9HWHULQDU\ 6FLHQFH Mucosal mast cell-derived chondroitin sulphate levels in and worm expulsion from FcR γ-knockout mice following oral challenge with Strongyloides venezuelensis Denis
Trang 19HWHULQDU\ 6FLHQFH
Mucosal mast cell-derived chondroitin sulphate levels in and worm
expulsion from FcR γ-knockout mice following oral challenge with
Strongyloides venezuelensis
Denis Nnabuike Onah 1,
*, Yukifumi Nawa 2
1Department of Veterinary Parasitology & Entomology, University of Nigeria, Nsukka, Nigeria
2
Department of Parasitology, Miyazaki Medical College, Kiyotake, Miyazaki 889-1692, Japan
Mucosal mast cell-derived chondroitin sulphates
(sulphated proteoglycans) were assayed in gut washings
and homogenate of FcR γ-knockout (KO) and wild-type
(WT) C57BL/6 mice challenged with Strongyloides
venezuelensis in order to assess their possible role in
secondary immunity against enteric nematodes Groups
of immune KO and WT mice were challenged by oral
gavage with 300 infective larvae (L 3 ) Establishment of
infection was assessed by daily faecal analysis to
determine the number of eggs per gram of faeces (EPG)
and by adult worm recovery on days 5 and 13 post
challenge Mucosal mast cell (MMC) counts were done on
days 5 and 13 post challenge while MMC-derived
chondroitin sulphates in gut washings (days 1 and 5) and
homogenate (day 8) were assayed by high performance
liquid chromatography (HPLC) Results showed that
patent infection occurred in challenged KO but not WT
mice despite significantly higher mastocytosis in jejunal
sections of KO than WT mice (p < 0.001) Similarly but
against prediction, significantly higher concentration of
MMC-derived chondroitin sulphates was observed in gut
homogenate of KO than WT mice (p < 0.05) In contrast,
significantly higher concentration of chondroitin
sulphates was observed in gut washings of WT than KO
mice (p < 0.05) These results suggest that MMC in KO
mice failed to release sufficient amount of sulphated
proteoglycans into the gut lumen as did the WT mice,
which may have been part of the hostile environment that
prevented the establishment in and eventual expulsion of
adult S venezuelensis from the gut of WT mice following
challenge.
Key words: Strongyloides venezuelensis, mast cells, mucosal
immunity, chondroitin sulphates, gut washings, mice
Introduction
The immune expulsion of adult Strongyloides species
from the small intestine of mice and rats is associated with intraepithelial mast cell hyperplasia [1,2,7,14] Mast cells are thought to mediate this expulsion by creating an environment hostile to the establishment and survival of gastrointestinal nematodes through inflammation-associated changes and release of inflammatory mediators [19] The release of these mediators is induced by mast cell degranulation, which in turn is induced by mast cell activation triggered by cross-linking of the high affinity immunoglobulin Fc receptor (FcR) for IgE (FcεRI) with an antigen-IgE immune complex [3,8,11,13] This type of acute inflammation, also known as type I hypersensitivity is therefore thought to depend on mast cells, its FcεRI and on IgE Perturbation in any one of these three components should result in the loss of type I hypersensitivity responses [18] This, in fact, is the case with FcεRI To be expressed on the surface of cells and for signal transduction into the interior of the cell, FcεRI requires the homodimeric γ subunit of FcRs (FcRγ) [5,10,17] Ablation of the γ subunit
by targeted gene deletion results in the loss of FcγRI expression on mast cells coupled with loss of mast cell functions including degranulation and mediator release in the mutant mouse [23] Using these mice we showed that while the wild type (WT) counterparts were able to expel a
primary Strongyloides venezuelensis infection, FcR γ-knockout (KO) mice failed to do so [16] However, the confounding aspect of our study was that both intestinal mastocytosis and serum mouse mast cell protease-I (mMCP-I), the levels of which have negative correlation with nematode burden in sheep [22] were similar Since FcRγ-KO mice fail to assemble FcγRI on their mast cells and therefore are unable to express mast cell degranulation and mediator release [23], we concluded that mMCP-1 may
be released spontaneously without requiring the mast cell
FcγRI cross-linking with the immune complex formed by the parasite antigen/IgE and that mMCP-1 is not involved in
*Corresponding author
Tel: +234-42-770106
E-mail: denisonah2003@yahoo.co.uk
Trang 2adult S venezuelensis expulsion [16] However, because
mastocytosis has a strong association and often corresponds
with the time of worm expulsion [14], we stated that mast
cells must be involved in the process in a manner yet to be
elucidated We then speculated that failure of FcRγ-KO mice
to expel S venezuelensis might be related to inability of their
mast cells to degranulate and release inflammatory
mediators other than mMCP-1 [16] Sulphated goblet cell
mucins [7] and experimental introduction of glycosaminoglycans
of the type produced by mucosal mast cells [12] have been
shown to prevent the establishment of S venezuelensis
infection and mediate its expulsion from infected mice
Chondroitin sulphates are the major proteoglycans
contained by mucosal mast cells in mouse [25] In this study
therefore, we assayed the amounts of chondroitin sulphates
in gut homogenate and washings of FcRγ-KO mice and their
WT counterparts following oral challenge with S.
