Nuclear cGAS: sequestration and beyond

The cyclic GMP-AMP (cGAMP) synthase (cGAS) has been identified as a cytosolic double stranded DNA sensor that plays a pivotal role in the type I interferon and inflammation responses via the STING-dependent signaling pathway. In the past several years, a growing body of evidence has revealed that cGAS is also localized in the nucleus where it is associated with distinct nuclear substructures such as nucleosomes, DNA replication forks, the double-stranded breaks, and centromeres, suggesting that cGAS may have other functions in addition to its role in DNA sensing. However, while the innate immune function of cGAS is well established, the non-canonical nuclear function of cGAS remains poorly understood. Here, we review our current understanding of the complex nature of nuclear cGAS and point to open questions on the novel roles and the mechanisms of action of this protein as a key regulator of cell nuclear function, beyond its well-established role in dsDNA sensing and innate immune response.

TRIM35 mediates protection against influenza infection by activating TRAF3 and degrading viral PB2

Tripartite motif (TRIM) family proteins are important effectors of innate immunity against viral infections. Here we identified TRIM35 as a regulator of TRAF3 activation. Deficiency in or inhibition of TRIM35 suppressed the production of type I interferon (IFN) in response to viral infection. Trim35-deficient mice were more susceptible to influenza A virus (IAV) infection than were wild-type mice. TRIM35 promoted the RIG-I-mediated signaling by catalyzing Lys63-linked polyubiquitination of TRAF3 and the subsequent formation of a signaling complex with VISA and TBK1. IAV PB2 polymerase countered the innate antiviral immune response by impeding the Lys63-linked polyubiquitination and activation of TRAF3. TRIM35 mediated Lys48-linked polyubiquitination and proteasomal degradation of IAV PB2, thereby antagonizing its suppression of TRAF3 activation. Our in vitro and in vivo findings thus reveal novel roles of TRIM35, through catalyzing Lys63- or Lys48-linked polyubiquitination, in RIG-I antiviral immunity and mechanism of defense against IAV infection.

T cell receptor signaling pathway is overexpressed in CD4 + T cells from HAM/TSP individuals

ABSTRACT Human T-lymphotropic virus type 1 (HTLV-1) is a human retrovirus related to the chronic neuroinflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). CD4+ T cells activation appears to play a key role on HTLV-1 infection. Here we investigated the expression of genes associated to T cell activation CD3e molecule, epsilon (CD3?), lymphocyte-specific protein tyrosine kinase (LCK), vav 1 guanine nucleotide exchange factor (VAV1), and zeta-chain (TCR) associated protein kinase 70 kDa (ZAP70) on T lymphocytes of HTLV-1-infected individuals and compared to healthy uninfected individuals (CT). We observed that CD3?, LCK, ZAP70, and VAV1 gene expression were increased in CD4+ T cells from HAM/TSP group compared to HTLV-1 asymptomatic patients (HAC). Moreover, ZAP70 and VAV1 were also upregulated in HAM/TSP compared to CT group. We detected a positive correlation among all these genes. We also observed that CD3?, LCK, and VAV1 genes had a positive correlation with the proviral load (PVL) and Tax expression. These results suggest that PVL and Tax protein could drive CD3?, LCK, and VAV1 gene expression in CD4+ T cells, and these genes function on a synchronized way on the CD4+ T cell activation. The elucidation of the mechanisms underlying T cell receptor signaling pathway is of considerable interest and might lead to new insights into the mechanism of HAM/TSP.

