Modulation of STAT-1, STAT-3, and STAT-6 activities in THP-1 derived macrophages infected with two Trypanosoma cruzi strains.

Trypanosoma cruzi is the causative protozoan of Chagas' Disease, a neglected tropical disease that affects 6-7 million people worldwide. Interaction of the parasite with the host immune system is a key factor in disease progression and chronic symptoms. Although the human immune system is capable of controlling the disease, the parasite has numerous evasion mechanisms that aim to maintain intracellular persistence and survival. Due to the pronounced genetic variability of T. cruzi, co-infections or mixed infections with more than one parasite strain have been reported in the literature. The intermodulation in such cases is unclear. This study aimed to evaluate the co-infection of T. cruzi strains G and CL compared to their individual infections in human macrophages derived from THP-1 cells activated by classical or alternative pathways. Flow cytometry analysis demonstrated that trypomastigotes were more infective than extracellular amastigotes (EAs) and that strain G could infect more macrophages than strain CL. Classically activated macrophages showed lower number of infected cells and IL-4-stimulated cells displayed increased CL-infected macrophages. However, co-infection was a rare event. CL EAs decreased the production of reactive oxygen species (ROS), whereas G trypomastigotes displayed increased ROS detection in classically activated cells. Co-infection did not affect ROS production. Monoinfection by strain G or CL mainly induced an anti-inflammatory cytokine profile by decreasing inflammatory cytokines (IFN-γ, TNF-α, IL-1ß) and/or increasing IL-4, IL-10, and TGF-ß. Co-infection led to a predominant inflammatory milieu, with reduced IL-10 and TGF-ß, and/or promotion of IFN-γ and IL-1ß release. Infection by strain G reduced activation of intracellular signal transducer and activator of transcription (STAT) factors. In EAs, monoinfections impaired STAT-1 activity and promoted phosphorylation of STAT-3, both changes may prolong cell survival. Coinfected macrophages displayed pronounced activation of all STATs examined. These activations likely promoted parasite persistence and survival of infected cells. The collective results demonstrate that although macrophages respond to both strains, T. cruzi can modulate the intracellular environment, inducing different responses depending on the strain, parasite infective form, and co-infection or monoinfection. The modulation influences parasite persistence and survival of infected cells.

IL-27 regulates IL-4-induced chemokine production in human bronchial epithelial cells.

IL-4 coordinates the Th2-type immune response in inflammatory diseases such as asthma. IL-27 can inhibit the development of both Th2 and Th1 cells. However, IL-27 can also drive naïve T cells to differentiate toward the Th1 phenotype. In this study, we investigated the effects of IL-27 on the activation of IL-4-induced human bronchial epithelial cells (BEAS-2B). Compared to controls, both IL-4 and IL-27 (25-100 ng/mL) increased the concentrations of CCL2 and IL-8 in a dose-dependent manner. However, compared to cells stimulated individually with IL-4 or IL-27, treatment with a combination of both cytokines reduced CCL2 and IL-8 concentrations in a dose- and time-dependent manner. IL-4 increased the activation of p38 MAPK, ERK1/2, STAT6 and NF-κB, while IL-27 increased the activation of p38 MAPK and ERK1/2 but not STAT6 and NF-κB. Compared to IL-4-stimulated cells, cells treated with both IL-27 and IL-4 displayed decreased activation of STAT6 and NF-κB but not ERK1/2 and p38 MAPK. Taken together, these results suggest that IL-27 plays a pro-inflammatory role when administered alone but downregulates bronchial epithelial cell activation when combined with IL-4. Therefore, IL-27 may be an interesting target for the treatment of Th2 inflammatory diseases.

Genome-wide transcriptome analysis of the STAT6-regulated genes in advanced-stage cutaneous T-cell lymphoma.

