The Effects Of Specific Immune Response On The Development Of Worms And Granuloma Formation In Mice Infected With Schistosoma Japonicum

Traditional opinion concerning the relationship between hosts and parasites is thatparasites damage their hosts and meanwhile the hosts bring up resistance against theinvading parasites through activation of immune response.However, some new study evidences have showed that the growth and oviposition ofschistosome was impeded in immune suppressed or immune deficient host. It suggests thatthe effect of host immune responses to schistosome infection is not only resistance and thereaction of schistosome to host immune attack is more than escape.It's our assumption that during the long period of co-evolution of schistosome with itshost, it is possible that schistosome has developed adaptation to the inner environment of itshost and even receives some host-derived immune signals to complete its own development.However, it is still not clear that which type of immune response or immune factors mightfacilitate the growth and development of the invading parasites.So far the rationale of research on schistosome vaccine is based rather narrowly on thetraditional opinion that host's immune response to schistosome infection is exclusivelyagainst the development of the parasite. However, this rationale may ignore the possibilitythat some immune factors in host might be benefit to the parasite. Thus, renewing theimmumological opinion on schistosome infection is m¹³ uch important. The purpose of this study is to explore the impact of a deficient immune system on thedevelopment of schistosoma japonicum and granuloma formation. We observe the growthand egg-laying of S.japonicum in a mouse model of specific immune deficiency, andaccordingly clarify the effects of those immune factors on the development of worms andgranuloma formation.The main contents of this thesis include:Part one. The effects of T cell on the development of worms and granuloma formation inmice infected with Schistosoma japonicumPart two. The effects of Signal transducers and activators of transcription (Stat)-4 andStat-6 on the development of worms and granuloma formation in mice infectedSchistosoma japonicumPart three. The effects of cytokines Interleukin (IL)-12 and IL-4 on the development ofworms and granuloma formation in mice infected Schistosoma japonicumPart oneThe effects of T-cell deficiency on the development of worms andgranuloma formation in mice infected with Schistosoma japonicumNude mice have a mutated homozygous recessive Nu gene and lack a thymus which isessential for the production of T cells. These mice are unable to mount most types ofimmune responses, which allowed us to explore the impact of a T cell-deficient immunesystem on the development of the parasites and the granuloma formation.Materials and methodsIntact BALB/c mice and nude mice in BALB/c background were infected with 25S.japonicum cercariae. The growth and development of the parasites and host tissuepathological change were compared between two groups 28, 35 and 42 days post infection. Worms were collected from the hepatic portal system by perfusion with physiologic saline.The total number of worms and the mean number of worm pairs were counted under adissecting microscope. After fixed by 10%formalin, all worms were stained by carmine andobserved under a compound microscope. Digital images of each worm were obtained usinga digital camera. Parasite length was measured from the digital images using "imagepro5.0"software. Female worms were examined under the microscope and the eggs in thegravid uteri were counted.Infected mice at day 42 post infection were evaluated for liver granuloma formationinduced by eggs. Liver was removed and the right lobe was fixed in 10% bufferedformaldehyde, embedded in paraffin and cut into sections. The sections were stained withhaematoxylin and eosin (HE). Representative HE-stained liver sections from each animalwere scanned under microscope. For every granuloma containing a single egg, thegranuloma size was determined by the measurement of the diameter at themid-transmiracidial level of the egg. The remaining part of the liver was digested in 5%KOH for counting the total number of eggs per liver and calculating the liver eggs perworm pair.Results were expressed as mean±standard deviation ((?)