Persistent infection of Strongyloides venezuelensis and normal expulsion of Nippostrongylus brasiliensis in Mongolian gerbils, Meriones unguiculatus, with reference to the cellular responses in the intestinal mucosa

The kinetics of daily faecal egg count, worm burdens, and intestinal cellular responses were examined in Mongolian gerbils after infection with either Strongyloides venezuelensis or Nippostrongylus brasiliensis alone, or concurrently with both parasites. The results show that, both in individual and concurrent infections, S. venezuelensis infection persisted for over 10 weeks and elicited a gradual increase in number of mast cells in the jejunal mucosa. On the other hand, N. brasiliensis worms were expelled by 3 weeks in association with goblet cell hyperplasia. These results suggest that effector/regulator cells involved in worm expulsion are different and highly selective depending on the genus of intestinal helminths.     

Inhibitory effects of concurrently present 'normal'Nippostrongylus brasiliensis worms on expulsion of 'damaged'worms and associated goblet cell changes in rats

Nippostrongylus brasiliensis worms obtained from the stable phase of a primary infection (‘normal’worms) are able to establish and survive in naïve recipient rats into which they have been adoptively transferred, whereas those obtained at the time of expulsion (‘damaged’worms) are rapidly expelled. Expulsion is associated with quantitative/ qualitative goblet cell changes. Thus, N. brasiliensis adult worms seem to stimulate the expulsion process only after being ‘damaged’by the host's immunity, whereas it is possible that ‘normal’worms may inhibit the expulsion process. To see whether ‘normal’worms have such inhibitory effects, the fate of concurrently implanted ‘normal’and ‘damaged’worms was examined with reference to goblet cell changes in the intestine. By using either male or female worms as ‘normal’or ‘damaged’populations, it was possible to determine the origin of worms remaining in the intestine. The results show that not only ‘normal’worms but also ‘damaged’worms, which would normally be expelled, remained in the intestines of the recipients on Day five. Goblet cell hyperplasia and alteration of terminal sugars of mucins, which was induced within five days after implantation of ‘damaged’worms, were completely inhibited by the concurrent presence of ‘normal’worms. The inhibitory effects of ‘normal’worms were destroyed when they were killed by heating before implantation. Possible mechanisms leading to expulsion of N. brasiliensis are discussed.     

Kinetics of expulsion of the nematode, Nippostrongylus brasiliensis, in mast-cell deficient W/Wv mice

Mucosal mast-cell hyperplasia is frequently observed in intestinal nematode infections and it has been suggested that mast-cell responses to parasite antigens are involved in worm expulsion (self cure). To evaluate the importance of this mechanism, the course of infection and expulsion of Nippostrongylus brasiliensis was compared in mast-cell deficient W/WV and normal (+/+) mice. Initial infectivity rates were similar, but the subsequent kinetics of expulsion of adult worms differed principally in that the onset of expulsion in mast-cell deficient mice appeared to occur 24-36 h later than that in normal mice. Expulsion was complete by the 14th day post infection in both W/WV and normal mice. Worm fertility (as estimated by faecal egg output) also differed in W/WV and normal mice, with maximal egg output in W/WV mice occurring 24 h later than that in normal mice. Although a few mast cells were present in the intestinal mucosa and tongue of W/WV mice, their numbers did not change during the course of infection with N. brasiliensis. In contrast, worm expulsion in normal mice was associated with a moderate increase in numbers of intestinal mast cells, commencing at the onset of expulsion and peaking several days after expulsion was completed.     

Different susceptibility to the IL-3 induced-protective effects between Strongyloides ratti and Nippostrongylus brasiliensis in C57BL/6 mice

Repetitive administration of recombinant IL-3 induced protection against Strongyloides ratti but not against Nippostrongylus brasiliensis in C57BL/6 mice. Numbers of S. ratti were negligible from day 4 to day 6 post-infection in mice injected with IL-3, whereas N. brasiliensis burdens were almost equal from day 4 to day 6 between mice injected with IL-3 or with medium. Mice treated with IL-3 and then concurrently infected with S. ratti and N. brasiliensis were protected from intestinal S. ratti but not from N. brasiliensis. The numbers of intestinal mucosal mast cells were increased by the repetitive IL-3 treatment on one day after the final injection and was augmented by subsequent infection with both nematodes.     