venezuelensis This was in order to ascertain (i) if
differences exit in their concentrations in the gut of KO and
WT mice and (ii) whether differences in worm
establishment and expulsion following challenge might be
related to any differences in their concentrations within the
gut niche occupied by the parasite in the two mouse types
Materials and Methods
Animals
FcRγ-knockout C57BL/6 male mice of 8 weeks old were
generous gifts from Professor Toshiyuki Takai and Dr
Masao Ono (Tohoku University, Sendai, Japan)
Age-matched specific pathogen free wild type C57BL/6 mice of
the same sex were purchased from Japan SLC (Shizuoka,
Japan) All animals were kept in our laboratory animal unit
for 2 weeks to acclimatize before use at 10 weeks old
Before and during the period of the experiment they were
supplied with feed and water ad libitum
Parasite
The strain of S venezuelensis originally isolated from a
brown rat in Okinawa Prefecture, Japan [6] and established
as a laboratory strain [20] was used in this study Infective
third stage larvae (L3) used for infecting experimental
animals orally (oral gavage) were obtained from the lungs of
mice given primary infection and sacrificed on day 3 post
infection Briefly, 10 C57BL/6 male mice were each
infected with 5000 L3 of S venezuelensis obtained by the
filter-paper faecal culture method and sacrificed on day 3
post-infection by anaesthetic overdose using diethyl ether
The lungs were removed by dissection, shredded with
fine-tipped thumb forceps in fine-meshed coffee strainers placed
in a beaker of warm PBS and incubated at 37o
C for 3 h
Emerged lung L3 were recovered by centrifugation,
re-suspended and washed three time in fresh warm (37o
C) PBS, counted and adjusted to 1500 lung L/ml
Infection of experimental animals
All animal groups for challenge were primed with 2000 L3
subcutaneously 30 days before challenge For uniform treatment, every mouse in both the challenge and challenge control (primary infection) groups was treated orally with 20-mg/kg mebendazole (Sigma, St Louis, MO) on day 26 post priming to get rid of any residual adult worms from the priming dose On day 30 post priming, each animal in the challenge and control groups was infected with 300 lung-recovered L3 contained in 200µl PBS and introduced directly into the stomach using a stomach needle with blunt oval tip
Experimental protocol
Thirty knockout (KO) and thirty wild-type (WT) mice were used for the experiment Twenty each of the KO and
WT mice were primed as stated above For challenge, primed KO and WT mice with 10 each of their nạve controls were infected as described above The challenge groups were sacrificed 5 mice each on day 1, 5, 8 and 13 post challenge for sample collection while 5 each of their controls were sacrificed on day 5 and 13 post infection Daily faecal egg counts expressed as eggs per gram of faeces (EPG) and adult worm recovery to establish whether patent infection occurred were carried out until day 13, and on day
5 and 13 post infection respectively in both challenge and control animals In the challenge animals only, histology for MMC numbers was carried out on day 5 and 13, while various chondroitin sulphates and mouse mast cell
protease-1 (mMCP-protease-1) in gut washings were assayed on day protease-1 and 5 post challenge Chondroitin sulphates in gut homogenate were assayed on day 8 post challenge
EPG and adult worm recovery
As stated above, faecal samples for daily EPG was collected only from individual animal of the groups sacrificed on day 13 post challenge To ascertain worm establishment, adult worms were recovered from sacrificed challenge and control animals on day 5 and 13 post infection The entire small intestine of each sacrificed animal was isolated and processed for adult worm recovery The methods used for EPG and adult worm recovery, were
as previously described [9,20]
Histology and serology
Jejunal pieces were taken and histological sections prepared, stained and mucosal mast cells were enumerated
as previously described [16] on day 5 and 13 post challenge Also samples of gut washings as described below were collected on days 1 and 5 post challenge and analysed for mMCP-1 concentration by ELISA as described [16]
Proteoglycan assay in gut washings and homogenate
The entire small intestine isolated from each of the
Trang 3sacrificed animal on the days specified above was washed
out twice with a total volume of 10 ml sterile distilled water
Each sample was then centrifuged at 350 g for 10 min to
remove debris The supernatant was frozen until used For
gut homogenate, approximately 15 cm of the upper small
intestine was cut off, washed out as above and minced using
a homogeniser (Polytron homogeniser, Kinematika AG,
Littau, Switzerland) The minced samples were suspended
in 5 ml sterile PBS, centrifuged as above and the supernatant
was frozen until used For the assay of the various
chondroitin sulphates, each sample was removed from the
freezer, thawed under room temperature and was then
processed and analysed by high-performance liquid
chromatography according to the methods of Yoshida et al.
