Absence of Tec family kinases interleukin-2 inducible T cell kinase (Itk) and Bruton's tyrosine kinase (Btk) severely impairs Fc epsilonRI-dependent mast cell responses

Mast cells are critical effector cells in the pathophysiology of allergic asthma and other IgE-mediated diseases. The Tec family of tyrosine kinases Itk and Btk serve as critical signal amplifiers downstream of antigen receptors. Although both kinases are expressed and activated in mast cells following FcεRI stimulation, their individual contributions are not clear. To determine whether these kinases play unique and/or complementary roles in FcεRI signaling and mast cell function, we generated Itk and Btk double knock-out mice. Analyses of these mice show decreased mast cell granularity and impaired passive systemic anaphylaxis responses. This impaired response is accompanied by a significant elevation in serum IgE in Itk/Btk double knock-out mice. In vitro analyses of bone marrow-derived mast cells (BMMCs) indicated that Itk/Btk double knock-out BMMCs are defective in degranulation and cytokine secretion responses downstream to FcεRI activation. These responses were accompanied by a significant reduction in PLCγ2 phosphorylation and severely impaired calcium responses in these cells. This defect also results in altered NFAT1 nuclear localization in double knock-out BMMCs. Network analysis suggests that although they may share substrates, Itk plays both positive and negative roles, while Btk primarily plays a positive role in mast cell FcεRI-induced cytokine secretion.     

Transforming growth factor-beta-activated kinase 1 regulates natural killer cell-mediated cytotoxicity and cytokine production

Carma1, a caspase recruitment domain-containing membrane-associated guanylate kinase, initiates a unique signaling cascade via Bcl10 and Malt1 in NK cells. Carma1 deficiency results in reduced phosphorylation of JNK1/2 and activation of NF-κB that lead to impaired NK cell-mediated cytotoxicity and cytokine production. However, the precise identities of the downstream signaling molecules that link Carma1 to these effector functions were not defined. Here we show that transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) is abundantly present in NK cells, and activation via NKG2D results in its phosphorylation. Lack of Carma1 considerably reduced TAK1 phosphorylation, demonstrating the dependence of TAK1 on Carma1 in NKG2D-mediated NK cell activations. Pharmacological inhibitor to TAK1 significantly reduced NK-mediated cytotoxicity and its potential to generate IFN-γ, GM-CSF, MIP-1α, MIP-1β, and RANTES. Conditional in vivo knockdown of TAK1 in NK cells from Mx1Cre(+)TAK1(fx/fx) mice resulted in impaired NKG2D-mediated cytotoxicity and cytokine/chemokine production. Inhibition or conditional knockdown of TAK1 severely impaired the NKG2D-mediated phosphorylation of ERK1/2 and JNK1/2 and activation of NF-κB and AP1. Our results show that TAK1 links Carma1 to NK cell-mediated effector functions.     

NK Cells Regulate CD8+ T Cell Priming and Dendritic Cell Migration during Influenza A Infection by IFN-γ and Perforin-Dependent Mechanisms

An effective immune response against influenza A infection depends on the generation of virus-specific T cells. NK cells are one of the first-line defenses against influenza A infection. We set out to delineate the role of NK cells in T cell immunity using a murine model of influenza A infection with A/PR/8/34. We show that early T cell recruitment mainly occurs in the posterior mediastinal lymph node (pMLN). Depletion of NK cells significantly impaired both dendritic cell (DC) and T cell recruitment into the pMLN. A similar reduction of T cell recruitment was observed when migration was blocked by pertussis toxin, suggesting that migration of pulmonary NK cells and DCs regulates cell recruitment to the pMLN. T cell recruitment was dependent on IFN-γ, and transfer of IFN-γ-competent naive NK cells into IFN-γ(-/-) mice restored T cell recruitment, whereas IFN-γ-deficient NK cells failed to do so. In addition, NK cell depletion reduced the uptake and transport of influenza A virus by DCs, and significantly impaired the virus-specific T cell response. Both IFN-γ(-/-) and perforin(-/-) mice showed reduced viral Ag transport by DCs, suggesting that the ability of NK cells to influence virus transport depends on IFN-γ and perforin. In summary, our data suggest that NK cells play a critical role in the initiation and shaping of the T cell response after influenza A infection.     