Propulsive activity of the rat small intestine during infection with the nematode Nippostrongylus brasiliensis

Summury Intestinal propulsive motility was measured in rats infected with 4000 Nippostrongylus brasiliensis larvae by following the transit of radioactive chromium (⁵¹Cr) through the gut. On days 6, 8, 10, 12 and 14 post-infection, ⁵¹Cr was injected through an indwelling catheter into the duodenum. The animals were killed 15 min later and the distribution of radioactivity in the small intestine measured. A group of uninfected, catheterized animals served as controls. Intestinal propulsive activity was increased significantly on day 8 post-infection. No significant difference in the overall intestinal transit occurred on days 6,10,12 and 14 post-infection, although it appeared that it may have been decreased in the upper small intestine on day 6. The significance of these results is discussed.     

Protective mechanisms against the intestinal nematode Strongyloides venezuelensis in Schistosoma japonicum-infected mice

Mice infected with Schistosoma japonicum were resistant to the intestinal nematode, Strongyloides venezuelensis. The numbers of adult S. venezuelensis recovered from mice were significantly decreased when infections were given from 6 weeks after S. japonicum infection. Larval recovery from the lungs showed that significant numbers of subcutaneously inoculated S. venezuelensis larvae were eliminated by 3 days in S. japonicum-infected mice (P < 0.0001), while histology revealed that this was associated with massive eosinophilic infiltration in the lungs. In addition, adult S. venezuelensis worms implanted in the duodenum of S. japonicum-infected mice could not establish in the intestine. This failure was associated with mucosal mastocytosis. Activation of eosinophils and intestinal mast cells was correlated with elevated expression of mRNA for interleukin (IL)-3, IL-4, and IL-5 in S. japonicum-infected mice. Sera from S. japonicum-infected mice recognized S. venezuelensis larva antigens as strongly as those from S. venezuelensis-infected mice, although transfer of sera from S. japonicum-infected mice to normal recipient mice did not protect them from S. venezuelensis challenge infection. It was concluded that the mechanisms for larval killing and adult worm expulsion of S. venezuelensis in S. japonicum-infected mice were identical to those seen in infections with S. venezuelensis only.     

A sticky end for gastrointestinal helminths; the role of the mucus barrier

Gastrointestinal (GI) nematodes are a group of successful multicellular parasites that have evolved to coexist within the intestinal niche of multiple species. It is estimated that over 10% of the world's population are chronically infected by GI nematodes, making this group of parasitic nematodes a major burden to global health. Despite the large number of affected individuals, there are few effective treatments to eradicate these infections. Research into GI nematode infections has primarily focused on defining the immunological and pathological consequences on host protection. One important but neglected aspect of host protection is mucus, and the concept that mucus is just a simple barrier is no longer tenable. In fact, mucus is a highly regulated and dynamic‐secreted matrix, underpinned by a physical hydrated network of highly glycosylated mucins, which is increasingly recognized to have a key protective role against GI nematode infections. Unravelling the complex interplay between mucins, the underlying epithelium and immune cells during infection are a major challenge and are required to fully define the protective role of the mucus barrier. This review summarizes the current state of knowledge on mucins and the mucus barrier during GI nematode infections, with particular focus on murine models of infection.     

Suppression of muscosal mastocytosis by infection with the intestinal nematode Nematospiroides dubius

Mice exposed to primary infections with the parasite intestinal nematode Nematospiroides dubius failed to show the mucosal mast cell (MMC) response which is characteristic of infections with other species of intestinal nematode and which was readily induced in these mice by infections with Nippostrongylus brasiliensis or Trichinella spiralis. The failure to generate a mucosal mastocytosis was independent of host strain or sex. When infections with N. dubius were established before, or concurrently with, T. spiralis or N. brasiliensis, the MMC response elicited by these species was delayed and/or depressed as was expulsion of the worms themselves. Infection with N. dubius given when a MMC response was already established, by exposure to T. spiralis, had no effect on MMC numbers. The possibility that the effects of N. dubius upon MMC responses reflect a lack of mastocytopoietic potential, rather than an active interference, was excluded by showing that SJL mice, which expel primary infections with N. dubius and express strong immunity to reinfection, developed marked mastocytosis during secondary infections. The depression of MMC responses by N. dubius is discussed in relation to the known immunosuppressive properties of this parasite and in relation to the T cell mediated control of MMC development.     

