Multipotential acceptance of Peyer's patches in the intestine for both thymus-derived T cells and extrathymic T cells in mice

Peyer's patches (PP) are important inductive sites for the mucosal immune response. It is well known that lymphocytes that migrate into PP are mainly of T-cell lineage from thymus-derived cells (i.e. alphabetaTCR(high) cells). In this study, we further characterized the properties of PP lymphocytes in mice using a mouse model of colitis induced by dextran sulphate sodium (DSS). Although the major site of the inflammation induced by DSS is known to be the large intestine, the small intestine was also damaged. When mice developed DSS-induced colitis, CD3+CD8+B220+ gammadelta T cells increased in PP in the small intestine. These gammadelta T cells, which are not seen in the PP of normal mice, resembled intraepithelial lymphocytes (IEL) in the small intestine in terms of their expression of CD5, CD103 and Thy1.2. In addition, the Vgamma/delta repertoire of these gammadelta T cells was similar to that of gammadelta IEL. When DSS-treated mice were injected with IEL isolated from normal mice, IEL including gammadelta T cells preferentially migrated to PP, raising the possibility that B220+ T cells seen in PP of diseased mice may derive from IEL in the small intestine. Our present study suggests that PP might be able to accept T-cell lineages from intestinal IEL as well as from thymus-derived T cells.     

Lymphokine production by mitogen and antigen activated mouse intraepithelial lymphocytes

Although most intraepithelial lymphocytes (IEL) in mouse small intestine bear surface markers classically associated with T lymphocytes, the T-cell nature of these cells remains controversial. In the present study IEL from normal mice, or from mice infected with the gut nematode Trichinella spiralis, were therefore tested for their ability to produce T-cell-derived lymphokines in response to in vitro stimulation with concanavalin A (Con A) or with specific worm antigens. The data show that Con A-stimulated IEL produce minimal amounts of IL-2, and intermediate levels of IFN-gamma and IL-3 in comparison to the levels produced by spleen T cells. The FDC-P2 cell line, which proliferates in response to both IL-3 and GM-CSF, was identified as the most sensitive and reproducible indicator of lymphokine activity in supernatants from mitogen-stimulated IEL from normal mice. IEL isolated from mice infected with T. spiralis also produced high levels of FDC-P2 growth factors when challenged in vitro with Trichinella-derived antigens; however, normal IEL did not respond to this stimulus. The data thus provide evidence that antigen-sensitive T cells can arise in (or migrate to) the gut epithelium during gut infection.     

The characterization of intraepithelial lymphocytes, lamina propria leukocytes, and isolated lymphoid follicles in the large intestine of mice infected with the intestinal nematode parasite Trichuris muris

Despite a growing understanding of the role of cytokines in immunity to the parasitic helminth Trichuris muris, the local effector mechanism culminating in the expulsion of worms from the large intestine is not known. We used flow cytometry and immunohistochemistry to characterize the phenotype of large intestinal intraepithelial lymphocytes (IEL) and lamina propria leukocytes (LPL) from resistant and susceptible strains of mouse infected with T. muris. Leukocytes accumulated in the epithelium and lamina propria after infection, revealing marked differences between the different strains of mouse. In resistant mice, which mount a Th2 response, the number of infiltrating CD4+, CD8+, B220+, and F4/80+ IEL and LPL was generally highest around the time of worm expulsion from the gut, at which point the inflammation was dominated by CD4+ IEL and F4/80+ LPL. In contrast, in susceptible mice, which mount a Th1 response, the number of IEL and LPL increased more gradually and was highest after a chronic infection had developed. At this point, CD8+ IEL and F4/80+ LPL were predominant. Therefore, this study reveals the local immune responses underlying the expulsion of worms or the persistence of a chronic infection in resistant and susceptible strains of mouse, respectively. In addition, for the first time, we illustrate isolated lymphoid follicles in the large intestine, consisting of B cells interspersed with CD4+ T cells and having a central zone of rapidly proliferating cells. Furthermore, we demonstrate the organogenesis of these structures in response to T. muris infection.     

