Ltd, Parasite Immunology, 36, 207–217
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B-1 cells play an important role in the outcome of infection in experimental filariasis and schistosomiasis. Analysis
of profile of B-1 cells in the clinical spectrum of lymphatic filariasis offered interesting leads to understand the role of B-1 cells in the parasitological outcome of filarial infection. Our investigation revealed a significant difference in levels of B-1 cells between MF carriers and other clinical groups. We found that B-1 cell population is significantly low in MF carriers compared with endemic normals and patients with chronic pathology. Mice lacking B lymphocytes were shown to be permissive for Brugian infections (11). Al-Qaoud et al. (13) have shown that BALB.Xid mice support higher L. sigmodontis adult worm and MF burdens compared with wild-type mice. Although, several studies have shown that B-1 cells play an important role in the outcome of infection in experimental filariasis (11, 13) and schistosomiasis (14), the exact role of B-1 cells during human filarial infection is yet to be understood. How B-1 lymphocytes contribute to protection against this large, extracellular metazoan parasite is largely unknown. It could be attributed to the ability of B cells to act as (i) early antigen-presenting cells which might contribute to initiation of T-cell response; (ii) a source of IL10 (24) that suppresses Th1 immune responses, believed to be important in host defence; and (iii) a source of antibodies for mediating humoral immunity against filarial parasites. From the above possibilities, we examined antibody production as well as IL-10 levels. B-1 lymphocytes are committed to the production of polyreactive natural antibodies, mainly IgM, which binds to a variety of selfantigens in humans (25). We found significantly decreased levels of IgM antibodies to various antigens in microfilaraemic individuals. This finding is consistent with the generally dampened immune response commonly seen in microfilaraemic individuals (26). Inability to respond to various antigens may result in survival of MF in this group. It has been demonstrated that mice unable to make naturally occurring antibodies as a result of inheriting the X-linked deficiency are found to be susceptible to schistosomiasis and filariasis (11–13). Jirds are the most susceptible animal models for a variety of human and animal filarial parasites and have been shown to be significantly incompetent in eliciting antibodies against ss-DNA, LPS and phospholipids. In the absence of B-1-derived natural anti-influenza IgM antibodies, delayed T-dependent IgG2a response and increased mortality were reported in murine models (27). Although these studies did not relate directly to naturally occurring antibody responses, the absence of naturally occurring antibodies might play a crucial role in making the hosts susceptible to filarial infection. Furthermore, the data presented in this communication demonstrated that the anti-ss-DNA antibodies observed during filarial infection have multiple reactivity because: (i) a positive correlation was found between antifilarial
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Figure 4 Correlation between percentage of B-1 cell (CD5+CD19+) and plasma levels of anti-ss-DNA IgM antibodies in human filariasis. The IgM antibody levels to ss-DNA of 1/400 diluted plasma were compared with percentage of B-1 cells. Percentage of B-1 cells were significantly correlated with levels of anti-ss-DNA IgM antibodies in human filarial plasma (r = 0548, P < 0000; n = 71).
anti-ss-DNA antibodies were found to react with actin, myosin, LPS and filarial antigens (Figure 7), further confirming the polyreactive nature of anti-ss-DNA antibodies in human filarial plasma. However, there was no significant reactivity of affinity-purified anti-ss-DNA antibodies to human serum albumin (data not shown).
Affinity determination It is evident from the above experiments that the anti-ssDNA antibodies detected in plasma of human filariasis are polyreactive in nature. We examined relative binding of affinity-purified anti-ss-DNA antibodies to ss-DNA, actin, myosin, LPS and filarial antigens (Table 2). These affinity-purified antibodies to ss-DNA from human plasma at a dilution of 1 : 10 have a lower affinity for filarial antigens and other autoantigens. Significant dissociation of the antibody reactivity against ss-DNA, actin, myosin, LPS and filarial antigens occurred only at a much lower concentration of 2 M potassium thiocyanate. These results were further confirmed when the affinity-purified antibodies to ss-DNA were diluted 20 times and tested against various autoantigens. Similarly, significant dissociation of the antibody reactivity was observed at a lower concentration of potassium thiocyanate indicating a lower affinity of purified antibodies to ss-DNA.
DISCUSSION
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(a)
(b)
(c)
(d)
Figure 5 Correlation between plasma levels of anti-ss-DNA IgM antibodies and IgM antibody levels to filarial antigens and various autoantigens such as actin, myosin and lipopolysaccharides (LPS). (a) Levels of anti-ssDNA IgM antibodies were positively correlated with levels of antifilarial IgM antibodies in plasma of different clinical categories (r = 0599, P < 00001; n = 85). (b) A positive correlation between levels of anti-ssDNA IgM antibodies and anti-actin IgM antibodies was observed (r = 0558, P < 00001, n = 85). (c) A similar correlation for levels of anti-ssDNA IgM antibodies and antimyosin IgM antibodies (r = 0674, P < 00001, n = 127) and (d) levels of antissDNA IgM and anti-LPS IgM levels (r = 0605, P < 00001, n = 141) was observed.
antibodies and anti-ss-DNA antibodies in plasma of both normal and infected individuals; (ii) the antibody reactivity to DNA could be effectively absorbed from plasma by pre-incubation with soluble actin, myosin, filarial antigens; and (iii) the antibodies eluted from ss-DNA-Sepharose column reacted with various antigens. CD5+ B lymphocytes have been shown to be preferentially involved in the synthesis of naturally occurring antibodies. Monoreactive, high affinity autoantibodies of the IgG class are produced by CD5 B cells and low affinity polyreactive autoantibodies arise from the B-1 subset. It has been shown that the polyreactive autoantibodies normally produced by CD5+ B cells differ functionally from the monoreactive autoantibodies that are characteristic of various autoimmune diseases (25, 28). We demonstrated cross-reactivity of affinity-purified antibodies to filarial
antigens as well as to various autoantigens such as actin, myosin tubulin and LPS. Thus, they appear to be nonpathogenic antibodies. Mice deficient in circulating IgM but otherwise normal in their humoral responses manifest a significant impairment in worm elimination (8). Further, the substantial deficit of IgM in Xid mice could account for the delayed clearance of infection (8). Although lack of B-1 cells increases filarial worm burden in animal models (13), the down-regulation of B-1 cells and IgM antibodies may prevent helminth-induced pathology. The observed low levels of IgM antibodies in microfilaraemic cases may not eliminate parasites in the host but increase susceptibility to infection. Interestingly, treatment of microfilaraemic patients with antifilarial drugs significantly increased IgM antibodies to various autoantigens. These observations pointed towards the possibility of naturally
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(a)
(b)
Figure 7 Polyreactivity of affinity-purified ss-DNA antibodies. Pooled plasma of different clinical categories was precipitated with 50% ammonium sulphate and passed through a column of ss-DNA-Sepharose. The bound immunoglobulins were eluted with elution buffer (20 mM sodium carbonate, PH (105), containing 5% vol. dimethyl sulfoxide. Reactivity of the purified antibodies against ssDNA, actin, myosin, lipopolysaccharides, tubulin and filarial antigen was evaluated.