The signal transducer and activator of transcription 6 (STAT6) is a critical up-stream mediator of interleukin-13 (IL-13) and IL-4 signaling and is constitutively activated in malignant lymphocytes from Sezary syndrome (SS) and mycosis fungoides (MF), the most common subtypes of cutaneous T-cell lymphomas. By combining genome-wide expression profiling with pharmacological STAT6 inhibition, we have identified the genes regulated by STAT6 in MF/SS tumors. We found that STAT6 regulates several common pathways in MF/SS malignant lymphocytes that are associated with control of cell-cycle progression and genomic stability as well as production of Th2 cytokines. Using ex vivo skin explants from cutaneous MF tumors as well as Sezary cells derived from the blood of SS patients, we demonstrated that inhibition of STAT6 activation downregulates cytokine production and induces cell-cycle arrest in MF/SS malignant lymphocytes, inhibiting their proliferation but not their survival. Furthermore, we show that STAT6 promotes the protumoral M2-like phenotype of tumor-associated macrophages in the tumor microenvironment of advanced stage MF by upregulating the expression of genes associated with immunosuppression, chemotaxis, and tumor matrix remodeling. Thus, we show STAT6 to be a major factor in the pathogenesis and progression of MF/SS, promoting proliferation and invasion of the malignant lymphocytes while inducing a progressive depression of the antitumor immune response. Together, our results provide new insights into disease pathogenesis and offer new prospective targets for therapeutic intervention.

Use of STAT6 Phosphorylation Inhibitor and Trimethylglycine as New Adjuvant Therapies for 5-Fluorouracil in Colitis-Associated Tumorigenesis.

Colorectal cancer (CRC) is one of the most widespread and deadly types of neoplasia around the world, where the inflammatory microenvironment has critical importance in the process of tumor growth, metastasis, and drug resistance. Despite its limited effectiveness, 5-fluorouracil (5-FU) is the main drug utilized for CRC treatment. The combination of 5-FU with other agents modestly increases its effectiveness in patients. Here, we evaluated the anti-inflammatory Trimethylglycine and the Signal transducer and activator of transcription (STAT6) inhibitor AS1517499, as possible adjuvants to 5-FU in already established cancers, using a model of colitis-associated colon cancer (CAC). We found that these adjuvant therapies induced a remarkable reduction of tumor growth when administrated together with 5-FU, correlating with a reduction in STAT6-phosphorylation. This reduction upgraded the effect of 5-FU by increasing both levels of apoptosis and markers of cell adhesion such as E-cadherin, whereas decreased epithelial-mesenchymal transition markers were associated with aggressive phenotypes and drug resistance, such as ß-catenin nuclear translocation and Zinc finger protein SNAI1 (SNAI1). Additionally, Il-10, Tgf-ß, and Il-17a, critical pro-tumorigenic cytokines, were downmodulated in the colon by these adjuvant therapies. In vitro assays on human colon cancer cells showed that Trimethylglycine also reduced STAT6-phosphorylation. Our study is relatively unique in focusing on the effects of the combined administration of AS1517499 and Trimethylglycine together with 5-FU on already established CAC which synergizes to markedly reduce the colon tumor load. Together, these data point to STAT6 as a valuable target for adjuvant therapy in colon cancer.

Influence of Two Major Toxoplasma Gondii Virulence Factors (ROP16 and ROP18) on the Immune Response of Peripheral Blood Mononuclear Cells to Human Toxoplasmosis Infection.

Toxoplasma gondii ROP16 and ROP18 proteins have been identified as important virulence factors for this parasite. Here, we describe the effect of ROP16 and ROP18 proteins on peripheral blood mononuclear cells (PBMCs) from individuals with different clinical status of infection. We evaluated IFN-γ, IL-10, and IL-1ß levels in supernatants from PBMCs cultures infected with tachyzoites of the T. gondii wild-type RH strain or with knock-out mutants of the rop16 and rop18 encoding genes (RHΔrop16 and RHΔrop18). Cytokine secretion was compared between PBMCs obtained from seronegative individuals (n = 10), with those with chronic asymptomatic (n = 8), or ocular infection (n = 12). We also evaluated if polymorphisms in the genes encoding for IFN-γ, IL-10, IL-1ß, Toll-like receptor 9 (TLR9), and purinoreceptor P2RX7 influenced the production of the encoded proteins after ex vivo stimulation. In individuals with chronic asymptomatic infection, only a moderate effect on IL-10 levels was observed when PBMCs were infected with RHΔrop16, whereas a significant difference in the levels of inflammatory cytokines IFN-γ and IL-1ß was observed in seronegative individuals, but this was also dependent on the host's cytokine gene polymorphisms. Infection with ROP16-deficient parasites had a significant effect on IFN-γ production in previously non-infected individuals, suggesting that ROP16 which is considered as a virulence factor plays a role during the primary infection in humans, but not in the secondary immune response.