Effect of testosterone on the mucosal defence against intestinal helminths in Indian soft-furred rats, Millardia meltada with reference to goblet and mast cell responses

Effects of testosterone on the mucosal defence mechanisms against intestinal helminths were examined in Millardia meltada. When female M. meltada were treated with testosterone at the pharmacological dose, Nippostrongy-lus brasiliensis infection persisted for over seven weeks with prominent biphasic pattern of faecal egg production, whereas almost complete expulsion was observed by two weeks in untreated controls. In spite of a biphasic pattern of faecal egg production, the worm burden of testosterone-treated animals remained constant up to three weeks and then slowly decreased by seven weeks. To see whether or not this delayed expulsion in testosterone treated animals was due to altered cellular responses of the intestinal mucosa, goblet and mast cell responses were examined histologically. At two weeks post-infection, goblet cell responses at the infected site were significantly lower in testosterone-treated animals than in controls. In contrast mast cell hyperplasia was comparable between testosterone-treated and control animals. When Strongyloides venezuelensis, in which expulsion is dependent on mucosal mast cells, were infected concurrently with N. brasiliensis, testosterone-treated animals could expel S. venezuelensis worms by Day 18, but failed to expel N. brasiliensis. Histologically, mast cell hyperplasia was associated with expulsion ofS. venezuelensis, while goblet cell responses were suppressed. From these results, testosterone seems to suppress proliferation/function of goblet cells but does not affect mast cells ofM. meltada.     

Immune-mediated damage is not essential for the expulsion of Nippostrongylus brasiliensis adult worms from the small intestine of mice

Nippostrongylus brasiliensis adult worms are expelled from the rat small intestine during a primary infection by two steps. First, host immune responses cause damage to the worms, and then a nonspecific inflammatory response initiates expulsion. We have tested the two-step expulsion hypothesis in mice infected with N. brasiliensis. After a primary infection in C57BL/6 mice, adult worms started to lay eggs on day 5 postinfection (p.i.) and were expelled around day 9-10 p.i. According to the rat system, 5 day- and 8-day-old worms were assumed to be 'normal' and 'damaged', respectively. When 5 day- and 8 day-old worms obtained from C57BL/6 mice were transferred surgically into the small intestine of naive C57BL/6 mice, both 5 day- and 8 day-old worms were almost simultaneously expelled by day 6 postworm implantation (p.w.i.). In contrast, when 5 day- and 8 day-old worms of mouse origin were implanted into naive Wistar rats, 8 day-old worms were expelled by day 5 p.w.i., while 5 day-old worms were expelled by day 8 p.w.i. Similar results were obtained when BALB/c mice were used. Therefore, mice can expel N. brasiliensis adult worms as rapidly as rats expel 'damaged' worms, regardless of the status of the worms ('normal' or 'damaged'). Stat6-deficient mice were unable to expel implanted 5 day-old worms up to day 10 p.w.i., suggesting that cellular mechanisms depending on Stat6-signalling system are necessary for the expulsion. When N. brasiliensis adult worms obtained from Stat6-deficient mice 5 and 15 days after a primary infection were implanted into Wistar rats, the former established in the recipient rats for approximately 1 week and were then expelled by day 10 p.w.i., whereas the latter were expelled by day 4 p.w.i. These results suggest that immune-mediated damage of N. brasiliensis adult worms (first step) is not a prerequisite for expulsion from the small intestine of mice, although adult worms are actually damaged by Stat6-independent immune mechanisms.     