IFN-gamma-producing dendritic cells are important for priming of gut intraepithelial lymphocyte response against intracellular parasitic infection

The importance of intraepithelial lymphocytes (IEL) in immunoprotection against orally acquired pathogens is being increasingly recognized. Recent studies have demonstrated that Ag-specific IEL can be generated and can provide an important first line of defense against pathogens acquired via oral route. However, the mechanism involved in priming of IEL remains elusive. Our current study, using a microsporidial model of infection, demonstrates that priming of IEL is dependent on IFN-gamma-producing dendritic cells (DC) from mucosal sites. DC from mice lacking the IFN-gamma gene are unable to prime IEL, resulting in failure of these cells to proliferate and lyse pathogen-infected targets. Also, treatment of wild-type DC from Peyer's patches with Ab to IFN-gamma abrogates their ability to prime an IEL response against Encephalitozoon cuniculi in vitro. Moreover, when incubated with activated DC from IFN-gamma knockout mice, splenic CD8(+) T cells are not primed efficiently and exhibit reduced ability to home to the gut compartment. These data strongly suggest that IFN-gamma-producing DC from mucosal sites play an important role in the generation of an Ag-specific IEL response in the small intestine. To our knowledge, this report is the first demonstrating a role for IFN-gamma-producing DC from Peyer's patches in the development of Ag-specific IEL population and their trafficking to the gut epithelium.     

Mucosal Immune Responses of Mice Experimentally Infected with Pygidiopsis summa (Trematoda: Heterophyidae)

Mucosal immune responses against Pygidiopsis summa (Trematoda: Heterophyidae) infection were studied in ICR mice. Experimental groups consisted of group 1 (uninfected controls), group 2 (infection with 200 metacercariae), and group 3 (immunosuppression with Depo-Medrol and infection with 200 metacercariae). Worms were recovered in the small intestine at days 1, 3, 5, and 7 post-infection (PI). Intestinal intraepithelial lymphocytes (IEL), mast cells, and goblet cells were counted in intestinal tissue sections stained with Giemsa, astra-blue, and periodic acid-Schiff, respectively. Mucosal IgA levels were measured by ELISA. Expulsion of P. summa from the mouse intestine began to occur from days 3-5 PI which sustained until day 7 PI. The worm expulsion was positively correlated with proliferation of IEL, mast cells, goblet cells, and increase of IgA, although in the case of mast cells significant increase was seen only at day 7 PI. Immunosuppression suppressed all these immune effectors and inhibited worm reduction in the intestine until day 7 PI. The results suggested that various immune effectors which include IEL, goblet cells, mast cells, and IgA play roles in regulating the intestinal mucosal immunity of ICR mice against P. summa infection.     

Response of Ig-positive cells to Enteromyxum scophthalmi (Myxozoa) experimental infection in turbot, Scophthalmus maximus (L.): A histopathological and immunohistochemical study

In recent years, a new parasite that causes severe losses has been detected in farmed turbot, Scophthalmus maximus (L.), in Northwestern Spain. Dead fish showed emaciation and cachexia caused by severe necrotizing enteritis that affected all portions of the digestive tract. The parasite was classified as a myxosporean and named Enteromyxum scophthalmi. This study was set up to gain insights into the immune response of fish against this parasitic infection. The kinetics of immunoglobulin positive (Ig+) cells in spleen, kidney and intestine from turbot experimentally infected with E. scophthalmi was studied. For evaluating both the progress of infection and the lesions induced by the parasite, we performed histopathological studies and for detecting Ig+ cells in situ we used an indirect immunohistochemical method. In fish exposed to the parasite, the number of Ig+ cells significantly increased in the intestine, the target organ of the parasite, whereas in spleen and kidney, haematopoietic organs where the parasite was not detected, the number of Ig+ cells decreased. Furthermore, the pattern of distribution of Ig+ cells changed in all three organs examined in recipient/infected fish compared with control fish (not exposed to the parasite). The results obtained in this study indicate that the infection by E. scophthalmi in turbot induced an immune response that involved changes in the number and distribution of Ig+ cells.     