[24] and Shinmei et al [21].
Statistical analysis
Data were compiled and subjected to descriptive statistics
while differences between group-means were obtained by
the Students t-test using Microsoft Excel Statistical
Toolpack Differences at p = 0.05 were considered significant.
Results
Eggs per gram of faeces (EPG)
The mean daily EPG of animals sacrificed on day 13 post
challenge are presented in Fig 1 All control WT and KO
animals as well as all challenged KO mice persistently shed
eggs in their faeces until the day of sacrifice In contrast,
there were no eggs in the faeces of all challenged WT mice
One significant observation however, is that the oral route of
infection with lung-recovered L3 does not seem to be an
efficient means of establishing patent infection judging from
the level of EPG of the animals (less than 1000 at peak EPG)
when compared with the EPG of mice infected by the
subcutaneous route in which peak EPG usually runs in tens
of thousands [15,16]
Adult worm recovery
Consistent with the EPG result, control WT and KO as
well as challenged KO mice developed persistent patent S venezuelensis infection The mean number of adult worms
recovered from animals sacrificed on days 5 and 13 are shown in Fig 2 As expected, no adult worms were recovered from any of the challenged WT mice on both days, which agreed with the zero EPG recorded for this group Again, the very few number of adult worms recovered from these mice indicate that oral implantation of third stage larvae is not very efficient in establishing patent infection
Intestinal mast cell numbers and serum mMCP-1 concentration
Mast cells were enumerated in jejunal sections prepared
on days 5 and 13 post oral challenge Results (Table 1a) show that significantly higher numbers of mast cells were
counted in KO than WT mice on day 5 (p < 0.01) and day
13 (p < 0.001) On the other hand, there was no significant difference (p > 0.05) in the amount of mMCP-1 concentration
in gut washings of challenged KO and WT mice on days 1 and 5 post challenge (Table 1b)
Sulphated proteoglycan concentration in gut washings and homogenate
Results of the assay for various sulphated proteoglycans (Chondroitin sulphates A, C, D, E, and total chondroitin sulphate) in gut washings of challenged KO and WT mice sacrificed on days 1 and 5 are presented in Figs 3a and 3b respectively The results show that significantly higher concentrations of the chondroitin sulphates occurred in the
gut washings of WT than KO mice on these days (p < 0.05).
In contrast, significantly higher concentrations of these
Table 1.
(a) Mucosal mast cell number/10 villus crypt unit (Mean ± SD) in FcRγ-KO and WT mice challenged orally with 300
lung-recovered S venezuelensis L3
(b) Mean ± SD of mMCP-1 level (ng/ml) in gut washings of FcRγ-KO and WT mice challenged orally with 300 L3 of S.
venezuelensis
mMCP-1 Concentration (ng/ml)
Trang 4sulphated sugars were obtained in gut homogenate
preparations of KO than WT mice on day 8 post-challenge
(p < 0.05, Fig 3c).
Discussion
Primary infection of mice with S ratti and S.