Risk behaviors for sexually transmitted diseases among crack users / Comportamentos de risco para doenças sexualmente transmissíveis em usuários de crack / Conductas de riesgo para enfermedades de transmisión sexual entre usuarios de crack

AbstractObjectives: to investigate the prevalence and risk behaviors by means of reporting of sexually transmitted diseases among crack users.Method: cross-sectional study carried out with 588 crack users in a referral care unit for the treatment of chemical dependency. Data were collected by means of face-to-face interview and analyzed using Stata statistical software, version 8.0.Results: of the total participants, 154 (26.2%; 95% CI: 22.8-29.9) reported antecedents of sexually transmitted diseases. Ages between 25 and 30 years (RP: 2.1; 95% CI: 1.0-4.0) and over 30 years (RP: 3.8; 95% CI: 2.1-6.8), alcohol consumption (RP: 1.9; 95% CI: 1.1-3.3), antecedents of prostitution (RP: 1.9; 95% CI: 1.3-2.9) and sexual intercourse with person living with human immunodeficiency virus/AIDS (RP: 2.7; 95% CI: 1.8-4.2) were independently associated with reporting of sexually transmitted diseases.Conclusion: the results of this study suggest high risk and vulnerability of crack users for sexually transmitted diseases.

ResumoObjetivos:investigar a prevalência e comportamentos de risco através do relato de doenças sexualmente transmissíveis em usuários de crack.Método:estudo transversal, realizado com 588 usuários de crack, de uma unidade de referência para tratamento de dependência química. Os dados foram obtidos por meio de entrevista face a face e analisados em programa estatístico Stata, versão 8.0.Resultados:do total de participantes, 154 (26,2%; IC 95%: 22,8-29,9) referiram antecedentes de doenças sexualmente transmissíveis. Idade entre 25 e 30 anos (RP: 2,1; IC 95%: 1,0-4,0) e superior a 30 anos (RP: 3,8; IC 95%: 2,1-6,8), consumo de álcool (RP: 1,9; IC 95%: 1,1-3,3), antecedentes de prostituição (RP: 1,9; IC 95%: 1,3-2,9) e relação sexual com pessoa vivendo com o vírus da imunodeficiência humana/aids (RP: 2,7; IC 95%: 1,84,2) foram independentemente associados ao relato de doenças sexualmente transmissíveis.Conclusão:os resultados deste estudo sugerem elevado risco e vulnerabilidade dos usuários de crackpara as doenças sexualmente transmissíveis.

ResumenObjetivos:investigar la prevalencia y las conductas de riesgo a través del informe de las enfermedades de transmisión sexual entre los usuarios de crack.Método:estudio transversal con 588 usuarios de crack, de una unidad de referencia para el tratamiento de la dependencia química. Los datos fueron obtenidos a través de entrevista cara a cara y se analizaron utilizando el programa estadístico Stata, versión 8.0.Resultados:del total de participantes, 154 (26,2%; IC 95%: 22,8-29,9) informaron antecedentes de enfermedades de transmisión sexual. Edad entre 25 y 30 años (RP: 2,1; IC9 5%: 1,0-4,0) y superior a 30 años (RP: 3,8; IC 95%: 2,1-6,8), consumo de alcohol (OR: 1,9; IC 95%: 1,1-3,3), antecedentes de prostitución (RP: 1,9; IC 95%: 1,3-2,9) y relaciones sexuales con persona viviendo con el virus de inmunodeficiencia humana/ SIDA (RP: 2,7; IC 95%: 1,8-4,2) se asociaron de forma independiente con la notificación de las enfermedades de transmisión sexual.Conclusión:los resultados de este estudio sugieren alto riesgo y la vulnerabilidad de los usuarios de crackpara las enfermedades de transmisión sexual.

Leishmania donovani secretory serine protease alters macrophage inflammatory response via COX-2 mediated PGE-2 production.

Leishmania parasites determine the outcome of the infection by inducing inflammatory response that suppresses macrophage’s activation. Defense against Leishmania is dependent on Th1 inflammatory response by turning off macrophages’ microbicidal property by upregulation of COX-2, as well as immunosuppressive PGE-2 production. To understand the role of L. donovani secretory serine protease (pSP) in these phenomena, pSP was inhibited by its antibody and serine protease inhibitor, aprotinin. Western blot and TAME assay demonstrated that pSP antibody and aprotinin significantly inhibited protease activity in the live Leishmania cells and reduced infection index of L. donovani-infected macrophages. Additionally, ELISA and RT-PCR analysis showed that treatment with pSP antibody or aprotinin hold back COX-2-mediated immunosuppressive PGE-2 secretion with enhancement of Th1 cytokine like IL-12 expression. This was also supported in Griess test and NBT assay, where inhibition of pSP with its inhibitors elevated ROS and NO production. Overall, our study implies the pSP is involved in down-regulation of macrophage microbicidal activity by inducing host inflammatory responses in terms of COX-2-mediated PGE-2 release with diminished reactive oxygen species generation and thus suggests its importance as a novel drug target of visceral leishmaniasis.