Mucosal T cells bearing TCRgammadelta play a protective role in intestinal inflammation

Intestinal intraepithelial lymphocytes (IEL) bearing TCRgammadelta represent a major T cell population in the murine intestine. However, the role of gammadelta IEL in inflammatory bowel diseases (IBD) remains controversial. In this study, we show that gammadelta IEL is an important protective T cell population against IBD. gammadelta T cell-deficient (Cdelta(-/-)) mice developed spontaneous colitis with age and showed high susceptibility to Th1-type 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis at a young age. Transfer of gammadelta IEL to Cdelta(-/-) mice ameliorated TNBS-induced colitis, which correlated with decrease of IFN-gamma and TNF-alpha production and an increase of TGF-beta production by IEL. Furthermore, a high level of IL-15, which inhibits activation-induced cell death to terminate inflammation, was expressed more in intestinal epithelial cells (EC) from TNBS-treated Cdelta(-/-) mice than in those from wild-type mice. EC from wild-type mice significantly suppressed the IFN-gamma production of IEL from TNBS-treated Cdelta(-/-) mice, whereas EC from TNBS-treated Cdelta(-/-) mice did not. These data indicate that gammadelta IEL play important roles in controlling IBD by regulating mucosal T cell activation cooperated with EC function. Our study suggests that enhancement of regulatory gammadelta T cell activity is a possible new cell therapy for colitis.     

Stem cell factor (SCF) can regulate the activation and expansion of murine intraepithelial lymphocytes

Murine intraepithelial lymphocytes (IEL) express c-kit, the receptor for stem cell factor (SCF). SCF induced a low but significant proliferative response in IEL, but not in splenic T cells. SCF stimulation of IEL resulted in an expansion of the c-kit(+), TCRgammadelta(+)cell population. SCF-induced proliferation was dependent upon SCF-c-kit interactions, since antibody to c-kit blocked this response, and IEL obtained from c-kit mutant (W/W(v)) mice failed to respond to SCF. SCF acted synergistically with anti-TCRgammadelta and with concavalin A (Con A) to induce proliferation and interferon gamma (IFN-gamma) production in IEL. Finally, mice injected with SCF had a significant increase in the number of IEL in the small intestine. SCF-treated mice had increased numbers of TCRalphabeta(+)and TCRgammadelta(+)cell populations, as well as increased numbers of c-kit(+)and c-kit(-)IEL. These data suggest that SCF-c-kit interactions play an important role in regulating IEL expansion and activation.     

Systemic dissemination and persistence of Th2 and type 2 cells in response to infection with a strictly enteric nematode parasite

Oral infection with the nematode parasite Heligmosomoides polygyrus H. polygyrus is entirely restricted to the small intestine. Although the evoked Th2 response has been extensively studied in secondary lymphoid organs, little is known about the systemic dissemination of Th2 cells or type 2 associated eosinophils and basophils. In this study we use bicistronic 4get IL-4 reporter mice to directly visualize the type 2 response to H. polygyrus infection. We observed that CD4(+)/GFP(+) Th2 cells spread systemically and found that these cells accumulated in nonlymphoid "hot spots" in the liver, the lung airways, and the peritoneal cavity. Interestingly, the total number of Th2 cells in the peritoneal cavity was comparable to those found in the draining mesenteric lymph node or the spleen. Peritoneal Th2 cells were distinguished by an exceptionally low apoptotic potential and high expression of the intestinal homing receptor alpha(4)beta(7) integrin. CD4(+)/GFP(+) Th2 cells from these peripheral sites were fully functional as indicated by rapid IL-4 production upon polyclonal or Ag-specific restimulation. Th2 cells persisted in the intestinal tissue and the peritoneal cavity of drug-cured mice for weeks. The presence of peripheral memory Th2 cells in the intestine might be crucial for immunity to recall infections. These findings have important implications for the design of vaccination strategies because it may be necessary to establish and maintain memory CD4(+) T cells at the potential future site of infection.     

