Shin Ae Kang

Parasitic Helminth Cystatin Inhibits DSS-Induced Intestinal Inflammation Via IL-

Many immune down-regulatory molecules have been isolated from parasites, including cystatin (cystain protease inhibitor). In a previous study, we isolated and characterized Type I cystatin (CsStefin-1) of the liver fluke, Clonorchis sinensis. To investigate whether the CsStefin-1 might be a new host immune modulator, we induced intestinal inflammation in mice by dextran sodium sulfate (DSS) and treated them with recombinant CsStefin-1 (rCsStefin-1). The disease activity index (DAI) increased in DSS only-treated mice. In contrast, the DAI value was significantly reduced in rCsStefin-1-treated mice than DSS only-treated mice. In addition, the colon length of DSS only-treated mice was shorter than that of rCsStefin-1 treated mice. The secretion levels of IFN-γ and TNF-α in the spleen and mesenteric lymph nodes (MLNs) were significantly increased by DSS treatment, but the level of TNF-α in MLNs was significantly decreased by rCsStefin-1 treatment. IL-10 production in both spleen and MLNs was significantly increased, and IL-10+F4/80+ macrophage cells were significantly increased in the spleen and MLNs of rCsStefin-1 treated mice after DSS treatment. In conclusion, rCsStefin-1 could reduce the intestinal inflammation occurring after DSS treatment, these effects might be related with recruitment of IL-10 secreting macrophages.

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Infection by Trichinella spiralis takes place in two distinct phases: one is the intestinal phase and the other is the muscle phase. To evaluate alterations in cytokine production during a T. spiralis infection, we periodically assessed the cytokine production of splenocytes in mice after infection (AI). The levels of Th2-related cytokines immediately increased after the initiation of T. spiralis larval intestinal invasion (1 week AI). These early elevations in the Th2 response might be associated with the innate immune responses of intestine epithelial cells against T. spiralis larval invasion. IL-4 and IL-13 levels reached a peak prior to the initiation of nurse cell formation (2 weeks AI). Additionally, all Th17-related cytokines, except for IL-17, increased slightly until 2 weeks AI. However, expression levels for all of the Th2 and Th17-related cytokines began to decrease after the initiation of nurse cell formation and reached basal levels at 4 weeks AI, except for IL-5. At the same time, the CD4(+)CD25(+)Foxp3(+) T (regulatory T, T(reg)) cell population increased significantly in the spleen. Additionally, the number of cells in the peripheral lymph nodes increased. In conclusion, T. spiralis larva intestinal invasion induced the production of Th2 and Th17 cell-related cytokines, and the cytokines decreased with T(reg) cell-related cytokine.

Macrophage Recruitment

In order to get a better understanding of the role of protease-activated receptor 2 (PAR2) in type 2 helper T (Th2) cell responses against Trichinella spiralis infection, we analyzed Th2 responses in T. spiralis-infected PAR2 knockout (KO) mice. The levels of the Th2 cell-secreted cytokines, IL-4, IL-5, and IL-13 were markedly reduced in the PAR2 KO mice as compared to the wild type mice following infection with T. spiralis. The serum levels of parasite-specific IgE increased significantly in the wild type mice as the result of T. spiralis infection, but this level was not significantly increased in PAR2 KO mice. The expression level of thymic stromal lymphopoietin, IL-25, and eotaxin gene (the genes were recently known as Th2 response initiators) of mouse intestinal epithelial cells were increased as the result of treatment with T. spiralis excretory-secretory proteins. However, the expression of these chemokine genes was inhibited by protease inhibitor treatments. In conclusion, PAR2 might involve in Th2 responses against T. spiralis infection.

Alteration of helper T-cell related cytokine production in splenocytes during Trichinella spiralis infection.

In order to know the effect of pre-existing Trichinella spiralis infection on experimentally induced intestinal inflammation and immune responses, we induced colitis in T. spiralis-infected mice and observed the severity of colitis and the levels of Th1, Th2, and regulatory cytokines and recruitment of CD4+CD25+Foxp3+ T (regulatory T; Treg) cells. Female C57BL/6 mice were infected with 250 muscle larvae; after 4 weeks, induction of experimental colitis was performed using 3% dextran sulfate sodium (DSS). During the induction period, we observed severity of colitis, including weight loss and status of stool, and evaluated the disease activity index (DAI). A significantly low DAI and degree of weight loss were observed in infected mice, compared with uninfected mice. In addition, colon length in infected mice was not contracted, compared with uninfected mice. We also observed a significant increase in production of pro-inflammatory cytokines, IL-6 and IFN-γ, in spleen lymphocytes treated with DSS; however, such an increase was not observed in infected mice treated with DSS. Of particular interest, production of regulatory cytokines, IL-10 and transforming growth factor (TGF)-β, in spleen lymphocytes showed a significant increase in mice infected with T. spiralis. A similar result was observed in mesenteric lymph nodes (MLN). Subsets of the population of Treg cells in MLN and spleen showed significant increases in mice infected with T. spiralis. In conclusion, T. spiralis infection can inhibit the DSS-induced colitis in mice by enhancing the regulatory cytokine and Treg cells recruitment.

