Demyelination and microglia activation were observed in brain tissue of mice during A. cantonensis infection
After A. cantonensis infect mice, they could invade body through blood circulation. Larvae III of A. cantonensis move into intestinal mucosa of small intestine, migrate into liver and then lung, ultimately arrive into brain, cause severe central nervous system damage, the most severe damage is myelin sheath. Transmission electron microscope (TEM) images of corpus callosum showed obvious demyelination at 14 days post infection (14 dpi) and this damage was more pronounced at 21 dpi. Myelin sheath structure became incompact and thickness continuously decreased. The myelin G ratio, in infected animals, was higher than their controls, which is contrary to myelin thickness. And axon arrangement also became disordered (Fig. 1a). A. cantonensis infection induced not only demyelination injury but also inflammatory cytokine storm. Earlier research showed brain damage due to mechanical damage caused by parasite movement, but recent evidences indicate inflammatory injury may be more important [17].
Flow cytometric analysis of brain immune cell revealed increased infiltration of blood-borne cells (CD45+) after 7 dpi in infected mice. Although leukocytes were increased, the number of microglia did not show prominently change (Fig. 1b). Even so, microglia occupy the main position of inflammatory cells in the brain, we detected the expression pattern of cell type that might be involved in eosinophils recruitment. Real-time PCR result showed increased levels of markers for M1 (CD86, iNOS, CD11b) and M2 (CD206, Arg-1, YM1) microglia, with the highest levels at 21 dpi (Fig. 1c). We also detected the protein levels of iNOS and Arg-1 of infected brain tissue. iNOS expression persistently increased and peaked at 21 dpi, whereas Arg-1 was highly expressed beginning at 14 dpi and continued to increase until 21 dpi (Fig. 1d). Double immunofluorescence for Iba-1/iNOS and Iba-1/Arg-1 showed obvious increases in iNOS and Arg-1 expression in brain tissue, especially around corpus callosum (Fig. 1e). Collectively, these results indicate that A. cantonensis infection could activate both M1 and M2 microglia.
IL-17A and IL-17RA expression level obviously rose in brain after A. cantonensis infection
To evaluate the impact of inflammatory factor on the brain, we compared the level of interleukin in A. cantonensis-infected versus WT mice injected with normal saline. Among several types of interleukin, we focused on IL-17A as it had been implicated in EAE and multiple sclerosis (MS) [18]. We previously described the level of some interleukins increased during A. cantonensis infection [19], but we did not explore their effect to the brain damages and inflammatory responses. The transcriptional level of IL-17A and IL-17RA in brain were greater in infected mice than in control mice treated with saline (Fig. 2a). IL-17A protein production was assessed after infection, but was detectable at low amounts in the first seven days. Intracellular cytokine staining (ICS) confirmed that A. cantonensis induced IL-17A production, the content of IL-17A increased with lastingness of infection (Fig. 2b).
Furthermore, to determine the location of IL-17RA within the brain, we examine intact brain sections of mice using immunofluorescence. Some IL-17RA were observed on the oligodendrocyte, especially on the axon, this change was particularly pronounced at 14 dpi (Fig. 2c upper). IL-17A influences oligodendrocyte lineage cell proliferation and differentiation through multiple pathways mediated by IL-17RA in inflammatory disease[20]. It is known microglia as a type of macrophage can secret IL-17, and in the meanwhile, it can be active by IL-17, but it was unclear whether IL-17 plays a role during A. cantonensis-infection. To clarify the relationship between IL-17 and demyelination, microglial IL-17RA expression was also detected with immunofluorescence double labeling. IL-17RA was expressed in Iba-1-positive microglia (Fig. 2c inferior), suggesting a possible link between IL-17A and microglia. Whereas another important glia cell—astrocyte,which is indicated by GFAP༌was devoid of IL17RA (data not shown). It has previously reported microglia produce IL-1β, IL-6 and TNFα when cocultured with IL-17 producing Th1/Th17 cells, and then promote tissue damage[21]. Demyelination injury as typical injury of A. cantonensis infection, partly because the attack of direct damage of IL-17A on oligodendrocyte, and may also due to the microglia effects.