Increase in secreted airway mucins and partial Muc5b STAT6/FoxA2 regulation during Pneumocystis primary infection.

Airway mucus responses to subclinical infections may explain variations in progression of chronic lung diseases and differences in clinical expression of respiratory infections across individuals. Pneumocystis associates to more severe Chronic Obstructive Pulmonary Disease (COPD), asthma, respiratory distress of premature newborns, and is a consistent subclinical infection between 2 and 5 months of age when hospitalizations for respiratory cause and infant mortality are higher. This atypical fungus associates to increased mucin 5AC (MUC5AC), a central effector of Th2-type allergic inflammation, in infant lungs. However, mucus progression, expression of MUC5B essential for airway defense, and potential for pharmacologic modulation of mucus during Pneumocystis infection remain unknown. We measured MUC5B and Pneumocystis in infant lungs, and progression of mucin levels and effect of inhibition of the STAT6/FoxA2 mucus pathway using Kaempferol, a JAK/STAT6 inhibitor, in immunocompetent rats during Pneumocystis primary infection. Pneumocystis associated to increased MUC5B in infant lungs. Muc5b increased earlier and more abundantly than Muc5ac during experimental primary infection suggesting an acute defensive response against Pneumocystis as described against bacteria, while increased Muc5ac levels supports an ongoing allergic, Th2 lymphocyte-type response during primary Pneumocystis infection. Kaempferol partly reversed Muc5b stimulation suggesting limited potential for pharmacological modulation via the STAT6-FoxA2 pathway.

Impaired wound healing in type 1 diabetes is dependent on 5-lipoxygenase products.

Type 1 diabetes is associated with systemic low grade inflammation (LGI). We have previously shown that LGI in diabetic mice depends on systemic circulation of leukotriene (LTB4) which potentiates the toll-like/IL1ß receptors response in macrophages. Impaired wound healing is an important co-morbidity in diabetes, and macrophages play a key role in this process. Here, we investigated the role of leukotrienes on monocytes and macrophages phenotype and in the impaired wound healing in diabetic mice. Type 1 diabetes was induced with streptozotocin in 129SvE wild-type (WT) and leukotrienes-deficient 5LO-/- (5-lipoxygenase knockout) mice. In diabetics, the systemic levels of LTB4, TNF-α, IL-6, IL-10, IL-12 and IFNγ were increased as well as the frequency of pro-inflammatory monocytes (CD11b+Ly6ChighLy6G-) compared to healthy mice. In diabetic 5LO-/- mice, these parameters were similar to those in healthy mice. Resident peritoneal macrophages from diabetic WT mice showed a classically activated M1-like phenotype (high Nos2, Stat and Il12 expression, and nitrite levels). Macrophages from diabetic 5LO-/- mice presented alternatively activated M2-macrophages markers (high Arg1 and Chi3l3 expression and arginase activity) and when stimulated with IL4, enhanced phosphorylated-STAT6. Cutaneous wound healing in diabetic WT mice was impaired, which correlated with the decreased frequency of M2-macrophages (CD45+F4/80+CD206+) in the lesions. In diabetic 5LO-/- mice, the frequency of M2-macrophages in the wound was similar to that in healthy mice, suggesting that the impaired healing of diabetic mice depends on 5LO products. The inhibition of leukotrienes or antagonism of its receptors could be a therapeutic alternative for diabetic patients with impaired healing.

Kcnn4 is a modifier gene of intestinal cystic fibrosis preventing lethality in the Cftr-F508del mouse.