Figure 6 (a) Levels of anti-ss-DNA IgM antibodies in plasma were absorbed with actin, myosin and lipopolysaccharides (LPS). Ten plasma samples were absorbed with soluble actin, myosin and LPS, and then, anti-ss-DNA IgM antibody levels were measured by ELISA. Pre- and postabsorption IgM levels were compared. (b) Filarial plasma was pre-absorbed with soluble ss-DNA, and then, IgM levels against actin, myosin and LPS were measured by ELISA. (c) Filarial plasma was pre-absorbed with soluble ssDNA, actin, myosin, and LPS, and then, IgM levels against filarial antigens were measured by ELISA.
occurring polyreactive antibodies playing a significant role in the parasitological outcome of exposed population. Filarial parasites polarize host immune response towards the Th2 phenotype with consequent suppression of T-cell proliferation and are important in maintaining parasitaemia. The IL-10 production has long been associated with filarial infection wherein the parasites down-regulate the host immune response to establish infection by inducing IL-10-mediated T-cell suppression (26). Animal models of filarial infections have demonstrated that depletion of IL-10 or introduction of IL-10 deficiency accelerates worm clearance (26, 29). Several studies have shown that high levels of spontaneous and parasite antigen-driven IL-10 production are associated with antigen-specific hyporesponsiveness in human lymphatic filariasis (29). Peripheral blood mononuclear cells (PBMC) of microfilaraemic individuals have been shown to release higher levels of IL-10 than in patients with chronic pathology. In this study, increased plasma levels of IL-10 and decreased frequency of B-1 cell population were detected in microfilaraemic individuals. IL-10 production appears not to be affected by reduced levels of B-1 cells in microfilaraemic individuals. It may be possible that despite the drop of overall B-1 cells, microfilaraemic patients have an increased cell numbers in an IL-10 producing B-cell subset. Several other cell types including CD4+CD25+ and CD4+CD25 regulatory T cells, Th1 CD4+ T cells, CD8+ T cells, monocytes/macrophages, NK cells have been shown to involve in enhanced IL-10 production (30).
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Table 2 ELISA: affinity determination of affinity-purified anti-ss-DNA antibodies to ss-DNA, actin, myosin and lipopolysaccharides (LPS) Absorbance at 492 nm Autoantigensa
Autoantigensb
Treatment
ss-DNA
Actin
Myosin
LPS
Filarial antigen
ss-DNA
Actin
Myosin
LPS
Filarial antigen
None 2 M KSCN 4 M KSCN 6 M KSCN
0866 0232 0121 0126
0434 0146 0061 0098
0504 0350 0252 0234
0314 0104 0060 0030
2019 0623 0411 0248
0515 0134 0115 0104
0257 0067 0028 0034
0330 0264 0234 0220
0124 0033 0007 0057
1009 0422 0348 0187
KSCN, potassium thiocyanate; a10-fold diluted sample against autoantigens; b20-fold diluted sample against autoantigens.
The mechanism by which B-1 cells and polyreactive IgM antibody production was down-regulated in microfilaraemic patients is largely unknown. IL-5 induces differentiation of activated conventional B (B-2) cells into immunoglobulinsecreting cells and induces the growth of progenitors of CD5+ B (B-1) cells and IgM production by B-1 cells (31). It has been shown that mice constitutively overexpressing IL-5 have an increased eosinophilia that causes a more rapid clearance of L. sigmodontis (32). Conversely, the parasites survive and reproduce well beyond their normal lifespan in IL-5 KO mice (33). As IL-5 responses were reported to be low in microfilaraemic individuals, low levels of IL-5 may enable these individuals to down-regulate B-1 cells and polyreactive IgM antibody production. The demonstration of the polyreactive property of anti-ss-DNA antibodies in this study indicates an important role of these antibodies to provide host protection against filarial infection. As naturally occurring polyreactive antibodies have been demonstrated to play a role in innate immunity as well as in immune regulation, our laboratory is now directed towards investigating the role of CD5+ B-1 cells in polarizing cytokine responses by filarial parasites, an area which has not been explored so far.
ACKNOWLEDGEMENTS The authors are grateful to the patients who participated in the study. We thank Dr. S.K. Kar, the Director of Regional Medical Research Centre, Bhubaneswar, for sustained support and for providing the facilities for conducting the work.
FUNDING This work was supported by Department of Science & Technology, Govt of India: SR/SO/HS 2008 (IFD diary no.100/IFD/10887/2009-12010); The Regional Medical Research Centre in Bhubaneswar is funded by the Indian Council of Medical Research, New Delhi.
AUTHOR CONTRIBUTIONS AKS: conceived and designed the experiments. RM: performed the experiments. SM and KG: involved in the acquisition of data. PKS, BD and AKS: analysed the data. SKK revised the manuscript. RM and AKS: wrote the paper.
REFERENCES 1 World Health Organization. Global programme to eliminate lymphatic filariasis. Wkly Epidemiol Rec 2006; 81: 221–232. 2 Babu S, Blauvelt CP, Kumaraswami V & Nutman TB. Regulatory networks induced by live parasites impair both Th1 and Th2 pathways in patent lymphatic filariasis: implications for parasite persistence. J Immunol 2006; 176: 3248–3256. 3 Metenou S, Dembele B, Konate S, et al. Patent filarial infection modulates malariaspecific type 1 cytokine responses in an IL-10-dependent manner in a filaria/malariacoinfected population. J Immunol 2009; 183: 916–924. 4 Ravindran B, Satapathy AK, Sahoo PK & Mohanty MC. Protective immunity in
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human lymphatic filariasis: problems and prospects. Med Microbiol Immunol 2003; 192: 41–46. Berland R & Wortis HH. Origins and functions of B-1 cells with notes on the role of CD5. Annu Rev Immunol 2002; 20: 253–300. Casali P & Schettino EW. Structure and function of natural antibodies. Curr Top Microbiol Immunol 1996; 210: 167–179. Mohanty MC, Satapathy AK, Sahoo PK & Ravindran B. Human bancroftian filariasis – a role for antibodies to parasite carbohydrates. Clin Exp Immunol 2001; 124: 54–61. Rajan B, Ramalingam T & Rajan TV. Critical role for IgM in host protection in experimental filarial infection. J Immunol 2005; 175: 1827–1833.
