Pathogenic helminths have the ability to downregulate the immune responses of their hosts (14
). Indeed, helminths have been shown to ameliorate autoimmune diseases in several animal studies (24
), and in humans, egg preparations of the pig whipworm (Trichuris suis
) have been used successfully to treat severe inflammatory bowel disease (35
Whereas most of these studies have focused on the effects helminths have on the adaptive immune response, in this study we evaluated the effects helminths have on the innate immune system. Through the use of an LPS sepsis model, we have demonstrated that helminths substantially alter innate immune responses and that the type of immune modulation observed is specific to the stage of the parasite.
One of the most striking findings in our study is that female adult worms from prepatent infections (microfilaria negative) seemed to diminish the inflammatory response induced by LPS challenge. Implantation of female adult worms from prepatent infections resulted in decreased concentrations of the proinflammatory cytokines IFN-γ, TNF-α, and IL-12p40 in the peripheral blood samples of mice compared to the levels for sham-treated controls after LPS challenge. Therefore, we suggest that the implanted worms initiated an immunosuppressive milieu in mice which dampened the immune response to LPS. Possible mechanisms for this phenomenon include induction of higher frequencies of regulatory T cells, development of alternatively activated macrophages, diminished antigen presentation, and a more pronounced Th2 immune response. All of these mechanisms are known to be provoked by helminth infections, and all have the potential to reduce proinflammatory immune responses to LPS. Another possible explanation for the protective effects that prepatent female adult worms had on LPS challenge is the release of helminth products that directly impede the immune response triggered by LPS, as earlier studies have demonstrated that helminths can secrete products that reduce the responses of Toll-like receptors (TLRs) to LPS. Soluble extracts from Brugia malayi
and Schistosoma mansoni
reduced the TLR4 expression on macrophages and the ability of TLRs from dendritic cells to respond to LPS (21
). Further studies with female adult L. sigmodontis
worms will investigate these possible protective mechanisms with respect to LPS-induced sepsis.
Interestingly, the implantation of female adult worms from postpatent infections (adult female worms which no longer release microfilariae into the peripheral blood samples of their hosts) or of male adult worms seemed not to decrease the proinflammatory response induced by LPS challenge. The loss of the beneficial effect seen in mice infected with female adult worms from prepatent infections after LPS challenge was probably due to the fact that the female adult worms from postpatent infections still released a few microfilariae, which were detected in peritoneal smears but not in the peripheral blood. Alternatively, this finding could be due to the higher ages of the worms and decreasing abilities of immunomodulation. While we can only speculate, the lack of a protective response from adult male worms suggests that perhaps adult female worms release immunomodulatory substances to protect microfilariae from immune destruction.
In contrast to the observed protective effects associated with implantation of adult female worms from prepatent infections, the presence of microfilariae in the peripheral blood, following either direct injection or implantation of microfilaria-releasing female adult worms, resulted in a marked reduction of survival after LPS challenge. This increased mortality after LPS challenge appears to be due to an augmentation in the inflammatory response toward LPS in the setting of microfilariae. Specifically, the presence of microfilaremia was associated with increased levels of proinflammatory cytokines and chemokines and with a greater reduction in body temperature in response to LPS challenge than for uninfected mice.
After LPS challenge, concentrations of TNF-α and IL-1, which play central roles in the innate immune response to LPS (2
), were significantly greater in microfilaria-infected mice than in control mice. In addition, the most abundant cytokine in the peripheral blood samples of microfilaremic mice after LPS injection was IL-6, which enhances the acute-phase response of the liver and is a marker for sepsis that correlates with severity of inflammation (28
The fatal course of disease observed when microfilaremic mice were given a sublethal dose of LPS may be due to IFN-γ and IL-12 induced by microfilariae. A single injection of microfilariae resulted in a Th1-type immune response characterized by elevated numbers of monocytes, granulocytes, and NK cells in the peripheral blood and higher plasma concentrations of IFN-γ, IL-12, and MIG. These results are similar to those reported earlier by Lawrence et al. for B. malayi
), a related filarial nematode. In B. malayi
infections, a serine protease inhibitor (serpin, BM-SPN-2) was shown to be partly responsible for IFN-γ production by host cells (42
), suggesting that in our model specific microfilarial antigens may have been responsible for increases in IFN-γ.
The elevated levels of IFN-γ could also have been driven by Wolbachia
organisms, intrahelminthic bacteria present in L. sigmodontis
. These endosymbiotic organisms, which have been shown to be released along with microfilariae by mature worms or by dying microfilariae, induce proinflammatory immune responses (11
). Further, the Wolbachia
surface protein WSP has been shown to induce IL-12 production by acting through TLR2 and -4 (4
). However, fatal effects mediated by microfilaria-derived components other than Wolbachia
products have been demonstrated using filarial species that do not contain Wolbachia
). In addition to direct stimulation of cytokine production by microfilariae or Wolbachia
, it is also possible that the higher concentrations of IL-6, IL-12, TNF-α, MCP-1, and MIP-1α observed in LPS-challenged mice infected with microfilariae were due to the elevated numbers of monocytes induced by the microfilaremia.
