Differential localization of colitogenic Th1 and Th2 cells monospecific to a microflora-associated antigen in mice

BACKGROUND & AIMS: Clonal expansion of T cells is associated with inflammatory bowel diseases, which indicates antigenic activation of the T cells. We investigated whether the introduction of CD4 T cells specific to a microflora would initiate colitis and assessed the cytokine requirements for colitogenic CD4 T cells. METHODS: Severe combined immunodeficiency disease (SCID) mice were reconstituted with CD4 T cells, which were either deficient in interleukin (IL)-4/interferon (IFN)-gamma production or differentiated in vitro to T-helper (Th) 1/Th 2 and bearing a transgenic T-cell receptor (TCR) specific to ovalbumin (OVA), and then inoculated with an Escherichia coli-producing OVA (ECOVA). Clinical and histologic manifestations of colitis were assessed. RESULTS: Mice with ECOVA colonization and OVA-specific CD4 T cells developed colitis with histologic features of focal infiltration by mononuclear cells, destruction of crypts, and loss of goblet cells. Further, infiltration was initiated in pre-existing lymph follicles. Th1- and IL-4 deficient T cells were diffusely localized in the lamina propria and submucosa, whereas Th2- and IFN-gamma-deficient T cells were localized preferentially in lymph follicles. CONCLUSIONS: A microbe-associated antigen, non-cross-reactive to colonic tissue, can drive antigen-specific CD4 T cells to cause colitis in SCID mice. Although the presence of IFN-gamma and IL-4 in the effector CD4 T cells was not an absolute requirement for the development of colitis, they seemed to regulate it in part by modulating migration of the effector T cells.     

Fasciola hepatica infection downregulates Th1 responses in mice

Immune responses induced with helminth parasites have been extensively studied, but there is limited information on those to Fasciola hepatica, especially on the subtype of T cell induced with this parasite. We investigated the local and systemic T cell responses of different strains of mice following oral infection with doses of metacercariae from F. hepatica. Spleen cells from BALB/c and 129Sv/Ev mice given a low-dose (5 metacercariae) infection exhibited a Th2 response, producing high levels of the cytokines IL-4 and IL-5, and low levels of IFN-gamma and IL-2. In contrast, C57BL/6 mice showed a mixed Th1/Th2 response. A more marked polarization to a Th2 response was observed in BALB/c, 129Sv/Ev exposed to a high-dose (15 metacercariae) infection and the C57BL/6 mice also exhibited a clear Th2 response. IL-4 defective (IL-4-/-) C57BL/6 mice infected with 5 metacercariae produced less IFN-gamma and more IL-5 compared to their wild-type C57BL/6 counterparts, suggesting that IL-4 is important in establishing the Th2 type response in murine fasciolosis. However, the secretion of IFN-gamma and IL-2 was completely suppressed in the high-dose infection and this was also observed in IL-4-/- mice. Thus, liver flukes may secrete molecules that downregulate Th1 responses. T cell responses in the mesenteric (MLN) and hepatic lymph nodes (HLN) were also examined since newly excysted juveniles infect through the intestinal wall of their host before migrating to the hepatic tissue. Cells from both MLN and HLN secreted higher levels of IL-4 and IL-5 compared to spleen cells. We also observed a difference in cytokine profiles secreted by the MLN and HLN, which may reflect responses to antigens liberated by newly excysted juveniles and hepatic stage parasites, respectively.     

Disruption of T helper 2-immune responses in Epstein-Barr virus-induced gene 3-deficient mice