Nearly 70% of cystic fibrosis (CF) patients bear the phenylalanine-508 deletion but disease severity differs greatly, and is not explained by the existence of different mutations in compound heterozygous. Studies demonstrated that genes other than CFTR relate to intestinal disease in humans and CF-mouse. Kcnn4, the gene encoding the calcium-activated potassium channel KCa3.1, important for intestinal secretion, is present in a locus linked with occurrence of intestinal CF-disease in mice and humans. We reasoned that it might be a CF-modifier gene and bred a CF-mouse with Kcnn4 silencing, finding that lethality was almost abolished. Silencing of Kcnn4 did not improve intestinal secretory functions, but rather corrected increased circulating TNF-α level and reduced intestinal mast cell increase. Given the importance of mast cells in intestinal disease additional double mutant CF-animals were tested, one lacking mast cells (C-kitW-sh/W-sh) and Stat6-/- to block IgE production. While mast cell depletion had no effect, silencing Stat6 significantly reduced lethality. Our results show that Kcnn4 is an intestinal CF modifier gene partially acting through a STAT6-dependent mechanism.

Macrophage alternative activation confers protection against lipotoxicity-induced cell death.

OBJECTIVE: Alternative activation (M2) of adipose tissue resident macrophage (ATM) inhibits obesity-induced metabolic inflammation. The underlying mechanisms remain unclear. Recent studies have shown that dysregulated lipid homeostasis caused by increased lipolysis in white adipose tissue (WAT) in the obese state is a trigger of inflammatory responses. We investigated the role of M2 macrophages in lipotoxicity-induced inflammation. METHODS: We used microarray experiments to profile macrophage gene expression regulated by two M2 inducers, interleukin-4 (Il-4), and peroxisome proliferator-activated receptor delta/gamma (Pparδ/Pparγ) agonists. Functional validation studies were performed in bone marrow-derived macrophages and mice deprived of the signal transducer and activator of transcription 6 gene (Stat6; downstream effector of Il-4) or Pparδ/Pparγ genes (downstream effectors of Stat6). Palmitic acid (PA) and ß-adrenergic agonist were employed to induce macrophage lipid loading in vitro and in vivo, respectively. RESULTS: Profiling of genes regulated by Il-4 or Pparδ/Pparγ agonists reveals that alternative activation promotes the cell survival program, while inhibiting that of inflammation-related cell death. Deletion of Stat6 or Pparδ/Pparγ increases the susceptibility of macrophages to PA-induced cell death. NLR family pyrin domain containing 3 (Nlrp3) inflammasome activation by PA in the presence of lipopolysaccharide is also increased in Stat6-/- macrophages and to a lesser extent, in Pparδ/γ-/- macrophages. In concert, ß-adrenergic agonist-induced lipolysis results in higher levels of cell death and inflammatory markers in ATMs derived from myeloid-specific Pparδ/γ-/- or Stat6-/- mice. CONCLUSIONS: Our data suggest that ATM cell death is closely linked to metabolic inflammation. Within WAT where concentrations of free fatty acids fluctuate, M2 polarization regulated by the Stat6-Ppar axis enhances ATM's tolerance to lipid-mediated stress, thereby maintaining the homeostatic state.

Lack of STAT6 Attenuates Inflammation and Drives Protection against Early Steps of Colitis-Associated Colon Cancer.

Colitis-associated colon cancer (CAC) is one of the most common malignant neoplasms and a leading cause of death. The immunologic factors associated with CAC development are not completely understood. Signal transducer and activator of transcription 6 (STAT6) is part of an important signaling pathway for modulating intestinal immune function and homeostasis. However, the role of STAT6 in colon cancer progression is unclear. Following CAC induction in wild-type (WT) and STAT6-deficient mice (STAT6-/-), we found that 70% of STAT6-/- mice were tumor-free after 8 weeks, whereas 100% of WT mice developed tumors. STAT6-/- mice displayed fewer and smaller colorectal tumors than WT mice; this reduced tumorigenicity was associated with decreased proliferation and increased apoptosis in the colonic mucosa in the early steps of tumor progression. STAT6-/- mice also exhibited reduced inflammation, diminished concentrations COX2 and nuclear ß-catenin protein in the colon, and decreased mRNA expression of IL17A and TNFα, but increased IL10 expression when compared with WT mice. Impaired mucosal expression of CCL9, CCL25, and CXCR2 was also observed. In addition, the number of circulating CD11b+Ly6ChiCCR2+ monocytes and CD11b+Ly6ClowLy6G+ granulocytes was both decreased in a STAT6-dependent manner. Finally, WT mice receiving a STAT6 inhibitor in vivo confirmed a significant reduction in tumor load as well as less intense signs of CAC. Our results demonstrate that STAT6 is critical in the early steps of CAC development for modulating inflammatory responses and controlling cell recruitment and proliferation. Thus, STAT6 may represent a promising target for CAC treatment. Cancer Immunol Res; 5(5); 385-96. ©2017 AACR.