TLR4-mediated activation of MyD88 signaling induces protective immune response and IL-10 down-regulation in Leishmania donovani infection.

In visceral leishmaniasis, a fragmentary IL-12 driven type 1 immune response along with the expansion of IL-10 producing T-cells correlates with parasite burden and pathogenesis. Successful immunotherapy involves both suppression of IL-10 production and enhancement of IL-12 and nitric oxide (NO) production. As custodians of the innate immunity, the toll-like receptors (TLRs) constitute the first line of defense against invading pathogens. The TLR-signaling cascade initiated following innate recognition of microbes shapes the adaptive immune response. Whereas numerous studies have correlated parasite control to the adaptive response in Leishmania infection, growing body of evidence suggests that the activation of the innate immune response also plays a pivotal role in disease pathogenicity. In this study, using a TLR4 agonist, a Leishmania donovani (LD) derived 29 kDa β 1,4 galactose terminal glycoprotein (GP29), we demonstrated that the TLR adaptor myeloid differentiation primary response protein-88 (MyD88) was essential for optimal immunity following LD infection. Treatment of LD-infected cells with GP29 stimulated the production of IL-12 and NO while suppressing IL-10 production. Treatment of LD-infected cells with GP29 also induced the degradation of IKB and the nuclear translocation of NF-kB, as well as rapid phosphorylation of p38 MAPK and p54/56 JNK. Knockdown of TLR4 or MYD88 using siRNA showed reduced inflammatory response to GP29 in LD-infected cells. Biochemical inhibition of p38 MAPK, JNK or NF-kB, but not p42/44 ERK, reduced GP29-induced IL-12 and NO production in LD-infected cells. These results suggested a potential role for the TLR4-MyD88–IL-12 pathway to induce adaptive immune responses to LD infection that culminated in an effective control of intracellular parasite replication.

Phospholipid Remodeling and Eicosanoid Signaling in Colon Cancer Cells.

Phospholipid remodeling and eicosanoid synthesis are central to lipid-based inflammatory reactions. Studies have revealed that membrane phospholipid remodeling by fatty acids through deacylation/reacylation reactions increases the risk of colorectal cancers (CRC) by allowing the cells to produce excess inflammatory eicosanoids, such as prostaglandins, thromboxanes and leukotrienes. Over the years, efforts have been made to understand the lipid remodeling pathways and to design anti-cancer drugs targeting the enzymes of eicosanoid biosynthesis. Here, we discuss the recent progress in phospholipid remodeling and eicosanoid biosynthesis in CRC.

Regulation of Cardiac β3-Adrenergic Receptors in Hyperglycemia.

Beta-adrenoceptors (β-AR), members of the G protein-coupled receptors play important roles in the regulation of heart function. A positive inotropic action of catecholamines is mediated through their interaction with β-AR, located on the sarcolemma, while they can also mediate some deleterious effects, such as cardiac arrhythmias or myocardial apoptosis. The well-known β-AR-associated signaling in heart is composed of a coupled mechanism among both β1- and β2-AR and stimulatory G protein (Gs). This coupled mechanism further leads to the activation of adenylyl cyclase and thereby increases in intracellular cAMP level. However, recent studies have emphasized the contribution of constitutive β3-AR coupling to Gi proteins, thereby initiating additional signal transduction pathways, particularly under physiopathological conditions. Diabetic cardiomyopathy, as a distinct entity is recognized due to its diminished responsiveness to β1-AR agonist stimulation in the heart from diabetic rats with no important changes in the responses mediated with β2-AR. Furthermore, an upregulation of β3-AR has been shown in diabetic rat heart with a strong negative inotropic effect on left ventricular function. Experimental data provide evidences that the mechanisms for the negative inotropic effect with β3-AR activation appear to involve a pertussis toxin (PTX)-sensitive G protein and the activation of a nitric oxide synthase pathway. On the other hand, β-blockers demonstrate marked beneficial effects in heart dysfunction with scavenging free radicals and/or acting as an antioxidant with both sex- and dose-dependent manner. However, further investigations are needed to clarify the roles of both altered expression and/or responsiveness of β-AR and the benefits with β-blocker treatment in diabetes. This review discusses the role of β-AR activation, particularly β3-AR in cardiac pathological remodeling under hyperglycemia.