Epstein-Barr virus-induced gene 3 (EBI3) is a widely expressed IL-12p40-related protein that associates as a heterodimer with either IL-12p35 or an IL-12p35 homologue, p28, to create a new cytokine (IL-27). To define the function of EBI3 in vivo, we generated knockout mice in which the ebi3 gene was targeted by homologous recombination. EBI3-/- mice exhibited normal numbers of both naive and mature CD4+ and CD8+ T cells and B cells, but markedly decreased numbers of invariant natural killer T cells (iNKT) as defined by staining with an alpha-galactosylceramide (alphaGalCer)-loaded CD1d-tetramer. iNKT cells from EBI3-/- mice exhibited decreased IL-4 and, to a lesser extent, IFN-gamma production after alphaGalCer stimulation in vitro. A sustained decrease in IL-4 production was also observed in EBI3-/- mice after alphaGalCer stimulation in vivo in contrast to IFN-gamma production, which was only transiently decreased under such stimulation. Notably, EBI3-/- mice were resistant to the induction of immunopathology associated with oxazolone-induced colitis, a colitis model mediated primarily by T helper (Th) 2-type cytokine production by iNKT cells. In contrast, trinitrobenzene sulfonic acid-induced colitis, a predominantly Th1-mediated colitis model, was unaffected. Thus, EBI3 plays a critical regulatory role in the induction of Th2-type immune responses and the development of Th2-mediated tissue inflammation in vivo, which may be mediated through the control of iNKT cell function.     

Cellular immune responses and cytokine production in BALB/c and C57BL/6 mice during the acute phase of Angiostrongylus costaricensis infection

In our experimental study we were able to show that the contrasting outcome of Angiostrongylus costaricensis infection in C57BL/6 and BALB/c mice, in respect of morbidity and mortality, can be explained by divergent cellular immune responses and a different cytokine pattern in each strain. In BALB/c mice (i.e. those with high mortality), the initial high proliferation of ConA or LPS stimulated spleen cells dropped to very low levels after 2 weeks post-infection (p.i.), whereas in C57BL/6 mice (i.e. those with low mortality), only a minor reduction in lymphoproliferative responses after mitogenic stimulation was observed. The specific proliferation of spleen cells after stimulation with A. costaricensis adult worm antigen remained low in BALB/c mice throughout the experiment, but showed an augmented proliferation in C57BL/6 mice, especially from 2 weeks p.i. onwards. The mitogen-induced production of Th2-type cytokines (IL-4, IL-5, IL-6, IL-10) in spleen cell cultures remained low in BALB/c mice until 4 weeks p.i., but production of Th1-type cytokines (IL-2, IFN-gamma) was highly elevated at 14 and 28 days p.i. In C57BL/6 mice, an upregulated and balanced production of both Th1- and Th2-type cytokines was measured during the course of infection. In summary, a polarization of the immune response towards cellular hyporesponsiveness and a predominantly Th1 cytokine profile was observed in A. costaricensis infected BALB/c mice, which may contribute to pathogenesis and increased morbidity.     

In vitro indomethacin administration upregulates interleukin-12 production and polarizes the immune response towards a Th1 type in susceptible BALB/c mice infected with Leishmania mexicana

The immune response in Leishmania infected BALB/c mice is associated with a Th2 type cellular response, which has been characterized by the absence of interleukin (IL)-12, interferon (IFN)-gamma, and nitric oxide (NO) and the presence of IL-10 and IL-4. Prostaglandins (PGs) can modulate the immune response inhibiting the development of Th1 response and enhancing the development of Th2 response. We investigated the production of PGs and their effects on cytokine and NO production by spleen cells from Leishmania mexicana infected BALB/c and C57BL/6 mice. Increased production of PGs was noted as early as 1 week after infection in BALB/c mice, whereas in infected C57BL/6 mice PGs were not detected. In vitro administration of indomethacin (INDO), a specific inhibitor of PGs synthesis, reduced PGs production at normal levels, and increased IL-12, IFN-gamma, and NO production in infected BALB/c mice. Whereas, IL-10 and IL-4 were not affected. Moreover, INDO did not modulate cytokine and NO production in infected C57BL/6. INDO addition induced the intracellular killing of parasites in infected BALB/c mice. Together, these results suggest that suppression of PGs by INDO may promote the development of a protective Th1 type response in susceptible mice by a mechanism, which involves an enhancement of IL-12, IFN-gamma and NO production. These findings were confirmed by smaller lesions in BALB/c mice, when treated with INDO.     

