Induction Of Th1 Immune Responses Research Articles

879 BLADDER EXPRESSION OF CYTOTOXIC EFFECTOR MOLECULES IN RESPONSE TO INTRAVESICAL MYCOBACTERIUM BOVIS BACILLUS CALMETTE-GUÉRIN (BCG)

You have accessJournal of UrologyBladder Cancer: Basic Research1 Apr 2011879 BLADDER EXPRESSION OF CYTOTOXIC EFFECTOR MOLECULES IN RESPONSE TO INTRAVESICAL MYCOBACTERIUM BOVIS BACILLUS CALMETTE-GUÉRIN (BCG) Matthew Knudson, Jonathan Henning, Michael O'Donnell, and Yi Luo Matthew KnudsonMatthew Knudson Iowa City, IA More articles by this author , Jonathan HenningJonathan Henning Iowa City, IA More articles by this author , Michael O'DonnellMichael O'Donnell Iowa City, IA More articles by this author , and Yi LuoYi Luo Iowa City, IA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.703AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder expression of cytokines and chemokines in response to intravesical (i.b.) BCG has been well documented. However, studies on BCG induction of bladder expression of cytotoxic effector molecules are underdeveloped. We investigated a panel of cytotoxic effector molecules, including IFN-γ, TNF-α, perforin, granzyme B, Fas ligand (FasL), and tumor necrosis factor related apoptosis-inducing ligand (TRAIL), in the BCG-treated bladders to determine whether expression of these molecules was correlated with the effect of BCG in treating bladder cancer. METHODS C57BL/6 female mice were treated i.b. with Pasteur strain BCG (0.1 OD/dose) twice weekly for a total of 7 treatments. Mice treated with PBS served as a control. Mice were sacrificed for bladder RNA extraction after treatments 2, 4, 6 and 7. RT-PCR was used to analyze mRNA levels of IFN-γ TNF-α, perforin, granzyme B, FasL and TRAIL. IP-10 (a Th1 chemokine) and TGF-β (an inhibitory cytokine) were included. GAPDH was used as an internal control. ImageJ software was used to quantify PCR products on agarose gels. In a parallel experiment C57BL/6 female mice were implanted i.b. with 5 X 105 syngeneic bladder cancer MB49 cells that expressed luciferase (MB49-Luc) at day 0 and treated i.b. with BCG (0.1 OD/dose) twice weekly starting at day 1 for a total of 6 treatments. Control mice were treated with PBS. Bioluminescence was measured once weekly with Xenogen IVIS. At day 23 mice were euthanized and bladder weights measured. Statistics was performed by one-way analysis of variance (ANOVA) of natural log transformed bladder weights followed by a post hoc Tukey's test. RESULTS I.b. BCG treatment led to an increase in bladder mRNAs of IFN-γ,TNF-α, IP-10 and perforin and a decrease in bladder mRNAs of TGF-β. There were no changes in bladder mRNAs of granzyme B, FasL and TRAIL. In MB49-Luc orthotopic tumor model, i.b. BCG treatment reduced bladder weight by 53% (p = 0.0503 vs. PBS-treated group). In addition, i.b. BCG treatment also led to a delay in tumor growth by bioluminescence compared to PBS-treated group. CONCLUSIONS Intravesical administration of BCG induced bladder expression of cytotoxic effector molecules IFN-γ, TNF-α and perforin, which was correlated with effective BCG treatment of bladder cancer. Increased expression of IP-10 and decreased expression of TGF-β, together with increased IFN-γ and TNF-α, support the role of BCG in the induction of Th1 immune responses in the bladders. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e352 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Matthew Knudson Iowa City, IA More articles by this author Jonathan Henning Iowa City, IA More articles by this author Michael O'Donnell Iowa City, IA More articles by this author Yi Luo Iowa City, IA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Th1 Cytokine-Secreting RecombinantMycobacterium bovisBacillus Calmette-Guérin and Prospective Use in Immunotherapy of Bladder Cancer