T and B cells are not required for clearing Staphylococcus aureus in systemic infection despite a strong TLR2-MyD88-dependent T cell activation

Staphylococcus aureus infection elicits through its mature lipoproteins an innate immune response by TLR2-MyD88 signaling, which improves bacterial clearing and disease outcome. The role of dendritic cells (DCs) and T cells in this immune activation and the function of T and B cells in defense against S. aureus infection remain unclear. Therefore, we first evaluated DC and T cell activation after infection with S. aureus wild type (WT) and its isogenic mutant, which is deficient in lipoprotein maturation, in vitro. Lipoproteins in viable S. aureus contributed via TLR2-MyD88 to activation of DCs, which promoted the release of IFN-γ and IL-17 in CD4(+) T cells. This strong effect was independent of superantigens and MHC class II. We next evaluated the function of T cells and their cytokines IFN-γ and IL-17 in infection in vivo. Six days after systemic murine infection IFN-γ, IL-17, and IL-10 production in total spleen cells were MyD88-dependent and their levels increased until day 21. The comparison of CD3(-/-), Rag2(-/-), and C57BL/6 mice after infection revealed that IFN-γ and IL-17 originated from T cells and IL-10 originated from innate immune cells. Furthermore, vaccination of mice to activate T and B cells did not improve eradication of S. aureus from organs. In conclusion, S. aureus enhances DC activation via TLR2-MyD88 and thereby promotes T(H)1 and T(H)17 cell differentiation. However, neither T cells and their MyD88-regulated products, IFN-γ and IL-17, nor B cells affected bacterial clearing from organs and disease outcome.     

Outer membrane protein A (OmpA) of Shigella flexneri 2a links innate and adaptive immunity in a TLR2-dependent manner and involvement of IL-12 and nitric oxide

We determine that OmpA of Shigella flexneri 2a is recognized by TLR2 and consequently mediates the release of proinflammatory cytokines and activates NF-κB in HEK 293 cells transfected with TLR2. We also observe that in RAW macrophages TLR2 is essential to instigate the early immune response to OmpA via NF-κB activation and secretion of cytokines and NO. Consistent with these results, TLR2 knockdown using siRNA abolishes the initiation of immune responses. Processing and presentation of OmpA depend on TLR2; MHCII presentation of the processed antigen and expression of CD80 significantly attenuated in TLR2 knockdown macrophages. The optimum production of IFN-γ by the macrophages:CD4(+) T cells co-culture depends on both TLR2 activation and antigen presentation. So, TLR2 is clearly recognized as a decisive factor in initiating host innate immune response to OmpA for the development of CD4(+) T cell adaptive response. Furthermore, we demonstrate in vivo that intranasal immunization of mice with OmpA selectively enhances the release of IFN-γ and IL-2 by CD4(+) T cells. Importantly, OmpA increases the level of IFN-γ production in Ag-primed splenocytes. The addition of neutralizing anti-IL-12p70 mAb to cell cultures results in the decreased release of OmpA-enhanced IFN-γ by Ag-primed splenocytes. Moreover, coincubation with OmpA-pretreated macrophages enhances the production of IFN-γ by OmpA-primed CD4(+) T cells, representing that OmpA may enhance IFN-γ expression in CD4(+) T cells through the induction of IL-12 production in macrophages. These results demonstrate that S. flexneri 2a OmpA may play a critical role in the development of Th1 skewed adaptive immune response.     