9 Yoder BJ & Goodrum KJ. Plasmodium chabaudi chabaudi: B-1 cell expansion correlates with semiresistance in BALB/cJ mice. Exp Parasitol 2001; 98: 71–82. 10 Haas KM, Poe JC, Steeber DA & Tedder TF. B-1a and B-1b cells exhibit distinct developmental requirements and have unique functional roles in innate and adaptive immunity to S. pneumoniae. Immunity 2005; 23: 7–18. 11 Paciorkowski N, Porte P, Shultz LD & Rajan TV. B1 B lymphocytes play a critical role in host protection against lymphatic filarial parasites. J Exp Med 2000; 191: 731–736. 12 Mukhopadhyay S, Sahoo PK, George A, Bal V, Rath S & Ravindran B. Delayed clearance of filarial infection and enhanced Th1
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immunity due to modulation of macrophage APC functions in xid mice. J Immunol 1999; 163: 875–883. Al-Qaoud KM, Fleischer B & Hoerauf A. The Xid defect imparts susceptibility to experimental murine filariosis–association with a lack of antibody and IL-10 production by B cells in response to phosphorylcholine. Int Immunol 1998; 10: 17–25. Gaubert S, Viana da Costa A, Maurage CA, et al. X-linked immunodeficiency affects the outcome of Schistosoma mansoni infection in the murine model. Parasite Immunol 1999; 21: 89–101. King CL. Immune regulation and the spectrum of filarial disease. In Klei TR, Rajan TV (eds): The Filaria. Kluwer, Boston, 2002: 127–142. Coutinho A, Kazatchkine MD & Avrameas S. Natural autoantibodies. Curr Opin Immunol 1995; 7: 812–818. Baumgarth N, Herman OC, Jager GC, Brown L, Herzenberg LA & Herzenberg LA. Innate and acquired humoral immunities to influenza virus are mediated by distinct arms of the immune system. Proc Natl Acad Sci USA 1999; 96: 2250–2255. Montecino-Rodriguez E & Dorshkind K. New perspectives in B-1 B cell development and function. Trends Immunol 2006; 27: 428–433.
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19 Choi YS & Baumgarth N. Dual role for B1a cells in immunity to influenza virus infection. J Exp Med 2008; 205: 3053–3064. 20 Mohanty MC & Ravindran B. Deficiency of antibody responses to T-independent antigens in gerbils—Meriones unguiculatus. Dev Comp Immunol 2002; 26: 385–391. 21 Gilliam AC, Lang D & LoSpalluto JJ. Antibodies to double-stranded DNA: purification and characterization of binding specificities. J Immunol 1980; 125: 874–885. 22 Gadgil H, Oak SA & Jarrett HW. Affinity purification of DNA-binding proteins. J Biochem Biophys Methods 2001; 49: 607–624. 23 Satapathy AK, Das MK & Ravindran B. Murine Malaria; Anti-erythrocytic antibodies recognize N-Acetyl neuraminic acid residues. Immunology 1993; 80: 546–552. 24 OGarra A, Chang R, Go N, Hastings R, Haughton G & Howard M. Ly-1 B (B-1) cells are the main source of B cell-derived interleukin 10. Eur J Immunol 1992; 22: 711– 717. 25 Casali P & Notkins AL. CD5+ B lymphocytes, polyreactive antibodies and the human B-cell repertoire. Immunol Today 1989; 10: 364–368. 26 Maizels RM, Balic A, Gomez-Escobar N, Nair M, Taylor MD & Allen JE. Helminth parasites–masters of regulation. Immunol Rev 2004; 201: 89–116.
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27 Baumgarth N, Herman OC, Jager GC, Brown LE, Herzenberg LA & Chen J. B-1 and B-2 cell-derived immunoglobulin M antibodies are nonredundant components of the protective response to influenza virus infection. J Exp Med 2000; 192: 271–280. 28 Kohler H, Bayry J, Nicoletti A & Kaveri SV. Natural autoantibodies as tools to predict the outcome of immune response? Scand J Immunol 2003; 58: 285–289. 29 Specht S, Volkmann L, Wynn T & Hoerauf A. Interleukin-10 (IL-10) counterregulates IL-4-dependent effector mechanisms in Murine Filariasis. Infect Immun 2004; 72: 6287– 6293. 30 Mitre E, Chien D & Nutman TB. CD4(+) (and not CD25+) T cells are the predominant interleukin-10-producing cells in the circulation of filaria-infected patients. J Infect Dis 2008; 197: 94–101. 31 Takatsu K. Interleukin-5. Curr Opin Immunol 1992; 4: 299–306. 32 Martin C, Le Goff L, Ungeheuer MN, Vuong PN & Bain O. Drastic reduction of a filarial infection in eosinophilic interleukin-5 transgenic mice. Infect Immun 2000; 68: 3651–3656. 33 Volkmann L, Bain O, Saeftel M, et al. Murine filariasis: interleukin 4 and interleukin 5 lead to containment of different worm developmental stages. Med Microbiol Immunol 2003; 192: 23–31.