Data integration for identification of important transcription factors of STAT6-mediated cell fate decisions.

Data integration has become a useful strategy for uncovering new insights into complex biological networks. We studied whether this approach can help to delineate the signal transducer and activator of transcription 6 (STAT6)-mediated transcriptional network driving T helper (Th) 2 cell fate decisions. To this end, we performed an integrative analysis of publicly available RNA-seq data of Stat6-knockout mouse studies together with STAT6 ChIP-seq data and our own gene expression time series data during Th2 cell differentiation. We focused on transcription factors (TFs), cytokines, and cytokine receptors and delineated 59 positively and 41 negatively STAT6-regulated genes, which were used to construct a transcriptional network around STAT6. The network illustrates that important and well-known TFs for Th2 cell differentiation are positively regulated by STAT6 and act either as activators for Th2 cells (e.g., Gata3, Atf3, Satb1, Nfil3, Maf, and Pparg) or as suppressors for other Th cell subpopulations such as Th1 (e.g., Ar), Th17 (e.g., Etv6), or iTreg (e.g., Stat3 and Hif1a) cells. Moreover, our approach reveals 11 TFs (e.g., Atf5, Creb3l2, and Asb2) with unknown functions in Th cell differentiation. This fact together with the observed enrichment of asthma risk genes among those regulated by STAT6 underlines the potential value of the data integration strategy used here. Thus, our results clearly support the opinion that data integration is a useful tool to delineate complex physiological processes.

Type 2 immunity-dependent reduction of segmented filamentous bacteria in mice infected with the helminthic parasite Nippostrongylus brasiliensis.

BACKGROUND: Dynamic interactions between the host and gastrointestinal microbiota play an important role for local and systemic immune homeostasis. Helminthic parasites modulate the host immune response, resulting in protection against autoimmune disease but also increased susceptibility to pathogen infection. The underlying mechanisms remain largely unknown. RESULTS: We showed that the type 2 immune response to enteric Nippostrongylus brasiliensis infection in mice was associated with altered intestinal mucin and AMP expression and shifts in microbiota composition. Most strikingly, infection reduced concentrations of intestinal segmented filamentous bacteria (SFB), known inducers of T helper 17 cells, and IL-17-associated gene expression. Infected mice deficient in IL-13 or STAT6 did not reduce SFB or IL-17, and exogenous IL-25 replicated the effects of parasite infection in wild type mice. CONCLUSIONS: Our data show that parasite infection acts through host type 2 immunity to reduce intestinal SFB and expression of IL-17, providing an example of a microbiota-dependent immune modulation by parasites.

Boswellic acid attenuates asthma phenotype by downregulation of GATA3 via nhibition of PSTAT6.

To study the role of boswellic acid in reducing asthma phenotype severity and the relationship between the expression of pSTAT6 and GATA3, thirty-six mice were randomly divided into normal control group, asthma group, and boswellic acid group (treatment group). The asthma model was established through an intraperitoneal injection of sensitization liquid (0.15 mL aluminum hydroxide gel at 88.67 mg/mL and 0.05 mg ovalbumin). pSTAT6 and GATA3 expression levels in peripheral blood were detected by reverse transcription-polymerase chain reaction and Western blot analysis. pSTAT6 and GATA3 gene expressions in the asthmatic group were significantly higher than in the normal control group; they were markedly lower in the treatment group than the asthma group, and there was no significant difference when compared with the normal control group. The pSTAT6 expressions in the asthma, control and treatment groups were 2.256 ± 0.125, 0.524 ± 0.210, and 0.897 ± 0.134 at gray level, respectively. The GATA3 expressions in the asthma, control, and treatment groups were 3.521 ± 0.631, 0.435 ± 0.136, and 0.743 ± 0.149 at gray level, respectively. pSTAT6 and GATA3 expression levels were similar in the treatment and control groups. GATA3 expression had a positive correlation with pSTAT6 expression. Boswellic acid may improve asthma symptoms by inhibiting pSTAT6 expression, which consequently reduces GATA3 expression.