Restoration of TLR3-activated myeloid dendritic cell activity leads to improved natural killer cell function in chronic hepatitis B virus infection

There is increasing evidence that the function of NK cells in patients with chronic hepatitis B (CHB) infection is impaired. The underlying mechanism for the impaired NK cell function is still unknown. Since myeloid dendritic cells (mDC) are potent inducers of NK cells, we investigated the functional interaction of mDC and NK cells in CHB and the influence of antiviral therapy. Blood BDCA1(+) mDC and NK cells were isolated from 16 healthy controls or 39 CHB patients at baseline and during 6 months of antiviral therapy. After activation of mDC with poly(I · C) and gamma interferon (IFN-γ), mDC were cocultured with NK cells. Phenotype and function were analyzed in detail by flow cytometry and enzyme-linked immunosorbent assay. Our findings demonstrate that on poly(I · C)/IFN-γ-stimulated mDC from CHB patients, the expression of costimulatory molecules was enhanced, while cytokine production was reduced. In cocultures of poly(I · C)/IFN-γ-stimulated mDC and NK cells obtained from CHB patients, reduced mDC-induced NK cell activation (i.e., CD69 expression) and IFN-γ production compared to those in healthy individuals was observed. Antiviral therapy normalized mDC activity, since decreased expression of CD80 and CD86 on DC and of HLA-E on NK cells was observed, while poly(I · C)/IFN-γ-induced cytokine production by mDC was enhanced. In parallel, successful antiviral therapy resulted in improved mDC-induced NK cell activation and IFN-γ production. These data demonstrate that CHB patients display a diminished functional interaction between poly(I · C)/IFN-γ activated mDC and NK cells due to impaired mDC function, which can be partially restored by antiviral therapy. Enhancing this reciprocal interaction could reinforce the innate and thus the adaptive T cell response, and this may be an important step in achieving effective antiviral immunity.     

Bruton's tyrosine kinase separately regulates NFkappaB p65RelA activation and cytokine interleukin (IL)-10/IL-12 production in TLR9-stimulated B Cells

B lymphocytes express both B cell receptor and Toll-like receptors (TLR). We show here that Bruton's tyrosine kinase (Btk), a critical component in B cell receptor signaling, is also involved in TLR9 signaling in B cells. Stimulation of B cells with TLR9 ligand CpG oligodeoxynucleotide (ODN) leads to transient phosphorylation of Btk, and in the absence of Btk, TLR9-induced proliferation of B cells is impaired. Interestingly, Btk(-/-) B cells secrete significantly more interleukin (IL)-12 but much less IL-10 compared with wild type B cells upon TLR9 stimulation. Immunization of Btk(-/-) mice with CpG ODN also leads to elevated levels of IL-12 in vivo and consequently, a greater -fold increment in the production of Th1 type IgG2b and IgG3 antibodies in these mice compared with wild type controls. The addition of exogenous recombinant IL-10 could suppress IL-12 production by TLR9-activated Btk(-/-) B cells, suggesting that in B cells, Btk negatively regulates IL-12 through the induction of autocrine IL-10 production. TLR9 signaling also leads to the activation of NFkappaB, including the p65RelA subunit in wild type B cells. The lack of Btk signaling affects the activation of NFkappaB and impairs the translocation of the p65RelA subunit to the nucleus of B cells upon TLR9 stimulation. However, p65RelA(-/-) B cells could respond similarly to wild type B cells in terms of IL-10 and IL-12 secretion when stimulated with CpG ODN, suggesting that the defect in NFkappaB p65RelA activation is additional to the impairment in cytokine production in TLR9-activated Btk(-/-) B cells. Thus, Btk plays an important role in TLR9 signaling and acts separately to regulate NFkappaB RelA activation as well as IL-10 and IL-12 production in B cells.     