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DOI: 10.1111/pim.12105
Bancroftian filariasis: circulating B-1 cells decreased in microfilaria carriers and correlate with immunoglobulin M levels R. MISHRA, P. K. SAHOO, S. MISHRA, K. G. ACHARY, B. DWIBEDI, S. K. KAR & A. K. SATAPATHY Regional Medical Research Centre (ICMR), Chandrasekharpur, Bhubaneswar, Odisha, India
SUMMARY B-1 cells play an important role in the outcome of infection in schistosomiasis, pneumonia and experimental filariasis. However, no information exists regarding status of B-1 cells in clinical manifestations of human filariasis. We investigated the levels of B-1 cells from the total B cells by flow cytometry. Significantly low levels of B-1 cells and IgM antibodies were detected against a wide variety of autoantigens in microfilariae carriers as compared to endemic controls and patients with chronic pathology. A positive correlation was found between IgM antibodies to actin and ss-DNA. Absorption of plasma with soluble actin, myosin and lipopolysaccharides (LPS) resulted in significant removal of antifilarial antibodies. Affinity-purified anti-ssDNA antibodies were found to be reactive to filarial antigens and various autoantigens. Further, a positive correlation was found between polyreactive antibodies and B-1 cells in filarial-infected human subjects. After antifilarial treatment, levels of IgM antibodies to ss-DNA, actin, LPS and filarial antigen increased significantly indicating a role of polyreactive naturally occurring antibodies in filarial infection. Our findings add to the existing evidence that the B-cell defect in BALB.Xid mice account for susceptibility to murine filarial infection and indicate an important role for these antibodies in providing host protection against filarial infection. Keywords B-1 cells, CD5+CD19+ lymphocyte, lymphatic filariasis, polyreactive antibodies
Correspondence: Ashok K. Satapathy, Regional Medical Research Centre (ICMR), Chandrasekharpur, Bhubaneswar, Odisha 751 023, India (e-mails: [email protected], aksatapathy1@ rediffmail.com). Disclosures: The authors declare that they have no conflict of interests. Received: 3 July 2013 Accepted for publication: 28 January 2014 © 2014 John Wiley & Sons Ltd
INTRODUCTION Lymphatic filariasis, a chronic and debilitating disease, is a major public health problem affecting about 120 million cases throughout the tropics and subtropics (1). It is caused by vector-borne nematode parasites such as Wuchereria bancrofti, Brugia malayi and Brugia timori and often leads to clinical manifestations (such as lymphedema, elephantiasis and/or hydrocele). Filarial infection in humans is characterized by polarization of T-cell responses. A Th1 type of hyperimmune response characterized by increased production of IFN-c by antigenspecific lymphocytes is associated with the absence of active filarial infection. In contrast, the hyporesponsive state observed in individuals harbouring worms is associated with higher levels of IL-10 secretion and severely down-regulated IFN-c production by filarial-specific lymphocytes (2–4). This modulation of immune response appears to play an important role in survival of the parasites as well as protection of the host from proinflammatory pathology. The protective potential of T lymphocytes in filarial infection is well documented. However, the role of B cells in host protection against filariasis remains unclear. B lymphocytes exist in two subsets termed as B-1 and B-2 cells. In normal humans and mice, B-1 cells are committed for the production of polyreactive natural antibodies. In contrast, conventional B-cells (B-2) are mainly involved in the production of antigen-driven antibodies (5, 6). Several findings reported in the literature pointed towards a protective role of antibodies in filariasis (7, 8). However, the role of B-1 cells in protection against filarial infection is not yet clearly understood. B-1 cells are considered to be a part of the natural resistance against a variety of diseases. B-1 cells significantly expand in the spleen and peritoneum of Plasmodium chabaudi chabaudi semiresistant BALB/cJ mice, but not in fully susceptible BALB/cByJ mice (9). Mice expressing high levels of B-1 cells (transgenic mice) have been demonstrated to be
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observations pointed towards the possibility of polyreactive antibodies playing a significant role in the parasitological outcome in the exposed population. Although a spectrum of clinical manifestations has been observed in human bancroftian filariasis, the profile of B-1 cells and polyreactive antibodies during filarial infection in human has not been explored. In this communication, we evaluated the profile of B-1 cell populations and its association with polyreactive antibodies in filarial-infected human subjects and report for the first time the presence of polyreactive antibodies to ss-DNA in plasma of individuals with bancroftian filariasis.
resistant to challenge with Streptococcus pneumoniae (10). The involvement of B-1 cells in the growth and development of filarial parasites in experimental animals have also been documented. Unlike immunocompetent mice, which are uniformly resistant to Brugian filariasis, the B cell-deficient mice are permissive to the infection. Reconstitution of immune-deficient recombination-activating gene (Rag)1/2 mice with B-1 cells reportedly confers resistance to filariasis even in the absence of conventional B-2 lymphocytes and most T cells (11). Further, Xid mice, which lack B-1 cells, also support the growth and development of filarial parasites. B cell-deficient mice and mice bearing the Btk mutation were found to be equally susceptible to filarial infection (12). The above results suggested that B-1 cells play a critical role in mediating host resistance to Brugia infection. Further, BALB.Xid mice support higher Litomosoides sigmodontis adult worm and microfilariae (MF) burden compared with wild-type mice (13). Similarly, the role of B-1 cells in the outcome of infections by other helminth parasites, that is, Schistosoma mansoni, was evaluated in Xid mice and found to be highly susceptible (14). In human lymphatic filariasis, a spectrum of clinical manifestations has been observed in filarial endemic areas. Some individuals display acute/chronic clinical manifestations, and others with circulating parasites are found without apparent clinical disease. The third group, that is, endemic controls (EN), is free from demonstrable patent infection and/or disease despite living in the endemic region for several years and is considered to be resistant to filarial infection (15). Although it appears that B-1 cells play an important role in the outcome of infection in experimental filariasis, the status of B-1 cell population in different clinical manifestations of human filariasis is yet to be investigated. B-1 cells are a small subset of B cells that secrete most, if not all, natural antibodies in the apparent absence of antigenic challenge. Natural antibodies are often polyreactive and bind to foreign antigens as well as to self-components (16). B-1 cell-derived natural antibodies are crucial for host survival from infections (17). Defects in their production cause increased mortality after infection with bacteria, such as Salmonella typhimurium and Streptococcus pneumoniae (18), and viruses, such as vesicular stomatitis virus, lymphocytic choriomeningitis virus, vaccinia virus and influenza virus (19). Absence of naturally occurring polyreactive antibodies (produced by B-1 cells) has been associated with susceptibility to filarial infection in Xid mice. Jirds, the most susceptible animal models for a variety of human and animal parasites, are found to be significantly deficient in eliciting antibodies to naturally occurring antigens, viz. single-stranded DNA (ss-DNA), lipopolysaccharides (LPS) and phospholipids (20). These
The study was conducted in the coastal districts of Odisha, India. Initially, a mass survey was undertaken in two villages (Tirumala and Kantia) by collection of 20 lL of nocturnal blood samples. Clinical assessments of subjects were carried out by both taking a detailed clinical history and physical examination. All individuals were negative for malaria. The individuals recruited were previously not treated for filarial infection. Approval for the study was obtained from the institutional ethical committee of the Regional Medical Research Center (Indian Council of Medical Research), Bhubaneswar, Odisha, and informed consent was obtained from the study subjects for the collection of blood samples. Subsequently, venous blood samples (4–5 mL) were collected in sterile heparinized tubes from those subjects who were included in the current investigation. The plasma samples were separated and stored at 70°C until further use. Subjects of both sexes, aged 15–50 years, were included in this study. The status of infection was evaluated by the examination of blood smear for MF, and the plasma samples were also tested by Og4C3 ELISA kit (Trop Bio) for circulating filarial antigen (CFA). The study subjects were categorized as follows: (i) EN – asymptomatic individuals residing in same filarial endemic area without demonstrable circulating Mf and CFA; (ii) MF carriers (AS) – individuals with circulating microfilariae of nocturnal periodicity without clinical symptoms; and (iii) chronic pathology (CH) – patients presenting with persistent lymphedema/elephantiasis or hydrocele for four or more years. Asymptomatic microfilaraemic individuals were treated with a single dose (6 mg/kg) of diethylcarbamazine (DEC). After 6 months, plasma samples were collected and evaluated for levels of IgM antibodies against ss-DNA (Sigma-Aldrich, St. Louis, MO, USA), LPS (Sigma-Aldrich), actin (Sigma-Aldrich) & myosin (Sigma-Aldrich).