±s). Student's two-tailed t-testwas used to determine the differences between the experimental and control groups.ResultsThere were no significant differences in worm burden between BALB/c mice andnude mice 28, 35 and 42 days post infection.The mean parasite length of either female or male worms in nude mice was foundsignificantly shorter than that in normal BALB/c mice 28 days after infection (p＜0.01).This difference lessened 35 days post infection (p＞0.05). And the mean parasite length forboth groups was found close to each other 42 days post infection.The number of eggs per worm pair in the liver of nude mice remained significantly less than that in BALB/c mice 28, 35 and 42 days post infection(p＜0.05). The number ofuterine eggs of ovigerous females in nude mice was always slightly lower than in BALB/cmice but, none of the differences was significant (p＞0.05). All female worms collectedfrom BALB/c mice at 28, 35 and 42 days after infection contained eggs in their uteri. Bycontrast, in nude mice, a few female worms had no eggs at 28 days (28.5%) or 35 days(4.7%) post infection.At day 42, histological examination showed that the granuloma size in nude mice wassignificantly smaller than that in BALB/c mice (p＜0.01). The cellular infiltration withingralunomas in nude mice was much less than that in normal BALB/c mice. A significantlyselective diminution in the number of eosinophils within the granulomas was noted in nudemice. In addition, a large area of hepatocytes necrosis in vicinity of eggs was observed innude mice. But this hepatocytes necrosis was minimal in normal BALB/c mice.ConclusionThis study indicates that the survival of S.japonicum within the host would not besignificantly affected by T cell deficiency of nude mice. Maturation could still be reached.But, maturation and oviposition of the parasites could be delayed in nude mice. It suggeststhat T cell induced immune response and related immune factors may be benefit for themigration or development of the worms. Through the dynamic observation, we found theeffects on the development of worms was temporary and especially exerted on the early stagy ofinfection.The deficiency of T cell dependent immune mechanisms could impair granulomaformation, especially the eosinophilia infiltration in the granuloma which is associated withinfection by S. japonicum. Part twoThe effects of Signal transducers and activators of transcription (Stat)-4and Star-6 on the development of worms and granuloma formation inmice infected Schistosoma japonicumRecent studies indicated that the differentiation of CD4⁺T cells in S. japonicum infectedmice, to Th1 cells or Th2 cells is crucially important for host protective immune responseand eggs induced granuloma immunopathology.In this part, we studied the development and fecundity of S. japonicum in Stat4^-/- miceand Stat6^-/- mice beginning 4 weeks after infection and accordingly explore the effects ofTh1-type immune deficiency and Th2-type immune deficiency on the development of S.japonicum and the granuloma formation respectively.Materials and methodsIntact BALB/c J mice and Stat4^-/-, Star6^-/- mice in BALB/c background were infectedwith 25 S japonicum cercariae. The growth and development of the parasites and host tissuepathological change were compared between two groups 28, 35 and 42 days post infection.Worms were collected from the hepatic portal system by perfusion with physiologic saline.The total number of worms and the mean number of worm pairs were counted. All wormswere stained by carmine and parasite length was measured from the digital images. Femaleworms were examined under the microscope and the eggs in the gravid uteri were counted.Infected mice at day 42 post infection were evaluated for liver granuloma formationinduced by eggs. Liver was removed and the right lobe was fixed in 10% bufferedformaldehyde, embedded in paraffin and cut into sections. The sections were stained withhaematoxylin and eosin (HE). Representative HE-stained liver sections from each animalwere scanned under microscope. For every granuloma containing a single egg, thegranuloma size was determined. The remaining part of the liver was digested in 5% KOH for counting the total number of eggs per liver and calculating the liver eggs per worm pair.Results were expressed as mean±standard deviation ((?)