The pathogenesis of filarial disease is characterized by acute and chronic inflammation. Inflammatory responses are thought to be generated by either the parasite, the immune response, or opportunistic infection. We show that soluble extracts of the human filarial parasite Brugia malayi can induce potent inflammatory responses, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and nitric oxide (NO) from macrophages. The active component is heat stable, reacts positively in the Limulus amebocyte lysate assay, and can be inhibited by polymyxin B. TNF-α, IL-1β, and NO responses were not induced in macrophages from lipopolysaccharide (LPS)-nonresponsive C3H/HeJ mice. The production of TNF-α after chemotherapy of microfilariae was also only detected in LPS-responsive C3H/HeN mice, suggesting that signaling through the Toll-like receptor 4 (TLR4) is necessary for these responses. We also show that CD14 is required for optimal TNF-α responses at low concentrations. Together, these results suggest that extracts of B. malayi contain bacterial LPS. Extracts from the rodent filaria, Acanthocheilonema viteae, which is not infected with the endosymbiotic Wolbachia bacteria found in the majority of filarial parasites, failed to induce any inflammatory responses from macrophages, suggesting that the source of bacterial LPS in extracts of B. malayi is the Wolbachia endosymbiont. Wolbachia extracts derived from a mosquito cell line induced similar LPS-dependent TNF-α and NO responses from C3H/HeN macrophages, which were eliminated after tetracycline treatment of the bacteria. Thus, Wolbachia LPS may be one of the major mediators of inflammatory pathogenesis in filarial nematode disease.

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The susceptibility of Brugia malayi and Onchocerca lienalis microfilariae to H2O2 and NO either in cell-free culture or from IFN gamma-activated macrophages was examined. In cell-free culture, O. lienalis microfilariae were highly susceptible to H2O2 induced toxicity, exhibiting rapid reductions in motility and viability. The addition of exogenous catalase abrogated H2O2-induced killing. In contrast, B. malayi microfilariae were relatively resistant to H2O2, with concentrations as high as 50 microM having no effect on motility or viability. On exposure to NO, both species showed reductions in motility within 5-30 min, but longer was required to see effects on the viability of microfilariae. Parasites incubated with IFN gamma-activated macrophages also exhibited marked reductions in motility and viability. In cultures with B. malayi and activated macrophages, inhibition of these effects was achieved by the addition of either L-NMMA, to abolish NO production, or neutralizing anti-TNF alpha antibodies. Attempts to inhibit parasite killing by the addition of catalase to macrophage cultures were ineffective. The results of this study show that B. malayi and O. lienalis microfilariae have different susceptibility to H2O2, but are equally affected by exposure to NO. Moreover both species are killed by IFN gamma-activated macrophages and in the case of B. malayi, killing is dependent on the generation of NO via TNF alpha.

Lymphatic filarial nematodes are infected with endosymbiotic Wolbachia bacteria. Lipopolysaccharide from these bacteria is the major activator of innate inflammatory responses induced directly by the parasite. Here, we propose a mechanism by which Wolbachia initiates acute inflammatory responses associated with death of parasites, leading to acute filarial lymphangitis and adverse reactions to antifilarial chemotherapy. We also speculate that repeated exposure to acute inflammatory responses and the chronic release of bacteria, results in damage to infected lymphatics and desensitization of the innate immune system. These events will result in an increased susceptibility to opportunistic infections, which cause acute dermatolymphangitis associated with lymphoedema and elephantiasis. The recognition of the contribution of endosymbiotic bacteria to filarial disease could be exploited for clinical intervention by the targeting of bacteria with antibiotics in an attempt to reduce the development of filarial pathology.