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MATERIALS AND METHODS
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Collection of Setaria digitata Peritoneal dwelling bovine filarial parasites were collected in phosphate-buffered saline (PBS) supplemented with 1% glucose, from a local abattoir. Freshly collected adult male and female parasites were transported to the laboratory. Parasites were washed extensively in PBS for the preparation of filarial antigens.
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commercially available ELISA kits (e-Bioscience). Briefly ELISA plates (NUNC, Maxisorp) were coated with capture antibodies. Subsequently, plasma samples of different clinical categories and recombinant human IL-10 (standard) were incubated for 2 h at 37°C. After washing, IL-10 levels were detected with biotinylated anti-human IL-10 antibodies followed by avidin-peroxidase. The plates were developed by TMB (tetramethylbenzidine, GeNei), and O.D. was measured at 450 nm.
Preparation of filarial antigens Filarial antigens were prepared from adult female S. digitata parasite by the method described elsewhere (7). The parasites were washed extensively in PBS, ground in a glass homogenizer and ultrasonicated (Artek sonic dismembrator; BioLogics, Inc., Manassas, VA, USA) for 15 min. The soluble antigens were collected by centrifuging at 2500 9 g for 10 min and kept frozen at 20°C for further use.
Circulating filarial antigen assay Plasma samples were examined for CFA. The commercially available Trop Bio ELISA kit was used for the quantification of antigens according to the manufacturers instructions (Tropical Biotechnology Pty. Ltd, Townsville, Australia) and expressed as antigen units.
ELISA Specific antibody responses were measured by solid-phase immunoassay using ss-DNA, LPS, actin, myosin and filarial antigens. Standard ELISA procedure was used to detect IgM levels against the above antigens. Briefly NUNC Maxisorp (Sigma-Aldrich, St. Louis, MO, USA) plates were coated with 1 lg/well antigens diluted in PBS and incubated at 37°C for 4 h followed by overnight incubation at 4°C. After blocking with 1% skimmed milk-PBS, human plasma samples were incubated for 2 h at 37°C. The plates were thoroughly washed and probed with anti-human IgM peroxidase (1 : 1000). Enzyme activity was measured using orthophenylenediamine (OPD) (Sigma-Aldrich), and absorbance was read at 492 nm using an ELISA reader. The results were expressed as arbitrary ELISA units using an internal standard. Total IgM in plasma samples of different clinical categories was measured by sandwich ELISA according to manufacturers instructions using commercially available ELISA kits (e-Bioscience, San Diego, CA, USA).
Flow cytometry Staining of cells with antibodies was conducted according to standard protocols. Fifty microlitre of freshly collected whole blood was incubated with 20 lL of specific monoclonal antibodies conjugated with FITC or PE along with appropriate isotype controls for 30 min at 4°C, lysed with 2 mL lysing solution (BD FACS lysing solution) followed by washing with sheath fluid. Cells were analysed by a flow cytometer (FACS Calibur; Becton & Dickinson, San Jose, CA, USA). Data were analysed using CELL QUEST PRO software (Becton & Dickinson).
Absorption of plasma Plasma samples were heat inactivated at 56°C for 30 min. Heat-inactivated (1/200) plasma was incubated with autoantigens (actin, myosin, ss-DNA and LPS) and filarial antigens for 2 h at 37°C. The aggregates were sedimented in a microfuge, and the supernatants were collected. Pre-and postabsorbed diluted plasma samples were then tested for levels of IgM antibodies to the above antigens by ELISA. The cross-reactivity of antibodies to ss-DNA was further assessed by inhibition with soluble DNA.
Affinity purification of anti-ss-DNA antibodies
Plasma samples were analysed for levels of IL-10 by sandwich ELISA as per manufacturers instructions using
Soluble ss-DNA was conjugated to cyanogen bromide-activated Sepharose 4B (Sigma-Aldrich) using poly-L-lysine (PLL, Sigma-Aldrich) by the method described by Gilliam, et al. (21) with minor modification. Anti-ss-DNA antibodies were purified by the procedure as described elsewhere (22). Anti-ss-DNA antibodies were isolated from a pool of 50% ammonium sulphate precipitated filarial plasma using a column of ss-DNA-Sepharose. About 3 mL of precipitated plasma samples was passed through a column of ss-DNA-Sepharose and washed extensively with PBS to remove the nonbound proteins. The bound antibodies were eluted with elution buffer [20 mM sodium carbonate, pH (105), containing 5% dimethyl sulfoxide] and adjusted to pH 70. The reactivity of the purified
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antibodies to a panel of autoantigens (actin, myosin, ssDNA and LPS) and filarial antigens was tested by ELISA.
distribution, median age, Mf status, Mf density, CFA status and CFA density for the different categories.
Frequency of B-1 cell population in human lymphatic filariasis
Affinity determination Relative affinity of antibodies to ss-DNA, actin, myosin, LPS and filarial antigens was determined as described elsewhere (23). Briefly, the ELISA plates were coated with actin, myosin, LPS and filarial antigens in PBS followed by incubation with affinity-purified anti-ss-DNA antibodies from human filarial plasma. The plates were washed with PBS-T and incubated with 2, 4 and 6 M of potassium thiocyanate for 1 h at 37°C. Plates were then washed extensively, and peroxidase-conjugated anti-human IgM was added to each well, and the enzyme activity was quantified using OPD.
Statistical analysis Data analyses were performed using GRAPH PAD PRISM v-3 (Graph Pad Software, Inc., San Diego, CA, USA).Twotailed unpaired Students t-test was used for comparison of two experimental groups. Coefficients of correlation were determined by the Pearson correlation test. Differences were considered as statistically significant when P < 005.
The profile of B-1 cells was evaluated by immunophenotyping. B-1 lymphocytes are a subset of B cells that express the surface marker CD5 together with CD19. Freshly collected blood samples from uninfected and filaria-infected subjects were stained with anti-CD5-FITC and anti-CD19PE and were analysed by flow cytometer. Lymphocytes gated on forward scatter (FSC) and side scatter (SSC) plot were used for quantification of B-1 cells (Figure 1a). Cells that appeared in the upper right quadrant, expressing both CD5 and CD19, are B-1 cells (Figure 1b). The frequency of B-1 cells was quantified from the total B cells. The frequencies of total B cells were not significantly different among the clinical groups (data not shown). Quantitative analysis of B-1 cells in the clinical spectrum of lymphatic filariasis revealed a significant difference in profile of B-1 cells between MF carriers and other clinical groups (Figure 1c). The profile of the B-1 cell population was significantly lower in MF carriers in comparison with EN and people displaying chronic pathology.