Identification of the synthetic cannabinoid R(+)WIN55,212-2 as a novel regulator of IFN regulatory factor 3 activation and IFN-beta expression: relevance to therapeutic effects in models of multiple sclerosis

β-Interferons (IFN-βs) represent one of the first line treatments for relapsing-remitting multiple sclerosis, slowing disease progression while reducing the frequency of relapses. Despite this, more effective, well tolerated therapeutic strategies are needed. Cannabinoids palliate experimental autoimmune encephalomyelitis (EAE) symptoms and have therapeutic potential in MS patients although the precise molecular mechanism for these effects is not understood. Toll-like receptor (TLR) signaling controls innate immune responses and TLRs are implicated in MS. Here we demonstrate that the synthetic cannabinoid R(+)WIN55,212-2 is a novel regulator of TLR3 and TLR4 signaling by inhibiting the pro-inflammatory signaling axis triggered by TLR3 and TLR4, whereas selectively augmenting TLR3-induced activation of IFN regulatory factor 3 (IRF3) and expression of IFN-β. We present evidence that R(+)WIN55,212-2 strongly promotes the nuclear localization of IRF3. The potentiation of IFN-β expression by R(+)WIN55,212-2 is critical for manifesting its protective effects in the murine MS model EAE as evidenced by its reduced therapeutic efficacy in the presence of an anti-IFN-β antibody. R(+)WIN55,212-2 also induces IFN-β expression in MS patient peripheral blood mononuclear cells, whereas down-regulating inflammatory signaling in these cells. These findings identify R(+)WIN55,212-2 as a novel regulator of TLR3 signaling to IRF3 activation and IFN-β expression and highlights a new mechanism that may be open to exploitation in the development of new therapeutics for the treatment of MS.     

Evaluating vaccinia virus cytokine co-expression in TLR GKO mice

Using Toll-like receptor (TLR) and MyD88 gene knock-out (GKO) mice the effect of TLRs and MyD88 on virus replication, interferon (IFN)-β production, natural killer (NK) cell and CD8T cell responses were assessed following ectromelia virus (ECTV) and recombinant vaccinia virus (rVV) infection. The capacity for rVVs encoding cytokines to restore immune function in MyD88(-/-) mice was clearly demonstrated. Results showed that TLR2(-/-), TLR4(-/-)and TLR7(-/-) mice survived ECTV infection whereas MyD88(-/-) and TLR9(-/-)mice, in contrast, were highly susceptible. Next, following infection with rVV, MyD88(-/-) mice elicited reduced serum IFN-β, NK cell and CD8T cell responses compared with wild-type mice, whereas TLR9(-/-) mice showed elevated CD8T cell responses. When MyD88(-/-)mice were infected with rVV co-expressing IFN-β these mice were able to restore IFN-β levels and CD8T cell responses but not NK cell activation. Interestingly, even though rVV co-expressing interleukin (IL)-2 enhanced NK cell activation in MyD88(-/-) mice, this was not associated with an antiviral effect, as observed in normal mice. Surprisingly, co-infection with rVV IL-2/rVV IL-12, but not rVV IL-2/rVV IFN-β, restored the attenuated phenotype of rVV IL-2 in MyD88(-/-) mice indicating that the IL-2/IL-12 combination promotes antiviral responses. Our results clearly show that the CD8T cell defect observed in MyD88(-/-) mice to vaccinia virus infection can be restored by rVV-encoding IFN-β demonstrating the critical role of this cytokine in T cell mediated immunity and illustrates that the model can provide an effective platform for the elucidation of cytokine immunobiology.     