Signaling Mechanisms Regulating Fibroblast Activation, Phenoconversion and Fibrosis in the Heart.

Cardiac fibroblasts (CFs) maintain the cardiac extracellular matrix (ECM) through myocardial remodelling. The remodelling process can become dysregulated during various forms of heart disease  which leads to an overall accumulation of ECM. This results in cardiac fibrosis which increases the risk of heart failure in many patients. During heart disease, quiescent CFs undergo phenoconversion to an activated cell type called cardiac myofibroblasts (CMFs). Factors influencing phenoconversion include transforming growth factor β (TGF-β) which via SMADs (small mothers against decapentaplegic) activates the myofibroblast marker gene αSMA (α smooth muscle actin). Signaling molecules as diverse as NAD(P)H oxidase 4 (Nox4) and Wnt have been found to interact with TGF-β signalling via SMADs. Pathways, including FAK/TAK/JNK and PI3K/Akt/rac have also been implicated in activating phenoconversion of fibroblasts. Another major contributor is mechanical stress exerted on CFs by ECM changes, which involves activation of ERK and subsequent αSMA expression. Other factors, such as the mast cell protease tryptase and the seeding density also affect the phenoconversion of fibroblast cultures in vitro. Further, reversal of myofibroblast phenotype has been reported by a negative regulator of TGF-β, Ski, as well as the hormone relaxin and the second messenger cAMP. Targeting the signaling molecules involved in promoting phenoconversion of CFs to CMFs presents a possible method of controlling cardiac fibrosis. Here, we provide a brief review of signaling mechanisms responsible for phenoconversion and identify critical targets for the treatment of cardiac fibrosis.

Regulation of Giα Protein Expression by Vasoactive Peptides in Hypertension: Molecular Mechanisms.

Guanine nucleotide regulatory proteins (G proteins) play a key role in the regulation of various signal transduction systems, including adenylyl cyclase/cAMP and phospholipase C (PLC)/phosphatidyl inositol (PI) turnover, which are implicated in the modulation of a variety of physiological functions, such as platelet functions, including platelet aggregation, secretion, and clot formation and cardiovascular functions, including arterial tone and reactivity. Several abnormalities in adenylyl cyclase activity, cAMP levels and G proteins have been shown to be responsible for the altered cardiac performance and vascular functions observed in cardiovascular disease states. The enhanced or unaltered levels of inhibitory G proteins (Giα) and mRNA have been reported in different models of hypertension, whereas Gsα levels are shown to be unaltered. The enhanced levels of Giα proteins precede the development of blood pressure and suggest that overexpression of Gi proteins may be one of the contributing factors for the pathogenesis of hypertension. The levels of vasoactive peptides including ET-1 and Ang II and growth factors are augmented in hypertension and contribute to the enhanced expression of Giα proteins in hypertension. In addition, oxidative stress due to enhanced levels of Ang II and ET-1 is enhanced in hypertension and may also be responsible for the enhanced expression of Giα proteins observed in hypertension. Furthermore, Ang II- and ET-1-induced transactivation of growth factor receptor through the activation of MAP kinase signaling is also shown to contribute to the augmented levels of Giα in hypertension. Thus, it appears that the enhanced levels of vasoactive peptides by increasing oxidative stress and transactivation growth factor receptors enhance MAP kinase activity that contribute to the enhanced expression of Giα proteins responsible for the pathogenesis of hypertension. In this review, we describe the role of vasoactive peptides and the signaling mechanisms responsible for the enhanced expression of Giα proteins in hypertension.