IgM antibodies to filarial antigens and autoantigens in clinical manifestations of filariasis
RESULTS Based on the status of microfilaraemia, antigenaemia and chronic disease manifestations, the study subjects were classified into three categories, as described in Materials and Methods. Table 1 shows the number of subjects, sex
Villages in Odisha included Tirumala and Kantia. Samples from Tirumala and Kantia were used for the data shown in this table; AS, asymptomatic microfilariae carriers; CH, subjects with chronic disease; EN, endemic controls (putatively immune subjects from areas of endemicity); CFA, circulating filarial antigen; GMI, geometric mean index for positive subjects; Mf, microfilariae.
As B-1 cells are committed to the production of polyreactive natural antibodies mostly of IgM, the levels of such antibodies that react with a wide variety of autoantigens such as actin, myosin, LPS, ss-DNA and filarial antigens were evaluated in human filariasis. Initially, the total IgM levels in plasma samples of different clinical groups of filariasis were measured. Plasma levels of total IgM in different clinical categories were comparable (Figure 2a). We quantified IgM antibodies reacting to ss-DNA in the spectrum of clinical manifestations. Levels of IgM antibodies to ss-DNA, actin, myosin, LPS and filarial antigens are shown in Figure 2(b). Significantly lower levels of IgM antibodies to ss-DNA were found in microfilaraemic subjects as compared to EN and patients with chronic manifestations. Similarly, levels of IgM antibodies to actin, myosin, LPS and filarial antigens were significantly lower in MF carriers compared to patients with chronic manifestations and EN (Figure 2b). We also assessed IgM antibody levels after antifilarial treatment. IgM antibody levels to ss-DNA, actin, myosin, LPS and filarial antigens in asymptomatic microfilaria positive individuals were increased significantly after treatment compared with untreated plasma (Figure 2c).
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Table 1 Details of the study population in different areas of Odisha endemic for bancroftian filariasis Characteristic
EN (n = 50)
CH (n = 50)
AS (n = 50)
Sex, no. male/no. female Age, median (range), years Mf status, % Mf density, GMI CFA status, % CFA density, GMI Clinical symptoms
41/9
46/4
47/3
26 (8–75)
335 (15–72)
285 (6–65)
0 0 0 0 No
0 0 2 160 Yes
100 153 100 138642 No
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Plasma levels of IL-10 in human filariasis (a)
Regulatory cytokines like IL-10 is known to be secreted by B-1 cells (24). To demonstrate the effect of B-1 cells on IL-10 production, plasma levels of IL-10 of different clinical categories of filariasis were measured. Increased levels of IL-10 were observed in microfilaraemic individuals as well as patients with chronic as compared to endemic normals (Figure 3). IL-10 production was not affected by reduced levels of B-1 cells in microfilaraemic individuals.
Relationship between antibodies to ss-DNA and frequency of B-1 cells
(b)
We demonstrated that during human filariasis, microfilaraemic individuals exhibited a significant decrease in the frequency of CD5+ B cells and developed low levels of IgM antibodies to various autoantigens as well as filarial antigens. These interesting findings led us to study the relationship between profile of B-1 cells and IgM antibodies to ss-DNA in different clinical manifestations. A positive correlation was found between levels of B-1 cells and IgM antibodies to ss-DNA (Figure 4), indicating that CD5+ B cells appear to be involved in the synthesis of polyreactive naturally occurring antibodies in human bancroftian filariasis.
Relationship between levels of IgM antibodies to ss-DNA and autoantigens (c)
Figure 1 Quantitative analysis of B-1 cells in the clinical spectrum of lymphatic filariasis. Freshly collected blood cells from individuals of filarial endemic areas were stained with anti-CD5FITC and anti-CD19-PE antibodies. (a) Lymphocytes gated (R1) on forward scatter (FSC) and side scatter (SSC) plot were examined for CD5 and CD19 co-expression. (b) The gated double-positive (CD5+CD19+) cell population of upper right quadrant were B-1 cells. The cell populations of both upper right and lower right quadrant denote the total B-cell population. (c) Percentage of B-1 cell population was quantified from the total B cells in three different clinical groups of filariasis endemic controls (EN; n = 35, CH; n = 35 and AS; n = 29). Subjects in the AS group had significantly lower levels of B-1 cells than subjects in the other two study populations, that is, EN and CH. (EN vs. AS, P < 001; CH vs. AS, P < 004). © 2014 John Wiley & Sons Ltd, Parasite Immunology, 36, 207–217
Plasma levels of IgM antibodies were found to be significantly lower in MF carriers compared with EN and patients with chronic manifestations. As reactivity to ss-DNA is the hallmark of polyreactive antibodies, plasma of different clinical manifestations was tested for levels of IgM antibodies to ss-DNA and filarial antigens by ELISA. A significant positive correlation was found between levels of IgM antibodies to filarial antigens and ss-DNA (n = 85, r = 0599, P < 00001; Figure 5a). The relationship between anti-ssDNA antibodies and antibodies to actin, myosin and LPS is shown in Figure 5(b–d), respectively. A positive correlation was also found between anti-ss-DNA and anti-actin IgM antibody levels (n = 85, r = 0558, P < 00001; Figure 5b). A similar significant positive correlation was found between levels of anti-ss-DNA antibodies and anti-myosin antibodies (n = 127, r = 0674, P < 00001 (Figure 5c), anti-ssDNA antibody levels and anti-LPS antibody levels (n = 141, r = 0605, P < 00001; Figure 5d).
Cross-reactivity of anti-ss-DNA antibodies The cross-reactivity of anti-ss-DNA antibodies was assessed by inhibition with soluble autoantigens.
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Figure 2 Plasma levels of total IgM antibodies and IgM antibodies to filarial antigens and various auto antigens [ss-DNA, actin, myosin, lipopolysaccharides (LPS)] of different clinical categories of filariasis. (a) Plasma levels of total IgM in endemic controls (EN), CH and AS groups were quantified by commercially available ELISA kit. Plasma levels of total IgM were comparable between different clinical groups of filariasis (n = 20). (b) IgM antibodies to filarial antigen and various autoantigens in EN, CH and AS groups were measured by ELISA. IgM antibodies to ss-DNA, actin, myosin, LPS and filarial antigens were significantly low in plasma of AS individuals as compared to EN and CH. (i) In case of IgM antibodies to ss-DNA, P < 0001 in EN vs. AS and P < 0003 in CH vs. AS; (ii) IgM antibodies to actin, P < 001 in EN vs. AS and P < 00001 in CH vs. AS; (iii) IgM antibodies to myosin, P < 0007 in EN vs. AS and P < 00001 in CH vs. AS; (iv) IgM antibodies to LPS, P < 0001 in EN vs. AS and P < 00001 in CH vs. AS. (v) IgM antibodies to filarial antigen, P < 00001 in EN vs. AS and P < 00004 in CH vs. AS (c) Plasma levels of IgM antibodies to filarial antigen and various auto antigens such as actin, myosin, LPS and ss-DNA of AS individuals (n = 20) were measured before and after antifilarial treatment by ELISA. A significant increase in levels of IgM antibodies to the above antigens was observed after antifilarial treatment.