Requirement of UNC93B1 reveals a critical role for TLR7 in host resistance to primary infection with Trypanosoma cruzi

UNC93B1 associates with TLR3, 7, and 9, mediating their translocation from the endoplasmic reticulum to the endolysosome, thus allowing proper activation by microbial nucleic acids. We found that the triple-deficient 3d mice, which lack functional UNC93B1 as well as functional endosomal TLRs, are highly susceptible to infection with Trypanosoma cruzi. The enhanced parasitemia and mortality in 3d animals were associated with impaired proinflammatory response, including reduced levels of IL-12p40 and IFN-γ. Importantly, the phenotype of 3d mice was intermediary between MyD88(-/-) (highly susceptible) and TLR9(-/-) (moderately susceptible), indicating the involvement of an additional UN93B1-dependent TLR(s) on host resistance to T. cruzi. Hence, our experiments also revealed that TLR7 is a critical innate immune receptor involved in recognition of parasite RNA, induction of IL-12p40 by dendritic cells, and consequent IFN-γ by T lymphocytes. Furthermore, we show that upon T. cruzi infection, triple TLR3/7/9(-/-) mice had similar phenotype than 3d mice. These data imply that the nucleic acid-sensing TLRs are critical determinants of host resistance to primary infection with T. cruzi.     

IMPDHII protein inhibits Toll-like receptor 2-mediated activation of NF-kappaB

Toll-like receptor 2 (TLR2) plays an essential role in innate immunity by the recognition of a large variety of pathogen-associated molecular patterns. It induces its recruitment to lipid rafts induces the formation of a membranous activation cluster necessary to enhance, amplify, and control downstream signaling. However, the exact composition of the TLR2-mediated molecular complex is unknown. We performed a proteomic analysis in lipopeptide-stimulated THP1 and found IMPDHII protein rapidly recruited to lipid raft. Whereas IMPDHII is essential for lymphocyte proliferation, its biologic function within innate immune signal pathways has not been established yet. We report here that IMPDHII plays an important role in the negative regulation of TLR2 signaling by modulating PI3K activity. Indeed, IMPDHII increases the phosphatase activity of SHP1, which participates to the inactivation of PI3K.     

Dendritic cells and NK cells stimulate bystander T cell activation in response to TLR agonists through secretion of IFN-alpha beta and IFN-gamma

Recognition of conserved features of infectious agents by innate pathogen receptors plays an important role in initiating the adaptive immune response. We have investigated early changes occurring among T cells after injection of TLR agonists into mice. Widespread, transient phenotypic activation of both naive and memory T cells was observed rapidly after injection of molecules acting through TLR3, -4, -7, and -9, but not TLR2. T cell activation was shown to be mediated by a combination of IFN-alphabeta, secreted by dendritic cells (DCs), and IFN-gamma, secreted by NK cells; notably, IFN-gamma-secreting NK cells expressed CD11c and copurified with DCs. Production of IFN-gamma by NK cells could be stimulated by DCs from TLR agonist-injected mice, and although soluble factors secreted by LPS-stimulated DCs were sufficient to induce IFN-gamma, maximal IFN-gamma production required both direct contact of NK cells with DCs and DC-secreted cytokines. In vitro, IFN-alphabeta, IL-18, and IL-12 all contributed to DC stimulation of NK cell IFN-gamma, whereas IFN-alphabeta was shown to be important for induction of T cell bystander activation and NK cell IFN-gamma production in vivo. The results delineate a pathway involving innate immune mediators through which TLR agonists trigger bystander activation of T cells.     

Homeostatic defects in interleukin 18-deficient mice contribute to protection against the lethal effects of endotoxin

Toll-like receptor-4-lipopolysaccharide (LPS)-mediated inflammation is used to delineate signals involved in cross-talk between antigen-presenting cells (APCs) and lymphocytes such as natural killer (NK) cells. Following APC stimulation and cytokine release, NK cells produce interferon (IFN)-γ. High levels of LPS induce endotoxicosis, a systemic inflammatory disease in which IFN-γ causes significant morbidity and mortality. Several studies have highlighted the role of interleukin (IL)-18, IL-1β, IL-17A and IFN-γ in the development of endotoxicosis, but whether these cytokines interact with each other is yet to be determined. Our data demonstrate that IL-18 and IL-17A have important roles in NK cell IFN-γ production during endotoxicosis. Importantly, we provide the first evidence that IL-18 also has a role in IL-17A production by T-cell receptor (TCR)-δ cells. Furthermore, we demonstrate that IL-18-deficient mice have a defect in γδ T-cell homeostasis and IL-1β production, both of which can contribute to the development of disease through induction of IL-17A. These results reveal novel requirements for IL-18 in innate immune cell homeostasis and activation, demonstrating that the role of IL-18 in innate immunity occurs at a level other than activation.     