Absence of donor Th17 leads to augmented Th1 differentiation and exacerbated acute graft-versus-host disease

Th17 is a newly identified T-cell lineage that secretes proinflammatory cytokine IL-17. Th17 cells have been shown to play a critical role in mediating autoimmune diseases such as EAE, colitis, and arthritis, but their role in the pathogenesis of graft-versus-host disease (GVHD) is still unknown. Here we showed that, in an acute GVHD model of C57BL/6 (H-2(b)) donor to BALB/c (H-2(d)) recipient, IL-17(-/-) donor T cells manifested an augmented Th1 differentiation and IFN-gamma production and induced exacerbated acute GVHD. Severe tissue damage mediated by IL-17(-/-) donor T cells was associated with increased Th1 infiltration, up-regulation of chemokine receptors by donor T cells, and enhanced tissue expression of inflammatory chemokines. Administration of recombinant IL-17 and neutralizing IFN-gamma in the recipients given IL-17(-/-) donor cells ameliorated the acute GVHD. Furthermore, the regulation of Th1 differentiation by IL-17 or Th17 may be through its influence on host DCs. Our results indicate that donor Th17 cells can down-regulate Th1 differentiation and ameliorate acute GVHD in allogeneic recipients, and that treatments neutralizing proinflammatory cytokine IL-17 may augment acute GVHD as well as other inflammatory autoimmune diseases.     

Macrophage-derived IL-18-mediated intestinal inflammation in the murine model of Crohn's disease

BACKGROUND & AIMS: Crohn's disease (CD) is associated with an increased number of infiltrating macrophages, which release a variety of proinflammatory cytokines. Interleukin (IL)-18 has been implicated in the modulation of mucosal CD4(+) T cells towards Th1 responses, which are implicated in the pathogenesis of CD. Here we assess the role of macrophages and of IL-18 in the murine model of intestinal inflammation that mimics the immunologic characteristics of human CD. METHODS: Colitis was induced in C57BL/6 mice immunized with 2,4,6-trinitrobenzene sulfonic acid (TNBS) followed by rectal administration of TNBS in ethanol. Mice were treated with either an antibody directed against macrophages conjugated to the ribosome-inactivating protein saporin (anti-Mac-1-saporin) or with a neutralizing antibody against IL-18. In addition, we assessed whether an identical TNBS immunization/challenge protocol could induce colitis in IL-18(-/-) mice. RESULTS: The colonic mucosa of TNBS-treated mice was marked by infiltration of Mac-1-positive macrophages and up-regulation of IL-18. The administration of the anti-Mac-1-saporin antibody or the neutralizing anti-IL-18 antibody resulted in a dramatic attenuation of mucosal inflammation in this model. In addition, TNBS was unable to induce significant colitis in the IL-18(-/-) mice. CONCLUSIONS: Our data underscore the pivotal role of macrophages, and the macrophage-derived IL-18, in the establishment of TNBS-induced colitis in mice. Our results highlight the potential use of therapy directed against IL-18 in the treatment of patients with CD.     

Amelioration of dextran sulfate sodium-induced colitis by anti-macrophage migration inhibitory factor antibody in mice

BACKGROUND & AIMS: We investigated the effects of macrophage migration inhibitory factor (MIF) antibodies in experimental colitis-induced dextran sulfate sodium (DSS) and trinitrobenzenesulfonic acid (TNBS) and examined whether plasma levels of MIF were elevated in patients with inflammatory bowel disease (IBD). METHODS: BALB/c or C57BL/6 mice were fed 4% DSS in their drinking water for up to 7 days with and without administration of an anti-MIF antibody every 2 days. The severity of inflammation in the cecum and colon was assessed by clinical signs and histologic scoring. Tissue levels of MIF, tumor necrosis factor (TNF)-alpha, interferon gamma (IFN-gamma), interleukin (IL)-4, and matrix metalloproteinase (MMP)-13 messenger RNA (mRNA) were measured. The effects of anti-MIF antibody on chronic colitis induced by TNBS was assessed in BALB/c mice. Plasma MIF concentrations were assayed in patients with Crohn's disease, ulcerative colitis, and healthy controls. RESULTS: During DSS-induced colitis, colonic MIF mRNA expression was increased. Clinical signs and histopathologic features were significantly improved in animals given anti-MIF antibody. DSS-induced up-regulation of colonic TNF-alpha and IFN-gamma were significantly suppressed in animals given the anti-MIF antibody. Colonic IL-4 was decreased during DSS but restored to baseline by the anti-MIF antibody. The anti-MIF antibody prevented MMP-13 up-regulation by DSS and ameliorated TNBS colitis. Plasma MIF was elevated in patients with Crohn's disease or ulcerative colitis compared with healthy controls. CONCLUSIONS: We conclude that anti-MIF antibodies reduce the severity of experimental colitis and limit the up-regulation of Th1-type cytokines. Anti-MIF antibodies are of potential therapeutic use in IBD.     