±s) and analyzed bySPSS 12.0. The data were analyzed using one-way analysis of variance (ANOVA).ResultsNo significant difference was found in the total number of worms and number ofworm pairs among Stat4^-/-, Stat6^-/- and BALB/c J mice at 28, 35 and 42 days post infection(p＞0.05). The parasite length among the three groups was also not significantly different(p＞0.05). The number of eggs per pair of worms in the liver of the three groups was notsignificantly different at 28 days post infection. However, this number in the livers ofStat4^-/- and Stat6^-/- mice was slightly higher than that in BALB/c J mice but still notstatistically significant at 42 days post infection (p＞0.05). The tendency of the number ofuterine eggs of ovigerous females in three groups was similar to the results of the liver eggsat all time points. The fibrosis around eggs in the liver was increased in Stat4^-/- mice. Theliver granulomas in Stat6^-/- mice were not well formed. No significant difference ingranuloma size around the single eggs in the liver was found among the threegroups(p＞0.05).ConclusionThe results indicate that the Th1 or Th2 immune deficiency has no conspicuous effecton the survival, development and fecundity of S. japonicum. Th1 immune deficiencyfacilitated the egg induced granuloma firosis. This suggests that Th1 immune responsecould inhibit the liver fribrosis which is induced by eggs. The deficiency of Th2 immuneresponse could impair granuloma formation. It suggests that Th2 immune response play animportant role for the granuloma formation. Part threeThe effects of cytokines Interleukin (IL)-12 and/or IL-4 deficiency on thedevelopment of worms and granuloma formation in mice infectedSchistosoma japonicumCytokines present during the primary stimulation were found to have an importantinfluence on naive Th cell differentiation. Differentiation of na(i|¨)ve CD4⁺ Th cell isreciprocally regulated by IL-12 and IL-4. IL-12 was shown to directly promote thedevelopment of Th1 cells, whereas IL-4 was found essential for the development of Th2cells. A great deal of research effort on schistosomiasis has also focused on the effects ofthese cytokines on schistosome infection.Intravenous injection of anti-cytokine monoclonal antibody is traditionally used forfunctional study of cytokines. Such an approach may not guarantee all the IL-4 and IL-12in the blood and tissue are blocked neutralized by the monoclonal antibodies, sinceschistosomes experience a long duration of migration and development and in vivoantibody degradation occurs over time. Direct inoculation of the animals with hybridomacell promises sustained antibody secretion which could be sufficient to block the effect ofcytokines on the development of schistosome. In the present study, we injected anti-mouseIL-12(C 17.8) and anti-mouse IL-4 (11B11) hybridoma cells subcutaneously into the upperbacks of mice to block the function IL-12 and IL-4 in vivo to observe the effects of IL-12and/or IL-4 deficiency on the development of S. japonicum.Materials and methodsThe identified Hybridoma cells producing anti-mouse IL-12(C 17.8) and/or anti-mouse IL-4(11B11)suspended in PBS was injected subcutaneously into the upper backs of BALB/c mice twice ata three week interval. Mice were divided into 3 experimental groups as follows:1) Anti-IL-12 (mice injected with anti-IL-12 Hybridoma cells)2) Anti-IL-4 (mice injected with anti-IL-4 Hybridoma cells)3) Intact control (mice injected with PBS)Each mouse was infected with 25 S. japonicum cercariae 4 to 5 days after the firstinjection of hybridoma cells. The growth and development of the parasites and host tissuepathological change were compared among four groups 28 and 42 days post infection. IL-4and IL-12 concentrations in serum were measured at 0, 28 and 42 days post infection byELISA. Worms were collected and worm number was counted under a dissectingmicroscope. All worms were stained by carmine and parasite length was measured from thedigital images using "imagepro 5.0"software. Female worms were examined under themicroscope and the eggs in the gravid uteri were counted. Livers were removed forcalculation of the liver eggs per worm pair. Infected mice at day 42 post infection wereevaluated for liver granuloma formation induced by eggs.Results were expressed as mean±standard deviation ((?)