Entamoeba histolytica: acute granulomatous intestinal lesions in normal and neutrophil-depleted mice

To study the role of neutrophils in the innate resistance to Entamoeba histolytica intestinal infection in mice, animals were treated with anti-neutrophil monoclonal antibodies prior to intracecal parasite inoculation and the resulting lesions were compared with normal mice that had been equally infected. In contrast to our previous finding that neutrophils are critical in eliminating E. histolytica infection in the liver, we show here that neutrophils are not absolutely required to eliminate E. histolytica infection from the intestine. Although the neutrophils are not critical for resolution of the E. histolytica infection, neutrophils do appear to provide some measure of protection as the intestinal amoeba burden was higher at early timepoints after infection in the neutropenic animals. In addition, we found that while both the normal and the neutrophil-depleted mice developed ulcerative lesions in the colon, the neutropenic mice had an increased frequency of granulomas that formed around the amoeba. Thus, our findings appear to be the first evidence showing that granulomatous inflammation can occur after intestinal infection in mice using axenically cultured amoeba.     

Endoplasmic reticulum stress response promotes cytotoxic phenotype of CD8αβ+ intraepithelial lymphocytes in a mouse model for Crohn's disease-like ileitis

Endoplasmic reticulum (ER) unfolded protein responses (UPR) are implicated in the pathogenesis of inflammatory bowel disease. Cytotoxic CD8αβ(+) intraepithelial lymphocytes (IEL) contribute to the development of Crohn's disease-like ileitis in TNF(ΔARE/+) mice. In this study, we characterized the role of ER-UPR mechanisms in contributing to the disease-associated phenotype of cytotoxic IEL under conditions of chronic inflammation. Inflamed TNF(ΔARE/+) mice exhibited increased expression of Grp78, ATF6, ATF4, and spliced XBP1 in CD8αβ(+) IEL but not in CD8αα(+) IEL or in lamina propria lymphocytes. Chromatin immunoprecipitation analysis in CD8αβ(+) T cells showed selective recruitment of ER-UPR transducers to the granzyme B gene promoter. Heterozygous Grp78(-/+) mice exhibited an attenuated granzyme B-dependent cytotoxicity of CD8αβ(+) T cells against intestinal epithelial cells, suggesting a critical activity of this ER-associated chaperone in maintaining a cytotoxic T cell phenotype. Granzyme B-deficient CD8αβ(+) T cells showed a defect in IL-2-mediated proliferation in Grp78(-/+) mice. Adoptively transferred Grp78(-/+) CD8αβ(+) T cells had a decreased frequency of accumulation in the intestine of RAG2(-/-) recipient mice. The tissue pathology in TNF(ΔARE/+) × Grp78(-/+) mice was similar to TNF(ΔARE/+) mice, even though the cytotoxic effector functions of CD8αβ(+) T cells were significantly reduced. In conclusion, ER stress-associated UPR mechanisms promote the development and maintenance of the pathogenic cytotoxic CD8αβ(+) IEL phenotype in the mouse model of Crohn's disease-like ileitis.     

Demonstration of functional role of TECK/CCL25 in T lymphocyte-endothelium interaction in inflamed and uninflamed intestinal mucosa

It has recently been suggested that C-C chemokines may play a role in the organ-specific homing of lymphocytes, but there is not enough in vivo evidence in intestinal mucosa. The aim of this study was to examine whether thymus-expressed chemokine (TECK)/CCL25 and its ligand CCR9 are involved in T-lymphocyte interaction with microvessels of murine intestinal mucosa. T lymphocytes from the small intestine were fluorescence labeled, and their adhesion to mucosal microvessels was observed by intravital microscopy. Lamina proprial lymphocytes (LPL) and intraepithelial lymphocytes (IEL) adhered to both the small intestine and colon, and desensitization of CCR9 with TECK/CCL25 or anti-TECK/CCL25 antibody significantly inhibited these adhesions only in small intestine. At both sites, TNF-alpha significantly increased LPL adhesion but not IEL adhesion. Desensitization of CCR9 or anti-TECK/CCL25 antibody also attenuated the TNF-alpha-induced LPL adhesion in the small intestine. Increased expression of TECK/CCL25 by TNF-alpha was observed in the lamina propria of small intestine. TECK/CCL25 may thus play an important role in the adherence of mucosal lymphocytes to the microvessels of the small intestine but not the colon under uninflamed as well as inflamed conditions.     