Anti-interleukin 12 treatment regulates apoptosis of Th1 T cells in experimental colitis in mice.

BACKGROUND & AIMS: Trinitrobenzene sulfonic acid (TNBS)-induced colitis is a T-helper 1 (Th1) T cell-mediated inflammation that is rapidly reversed by administration of anti-interleukin (IL) 12. This study sought to define the mechanism of this curative effect. METHODS: Cells and tissue from mice with TNBS colitis receiving treatment with anticytokines were analyzed for phenotype, cytokine production, and apoptosis. RESULTS: In initial studies, we found that treatment of mice with TNBS-induced colitis with anti-IL-12 was more effective than with anti-interferon (IFN)-gamma, and that anti-IL-12 led to complete normalization of IFN-gamma production by lamina propria T cells ex vivo, whereas anti-IFN-gamma did not. These data suggesting that anti-IL-12 leads to reversal of colitis by elimination of the Th1 T cells were substantiated by studies showing that anti-IL-12 treatment led to increased numbers of apoptotic cells in the lamina propria and spleen by both TUNEL staining of tissues and dispersed spleen cell populations. Finally, we found that the observed apoptosis was mediated by the Fas pathway because (1) MRL/MpJ-lpr(fas) mice lacking Fas function develop colitis that responds poorly to treatment with anti-IL-12; and (2) SJL/J mice with TNBS colitis that received Fas-Fc to block the Fas pathway were resistant to anti-IL-12 treatment. CONCLUSIONS: These studies show that a main mechanism of action of anti-IL-12 in TNBS-induced colitis is the induction of Fas-mediated apoptosis of the Th1 T cells, causing inflammation.     

日本血吸虫22.6kDa膜相关蛋白Th1型表位的免疫学鉴定

目的鉴定日本血吸虫22.6 kDa膜蛋白（Sj22.6）的Th1型表位,为构建短肽疫苗奠定基础。方法采用合成肽Sj22.6-P4、无关肽（对照）及PBS免疫C57BL/6小鼠2次（间隔7 d）,末次免疫后7～10 d取脾分离单个核细胞,用合成肽Sj22.6-P4、无关肽及PBS刺激培养,3H-TdR掺入法检测淋巴细胞的增殖效果,酶联免疫吸附试验（ELISA）检测其细胞培养上清中IFNγ-、IL-4及IL-2水平。运用流式细胞技术三色标记法检测经合成肽Sj22.6-P4、无关肽及PBS免疫2次的C57BL/6小鼠脾淋巴细胞内的Th1、Th2细胞因子。结果合成肽Sj22.6-P4可刺激经该抗原肽免疫2次的C57BL/6小鼠淋巴细胞增殖,与PBS组相比,增殖指数（SI）均〉2。与无关肽对照组相比,细胞培养上清中IL-2、IFN-γ分泌水平增高,其中IL-2分泌水平差异有统计学意义（P〈0.05）,IFN-γ差异无统计学意义（P〉0.05）,而IL-4分泌水平明显降低（P〈0.05）。合成肽Sj22.6-P4免疫组小鼠脾脏CD4＋T细胞中分泌IFN-γ细胞的百分比显著增高,分泌IL-4细胞的百分比显著降低（P均〈0.05）。结论合成肽Sj22.6-P4是C57BL/6小鼠特异的Th1型表位。     

Filaria/Wolbachia activation of dendritic cells and development of Th1-associated responses is dependent on Toll-like receptor 2 in a mouse model of ocular onchocerciasis (river blindness)