Role of T lymphocytes in intestinal mucosal injury

To determine the role of T cells versus mast cells in mucosal injury, we documented structural and functional changes in the intestine of congenitally athymic nude rats during infection with the enteric parasite,Nippostrongylus brasiliensis. Studies were conducted at days 4, 7, 10, and 21 postinfection; controls were uninfected. Villus damage was indicated by morphological abnormalities at days 7, 10, and 21 and reduced activities of disaccharidase enzymes at days 10 and 21. The activity of the proliferative enzyme, thymidine kinase, was increased only at day 21, at which time the crypts were elongated. Epithelial permeability increased significantly: 5-hr recovery (in urine and blood) of the probe molecule, [⁵¹Cr]EDTA, following injection into ligated jejunal segments, was elevated at days 7 and 10. Uptake of a protein antigen, ovalbumin, from lumen to blood followed a similar pattern. No evidence of functional T cells was demonstrated. However, mucosal mast-cell activation was indicated by elevated serum levels of rat mast-cell protease II at days 7 and 10. We conclude that the absence of thymus-derived T cells does not preclude mucosal damage involving impaired barrier and digestive function. Mucosal mast cells may be involved in causing the injury in this model.This research was supported by grants from the Medical Research Council of Canada and the Canadian Foundation for Ileitis and Colitis.     

Crosstalk between intestinal epithelial cell and adaptive immune cell in intestinal mucosal immunity

Constantly challenged by luminal bacteria, intestinal epithelium forms both a physical and biochemical defense against pathogens. Besides, intestinal epithelium senses dynamic and continuous changes in luminal environment and transmits signals to subjacent immune cells accordingly. It has been long accepted that adaptive immune cells fulfill their roles partly by modulating function of intestinal epithelial cells. Recent studies have brought up the proposal that intestinal epithelial cells also actively participate in the regulation of adaptive immunity, especially CD4+ adaptive T cells, which indicates that there is reciprocal crosstalk between intestinal epithelial cells and adaptive immune cells, and the crosstalk may play important role in intestinal mucosal immunity. This Review makes a comprehensive summary about crosstalk between intestinal epithelial cells and CD4+ adaptive T cells in intestinal immunity. Special attention would be given to their implications in inflammatory bowel disease pathogenesis and potential therapeutic targets.     

Regulation of type 2 immunity to helminths by mast cells

Recently, we demonstrated a novel role for gastrointestinal mast cells (MCs) in the early events that lead to the generation of Th2 immunity to helminth infection.¹ Hepworth MR, Dani?owicz-Luebert E, Rausch S, Metz M, Klotz C, Maurer M, et al. Mast cells orchestrate type 2 immunity to helminths through regulation of tissue-derived cytokines. Proc Natl Acad Sci U S A 2012; 109:6644 - 9; http://dx.doi.org/10.1073/pnas.1112268109; PMID: 22493240 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] Mice lacking MCs (Kit^W/Kit^W-v and Kit^W-Sh) showed a significant inhibition of Th2 cell priming following infection with the parasitic helminth Heligmosomoides polygyrus bakeri (Hp). We showed that MCs degranulate during the early stages of infection when the helminth larvae invade the small intestinal tissue. Furthermore, MC degranulation was required for the enhanced expression and production of the tissue-derived cytokines IL-25, IL-33 and TSLP, which are required for the optimal orchestration and priming of type 2 immunity. In this addendum we aim to address several questions raised by our findings — in particular, the mechanisms through which MCs may recognize helminth exposure in the early stages of infection and by which they may enhance expression of critical tissue cytokines thus, enabling Th2 priming. Furthermore, we will discuss these findings in the context of recently described novel innate immune cells, such as type 2 hematopoietic progenitors and type 2 innate lymphoid cells.     

Regulation of type 2 immunity to helminths by mast cells

Recently, we demonstrated a novel role for gastrointestinal mast cells (MCs) in the early events that lead to the generation of Th2 immunity to helminth infection. ( 1) Mice lacking MCs (Kit (W) /Kit (W-v) and Kit (W-Sh) ) showed a significant inhibition of Th2 cell priming following infection with the parasitic helminth Heligmosomoides polygyrus bakeri (Hp). We showed that MCs degranulate during the early stages of infection when the helminth larvae invade the small intestinal tissue. Furthermore, MC degranulation was required for the enhanced expression and production of the tissue-derived cytokines IL-25, IL-33 and TSLP, which are required for the optimal orchestration and priming of type 2 immunity. In this addendum we aim to address several questions raised by our findings - in particular, the mechanisms through which MCs may recognize helminth exposure in the early stages of infection and by which they may enhance expression of critical tissue cytokines thus, enabling Th2 priming. Furthermore, we will discuss these findings in the context of recently described novel innate immune cells, such as type 2 hematopoietic progenitors and type 2 innate lymphoid cells.     