Protease-activated receptor 2 is involved in Th2 responses against Trichinella spiralis infection.

In our previous studies, the recombinant type II macrophage migration inhibitory factor homologue (rAs‐MIF) secreted from Anisakis simplex suppressed experimental inflammation mouse model through IL‐10 production and CD4+CD25+Foxp3+ T‐cell recruitment. Also, TLR2 gene expression was significantly increased following rAs‐MIF treatment. To know the relation between TLR2 and amelioration mechanisms of rAs‐MIF, we induced allergic airway inflammation by ovalbumin and alum with or without rAs‐MIF under TLR2 blocking systems [anti‐TLR2‐specific antibody (α‐mTLR2 Ab) treatment and using TLR2 knockout mice]. As a result, the amelioration effects of rAs‐MIF in allergic airway inflammation model (diminished inflammation and Th2 response in the lung, increased IL‐10 secretion, CD4+CD25+Foxp3+ T‐cell recruitment) were diminished under two of the TLR2 blocking model. The expression of TLR2 on the surface of lung epithelial cell was significantly elevated by rAs‐MIF treatment or Pam3CSK (TLR2‐specific agonist) treatment, but they might have some competition effect on the elevation of TLR2 expression. In addition, the elevation of IL‐10 gene expression by rAs‐MIF treatment was significantly inhibited by α‐mTLR2 Ab or Pam3CSK pretreatment. In conclusion, anti‐inflammatory effects of the rAs‐MIF on OVA‐induced allergic airway inflammation might be closely related to TLR2.

Trichinella spiralis Infection Suppressed Gut Inflammation with CD4+CD25+Foxp3+ T Cell Recruitment

Acanthamoeba is a free-living amoeba commonly present in the environment and often found in human airway cavities. Acanthamoeba possesses strong proteases that can elicit allergic airway inflammation. To our knowledge, the aeroallergenicity of Acanthamoeba has not been reported. We repeatedly inoculated mice with Acanthamoeba trophozoites or excretory-secretory (ES) proteins intra-nasally and evaluated symptoms and airway immune responses. Acanthamoeba trophozoites or ES proteins elicited immune responses in mice that resembled allergic airway inflammation. ES proteins had strong protease activity and activated the expression of several chemokine genes (CCL11, CCL17, CCL22, TSLP, and IL-25) in mouse lung epithelial cells. The serine protease inhibitor phenyl-methane-sulfonyl fluoride (PMSF) inhibited ES protein activity. ES proteins also stimulated dendritic cells and enhanced the differentiation of naive T cells into IL-4-secreting T cells. After repeated inoculation of the protease-activated receptor 2 knockout mouse with ES proteins, airway inflammation and Th2 immune responses were markedly reduced, but not to basal levels. Furthermore, asthma patients had higher Acanthamoeba-specific IgE titers than healthy controls and we found Acanthamoeba specific antigen from house dust in typical living room. Our findings suggest that Acanthamoeba elicits allergic airway symptoms in mice via a protease allergen. In addition, it is possible that Acanthamoeba may be one of the triggers human airway allergic disease.

TLR2‐dependent amelioration of allergic airway inflammation by parasitic nematode type II MIF in mice

Background The recruitment of CD4+CD25+Foxp3+T (Treg) cells is one of the most important mechanisms by which parasites down-regulate the immune system. Methodology/Principal Findings We compared the effects of Treg cells from Trichinella spiralis-infected mice and uninfected mice on experimental allergic airway inflammation in order to understand the functions of parasite-induced Treg cells. After four weeks of T. spiralis infection, we isolated Foxp3-GFP-expressing cells from transgenic mice using a cell sorter. We injected CD4+Foxp3+ cells from T. spiralis-infected [Inf(+)Foxp3+] or uninfected [Inf(-)Foxp3+] mice into the tail veins of C57BL/6 mice before the induction of inflammation or during inflammation. Inflammation was induced by ovalbumin (OVA)-alum sensitization and OVA challenge. The concentrations of the Th2-related cytokines IL-4, IL-5, and IL-13 in the bronchial alveolar lavage fluid and the levels of OVA-specific IgE and IgG1 in the serum were lower in mice that received intravenous application of Inf(+)Foxp3+ cells [IV(inf):+(+) group] than in control mice. Some features of allergic airway inflammation were ameliorated by the intravenous application of Inf(-)Foxp3+ cells [IV(inf):+(-) group], but the effects were less distinct than those observed in the IV(inf):+(+) group. We found that Inf(+)Foxp3+ cells migrated to inflammation sites in the lung and expressed higher levels of Treg-cell homing receptors (CCR5 and CCR9) and activation markers (Klrg1, Capg, GARP, Gzmb, OX40) than did Inf(-)Foxp3+ cells. Conclusion/Significance T. spiralis infection promotes the proliferation and functional activation of Treg cells. Parasite-induced Treg cells migrate to the inflammation site and suppress immune responses more effectively than non-parasite-induced Treg cells. The adoptive transfer of Inf(+)Foxp3+ cells is an effective method for the treatment and prevention of allergic airway diseases in mice and is a promising therapeutic approach for the treatment of allergic airway diseases.