Intravesical instillation of Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been used for treating bladder cancer for 3 decades. However, BCG therapy is ineffective in approximately 30–40% of cases. Since evidence supports the T helper type 1 (Th1) response to be essential in BCG-induced tumor destruction, studies have focused on enhancing BCG induction of Th1 immune responses. Although BCG in combination with Th1 cytokines (e.g., interferon-α) has demonstrated improved efficacy, combination therapy requires multiple applications and a large quantity of cytokines. On the other hand, genetic manipulation of BCG to secrete Th1 cytokines continues to be pursued with considerable interest. To date, a number of recombinant BCG (rBCG) strains capable of secreting functional Th1 cytokines have been developed and demonstrated to be superior to BCG. This paper discusses current rBCG research, concerns, and future directions with an intention to inspire the development of this very promising immunotherapeutic modality for bladder cancer.

Induction of Th1 immune responses to Japanese cedar pollen allergen (Cry j 1) in mice immunized with Cry j 1 conjugated with CpG oligodeoxynucleotide

We determined whether a major Japanese cedar pollen allergen (Cry j 1) conjugated with CpG oligodeoxynucleotide would enhance allergen-specific Th1 responses in mice. Cry j 1 conjugated with CpG (Cry j 1-CpG) induced IL-12 in the spleen cells of naïve mice. Cry j 1-CpG immunization of BALB/c mice suppressed anti-Cry j 1 IgE response and enhanced anti-Cry j 1 IgG(2a) to subsequent Cry j 1 and alum adjuvant injection. CD4(+)T cells isolated from the spleens in mice immunized with Cry j 1-CpG produced higher IFN-γ levels than did CD4(+)T cells obtained from mice as negative controls. Our results suggested that Cry j 1-CpG immunization can induce Cry j 1-specific Th1 immune responses, thereby inhibiting IgE response to the pollen allergen.

Anti-allergic potential of oligomannose-coated liposome-entrapped Cry j 1 as immunotherapy for Japanese cedar pollinosis in mice

Administration of oligomannose-coated liposomes (OMLs) in mice can induce Th1 immune responses against antigens entrapped in the OMLs. In the present study, we investigated the anti-allergic effect of treatment with oligomannose-coated liposomes (OMLs) with entrapped Cry j 1, a major allergen of Japanese cedar pollen (Cry j 1/OMLs), in Balb/c mice sensitized with Cry j 1. Pretreatment of unsensitized mice with Cry j 1/OMLs repressed the elevation of total and allergen-specific IgE levels in sera elicited in response to subsequent sensitization with Cry j 1. Cry j 1-specific IgG1 in sera also decreased in Cry j 1/OML-treated mice, while the levels of Cry j 1-specific IgG2a in these mice significantly increased after sensitization with Cry j 1. In addition, Cry j 1/OML-treated mice showed high IFN-γ production from spleen cells and low IL-4/IFN-γ and IL-5/IFN-γ ratios in response to in vitro stimulation with Cry j 1. These results indicate that allergen-specific Th1 immune responses predominate over Th2 responses and control IgE elevation in Cry j 1/OML-treated mice. The anti-allergic effect of Cry j 1/OML determined by suppression of serum IgE levels was also observed in Cry j 1-presensitized mice after challenge with antigen. Thus, Cry j 1/OMLs may be useful for immunotherapeutic control of allergic reactions to Japanese cedar pollinosis.