CMRF-35-like molecule 3 preferentially promotes TLR9-triggered proinflammatory cytokine production in macrophages by enhancing TNF receptor-associated factor 6 ubiquitination

TLRs are critical innate immune sensors in the induction of proinflammatory cytokines to eliminate invading pathogens. However, the mechanisms for the full activation of TLR-triggered innate immune response need to be fully understood. The murine CMRF-35-like molecule (CLM)-3 is a representative of CLM family belonging to the Ig superfamily. Considering that CLM-3 is selectively expressed in macrophages and the roles of CLM members in innate immune response remain unclear, in this study we investigated the role of CLM-3 in the regulation of TLR-triggered innate response. We found that CLM-3 was an endosome/lysosome-localized molecule, and was downregulated in macrophages by stimulation with TLR9 ligand, but not TLR4 and TLR3 ligands. Interestingly, CLM-3 selectively promoted production of TNF-α and IL-6 in macrophages triggered by TLR9, but not TLR4 or TLR3. CLM-3 enhanced activation of MAPKs and NF-κB pathways in TLR9-triggered macrophages. Furthermore, CLM-3-transgenic mice were generated, and CLM-3 expression was confirmed by mAb against CLM-3 that we prepared. Accordingly, the macrophages derived from CLM-3-transgenic mice were more sensitive to TLR9 ligand stimulation, with more pronounced production of TNF-α, IL-6, and increased activation of MAPKs and NF-κB pathways. Moreover, ubiquitination of TNFR-associated factor 6, a crucial signaling transducer of TLR-triggered MAPKs and NF-κB activation, was found to be significantly promoted by CLM-3 in macrophages. Collectively, the endosome/lysosome-localized CLM-3 can promote full activation of TLR9-triggered innate responses by enhancing TNFR-associated factor 6 ubiquitination and subsequently activating MAPKs and NF-κB.     

NKT cells are required for complete Freund's adjuvant-mediated protection from autoimmune diabetes

Autoimmune diabetes in NOD mice can be prevented by application of Ags derived from Mycobacterium tuberculosis in the form of bacillus Calmette-Guérin or CFA. Disease protection by CFA is associated with a reduction in the numbers of pathogenic β-cell specific, self-reactive CTLs, a phenomenon dependent on the presence and function of NK cells. However, the mechanisms by which NK cells are activated and recruited by heat-killed M. tuberculosis within CFA are unclear. In this study, we report that CFA-mediated NK cell activation and mobilization is dependent on CD1d expression. The administration of M. tuberculosis from CFA results in rapid NKT cell activation and IFN-γ secretion both in vitro and in vivo. CFA-induced NKT cell activation is intact in MyD88(-/-) mice suggesting that the mechanism is independent of TLR signaling. Furthermore, CD1d expression was found to be essential for both M. tuberculosis-triggered NKT cell activation and CFA-mediated protection of NOD mice from diabetes. Collectively, these findings reveal hitherto previously unidentified roles for NKT cells in the adjuvant-promoting effects of CFA on innate and adaptive immunity.     