Involvement of the Early Growth Response Protein 1 in Vascular Pathophysiology: An Overview.

Hyperactivation of proliferative and growth promoting pathways underlies the progression of vessel remodeling, leading to vascular dysfunction. An upregulation of early growth response protein 1 (Egr-1), a zinc finger transcription factor has been observed in several models of vascular diseases. In the vasculature, Egr-1 expression can be induced by multiple hormonal, metabolic and external stimuli, such as growth factors, cytokines, reactive oxygen species, hyperglycaemia and stretch-induced stress. The structure of the Egr-1 promoter allows both its auto-regulation and its binding with several regulatory transcription cofactors like the serum response factor and the cAMP response element binding protein. Pharmacological and genetic studies have revealed the involvement of several signaling pathways that contribute to the expression of Egr-1. Among them, the mitogen-activated protein kinase pathway has emerged as a predominant signaling cascade that regulates Egr-1 transcription in response to various stimuli. Moreover, targeted deletion of Egr-1 by DNAzymes, antisense oligonucleotides or RNA interference has also helped in defining the importance of Egr-1 in the pathophysiology of vascular diseases. Neointimal formation and expression of genes directly linked with proinflammatory processes have been demonstrated to be enhanced by Egr-1 expression and activity. This review provides an overview on the signaling components implicated in Egr-1 expression and discusses its potential involvement in vascular pathophysiology.

Vascular Aneurysms: A Perspective.

Aneurysms develop as a result of chronic inflammation of vascular bed, where progressive destruction of structural proteins, especially elastin and collagen of smooth muscle cells has been shown to manifest. The underlying mechanisms are an increase in local production of proinflammatory cytokines and subsequent increase in proteases, especially matrix metalloproteinases (MMPs) that degrade the structural proteins. The plasminogen system: urokinase-type PA (u-PA), tissue-type PA (t-PA) and plasminogen activator inhibitor-1 (PAI-1) and the MMPs system-MMPs and TIMPs contribute to the progression and development of aneurysms. Recent studies suggest that aneurysms may be genetically determined. To date, most observable candidate genes for aneurysm (elastin, collagen, fibrillin, MMPs and TIMPs) have been explored with little substantiation of the underlying cause and effect. Recently, overexpression of the MMP-2 gene has been suggested as an important phenomenon for aneurysm formation. Along with MMPs, matrix formation also depends on JNK (c-Jun N-terminal kinase) as its activation plays important role in downregulating several genes of matrix production. Under stress, activation of JNK by various stimuli, such as angiotensin II, tumor necrosis factor-α and interleukin-1β has been noted significantly in vascular smooth muscle cells. Several therapeutic indications corroborate that inhibition of MMP-2 and JNK is useful in preventing progression of vascular aneurysms. This review deals with the role of proteases in the progression of vascular aneurysm.

Vascular endothelial growth factor-A enhances indoleamine 2,3-dioxygenase expression by dendritic cells and subsequently impacts lymphocyte proliferation

Dendritic cells (DCs) are antigen (Ag)-presenting cells that activate and stimulate effective immune responses by T cells, but can also act as negative regulators of these responses and thus play important roles in immune regulation. Pro-angiogenic vascular endothelial growth factor (VEGF) has been shown to cause defective DC differentiation and maturation. Previous studies have demonstrated that the addition of VEGF to DC cultures renders these cells weak stimulators of Ag-specific T cells due to the inhibitory effects mediated by VEGF receptor 1 (VEGFR1) and/or VEGFR2 signalling. As the enzyme indoleamine 2,3-dioxygenase (IDO) is recognised as an important negative regulator of immune responses, this study aimed to investigate whether VEGF affects the expression of IDO by DCs and whether VEGF-matured DCs acquire a suppressor phenotype. Our results are the first to demonstrate that VEGF increases the expression and activity of IDO in DCs, which has a suppressive effect on Ag-specific and mitogen-stimulated lymphocyte proliferation. These mechanisms have broad implications for the study of immunological responses and tolerance under conditions as diverse as cancer, graft rejection and autoimmunity.