venezuelensis results in the development of a strong
immunity, which completely aborts a patent infection
following secondary challenge of the animals with the parasites [4,20] This strong immunity depends on CD4+
T cells which make Th2 cytokines and induce mast cell-dependent gut inflammatory responses and changes in gut physiology, all of which act in concert to create an environment hostile to worm establishment in and their eventual expulsion from the intestinal niche [15] Our prediction was that an important part of this inflammatory response and change in gut physiology resulting in worm expulsion is the presence of sulphated proteoglycans released by degranulating mast cells Therefore, if FcRγ-KO mice are unable to undergo mast cell degranulation and mediator release [23], the enabling hostile environment for the ablation of secondary infections will be lacking in primed KO animals Consequently, if sulphated proteoglycans play a role in worm expulsion, then the introduction of migrating L3 into a hostile intestinal environment in primed
KO and WT mice would most likely result in a patent infection in the former but not in the latter Results of this study largely support our predictions First, significantly higher concentrations of chondroitin sulphates were present
in gut washings of WT, in which the challenge infection was sterile than in KO, in which persistent patent infection was established Secondly, there was no significant difference in the concentrations of mMCP-1 in gut washings taken from both mouse types irrespective of the fact that significantly higher numbers of intraepithelial mast cells were counted in
KO than in WT animals
Taken together, it can be inferred that (a) mMCP-1 may be released by mast cells in a manner not dependent on degranulation i.e., not requiring the antigen-IgE-FcεR cross-linking and triggering, (b) mMCP-1 may not be important in the mucosal immune mechanisms resulting in the expulsion
of adult S venezuelensis from the gut and (c) mucosal mast
cell-derived glycosaminoglycans (chondroitin sulphates) are apparently involved in and therefore play a role in the
prevention of the establishment of adult S venezuelensis in
and their eventual immune expulsion from the gut These
agree with the suggestions of Onah et al [16] regarding
mMCP-1 and its possible role in worm expulsion as well as with their speculation that FcRγ-KO mice are probably
unable to expel primary S venezuelensis infection as a result
of failure of their mast cells to degranulate and release mediators other than mMCP-1 In fact, that significantly higher concentrations of chondroitin sulphates were obtained from the homogenized gut tissue of KO (which contained more mast cells) than WT is an added support that the intraepithelial mast cells in KO contain them but are not releasing them in enough quantities into the gut lumen to effect worm expulsion In addition, we have similar evidence that when immune KO and WT mice are
challenged by subcutaneous introduction of 3000 S venezuelensis L3, fewer larvae are recovered from the lungs
of KO than in WT animals 3 days later yet patent infection
Fig 1 Daily numbers (mean ± SD) of eggs per gram of faeces
(EPG) in immune and control FcRγ-KO and WT mice
challenged orally with 300 lung-recovered L3 of S venezuelensis
and sacrificed on day 13 post challenge
Fig 2 Number of adult worms (mean ± SD) recovered from the
small intestines of immune and control FcRγ-KO and WT mice
challenged orally with 300 lung-recovered L3 of S venezuelensis
and sacrificed on days 5 and 13 post challenge
Trang 5occurs only in the latter animals In this infection protocol
serum mMCP-1 was also similar but sulphated
proteoglycans were found to be significantly higher in the
WT than KO animals Moreover, our results in this study
agree with and support those of Maruyama and his
associates [12] who showed that glycosaminoglycans of the
type produced by mucosal mast cells significantly inhibited
the invasion and establishment of adult S venezuelensis
implanted into the duodenum of nạve mice
In conclusion, defective secondary immunity against S.
venezuelensis in FcRγ-KO mice is associated with
significant decrease in the amount of mast cell-derived
chondroitin sulphates released into the gut lumen, suggesting that the sugars are intimately associated with worm expulsion It is our opinion that extensive further studies into the role of sulphated sugars of the type produced
by mucosal mast cells in parasitic gastrointestinal nematode expulsion is essential and worthy of support as they seem attractive candidates for anthelmintic drug investigation and development
Acknowledgments
We thank Mrs Eri Ono for excellent technical assistance
Fig 3 (A) Chondroitin sulphate (ChS) concentration (µg/ml) (mean ± SD) in gut washings of FcRγ-KO and WT mice challenged orally
with 300 lung-recovered L3 of S venezuelensis and sacrificed on day 1 post challenge ChS-A:E = Chondroitin sulphate A-E;
Us-Ch = Unsulphated chondroitin (B) Us-Chondroitin sulphate (Us-ChS) concentration (µg/ml) (mean ± SD) in gut washings of FcRγ-KO and
WT mice challenged orally with 300 lung-recovered L3 of S venezuelensis and sacrificed on day 5 post challenge
ChS-A:E = Chondroitin sulphate A-E; Us-Ch = Unsulphated chondroitin (C) Chondroitin sulphate (ChS) concentrations (µg/ml)
(mean ± SD) in gut homogenates of FcRγ-KO and WT mice challenged orally with 300 lung-recovered L3 of S venezuelensis and
sacrificed on day 8 post challenge ChS-A:E = Chondroitin sulphate A-E; Us-Ch = Unsulphated chondroitin
Trang 6and Professor Toshiyuki Takai and Dr Masao Ono (Tohoku
University, Sendai, Japan) for generously providing the
mutant mice used in this study DNO was a JSPS
postdoctoral research fellow and funding for this work was
provided by the Grant-in-Aid for scientific research from the
Ministry of Education, Culture, Sports, Science and
Technology, Japan We also thank the University of Nigeria,
Nsukka for granting DNO the study leave which enabled
him undertake the fellowship in Japan
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