Cytohesin-associated scaffolding protein (CASP) is involved in migration and IFN-γ secretion in Natural Killer cells

Natural Killer (NK) cells are highly mobile, specialized sub-populations of lymphocytic cells that survey their host to identify and eliminate infected or tumor cells. They are one of the key players in innate immunity and do not need prior activation through antigen recognition to deliver cytotoxic packages and release messenger chemicals to recruit immune cells. Cytohesin associated scaffolding protein (CASP) is a highly expressed lymphocyte adaptor protein that forms complexes with vesicles and sorting proteins including SNX27 and Cytohesin-1. In this study we show that by using stably integrated shRNA, CASP has a direct role in the secretion of IFN-γ, and NK cell motility and ability to kill tumor cells. CASP polarizes to the leading edge of migrating NK cells, and to the immunological synapse when engaged with tumor cells. However, CASP is not associated with cytotoxic granule mediated killing. CASP is a multi-faceted protein, which has a very diverse role in NK cell specific immune functions.     

The direct effects of Toll-like receptor ligands on human NK cell cytokine production and cytotoxicity

Toll-like receptor (TLR) ligands are potent inducers of the innate immune system, of which NK and NKT cells play an important role. We examined the direct activation of highly purified human NK and/or NKT cells with known TLR ligands. NK/NKT cells were positive for all known TLR mRNA (TLR1-10). Ligands for TLR2-5 induced production of significant amounts of IFN-gamma by purified NK cells. However, a TLR9 ligand failed to induce significant levels of the cytokine. NK cells were depleted from PBMCs to confirm that they were the main source of IFN-gamma following treatment with TLR ligands, which resulted in a significant decrease in cytokines. The direct effects of TLR ligands on NK cytotoxicity were determined using 51Cr-labeled K562 target cells. Ligands for TLR2-5 were potent inducers of NK cell cytotoxicity, a TLR9 ligand was not. Our results suggest that TLR ligands can directly stimulate and enhance NK cell cytokine production and induce cytotoxic activities.     

Suppression of CD4 T-Cells in the spleen of mice infected with Toxoplasma gondii KI-1 tachyzoites

Toxoplasma gondii KI-1, a recent new isolate from Korea, shows similar pathogenicity and infectivity to mice compared to the virulent RH strain. To understand characteristics of host immunity, including immune enhancement or suppression, we investigated proliferative responses and phenotypes of spleen cells. In addition, kinetics of IFN-γ, a Th1 cytokine, was examined in BALB/c mice up to day 6 post-infection (PI). Intraperitoneal injection of mice with 10(3) KI-1 tachyzoites induced significant decreases (P < 0.05) in proliferative responses of spleen cells. This occurred at days 2-6 PI even when concanavalin A (con A) was added and when stimulated with KI-1 antigen, suggesting suppression of the immunity. CD4(+) T-cells decreased markedly at day 2 PI (P < 0.05), whereas CD8(+) T-cells, NK cells, and macrophages did not show significant changes, except a slight, but significant, increase of CD8(+) T-cells at day 6 PI. The capacity of splenocytes to produce IFN-γ by con A stimulation dropped significantly at days 2-6 PI. These results demonstrate that intraperitoneal injection of KI-1 tachyzoites can induce immunosuppression during the early stage of infection, as revealed by the decrease of CD4(+) T-cells and IFN-γ.     

A let-7/Fas double-negative feedback loop regulates human colon carcinoma cells sensitivity to Fas-related apoptosis

Interferon-γ (IFN-γ) is considered essential for the regulation of anti-tumor reactions as it sensitizes Fas-related apoptosis in HT29 cells, but the mechanism is unclear. In the current study, our data demonstrated that IFN-γ stimulation and Fas activation suppressed Dicer processing and let-7 microRNA biogenesis, while let-7 microRNA strongly inhibited Fas expression by directly targeting Fas mRNA. Accordingly, our results indicate that Fas and let-7 microRNAs form a double-negative feedback loop in IFN-γ and Fas induced apoptosis in colon carcinoma cell line HT29, which may be an important synergistic mechanism in anti-tumor immune response. We also found that a let-7 microRNA inhibitor increased Fas expression and sensitized cells to Fas-related apoptosis, which may have future implications in colon carcinoma therapy.