Homing of mouse spermatogonial stem cells to germline niche depends on beta1-integrin

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis. In a manner comparable to hematopoietic stem cell transplantation, SSCs colonize the niche of recipient testes and reinitiate spermatogenesis following microinjection into the seminiferous tubules. However, little is known about the homing mechanism of SSCs. Here we examined the role of adhesion molecules in SSC homing. SSCs isolated from mice carrying loxP-tagged beta1-integrin alleles were ablated for beta1-integrin expression by in vitro adenoviral cre transduction. The beta1-integrin mutant SSCs showed significantly reduced ability to recolonize recipient testes in vivo and to attach to laminin molecules in vitro. In contrast, genetic ablation of E-cadherin did not impair homing, and E-cadherin mutant SSCs completed normal spermatogenesis. In addition, the deletion of beta1-integrin on Sertoli cells reduced SSC homing. These results identify beta1-integrin as an essential adhesion receptor for SSC homing and its association with laminin is critical in multiple steps of SSC homing.     

Intestinal IgA responses to Giardia muris in mice depleted of helper T lymphocytes and in immunocompetent mice

Immunocompetent mice infected with Giardia muris generate an intestinal antibody response to this parasite and clear G. muris infection. Previous work has shown that G. muris infection is prolonged in mice that have been depleted of helper (CD4+) T lymphocytes by treatment with a monoclonal antibody (mAb) directed against the murine CD4 antigen. The aim of the present study was to compare the intestinal anti-Giardia antibody response in immunocompetent mice and in mice depleted of helper T (Th) lymphocytes by treatment with anti-CD4 mAb. Immunocompetent mice generated an IgA response to G. muris, as judged by the presence of IgA on Giardia trophozoites harvested from the intestine of these animals more than 10 days after the start of the infection. The anti-Giardia IgA response was impaired in mice depleted of Th lymphocytes, as judged by virtual absence of immunofluorescent staining of trophozoites from these animals for surface-bound IgA. Clearance of G. muris infection was impaired by treatment of mice with anti-CD4 mAb. The results suggest that Th (CD4+) lymphocytes are important for the generation of a local IgA response against G. muris trophozoites in the mouse intestine and that IgA anti-trophozoite antibody may contribute to the clearance of G. muris from the intestine of immunocompetent mice.     

Modulation of CD8+ intraepithelial lymphocyte distribution by dietary fiber in the rat large intestine

We studied whether ingestion of dietary fiber modifies the distribution of intraepithelial lymphocytes (IEL) in a physiological condition. Male WKAH rats were fed diets either with fiber (sugar beet fiber or crystalline cellulose, 100 g/kg diet each) or without fiber for 3 weeks. The number of CD8(+), CD4(+), and NKR-P1(+) IEL per epithelial layer in the crypt section of the cecum, proximal colon, and distal colon were scored by immunohistochemical staining. We found that the proportion of CD8(+) IEL was greater in the cecal mucosa and was gradually reduced toward the distal large intestine in general. In contrast, there was no difference in the proportion of CD4(+) and NKR-P1(+) IEL in the large intestine. Dietary sugar beet fiber, but not crystalline cellulose, increased the proportion of CD8(+) IEL, especially in the cecal mucosa, but not the CD4(+) and NKR-P1(+) IEL. Analysis of cecal organic acid concentration confirmed higher concentrations of acetate and butyrate, and lower concentration of succinate and isovalerate, in the cecum of the rats fed sugar beet fiber than other diets. These results indicate that ingestion of some dietary fiber modulates local cell proliferation of a progenitor of CD8(+) IEL or promotes homing of CD8(+) T cells into the large intestinal epithelium, most likely via the fermentation in the luminal contents.     