Acanthamoeba Protease Activity Promotes Allergic Airway Inflammation via Protease-Activated Receptor 2

Health education has been shown to be effective in slowing the spread of the disease, infectious disease in particular. To evaluate the impact of health education on the prevalence and pattern of new infection of enterobiasis, children from 6 kindergartens in Ulsan city, South Korea, were recruited after undergoing a screening for enterobiasis, and then divided into three groups, including group medication (GM), education (Edu), and control group. All children in GM group received medical treatment with 500 mg albendazole twice, with 15 days interval. In the Edu group, only children diagnosed positive for Enterobius vermicularis eggs received medical treatment with 500 mg albendazole twice, with 15 days interval and all parents in the group received brochures providing information about enterobiasis. In the control group, only children diagnosed positive for E. vermicularis eggs received medical treatment with 500 mg albendazole twice, with 15 days interval, and no information about enterobiasis was provided to parents. Two post-treatment examinations were performed at three and six months after treatment. The infection rate in the GM group was dramatically decreased at 3 months, and this rate was almost the same as at 6 months after treatment. Infection rate of children in the Edu group was shown to drop from 9.9% to 3.0% at 3 months, and to 2.7% at 6 months after treatment; however, the infection rate in the control group continued to be higher than in the other two groups at both 3 and 6 months, with smaller change at 3 months compared to the other two groups. In addition, both new infection and re-infection cases in the Edu group were fewer, compared to those in the control group. In conclusion, although GM is the best method for eradication of enterobiasis, providing health information about enterobiasis to parents could reduce the prevalence, as well as the rate of new infection or re-infection with E. vermicularis in their children.

Parasitic nematode-induced CD4+Foxp3+T cells can ameliorate allergic airway inflammation.

Pinworm infection can occur through contact with contaminated surfaces followed by ingestion or even through inhalation of infective eggs. We have limited information regarding environmental contamination by eggs of Enterobius vermicularis. In order to determine environmental risk factors associated with the rate of E. vermicularis infection, we investigated possible environmental risk factors using a questionnaire from 46 kindergartens in 3 different cities of the southeast area of Korea. In total, using the cellotape anal swab technique, 3,422 children were examined for E. vermicularis infection. We evaluated E. vermicularis egg of books, educational materials, toys, room door handles, dusts of window edges, desks, chairs, tables, and dusts of classrooms. The overall egg-positive rate for E. vermicularis was 6.0%, and the prevalence of enterobiasis in each kindergarten ranged between 0% and 16.9%. We found that 78.9% of egg positive kindergartens were managed by private foundations, which was significantly higher, compared with kindergartens managed by public foundations or the nation. Compared with public or national kindergartens, most private kindergartens were located in residential areas and the number of children in these areas was significantly higher. In conclusion, numbers of children in kindergartens was found to be an environmental risk factor associated with transmission of enterobiasis in Korea.

Impact of health education on the prevalence of enterobiasis in Korean preschool students

Anisakis (Anisakidae) is one of the most important causes of helminth-induced allergic reactions and elicits clinical responses that include urticaria, rhinitis, bronco-constriction, cough, and/or gastrointestinal symptoms. More than 13 reactive allergens have been identified in the serum of Anisakis allergy patients, but the allergenicity of only a few of these have been evaluated in vivo using a mouse model. To evaluate the allergenicity of two important allergens, Ani s 1 and Ani s 9, we induced experimental allergic airway inflammation in a mouse model by repeated intranasal administration of the allergens. Both recombinant proteins (rAni s 1 and rAni s 9) elicited increased airway hyperresponsivity, airway infiltration by inflammatory cells (especially eosinophils), bronchial epithelial cell hyperplasia, all of which are characteristic of allergic airway inflammation. These allergens significantly increased the levels of Th2-related cytokines (IL-4, IL-5, IL-13, and IL-25) and Th17 related cytokines (IL-6 and IL-17) in both splenocytes and airway (except IL-17 in airway by rAni s 9). OVA-specific IgE and total IgE were increased in rAni s 1 and rAni s 9 treated mice as compared with controls treated with OVA alone. In addition, these two allergens induced gene expression of thymic stromal lymphopoietin (TSLP) and IL-25 (initiators of the Th2 response), as well as CXCL1 (initiator of the Th17 response) in mouse lung epithelial cells. In conclusion, repeated intranasal treatments with rAni s 1 and rAni s 9 induced airway inflammation in mice by elevating of Th2 and Th17 responses in the lung.