Biodegradable nanoparticle delivery of a Th2-biased peptide for induction of Th1 immune responses

The type of immune response developed against the hepatitis B virus (HBV) is crucial in determining the outcome of the disease. The protective effects of vaccine-induced antibody responses against subsequent exposure to HBV are well-established. After the establishment of chronic HBV infection, cell-mediated immune response is curative while humoral response is detrimental. A therapeutic vaccine that could switch the type of response could lead to disease resolution. Hepatitis B core antigen (HBcAg)(129-140) has been identified as a Th2-biased peptide in H-2(b) mice when it is administered along with complete Freund's adjuvant (CFA). We formulated HBcAg(129-140) along with monophosphoryl lipid A in poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles. Naive mice immunized with the nanoparticle formulation developed a strong Th1-type response while mice immunized with the control formulation of CFA and peptide did not. We then primed mice with CFA and peptide to establish a Th2-type immune response before administering the nanoparticle formulation. Mice receiving the nanoparticle formulation being primed with CFA still developed a strong Th1-type response, while mice that received incomplete Freund's adjuvant and peptide instead of nanoparticles did not. The ability of PLGA nanoparticles to alter the type of immune response elicited by a peptide, even in the context of an ongoing immune response, makes PLGA nanoparticles a strong candidate for the formulation of therapeutic vaccines.

Cutting Edge: Limiting MHC Class II Expression to Dendritic Cells Alters the Ability to Develop Th2- Dependent Allergic Airway Inflammation

In allergic airway inflammation, dendritic cells (DCs) are required for Th2 generation, recruitment, and activation in the respiratory tract. DCs have been shown to be necessary and sufficient for the induction of Th1 immune responses. In Th2 immunity and allergic airway inflammation, the ability of a DC to function as the sole APC has not been tested. We show that CD11c/A(beta)(b) mice with MHC class II expression restricted to CD11c-expressing DCs develop airway neutrophilia rather than allergic airway inflammation. Although CD11c/A(beta)(b) mice are capable of Th2 recruitment and activation in the lung, Th2 priming in CD11c/A(beta)(b) mice results in IFN-gamma production. Effective Th2 generation and allergic airway inflammation was achieved in CD11c/A(beta)(b) mice after treatment with anti-IFN-gamma. These studies show that DCs alone cannot drive the development of Th2 cells but require an additional MHC class II signal to stimulate effective Th2 immunity.

Interleukin-27 Activates Natural Killer Cells and Suppresses NK-Resistant Head and Neck Squamous Cell Carcinoma through Inducing Antibody-Dependent Cellular Cytotoxicity

Interleukin (IL)-27 is an IL-12 family cytokine playing a pivotal role in the induction of Th1 immune responses, although its action on natural killer (NK) cells has not been fully elucidated. Here, we show that IL-27 is capable of inducing phosphorylation of signal transducers and activators of transcription 1 and 3, as well as expression of T-bet and granzyme B in murine DX-5+ NK cells. IL-27 also enhances cytotoxic activity of NK cells both in vitro and in vivo, while the in vitro viability of NK cells is also improved by this cytokine. Therapeutic administration of the IL-27 gene drastically suppressed the growth of NK-unsusceptible SCCVII tumors that had been preestablished in syngenic mice, resulting in significant prolongation of the survival of the animals. This can likely be ascribed to the antibody-dependent cellular cytotoxicity machinery because IL-27 successfully induced tumor-specific IgG in the sera of the tumor-bearing mice, and supplementation of the sera enabled IL-27-activated NK cells to kill SCCVII cells in an Fcgamma receptor III-dependent manner. These findings strongly suggest that IL-27 may offer a powerful immunotherapeutic tool to eradicate head and neck squamous cell carcinoma and other poorly immunogenic neoplasms through activating NK cells and inducing tumor-specific immunoglobulin that may cooperatively elicit antibody-dependent cellular cytotoxicity activity.