In summary, we think that the lethal outcome of LPS-induced sepsis in microfilaria-positive mice was due to increased numbers of monocytes, granulocytes, and NK cells in the peripheral blood induced in response to the microfilariae. In addition, IFN-γ, which was elevated either due to Wolbachia
or due to the microfilaria itself, is able to upregulate the expression of TLR4 on monocytes, increase phosphorylation of IL-1 receptor-associated kinase, and enhance the DNA-binding capacity of NFκB, all of which results in increased IL-12 and TNF-α secretion by monocytes (3
). These actions further activate monocytes, natural killer cells, and neutrophils (2
) and can also be contributing to the strong LPS-induced inflammatory reaction observed in microfilaria-infected mice.
The usage of IFN-γR and TNFR1 knockout mice showed that the lethal course caused by LPS injection in microfilaria-infected mice was mediated by IFN-γ and TNF-α. Although we performed only a single experiment, with just three microfilaria-infected IFN-γR knockout mice, they showed the lowest measured cytokine levels after LPS challenge and relatively little decrease in body temperature, which likely enabled survival. Similarly, the survival rate of TNFR1 knockout mice infected with microfilariae was significantly greater than that of control microfilaria-infected mice.
The defect of MARCO, an LPS binding type A scavenger receptor expressed on macrophages, dendritic cells, and microglia cells (15
), seemed not to ameliorate the course of disease after LPS challenge of microfilaria-infected mice. Microfilaria-infected MARCO knockout mice had neither significantly increased body temperatures nor significantly decreased concentrations of IFN-γ, TNF-α, IL-12p40, and IL-6 compared to the immune-competent mice after LPS challenge, resulting in the deaths of three out of five infected MARCO knockout mice after LPS challenge. These findings suggest that MARCO may have a slight influence on LPS-induced sepsis but that other surface molecules that sense LPS, like TLR4, CD11b/CD18, and ion channels (10
), may be responsible for the observed lethal outcome after LPS injection.
The fatal outcome observed in our microfilaria-infected mice and in our animals implanted with female worms from patent infections does not mirror the expected outcome of sepsis under the conditions of a natural filarial infection. In contrast to our implantation experiments where microfilariae are released immediately in large numbers, the time course following natural infection allows the developing worms to down-modulate the immune response before they release microfilariae, thereby preventing an overwhelming immune response at this life cycle stage. When this regulated condition cannot be achieved, severe pathology, such as sowda in onchocerciasis or tropical pulmonary eosinophilia in lymphatic filariasis, may occur. While L. sigmodontis
infection in mice does not model the lymphatic damage observed in human filariasis, as the L. sigmodontis
adults reside in the pleural space and not in the lymphatics, it can be speculated that the presence of worms and their immunomodulatory effects may affect the outcome of disease in lymphatic filariasis as the modulation of the immune response by adult female worms from prepatent infections may reduce inflammatory responses in the host. The observation of two polar groups in lymphatic filariasis (30
), with one group having abundant microfilariae in circulation, but no symptoms or chronic pathology, and another group having elephantiasis and an absence of microfilariae, may therefore be possibly explained by successful immunosuppression by the helminths in the first group and a possible lack of immune modulation in the second.
Pathological effects due to a failure of immune down-regulation have also been observed in coinfections of BALB/c mice with L. sigmodontis
and Plasmodium chabaudi
where severe malaria occurred in animals that did not develop a patent filarial infection (13
). Moreover, these animals showed high IFN-γ levels, indicating a strong Th1-type response. Although the authors did not state whether the strong immune response observed in some of the coinfected animals was the cause or the consequence of the absence of circulating microfilariae, one can speculate that in mice with patent filariasis down-modulation of the immune response by female filariae may facilitate establishment of microfilaremia as well as prevent severe clinical manifestations of malaria.
Because our study utilized a model of LPS injection to study sepsis, we cannot conclude that infections with female adult worms from prepatent infections would protect against bacterium-induced sepsis. Theoretically, a downregulation of inflammatory responses by helminth parasites could be associated with an increased susceptibility to proliferating bacteria. Such a phenomenon was observed in coinfection studies with the Th2-inducing rodent helminth Heligmosomoides polygyrus
and the bacterium Citrobacter rodentium
In summary, we have demonstrated that different filarial life cycle stages of the filaria L. sigmodontis have differential impacts on LPS-induced inflammation in a murine sepsis model. While premature adult worms diminished the inflammatory response induced by LPS challenge in mice, microfilariae on the other hand worsened the course of LPS challenge. The field of immune modulation by helminths is an active one, with several research groups seeking to find which helminth antigens are responsible for immune modulation and some current studies even demonstrating success in treating autoimmune diseases with helminths. On the basis of our findings in this study, we suggest that future work for trying to determine the antigens responsible for helminth-induced downregulation should focus on molecules expressed in adult female worms and not in microfilariae.