AT-RvD1 modulates CCL-2 and CXCL-8 production and NF-κB, STAT-6, SOCS1, and SOCS3 expression on bronchial epithelial cells stimulated with IL-4.

Bronchial epithelial cells represent the first line of defense against microorganisms and allergens in the airways and play an important role in chronic inflammatory processes such as asthma. In an experimental model, both RvD1 and AT-RvD1, lipid mediators of inflammation resolution, ameliorated some of the most important phenotypes of experimental asthma. Here, we extend these results and demonstrate the effect of AT-RvD1 on bronchial epithelial cells (BEAS-2B) stimulated with IL-4. AT-RvD1 (100 nM) decreased both CCL2 and CXCL-8 production, in part by decreasing STAT6 and NF-κB pathways. Furthermore, the effects of AT-RvD1 were ALX/FRP2 receptor dependent, as the antagonist of this receptor (BOC1) reversed the inhibition of these chemokines by AT-RvD1. In addition, AT-RvD1 decreased SOCS1 and increased SOCS3 expression, which play important roles in Th1 and Th17 modulation, respectively. In conclusion, AT-RvD1 demonstrated significant effects on the IL-4-induced activation of bronchial epithelial cells and consequently the potential to modulate neutrophilic and eosinophilic airway inflammation in asthma. Taken together, these findings identify AT-RvD1 as a potential proresolving therapeutic agent for allergic responses in the airways.

Growth factor and Th2 cytokine signaling pathways converge at STAT6 to promote arginase expression in progressive experimental visceral leishmaniasis.

Host arginase 1 (arg1) expression is a significant contributor to the pathogenesis of progressive visceral leishmaniasis (VL), a neglected tropical disease caused by the intracellular protozoan Leishmania donovani. Previously we found that parasite-induced arg1 expression in macrophages was dependent on STAT6 activation. Arg1 expression was amplified by, but did not require, IL-4, and required de novo synthesis of unknown protein(s). To further explore the mechanisms involved in arg1 regulation in VL, we screened a panel of kinase inhibitors and found that inhibitors of growth factor signaling reduced arg1 expression in splenic macrophages from hamsters with VL. Analysis of growth factors and their signaling pathways revealed that the Fibroblast Growth Factor Receptor 1 (FGFR-1) and Insulin-like Growth Factor 1 Receptor (IGF-1R) and a number of downstream signaling proteins were activated in splenic macrophages isolated from hamsters infected with L. donovani. Recombinant FGF-2 and IGF-1 increased the expression of arg1 in L. donovani infected hamster macrophages, and this induction was augmented by IL-4. Inhibition of FGFR-1 and IGF-1R decreased arg1 expression and restricted L. donovani replication in both in vitro and ex vivo models of infection. Inhibition of the downstream signaling molecules JAK and AKT also reduced the expression of arg1 in infected macrophages. STAT6 was activated in infected macrophages exposed to either FGF-2 or IGF-1, and STAT6 was critical to the FGFR-1- and IGF-1R-mediated expression of arg1. The converse was also true as inhibition of FGFR-1 and IGF-1R reduced the activation of STAT6 in infected macrophages. Collectively, these data indicate that the FGFR/IGF-1R and IL-4 signaling pathways converge at STAT6 to promote pathologic arg1 expression and intracellular parasite survival in VL. Targeted interruption of these pathological processes offers an approach to restrain this relentlessly progressive disease.

Low-level laser therapy inhibits bronchoconstriction, Th2 inflammation and airway remodeling in allergic asthma.