Toll-like receptors (TLRs) regulate dendritic cell function and activate signals that mediate the nature of the adaptive immune response. The current study examined the role of TLRs in dendritic cell activation and in regulating T cell and antibody responses to antigens from the filarial parasites Onchocerca volvulus and Brugia malayi, which cause river blindness and lymphatic filariasis, respectively. Bone-marrow-derived CD11c(+) cells from C57BL/6 and TLR4(-/-) mice produced high levels of IL-6 and RANTES, and showed elevated surface CD40 expression, whereas CD11c(+) cells from myeloid differentiation factor 88(-/-) (MyD88(-/-)), TLR2(-/-) and TLR2/4(-/-) mice were not activated. Similarly, IFN-gamma production by splenocytes from immunized TLR2(-/-) mice was significantly impaired compared with splenocytes from C57BL/6 and TLR4(-/-) mice. In contrast, there was no difference among these strains in Th2-associated responses including IL-5 production by splenocytes from immunized animals, serum IgE and IgG(1), or eosinophil infiltration into the corneal stroma. Neutrophil recruitment to the cornea and CXC chemokine production was inhibited in immunized TLR2(-/-) mice compared with C57BL/6 and TLR4(-/-) mice. Taken together, these findings demonstrate an essential role for TLR2 in filaria-induced dendritic cell activation, IFN-gamma production and neutrophil migration to the cornea, but does not affect filaria-induced Th2-associated responses.     

Increased frequency of Th2-type cytokine-producing T cells in microfilaremic loiasis.

The frequency of cytokine-producing peripheral blood mononuclear cells was assessed in 28 subjects with microfilaremic loiasis and in 14 amicrofilaremic individuals. In addition, a subgroup of seven microfilaremic individuals coinfected with Plasmodium malariae was evaluated. By using flow cytometry for the intracellular detection of cytokines, a more pronounced T helper (Th)2 cell-type response with the expansion of interleukin (IL)-4, IL-10, and IL-13 expressing CD4+ cells in the microfilaremic compared with the amicrofilaremic group was noted. Expression of IL-5 was equivalent in both groups as was the frequency of Th2-type cytokines expressing CD8+ cells and of Th1-type cytokines (interferon [IFN]-gamma, IL-2, IFN-gamma/IL-2) producing CD4+ and CD8+ cells. Th0-type cytokine-expressing cells, represented by IL-4/IFN-gamma, IL-10/IFN-gamma, and IL-13/IFN-gamma, were equally distributed within groups. Coinfection of P. malariae did not significantly alter the cytokine expression compared with microfilaremic individuals without P. malariae infections. By identifying a large panel of cytokine-producing T cell subpopulations, a Th2-driven immune response in microfilaremic Loa loa patients was noted.     

The ICE Inhibitor Pralnacasan Prevents DSS-Induced Colitis in C57BL/6 Mice and Suppresses IP-10 mRNA but Not TNF-α mRNA Expression

Previously we demonstrated an ameliorating effect of the interleukin-1beta converting enzyme (ICE) inhibitor pralnacasan on dextran sulfate sodium (DSS)-induced colitis. This study investigates the effects of pralnacasan on cytokine expression in DSS-induced colitis. Colitis was induced by oral administration of DSS. Mice were treated intraperitoneally with the ICE inhibitor pralnacasan (50 mg/kg body weight twice daily). Body weight as well as the presence of occult blood or diarrhea was monitored daily. Subgroups were sacrificed at days 4, 8, and 11 after the beginning of DSS application. Cytokine profiles in colonic tissue were analyzed on the protein level by ELISA and on the mRNA level by real time RT-PCR. Administration of DSS led to an increase in IL-18, IL-12, TNF-alpha, and IFN-gamma protein as well as IP-10 and TNF-alpha mRNA. The increase in IL-18 and IFN-gamma was reduced by ICE inhibition. Pralnacasan prevented DSS-induced colitis in C57BL/6 mice. In C57BL/6 mice, the DSS-induced increase in IP-10 mRNA, but not TNF-alpha mRNA, was completely prevented by ICE inhibition. In conclusion, prevention of colitis in C57BL/6 mice was associated with a suppresion of IP-10 mRNA, but not TNF-alpha mRNA expression, indicating that IL-18-mediated cytokine production is a key element in the pathogenesis of DSS-induced colitis.     