Negative regulatory approaches to the attenuation of Toll-like receptor signaling

Toll-like receptors (TLRs) are pivotal components of the innate immune response, which is responsible for eradicating invading microorganisms through the induction of inflammatory molecules. These receptors are also involved in responding to harmful endogenous molecules and have crucial roles in the activation of the innate immune system and shaping the adaptive immune response. However, TLR signaling pathways must be tightly regulated because undue TLR stimulation may disrupt the fine balance between pro- and anti-inflammatory responses. Such disruptions may harm the host through the development of autoimmune and inflammatory diseases, such as rheumatoid arthritis and systemic lupus erythematosus. Several studies have investigated the regulatory pathways of TLRs that are essential for modulating proinflammatory responses. These studies reported several pathways and molecules that act individually or in combination to regulate immune responses. In this review, we have summarized recent advancements in the elucidation of the negative regulation of TLR signaling. Moreover, this review covers the modulation of TLR signaling at multiple levels, including adaptor complex destabilization, phosphorylation and ubiquitin-mediated degradation of signal proteins, manipulation of other receptors, and transcriptional regulation. Lastly, synthetic inhibitors have also been briefly discussed to highlight negative regulatory approaches in the treatment of inflammatory diseases.

MRP8 promotes Th17 differentiation via upregulation of IL-6 production by fibroblast-like synoviocytes in rheumatoid arthritis

Myeloid-related protein (MRP)8/MRP14 is an endogenous Toll-like receptor 4 (TLR4) ligand and is abundant in synovial fluid (SF) of rheumatoid arthritis (RA) patients. Belonging to damage-associated molecular patterns, it amplifies proinflammatory mediators and facilitates a wide range of inflammatory and autoimmune diseases. Interleukin (IL)-17-producing T-helper (Th)17 cells have a crucial role in RA pathogenesis, and IL-6 is the key factor promoting Th17 differentiation. We investigated whether the level of MRP8/MRP14 is positively associated with IL-6 and IL-17 levels in RA SF and found that MRP8/MRP14 level had a significant correlation with IL-6 and IL-17 levels in RA SF. We also observed that MRP8-induced IL-17 production by peripheral blood mononuclear cells but MRP14 did not. Upon stimulation with MRP8, IL-6 production was enhanced by RA fibroblast-like synoviocytes (FLS) and was further elevated by coculturing RA FLS with activated CD4+ T cells. Moreover, we demonstrated that MRP8-activated IL-6 production by RA FLS promoted differentiation of Th17 cells using the coculture system consisting of CD4+ T cells and RA FLS. In addition, IL-6 blockade attenuated Th17 polarization of CD4+ T cells in the cocultures. Inhibitor studies revealed that MRP8 increased IL-6 production in RA FLS via TLR4/phosphoinositide 3-kinase/nuclear factor-kappaB and mitogen-activated protein kinase signaling pathways. Our results show that MRP8 has a crucial role in stimulating IL-6 expression by RA FLS, and subsequently promotes Th17 differentiation in RA, suggesting that neutralizing MRP8 level in RA synovium may be an effective therapeutic strategy in RA treatment.

Regulation of B cell activating factor (BAFF) receptor expression by NF-kappaB signaling in rheumatoid arthritis B cells