Expulsion of Hymenolepis nana from mice with congenital deficiencies of IgE production or of mast cell development

The roles of IgE and mast cells on expulsion of adult Hymenolepis nana from the intestine were examined in mice. IgE-dependency was determined by comparing congenitally IgE-deficient SJA/9 and IgE-producing SJL/J mice infected with 50 H. nana eggs. Anti-H. nana IgE antibody was detected at three weeks post infection (p.i.) in SJL but not in SJA mice. The number of adult worms in the intestines of SJA and of SJL mice were similar at two weeks, but significantly more were found in SJA mice at three weeks p.i. Treatment of mice with anti-ɛ antibody also resulted in an increased worm burden at three weeks, suggesting participation of IgE in expulsion of H. nana. Intestinal mastocytosis was induced by infection regardless of the IgE status of the mice. Mast cell-dependency was tested in mast cell-deficient W/W^u and in normal littermate +/+ mice infected with 100 H. nana eggs. Anti-H. nana antibody was detected in both groups of mice at three weeks p.i. Worm expulsion seemed to be mast cell dependent because expulsion was less complete in W/W^u mice at three weeks p.i. Peripheral blood eosinophilia was comparable at three weeks p.i. in both IgE and mast cell sufficient and deficient mice. These results suggest that IgE and mast cells participate in the expulsion of H. nana adults from intestine in mice.     

Reconstitution of mucosal mast cells in W/Wv mice by adoptive transfer of bone marrow-derived cultured mast cells and its effects on the protective capacity to Strongyloides ratti-infection

Bone marrow-derived cultured mast cells (BMMC) were transferred intravenously into W/WV mice to examine if they could reconstitute defective mucosal mast cell response or defective protective capacity against infection with Strongyloides ratti. When mast cell growth factor-producing activity of W/WV mice were examined, mesenteric lymph node cells obtained at 7 to 14 days after infection could produce this factor in vitro by stimulation with S. ratti-adult worm antigen. A single injection of BMMC (1 X 10(7] on day 7 post-infection (p.i.) neither caused an increase in number of intestinal mucosal mast cells not altered the kinetics of faecal larval output (LPG). On the other hand, serial injections of BMMC (5 X 10(6] from day 5 to 10 p.i. (total 3 X 10(7) cells) resulted in the significant increase in number of intestinal mucosal mast cells. However, this treatment too could not alter the kinetics of LPG. Therefore, adoptive transfer of BMMC could cause the increase in number of histologically detectable-mucosal mast cells, but these cells are, by themselves, not sufficient to cause the expulsion of S. ratti adult worms from the intestine.     

Immunity to Strongyloides ratti in rats. 1. Adoptive transfer with mesenteric lymph node cells

Adoptive transfer of immunity against the enteral phase of Strongyloides ratti was monitored using four parameters; namely, number of worms recovered, position occupied in the host's small intestine, worm length and fecundity. When immune mesenteric lymph node cells (IMLNC) were transferred from donors infected for 20, 26 or 32 days into recipients infected for 3 days, a marked acceleration of worm expulsion was evident by day 16 post-infection (p.i.). IMLNC from day-16 p.i. donors did not transfer expulsion. In an experiment in which recipients were given 2 × 10⁸ or 1 × 10⁸ IMLNC from donors infected for 26 days, accelerated worm expulsion occurred only with the higher inoculum, although manifestations of direct worm immunity, i.e. altered position and reduced length and fecundity, were evident in both cases. Transfer of IMLNC appeared to have no effect upon worm establishment; when cells were transferred on the day of infection a period of 16 days was necessary to effect both direct anti-worm immunity and expulsion, although the former was evident on day 14 p.i. However, when IMLNC were transferred to rats already infected for 6 days, a very significant reduction of their worm burden was apparent 10 days after IMLNC transfer.     