(c)
Figure 3 Plasma levels of IL-10 of different clinical categories of filariasis. Plasma concentrations of IL-10 in endemic controls (EN), AS and CH groups were quantified by commercially available ELISA kit. Levels of IL-10 were significantly high in plasma of AS and CH groups compared with EN (P < 00001 in EN vs. AS and P < 00001 in EN vs. CH; n = 20).
Single-step absorption of plasma with soluble actin, myosin and LPS reduced anti-ss-DNA IgM antibodies (Figure 6a). Similarly, the reactivity to myosin, LPS and actin was effectively decreased by absorption with ss-DNA (Figure 6b), indicating that most of the ss-DNA-binding antibodies found in human plasma are cross-reactive. Further, the reactivity to ss-DNA, actin, myosin and LPS was effectively decreased by absorption with filarial antigens (Figure 6c), which also indicated that most of the soluble filarial antigen-binding antibodies cross-react with ss-DNA.
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Reactivity of affinity-purified anti-ss-DNA antibodies As naturally occurring polyreactive antibodies have been demonstrated to play a role in innate immunity as well as in immune regulation, the polyreactive property of DNAbinding antibodies was evaluated. Antibodies to ss-DNA were affinity purified from plasma of filarial patients using a column of ss-DNA-Sepharose. Affinity-purified anti-ssDNA antibodies were tested against a panel of antigens to evaluate their polyreactive nature. Affinity-purified © 2014 John Wiley & Sons Ltd, Parasite Immunology, 36, 207–217
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B-1 cells play an important role in the outcome of infection in experimental filariasis and schistosomiasis. Analysis
of profile of B-1 cells in the clinical spectrum of lymphatic filariasis offered interesting leads to understand the role of B-1 cells in the parasitological outcome of filarial infection. Our investigation revealed a significant difference in levels of B-1 cells between MF carriers and other clinical groups. We found that B-1 cell population is significantly low in MF carriers compared with endemic normals and patients with chronic pathology. Mice lacking B lymphocytes were shown to be permissive for Brugian infections (11). Al-Qaoud et al. (13) have shown that BALB.Xid mice support higher L. sigmodontis adult worm and MF burdens compared with wild-type mice. Although, several studies have shown that B-1 cells play an important role in the outcome of infection in experimental filariasis (11, 13) and schistosomiasis (14), the exact role of B-1 cells during human filarial infection is yet to be understood. How B-1 lymphocytes contribute to protection against this large, extracellular metazoan parasite is largely unknown. It could be attributed to the ability of B cells to act as (i) early antigen-presenting cells which might contribute to initiation of T-cell response; (ii) a source of IL10 (24) that suppresses Th1 immune responses, believed to be important in host defence; and (iii) a source of antibodies for mediating humoral immunity against filarial parasites. From the above possibilities, we examined antibody production as well as IL-10 levels. B-1 lymphocytes are committed to the production of polyreactive natural antibodies, mainly IgM, which binds to a variety of selfantigens in humans (25). We found significantly decreased levels of IgM antibodies to various antigens in microfilaraemic individuals. This finding is consistent with the generally dampened immune response commonly seen in microfilaraemic individuals (26). Inability to respond to various antigens may result in survival of MF in this group. It has been demonstrated that mice unable to make naturally occurring antibodies as a result of inheriting the X-linked deficiency are found to be susceptible to schistosomiasis and filariasis (11–13). Jirds are the most susceptible animal models for a variety of human and animal filarial parasites and have been shown to be significantly incompetent in eliciting antibodies against ss-DNA, LPS and phospholipids. In the absence of B-1-derived natural anti-influenza IgM antibodies, delayed T-dependent IgG2a response and increased mortality were reported in murine models (27). Although these studies did not relate directly to naturally occurring antibody responses, the absence of naturally occurring antibodies might play a crucial role in making the hosts susceptible to filarial infection. Furthermore, the data presented in this communication demonstrated that the anti-ss-DNA antibodies observed during filarial infection have multiple reactivity because: (i) a positive correlation was found between antifilarial
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Figure 4 Correlation between percentage of B-1 cell (CD5+CD19+) and plasma levels of anti-ss-DNA IgM antibodies in human filariasis. The IgM antibody levels to ss-DNA of 1/400 diluted plasma were compared with percentage of B-1 cells. Percentage of B-1 cells were significantly correlated with levels of anti-ss-DNA IgM antibodies in human filarial plasma (r = 0548, P < 0000; n = 71).
anti-ss-DNA antibodies were found to react with actin, myosin, LPS and filarial antigens (Figure 7), further confirming the polyreactive nature of anti-ss-DNA antibodies in human filarial plasma. However, there was no significant reactivity of affinity-purified anti-ss-DNA antibodies to human serum albumin (data not shown).
Affinity determination It is evident from the above experiments that the anti-ssDNA antibodies detected in plasma of human filariasis are polyreactive in nature. We examined relative binding of affinity-purified anti-ss-DNA antibodies to ss-DNA, actin, myosin, LPS and filarial antigens (Table 2). These affinity-purified antibodies to ss-DNA from human plasma at a dilution of 1 : 10 have a lower affinity for filarial antigens and other autoantigens. Significant dissociation of the antibody reactivity against ss-DNA, actin, myosin, LPS and filarial antigens occurred only at a much lower concentration of 2 M potassium thiocyanate. These results were further confirmed when the affinity-purified antibodies to ss-DNA were diluted 20 times and tested against various autoantigens. Similarly, significant dissociation of the antibody reactivity was observed at a lower concentration of potassium thiocyanate indicating a lower affinity of purified antibodies to ss-DNA.