Role of Th1 and Th2 cytokines in regulating the liver injury induced by delayed-type hypersensitivity to picryl chloride

AIMS/BACKGROUND: We have previously reported that a new model of liver injury induced in mice by delayed-type hypersensitivity (DTH) to picryl chloride (PCl) mimicks the pathogenesis of human hepatitis. This liver injury is mediated by CD4+ T cells. The interaction between lymphocyte function associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) is an essential process for hepatocyte (HC) damage. The present study was undertaken to reveal the role of Th1 and Th2-like cytokines in regulating the liver injury. METHODS: The kinetics of cytokine production were examined by ELISA and RT-PCR after the elicitation of liver injury for both serum protein and liver mRNA expression, respectively. A co-culture assay between liver nonparenchymal cells (NPC) and HC was conducted to evaluate the cytokine regulation on the cell-cell interaction. Expression of LFA-1 on NPC and ICAM-1 on HC were examined by FACScan and ELISA, respectively. RESULTS: Serum IL-2 and IFN-gamma showed a peak production at 6 and 12 h, while IL-5 and IL-4 reached their maximum levels at 18 and 24 h after induction of liver injury, respectively. Liver mRNA expression of IFN-gamma and IL-4 had a similar time course to their corresponding products. Both recombinant murine IFN-gamma and IL-2 triggered the hepatotoxicity of NPC or spleen cells at 0 h. In this case, an increased expression of both LFA-1 on NPC and ICAM-1 on HC was also observed. In contrast, IL-4 and IL-5 completely abolished the hepatotoxicity of NPC at 12 h without influencing the adhesion molecules. CONCLUSION: Th1 and Th2 may be involved in regulating liver injury. Th1/Th2 balance may critically contribute to the production of the liver injury or recovery from it.     

Role of interleukin 15 in colitis induced by dextran sulphate sodium in mice

BACKGROUND AND AIMS: Interleukin (IL)-15 is a member of the IL-2 family, stimulating dendritic cells, natural killer (NK) cells, NK T cells and memory CD8+ T cells. IL-15 levels were elevated in the intestinal mucosa of inflammatory bowel diseases. Here we investigated the involvement of IL-15 in the pathogenesis of acute and chronic dextran sulphate sodium (DSS) induced colitis. METHODS: IL-15 knockout (KO) mice and control C57BL/6 mice were used to induce colitis with DSS in their drinking water. Survival rate, clinical activity of diseases, extent of tissue damage, leucocyte population, and cytokine production of lamina propria (LP) cells of the large intestines were assessed. RESULTS: IL-15 KO mice exhibited resistance to DSS induced acute colitis, as reflected by lower lethality, weight loss, clinical scores, and histological scores compared with those in control mice (p<0.05). The proportions of CD44(high) CD8+ T cells and NK cells in LP cells and levels of interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, and IL-12p40 in culture supernatants of LP cells were reduced in IL-15 KO mice (p<0.05). In vivo depletion of CD8+ T cells and NK cells decreased levels of IFN-gamma, TNF-alpha, and IL-12p40 in culture supernatants of LP cells in C57BL/6 mice (p<0.01). In chronic colitis, weight loss and clinical scores were improved and levels of IFN-gamma, TNF-alpha, and IL-12p40 in culture supernatants of LP cells were also reduced in IL-15 KO mice (p<0.05). CONCLUSIONS: IL-15 plays an important role in the pathogenesis of both acute and chronic colitis induced by DSS in mice.     

Leishmania infantum released proteins specifically regulate cytokine expression and production patterns by CD4+ and CD8+ T cells

Specific immune responses by CD4+ and CD8+ T cells, from two infected mice strains (BALB/c and C57BL/6), induced by High, Inter and Low protein fractions released by Leishmania infantum, were assessed through the evaluation of IL-12, IFN-gamma and IL-10 mRNA by real-time PCR and respective protein production by ELISA. During infection establishment, High and Inter fractions directed both mice strains T cells subsets to increase the production of IFN-gamma, associated to IL-12 release. Later on, parasite replication augmented in BALB/c and stabilised in C57BL/6 mice. Inter fraction induced CD4+ T cells to maintain IFN-gamma production, with the simultaneous release of IL-12 by both cell subsets in BALB/c mice and by CD8+ T cells in C57BL/6 mice. These observations suggested a prophylactic potential for Inter fraction which was able to induce Th1 response with IL-12 involvement, required for the maintenance of memory cells, in mice strains with different parasitic evolution.