Immunopathology of intestinal helminth infection

The relationship between intestinal pathology and immune expulsion of gastrointestinal nematodes remains controversial. Parasite expulsion is associated with intestinal pathology in several model systems and both of these phenomena are T cell dependent. However, while immune expulsion of gastrointestinal helminth parasites is usually associated with Th2 responses, the effector mechanisms directly responsible for parasite loss have not been elucidated. In contrast, the intestinal pathology observed in many other disease models closely resembles that seen in helminth infections, but has been attributed to Th1 cytokines. We have used infection with the nematode Trichinella spiralis in mice defective for cytokines to demonstrate that although parasite expulsion is indeed IL-4 dependent, contrary to expectations, the enteropathy is also regulated by IL-4. Furthermore, abrogation of severe pathology in iNOS deficient and TNF receptor defective animals does not prevent parasite expulsion. TNF and iNOS are therefore involved in intestinal pathology in nematode infections, apparently under regulation by IL-4 and Th2 mediated responses. Therefore, it appears that the IL-4-dependent protective response against the parasite operates by a mechanism other than merely the gross degradation of the parasite's environment brought about by the immune enteropathy. However, it remains important to elucidate the protective mechanisms involved in parasite expulsion, which are still unclear.     

Reduced amount of intestinal mucus by treatment with anti-CD4 antibody interferes with the spontaneous cure of Nippostrongylus brasiliensis-infection in mice

Mechanism of spontaneous cure was studied in mice infected with mouse-nonadaptive Nippostrongylus brasiliensis. Adult BALB/c mice were cured spontaneously of infection with this strain of N. brasiliensis by Day 7 post-infection. Expulsion of intestinal worms was delayed dose-dependently by a treatment with anti-CD4 antibody. However, the treatment had no significant effect on larval recovery from the lungs. Treatment of mice with anti-IL-5 antibody suppressed intestinal tissue eosinophilia induced by the infection, but did not affect intestinal worm recovery. Antigen specific IgE antibody was not detected in the sera obtained from Days 5 to 15. Therefore, IL-5 and specific IgE antibody are probably not important in the spontaneous cure. Treatment of mice with anti-CD4 antibody had no significant effect on number of intestinal goblet cells or on expression of terminal sugars of goblet cell mucins. However, histological and quantitative analyses revealed that significantly less intestinal mucus was released in anti-CD4 antibody treated mice than in control mice. These results suggest that CD4+ lymphocytes control the amount of intestinal mucus and consequently the reduced mucus interferes with the spontaneous cure. Quantity of mucus released in the intestinal lumen may have an essential role in the spontaneous cure ofN. brasiliensis-in/fcfio/i of mice.     

Localization of mucosal mast cells in W/WV mice after reconstitution with bone marrow cells or cultured mast cells, and its relation to the protective capacity to Strongyloides ratti infection

Localization of mast cells in the intestinal epithelium, villous lamina propria and basal lamina propria of mast cell-deficient WBB6F1 (W/Wv) mice reconstituted with either bone marrow cells or with cultured mast cells (BMMC) was compared to that of mast cell-sufficient C57BL/6 or C57BL/6-bgj/bgj (beige) mice after infection with Strongyloides ratti. In mast cell-sufficient C57BL/6 or beige mice, the maximum number of intestinal mucosal mast cells (MMC) was more than 160 MMC/10 villus crypt units (VCU) and more than 90% of MMC were located in the intestinal epithelium. When W/Wv mice were reconstituted with bone marrow cells of beige mice, worm expulsion was hastened and the MMC response became comparable to that of mast cell-sufficient mice in terms of cell numbers and their intra-epithelial localization. On the other hand, when W/Wv mice were reconstituted with BMMC of beige mice, only a few donor type MMC were detected in the intestine. The proportion of intra-epithelial MMC was lower than that of mast cell-sufficient mice or of marrow-reconstituted W/Wv mice. Even repeated injection of BMMC could not fully restore the number of intra-epithelial MMC to the level of that observed in mast cell-sufficient mice. Since mast cell-growth factor-producing activity of W/Wv mice was comparable to that of mast cell-sufficient mice, the ineffectiveness of BMMC-transfer in restoring protective activity or MMC responses in W/Wv mice seems to be attributed to the functional immaturity or inactivity of BMMC.