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Figure 5 Correlation between plasma levels of anti-ss-DNA IgM antibodies and IgM antibody levels to filarial antigens and various autoantigens such as actin, myosin and lipopolysaccharides (LPS). (a) Levels of anti-ssDNA IgM antibodies were positively correlated with levels of antifilarial IgM antibodies in plasma of different clinical categories (r = 0599, P < 00001; n = 85). (b) A positive correlation between levels of anti-ssDNA IgM antibodies and anti-actin IgM antibodies was observed (r = 0558, P < 00001, n = 85). (c) A similar correlation for levels of anti-ssDNA IgM antibodies and antimyosin IgM antibodies (r = 0674, P < 00001, n = 127) and (d) levels of antissDNA IgM and anti-LPS IgM levels (r = 0605, P < 00001, n = 141) was observed.
antibodies and anti-ss-DNA antibodies in plasma of both normal and infected individuals; (ii) the antibody reactivity to DNA could be effectively absorbed from plasma by pre-incubation with soluble actin, myosin, filarial antigens; and (iii) the antibodies eluted from ss-DNA-Sepharose column reacted with various antigens. CD5+ B lymphocytes have been shown to be preferentially involved in the synthesis of naturally occurring antibodies. Monoreactive, high affinity autoantibodies of the IgG class are produced by CD5 B cells and low affinity polyreactive autoantibodies arise from the B-1 subset. It has been shown that the polyreactive autoantibodies normally produced by CD5+ B cells differ functionally from the monoreactive autoantibodies that are characteristic of various autoimmune diseases (25, 28). We demonstrated cross-reactivity of affinity-purified antibodies to filarial
antigens as well as to various autoantigens such as actin, myosin tubulin and LPS. Thus, they appear to be nonpathogenic antibodies. Mice deficient in circulating IgM but otherwise normal in their humoral responses manifest a significant impairment in worm elimination (8). Further, the substantial deficit of IgM in Xid mice could account for the delayed clearance of infection (8). Although lack of B-1 cells increases filarial worm burden in animal models (13), the down-regulation of B-1 cells and IgM antibodies may prevent helminth-induced pathology. The observed low levels of IgM antibodies in microfilaraemic cases may not eliminate parasites in the host but increase susceptibility to infection. Interestingly, treatment of microfilaraemic patients with antifilarial drugs significantly increased IgM antibodies to various autoantigens. These observations pointed towards the possibility of naturally
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Figure 7 Polyreactivity of affinity-purified ss-DNA antibodies. Pooled plasma of different clinical categories was precipitated with 50% ammonium sulphate and passed through a column of ss-DNA-Sepharose. The bound immunoglobulins were eluted with elution buffer (20 mM sodium carbonate, PH (105), containing 5% vol. dimethyl sulfoxide. Reactivity of the purified antibodies against ssDNA, actin, myosin, lipopolysaccharides, tubulin and filarial antigen was evaluated.
Figure 6 (a) Levels of anti-ss-DNA IgM antibodies in plasma were absorbed with actin, myosin and lipopolysaccharides (LPS). Ten plasma samples were absorbed with soluble actin, myosin and LPS, and then, anti-ss-DNA IgM antibody levels were measured by ELISA. Pre- and postabsorption IgM levels were compared. (b) Filarial plasma was pre-absorbed with soluble ss-DNA, and then, IgM levels against actin, myosin and LPS were measured by ELISA. (c) Filarial plasma was pre-absorbed with soluble ssDNA, actin, myosin, and LPS, and then, IgM levels against filarial antigens were measured by ELISA.
occurring polyreactive antibodies playing a significant role in the parasitological outcome of exposed population. Filarial parasites polarize host immune response towards the Th2 phenotype with consequent suppression of T-cell proliferation and are important in maintaining parasitaemia. The IL-10 production has long been associated with filarial infection wherein the parasites down-regulate the host immune response to establish infection by inducing IL-10-mediated T-cell suppression (26). Animal models of filarial infections have demonstrated that depletion of IL-10 or introduction of IL-10 deficiency accelerates worm clearance (26, 29). Several studies have shown that high levels of spontaneous and parasite antigen-driven IL-10 production are associated with antigen-specific hyporesponsiveness in human lymphatic filariasis (29). Peripheral blood mononuclear cells (PBMC) of microfilaraemic individuals have been shown to release higher levels of IL-10 than in patients with chronic pathology. In this study, increased plasma levels of IL-10 and decreased frequency of B-1 cell population were detected in microfilaraemic individuals. IL-10 production appears not to be affected by reduced levels of B-1 cells in microfilaraemic individuals. It may be possible that despite the drop of overall B-1 cells, microfilaraemic patients have an increased cell numbers in an IL-10 producing B-cell subset. Several other cell types including CD4+CD25+ and CD4+CD25 regulatory T cells, Th1 CD4+ T cells, CD8+ T cells, monocytes/macrophages, NK cells have been shown to involve in enhanced IL-10 production (30).
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Table 2 ELISA: affinity determination of affinity-purified anti-ss-DNA antibodies to ss-DNA, actin, myosin and lipopolysaccharides (LPS) Absorbance at 492 nm Autoantigensa
Autoantigensb
Treatment
ss-DNA
Actin
Myosin
LPS
Filarial antigen
ss-DNA
Actin
Myosin
LPS
Filarial antigen
None 2 M KSCN 4 M KSCN 6 M KSCN
0866 0232 0121 0126
0434 0146 0061 0098
0504 0350 0252 0234
0314 0104 0060 0030
2019 0623 0411 0248
0515 0134 0115 0104
0257 0067 0028 0034
0330 0264 0234 0220
0124 0033 0007 0057
1009 0422 0348 0187
KSCN, potassium thiocyanate; a10-fold diluted sample against autoantigens; b20-fold diluted sample against autoantigens.
The mechanism by which B-1 cells and polyreactive IgM antibody production was down-regulated in microfilaraemic patients is largely unknown. IL-5 induces differentiation of activated conventional B (B-2) cells into immunoglobulinsecreting cells and induces the growth of progenitors of CD5+ B (B-1) cells and IgM production by B-1 cells (31). It has been shown that mice constitutively overexpressing IL-5 have an increased eosinophilia that causes a more rapid clearance of L. sigmodontis (32). Conversely, the parasites survive and reproduce well beyond their normal lifespan in IL-5 KO mice (33). As IL-5 responses were reported to be low in microfilaraemic individuals, low levels of IL-5 may enable these individuals to down-regulate B-1 cells and polyreactive IgM antibody production. The demonstration of the polyreactive property of anti-ss-DNA antibodies in this study indicates an important role of these antibodies to provide host protection against filarial infection. As naturally occurring polyreactive antibodies have been demonstrated to play a role in innate immunity as well as in immune regulation, our laboratory is now directed towards investigating the role of CD5+ B-1 cells in polarizing cytokine responses by filarial parasites, an area which has not been explored so far.
ACKNOWLEDGEMENTS The authors are grateful to the patients who participated in the study. We thank Dr. S.K. Kar, the Director of Regional Medical Research Centre, Bhubaneswar, for sustained support and for providing the facilities for conducting the work.
FUNDING This work was supported by Department of Science & Technology, Govt of India: SR/SO/HS 2008 (IFD diary no.100/IFD/10887/2009-12010); The Regional Medical Research Centre in Bhubaneswar is funded by the Indian Council of Medical Research, New Delhi.
AUTHOR CONTRIBUTIONS AKS: conceived and designed the experiments. RM: performed the experiments. SM and KG: involved in the acquisition of data. PKS, BD and AKS: analysed the data. SKK revised the manuscript. RM and AKS: wrote the paper.
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