γδ T cell is the major source of IL-17A in A. cantonensis infected mice brain
To determine the source of IL-17A in infected mice brain, we measured several types of cell that produce IL-17, finally found γδ T cells are the major source of IL-17A. High diversity of γδ TCR, MHC-independent and antigen-independent process and presentation suggest that γδ T cell can be the first line of defense against infection. γδ T cells often appear in mucosal immunity but rarely in CNS neuroimmunity, similar to IL-17A, this cell is most extensively studied in stroke and EAE [22]. There were only a few γδ T cells detected in the normal mice brain. The frequency of brain γδ T cells was obviously increased after infection, as compared to that in sham operated mice (Fig. 3a). Approximately 60% of γδ T cells from infected mice brain expressed IL-17A. After 14dpi, IL17+ γδ T cells accounted the majority of IL17+ leukocyte (Fig. 3b). It has been reported A. cantonensis infection led to the immunosuppression of mice, the level of several important cytokines decreased, the total number of B and T cells declined at 21dpi [23], nevertheless, infection status of the brain continued worse, it may indicate a class of cells including γδ T cells still did the damage. The relation of γδ T cell with eosinophil had been studied, these two cells have synergistic effect in allergic response[24], eosinophilic meningeal encephalitis caused by A. cantonensis may also have a γδ T cell impact.
To determine whether the functional γδ T cells are directly from peripheral lymphoid organs or finish the differentiation in the leision, we detected the level of IL17+ γδ T cells in the thymus and spleen. Thymus is the birthplace of T cells, γδ T cells are different with αβ T cells, they may acquire the functions with the development of thymus[11]. We tested the level of γδ T cell and IL17+ cell in the thymus during infection, the result was different with brain, γδ T cells number maintained a high level even in wild type mice, and had a peak around 7 dpi, whereas IL17+ γδ T cells did not change appreciably even in the later phase of infection (data not show). Moreover, thymus morphology showed evident atrophy at 21dpi. Spleen as another lymphoid organ represented immunosuppressive status after infection, both γδ T cell (data not show) and IL17+ γδ cell numbers sharply fell at 21dpi. IL17+ γδ T cell merely rose at 7 dpi, which may be account of small intestinal infection (Fig. 3c). In order to acquire better survival environment, A. cantonensis suppress the body immunity function along with prolonged time. Moreover, we speculated γδ T cell owned corresponding function after migrated to the brain lesions caused by A. cantonensis.
Microglia Activation Weakened And Demyelination Relieved After IL-17 Neutralization
IL-17A is key factor involved in promoting the survival, recruitment and activation of other inflammatory cells via the regulation of cytokines and chemokines expression in several neuroimmune responses. Our above-mentioned data demonstrated that IL-17A expression is also outstanding in the A. cantonensis-infection, to test the real impact of IL-17A, we neutralized IL-17A by injecting specific blocking monoclonal antibodies (mAbs) through the intraperitoneal route. Both LFB (Luxol Fast Blue) staining and TEM were applied to examine whether demyelination condition improved, and myelin sheath thickness was estimated with measurement software. Previous results showed after 21 dpi of A. cantonensis infection, LFB staining of the mouse cerebral medulla decreased, indicating demyelination. While IL-17A neutralizing antibody was applied, the color of cerebral medulla was obviously darker, suggesting less demyelination. TEM results also support this conclusion. IL-17 neutralizing antibody treatment had no effect on myelin sheath thickness in normal mice (Fig. 4a). Because myelin damage can cause unpaired motor function, the neurobehavioral scores of mice were evaluated in each group. We found A. cantonensis-infected group got lower scores than WT group, whereas IL-17 neutralizing antibody attenuated this decrease (Fig. 4b). Given the relationship between demyelination and neurobehavioral scores, we proposed that IL-17A damage effect on myelin during infection cannot be ignored.
Previous research presented that inhibiting activation of microglia by minocycline can efficaciously relieve the injury from A. cantonensis [25]. Microglia maintain the steady statue of immune microenvironment of brain, the mRNA expression levels of CD86, iNOS, CD11b, Arg-1and YM1 were accordingly affected by IL-17 neutralization (Fig. 4c). On protein level, iNOS and Arg-1 had no alteration in the control mice after IL-17 inhibition but decreased in the A. cantonensis infected mice treated with the inhibitor (Fig. 4d). Moreover, the active state of microglia generally performs amebiform, this phenomenon also vanished by contrast (Fig. 4e). These findings implicate microglia inhibition in the ameliorating effect of IL-17 neutralizing antibody on demyelination caused by A. cantonensis infection.