Deficiency of gammadelta T lymphocytes contributes to mortality and immunosuppression in sepsis

Studies have indicated that gammadelta T lymphocytes play an important role in the regulation of immune function and the clearance of intracellular pathogens. We have recently reported that intraepithelial lymphocytes (IEL), which are rich in gammadelta T cells, within the small intestine illustrated a significant increase in apoptosis and immune dysfunction in mice subjected to sepsis. However, the contribution of gammadelta T cells to the host response to polymicrobial sepsis remains unclear. In this study, we initially observed that after sepsis induced by cecal ligation and puncture (CLP), there was an increase in small intestinal IEL CD8+gammadelta+ T cells in control gammadelta+/+ mice. Importantly, we subsequently found an increased early mortality in mice lacking gammadelta T cells (gammadelta-/- mice) after sepsis. This was associated with decreases in plasma TNF-alpha, IL-6, and IL-12 levels in gammadelta-/- mice compared with gammadelta+/+ mice after sepsis. In addition, even though in vitro LPS-stimulated peritoneal macrophages showed a reduction in IL-6 and IL-12 release after CLP, these cytokines were less suppressed in macrophages isolated from gammadelta-/- mice. Alternatively, IL-10 release was not different between septic gammadelta+/+ and gammadelta-/- mice. Whereas T helper (Th)1 cytokine release by anti-CD3-stimulated splenocytes was significantly depressed in septic gammadelta+/+ mice, there was no such depression in gammadelta-/- mice. However, gammadelta T cell deficiency had no effect on Th2 cytokine release. These findings suggest that gammadelta T cells may play a critical role in regulating the host immune response and survival to sepsis, in part by alteration of the level of IEL CD8+gammadelta+ T cells and through the development of the Th1 response.     

Chromosomal mapping of the host resistance locus to rodent malaria (Plasmodium yoelii) infection in mice

The disease outcome in malaria caused by the protozoan parasite Plasmodium is influenced by host genetic factors. To identify host genes conferring resistance to infection with the malaria parasite, we undertook chromosomal mapping using a whole-genome scanning approach in cross-bred mice. NC/Jic mice all died with high parasitemia within 8 days of infection with 1 x 10(5) parasitized erythrocytes. In contrast, 129/SvJ mice all completely excluded malaria parasites from the circulation and remained alive 21 days after infection. We performed linkage analysis in backcross [(NC/Jic x 129/SvJ)xNC/Jic] mice. The Pymr ( Plasmodium yoelii malaria resistance) locus was mapped to the telomeric portion of mouse Chromosome (Chr) 9. This locus controls host survival and parasitemia after infection. The Char1 locus ( P. chabaudi resistance locus 1), controlling host survival and peak parasitemia in P. chabaudi infection, was previously mapped to the same region. This host resistance locus mapping to Chr 9 may represent a ubiquitous locus controlling susceptibility to rodent malaria. Elucidation of the function of this gene will provide valuable insights into the mechanism of host defense against malaria parasite infection.     

Early-phase migration dynamics of Echinococcus multilocularis in two mouse strains showing different infection susceptibilities

The early-phase migration dynamics of Echinococcus multilocularis in the intermediate hosts remain largely unknown. We compared the parasite burden in the intestine, liver and faeces of DBA/2 and C57BL/6 mouse strains using parasite-specific quantitative PCR. Our results indicated that the parasites invaded mainly from the middle segments of the small intestine and completed migration to the liver within 24 h p.i. C57BL/6 mice had lower parasite DNA burdens in the intestine and liver but higher in the faeces than DBA/2 mice, suggesting that parasite invasion of the intestine may be a critical stage regulating susceptibility to E. multilocularis infection in mice.     

Importance of gender and sex hormones in regulation of susceptibility of the small intestine to peroral infection with Toxoplasma gondii tissue cysts.

This study was performed to investigate whether the gender and sex hormones play a role in susceptibility of the small intestine of mice to peroral infection with Toxoplasma gondii tissue cysts. Female animals died significantly earlier than males, and early mortality of females was associated with greater numbers of tachyzoites and severe necrosis in their small intestines. Treatment of female mice with testosterone resulted in markedly reduced intestinal parasite numbers and pathology. Thus, the gender and sex hormones are important factors for determining susceptibility of the small intestine to T. gondii infection.