Low-level laser therapy (LLLT) controls bronchial hyperresponsiveness (BHR) associated with increased RhoA expression as well as pro-inflammatory mediators associated with NF-kB in acute lung inflammation. Herein, we explore if LLLT can reduce both BHR and Th2 cytokines in allergic asthma. Mice were studied for bronchial reactivity and lung inflammation after antigen challenge. BHR was measured through dose-response curves to acetylcholine. Some animals were pretreated with a RhoA inhibitor before the antigen. LLLT (660 nm, 30 mW and 5.4 J) was applied on the skin over the right upper bronchus and two irradiation protocols were used. Reduction of BHR post LLLT coincided with lower RhoA expression in bronchial muscle as well as reduction in eosinophils and eotaxin. LLLT also diminished ICAM expression and Th2 cytokines as well as signal transducer and activator of transduction 6 (STAT6) levels in lungs from challenged mice. Our results demonstrated that LLLT reduced BHR via RhoA and lessened allergic lung inflammation via STAT6.

Signal transducer and activator of transcription factor 6 signaling contributes to control host lung pathology but favors susceptibility against Toxocara canis infection.

Using STAT6(-/-) BALB/c mice, we have analyzed the role of STAT6-induced Th2 response in determining the outcome of experimental toxocariasis caused by embryonated eggs of the helminth parasite Toxocara canis. Following T. canis infection wild-type BALB/c mice developed a strong Th2-like response, produced high levels of IgG1, IgE, and IL-4, recruited alternatively activated macrophages, and displayed a moderate pathology in the lungs; however, they harbored heavy parasite loads in different tissues. In contrast, similarly infected STAT6(-/-) BALB/c mice mounted a weak Th2-like response, did not recruit alternatively activated macrophages, displayed a severe pathology in the lungs, but efficiently controlled T. canis infection. These findings demonstrate that Th2-like response induced via STAT6-mediated signaling pathway mediates susceptibility to larval stage of T. canis. Furthermore, they also indicate that unlike most gastrointestinal helminths, immunity against larvae of T. canis is not mediated by a Th2-dominant response.

Differential monocyte STAT6 activation and CD4(+)CD25(+)Foxp3(+) T cells in kidney operational tolerance transplanted individuals.

In organ transplantation, the immunosuppression withdrawal leads, in most cases, to rejection. Nonetheless, a special group of patients maintain stable graft function after complete withdrawal of immunosuppression, achieving a state called "operational tolerance." The study of such patients may be important to understand the mechanisms involved in human transplantation tolerance. We compared the profile of CD4(+)CD25(+)Foxp3(+) T cells and the signaling pathways IL-6/STAT3 (signal transducers and activators of transcription) and IL-4/STAT6 in peripheral blood mononuclear cells of four kidney transplant groups: (i) operational tolerance (OT), (ii) chronic allograft nephropathy (CR), (iii) stable graft function under standard immunosuppression (Sta), (iv) stable graft function under low immunosuppression, and (v) healthy individuals. Both CR and Sta displayed lower numbers and percentages of CD4(+)CD25(+)Foxp3(+) T cells compared with all other groups (p < 0.05). The OT patients displayed a reduced activation of the IL-4/STAT6 pathway in monocytes, compared with all other groups (p < 0.05). The lower numbers of CD4(+)CD25(+)Foxp3(+) T cells observed in CR individuals may be a feature of chronic allograft nephropathy. The differential OT signaling profile, with reduced phosphorylation of STAT6, in monocytes' region, suggests that some altered function of STAT6 signaling may be important for the operational tolerance state.

Nitric oxide contributes to host resistance against experimental Taenia crassiceps cysticercosis.