±s) and analyzed bySPSS12.0. The data were analyzed using one-way analysis of variance (ANOVA).ResultsIL-12 and IL-4 were not detected in sera of the related hybridoma cell inoculated mice.No significant difference was found in the number of worms among the three groups at 28and 42 days post infection. However, larva worms were found in all the groups exceptanti-IL-12 group at 28 days post infection. Worms recovered from mice with anti-IL-12were of increased length compared with those from other groups at both 28 and 42 dayspost infection (p＜0.05). No significant difference was found in the length of both femaleand male worms between anti-IL-4 treated mice and intact control.The number of eggs per pair of worms in the liver of anti-IL-12 treated mice wassignificantly higher than that in anti-IL-4 treated mice and intact control mice at 28 days post infection (p＜0.05). But this difference was lessened and not statistically significant at42 days post infection. The number of uterine eggs of ovigerous females in anti-IL-12treated mice was significantly higher than that in intact control mice at 28 days postinfection (p＜0.05). All female worms collected from anti-IL-12 treated mice at 28 days postinfection contained eggs in their uteri. In comparison, a few female worms which bore noeggs were found in other two groups at day 28. The granuloma size around the eggs in theliver in anti-IL-12 group was significantly increased (p＜0.05) but slightly diminished inanti-IL-4 treated group (p＞0.05) compared to that in control group. The liver granulomas insingle anti-IL-4 treated mice were not well formed. The fibrosis around eggs in the liverwas dramatically increased in mice treated with anti-IL-12 but was decreased in anti-IL-4treated group.ConclusionInoculating the hybridoma cells subcutaneously into the upper backs of mice couldefficiently neutralize the related cytokines. Neutralization of IL-12 or IL-4 appeared not toinfluence the survival of S. japonicum. However, IL-12 may play an impeditive role in thegrowth of S. japonicum. IL-4 exerts no special effect on the growth of S. japonicum in host.Oviposition of mature worm pairs in anti-IL-12 was initially hastened at early stage ofinfection but not further enhanced with the time increasing. Anti-IL-4 treatment coulddecrease egg-induced hepatic fibrosis. Neutralization of IL-12 could promote thegranuloma formation and fibrosis.According to the three parts of study, the summary is as follows:1. In this study, maturation and oviposition of the S. japonicum could be delayed in nude mice. It suggests that T cell induced immune response and related immune factors may bebenefit for the migration or development of the worms. In addition, T cell may play animportant in granuloma formation, especially the eosinophilia infiltration in the granulomawhich is associated with infection by S. japonicum.Through the dynamic observation, we conclude the effect of T cell deficiency on thedevelopment of worms is temporary and especially exerts on the early stagy of infection.2. By studying the infection of S.japonicum in Stat4^-/- and Stat6^-/- mice, we confirm that theTh1 or Th2 immune deficiency has no conspicuous effect on the survival, development andfecundity of worms. Stat4 deficiency facilitated the egg induced granuloma firosis. Thissuggests that Th1 immune response could inhibit the liver fribrosis which is induced byeggs. The deficiency of Stat6 could impair granuloma formation. It suggests that Th2immune response play an important role for the granuloma formation.3. We injected anti-mouse-IL-12 and anti-mouse-IL-4 hybridoma cells subcutaneously intothe upper backs of mice to block the function of IL-12 and IL-4 in vivo to observe theeffects of IL- 12 and IL-4 deficiency on the development of S. japonicum. Neutralization ofIL-12 or IL-4 appeared not to influence the survival of S.japonicum. However, IL-12 mayplay an impeditive role in the development of S.japonicum but this effect is only temporary.IL-12 could inhibit the egg-induced hepatic fibrosis. IL-4 exerts no special effect on thedevelopment of S.japonicum in host but could promote granuloma formation and hepaticfibrosis.In conclusion, from this study we hypothesize that the type of Th cell induced immuneresponse and related cytokines are associated with granuloma formation and fibrosis. Thedevelopment and oviposition of S. japonicum may much more depends on the level of someimmune factors such as cytokines involved in schistosome infection but not essentiallyassociates to a high polarization of Th1 or Th2 type response. However, the mechanism onhow cytokines exert effects on the parasite development is not yet clear.