B cells play an important role in the pathogenesis of rheumatoid arthritis (RA). High levels of B cell activating factor (BAFF) are detected in autoimmune diseases. BAFF and BAFF receptor (BAFF-R) are expressed in B and T cells of RA synovium. The study was undertaken to identify the NF-kappaB signal pathway involved in the induction of BAFF-R in human B cells. Immunohistochemical staining of NF-kappaB p65, NF-kappaB p50, BAFF, and BAFF-R was performed on sections of synovium from severe and mild RA and osteoarthritis (OA) patients. Peripheral blood mononuclear cells (PBMCs) were isolated from control and RA patients and B cells were isolated from controls. BAFF-R was analyzed by flow cytometry, realtime PCR and confocal staining after treatment with NF-kappaB inhibitors. NF-kappaB p65, NF-kappaB p50, BAFF, and BAFF-R were highly expressed in severe RA synovium relative to mild RA synovium or OA synovium. BAFF-R expression was reduced by NF-kappaB inhibitors in PBMCs and B cells from normal controls. We also showed reduction in expression of BAFF-R via inhibition of the NF-kappaB pathway in PBMCs of RA patients. BAFF/BAFF-R signaling is an important mechanism of pathogenesis in RA and that BAFF-R reduction by NF-kappaB blocking therapy is another choice for controlling B cells in autoimmune diseases such as RA.

IL-12-STAT4-IFN-gamma axis is a key downstream pathway in the development of IL-13-mediated asthma phenotypes in a Th2 type asthma model

IL-4 and IL-13 are closely related cytokines that are produced by Th2 cells. However, IL-4 and IL-13 have different effects on the development of asthma phenotypes. Here, we evaluated downstream molecular mechanisms involved in the development of Th2 type asthma phenotypes. A murine model of Th2 asthma was used that involved intraperitoneal sensitization with an allergen (ovalbumin) plus alum and then challenge with ovalbumin alone. Asthma phenotypes, including airway-hyperresponsiveness (AHR), lung inflammation, and immunologic parameters were evaluated after allergen challenge in mice deficient in candidate genes. The present study showed that methacholine AHR and lung inflammation developed in allergen-challenged IL-4-deficient mice but not in allergen-challenged IL-13-deficient mice. In addition, the production of OVA-specific IgG2a and IFN-gamma-inducible protein (IP)-10 was also impaired in the absence of IL-13, but not of IL-4. Lung-targeted IFN-gamma over-expression in the airways enhanced methacholine AHR and non-eosinophilic inflammation; in addition, these asthma phenotypes were impaired in allergen-challenged IFN-gamma-deficient mice. Moreover, AHR, non-eosinophilic inflammation, and IFN-gamma expression were impaired in allergen-challenged IL-12Rbeta2- and STAT4-deficient mice; however, AHR and non-eosinophilic inflammation were not impaired in allergen-challenged IL-4Ralpha-deficient mice, and these phenomena were accompanied by the enhanced expression of IL-12 and IFN-gamma. The present data suggest that IL-13-mediated asthma phenotypes, such as AHR and non-eosinophilic inflammation, in the Th2 type asthma are dependent on the IL-12-STAT4-IFN-gamma axis, and that these asthma phenotypes are independent of IL-4Ralpha-mediated signaling.

4-1BB (CD137) signals depend upon CD28 signals in alloimmune responses

Our previous study has demonstrated that there is a significant delay of Balb/c cardiac allograft rejection in the C57BL/6 4-1BB-deficient knockout recipient. In this study, we examined the effect of combined blockade of the 4-1BB and CD28 costimulatory pathways on cardiac allograft rejection in the C57BL/6-->Balb/c model. A long-term cardiac allograft survival was induced in CD28/4-1BB- deficient mice (>100 days survival in 3 of 4 mice), which was comparable with CD28-deficient mice (>100 days survival in 2 of 5 mice; P<0.2026). There was no long-term cardiac allograft survival in either wild-type (WT) or 4-1BB-deficient mice, even though 4-1BB-deficient recipients showed a significant delay of cardiac allograft rejection than WT mice. An in vitro mixed leukocyte reaction (MLR) assay showed that 4-1BB-deficient and WT mouse T cells had a similar responsiveness to allostimulation, whereas CD28- and CD28/4-1BB-deficient mouse T cells had a defective responsiveness to allostimulation. Furthermore, 4-1BB-deficient mice showed a similar CTL but an elevated Ab response against alloantigens as compared to WT mice, and the alloimmune responses of 4-1BB-deficient mice were abrogated in the CD28-deficient background. Overall, these results indicate that the CD28 costimulatory pathway plays a major role in the alloimmune response and that 4-1BB signals are dependent upon CD28 signals.