The immune mechanisms that underlie resistance and susceptibility to cysticercosis are not completely understood. In this paper, using susceptible BALB/c mice and resistant STAT6-/-BALB/c mice, we have analyzed the role of nitric oxide (NO) in determining the outcome of murine cysticercosis caused by the cestode Taenia crassiceps. After T. crassiceps infection, wild-type BALB/c mice developed a strong Th2-like response, produced high levels of IgG1, IgE, IL-5, IL-4, and discrete levels of NO, and remained susceptible to T. crassiceps infection. In contrast, similarly infected BALB/c mice treated with N(omega)-nitro-L-arginine methyl ester (L-NAME, an inhibitor of NO synthase) mounted a similar immune response but with lower levels of NO and harbored nearly 100% more parasites than N(omega)-nitro-D-arginine methyl ester (D-NAME, inactive enantiomer)-treated mice. To further analyze the role of NO in murine cysticercosis, we treated STAT6-/-male mice (known to be highly resistant to T. crassiceps) with L-NAME during 8 weeks of infection. As expected, STAT6-/-mice mounted a strong Th1-like response, produced high levels of IgG2a, IFN-gamma, and IL-17, whereas their macrophages displayed increased transcripts of tumor necrosis factor (TNF)-alpha as well as inducible nitric oxide synthase (iNOS) and efficiently controlled T. crassiceps infection. However, STAT6-/-male mice receiving L-NAME mounted a similar immune response but with lower iNOS transcripts concomitantly with decreased levels of NO in sera and displayed significantly higher parasite burdens. These findings suggest that macrophage activation and NO production are effector mechanisms that importantly contribute in host resistance to T. crassiceps infection. The immune mechanisms that underlie resistance and susceptibility to cysticercosis are not completely understood. In this paper, using susceptible BALB/c mice and resistant STAT6-/-BALB/c mice, we have analyzed the role of nitric oxide (NO) in determining the outcome of murine cysticercosis caused by the cestode Taenia crassiceps. After T. crassiceps infection, wild-type BALB/c mice developed a strong Th2-like response, produced high levels of IgG1, IgE, IL-5, IL-4, and discrete levels of NO, and remained susceptible to T. crassiceps infection. In contrast, similarly infected BALB/c mice treated with N(omega)-nitro-L-arginine methyl ester (L-NAME, an inhibitor of NO synthase) mounted a similar immune response but with lower levels of NO and harbored nearly 100% more parasites than N(omega)-nitro-d-arginine methyl ester (D-NAME, inactive enantiomer)-treated mice. To further analyze the role of NO in murine cysticercosis, we treated STAT6-/-male mice (known to be highly resistant to T. crassiceps) with L-NAME during 8 weeks of infection. As expected, STAT6-/-mice mounted a strong Th1-like response, produced high levels of IgG2a, IFN-gamma, and IL-17, whereas their macrophages displayed increased transcripts of tumor necrosis factor (TNF)-alpha as well as inducible nitric oxide synthase (iNOS) and efficiently controlled T. crassiceps infection. However, STAT6-/-male mice receiving L-NAME mounted a similar immune response but with lower iNOS transcripts concomitantly with decreased levels of NO in sera and displayed significantly higher parasite burdens. These findings suggest that macrophage activation and NO production are effector mechanisms that importantly contribute in host resistance to T. crassiceps infection.

Expression of IL-4 receptor on non-bone marrow-derived cells is necessary for the timely elimination of Strongyloides venezuelensis in mice, but not for intestinal IL-4 production.

In rodents and in humans, Strongyloides infection induces an immune response which is predominantly Th2 in nature. In an attempt to understand the role of the IL-4R/STAT6 signaling pathway, the pathway activated by the Th2 cytokines IL-4 and IL-13, in the induction of protection during Strongyloides venezuelensis infection, we have carried out experiments in mice lacking the IL-4Ralpha chain. Experiments were also carried out in STAT6 (STAT6(-/-)) and IL-12-deficient (IL-12(-/-)) mice for comparison. There was enhancement of IL-13 and abolition of IFN-gamma production in the small intestine of 7 day-infected IL-12(-/-) animals but worm elimination proceeded with very similar kinetics to those of wild-type mice. In IL-4Ralpha- or STAT6-deficient mice, there was a delay in parasite elimination and a large number of S. venezuelensis adult worms was still present in the small intestine 14 days after infection. Moreover, IgE production was completely abolished in IL-4Ralpha- or STAT6-deficient mice but tissue eosinophilia was normally induced by the parasite infection in deficient mice. Bone marrow transfer experiments showed that worm elimination occurred when a functional IL-4 receptor was present only in non-bone marrow-derived cells but not when IL-4R was only expressed in bone marrow cells. The induction of IL-4, but not IL-13, occurred independently of IL-4R. We believe these results are the first direct evidence that the mechanism responsible for the timely elimination of S. venezuelensis is dependent on the activation of IL-4R and STAT6. Moreover, a functional protective response is dependent on the expression of IL-4Ralpha on non-bone marrow-derived cells.