EFFECTS OF COMBINED BACILLE CALMETTE-GUÉRIN AND DEHYDROEPIANDROSTERONE TREATMENT ON ESTABLISHED ASTHMA IN MICE

Both bacille Calmette-Guérin (BCG) and dehydroepiandrosterone induce Th1 immune responses and suppress Th2 allergic reactions. To investigate whether a combined administration of BCG and dehydroepiandrosterone treat asthma more effectively, BALB/c mice (n = 8 per group) with established airway hyper-responsiveness were treated with BCG and/or dehydroepiandrosterone. Combined treatment with 2 × 105 colony-forming units (CFUs) of BCG and 0.01% dehydroepiandrosterone was the most effective one at suppressing eosinophilia in bronchoalveolar lavage fluids. In addition, this combination also was better at suppressing hypersensitivity as compared to BCG alone (13.7 ± 4.0- versus 3.6 ± 0.5-fold increase in the sensitivity index; P <. 05) in male mice. Similarly, the effect of the combined treatment was superior to that of individual treatments at decreasing the serum ovalbumin-specific immunoglobulin E (IgE) level. However, the addition of 0.1% dehydroepiandrosterone to BCG significantly decreased the efficacy of BCG on hypersensitivity in female mice. In male mice, the suppressive effect of the treatments on hypersensitivity tended to be lower, and the baseline interferon-γ /interleukin-5 (IFN-γ /IL-5) ratio in the splenocyte supernatant was significantly higher as compared to female mice. In conclusion, treatment with an appropriate combination of BCG and dehydroepiandrosterone had additive therapeutic effects on mice with established asthma. Androgens in males and dehydroepiandrosterone overdose might reduce the efficacy of BCG.

Induction of multispecific Th-1 type immune response against HCV in mice by protein immunization using CpG and Montanide ISA 720 as adjuvants

Recent studies demonstrate that Th1-type immune responses against a broad spectrum of hepatitis C virus (HCV) gene products are crucial to the resolution of acute HCV infection. We investigated new vaccine approaches to augment the strength of HCV-specific Th1-type immune responses. ELISPOT assay revealed that single or multiple protein immunization using both CpG ODN and Montanide ISA 720 as adjuvants induced much stronger IFN-γ-producing Th1 responses against core, NS3 and NS5b targets than did the formulation without these adjuvants. Protein vaccination using CpG ODN and Montanide ISA 720 as adjuvants also greatly enhanced humoral responses to HCV core, E1/E2 and NS3. When specific IgG isotypes were assayed, protein immunization using CpG ODN and Montanide ISA 720 as adjuvants produced higher titers of IgG2a dominant antibodies than did protein immunization alone, indicating a more Th1-biased pathway. This increase in IgG2a is consistent with the induction of Th1 cells secreting IFN-γ demonstrated by ELISPOT assay. In conclusion, protein immunization using CpG ODN and Montanide ISA 720 as adjuvants greatly enhanced cellular (Th1 type) as well as humoral immune responses against HCV in Balb/c mice. The use of adjuvants appears critical to the induction of Th1 immune responses during HCV vaccination with recombinant proteins.

IL-12-Impaired and IL-12-Secreting Dendritic Cells Produce IL-23 upon CD154 Restimulation

Experimental studies in monkeys on the basis of ex vivo-generated, reinjected dendritic cells (DCs) allow investigations of primate DC biology in vivo. To study in vitro and in vivo properties of DCs with a reduced capacity to produce IL-12, we adapted findings obtained in vitro with human cells to the rhesus macaque model. Following exposure of immature monocyte-derived monkey DCs to the immunomodulating synthetic polypeptide glatiramer acetate (GA) and to dibutyryl-cAMP (d-cAMP; i.e., a cAMP enhancer that activates DCs but inhibits the induction of Th1 immune responses), the resulting DCs displayed a mature phenotype with enhanced Ag-specific T cell stimulatory function, notably also for memory Th1 cells. Phosphorylation of p38 MAPK was not induced in GA/d-cAMP-activated DCs. Accordingly, these cells secreted significantly less IL-12p40 (p < or = 0.001) than did cytokine-activated cells. However, upon restimulation with rhesus macaque CD154, GA/d-cAMP-activated DCs produced IL-12p40/IL-23. Additionally, DCs activated by proinflammatory cytokines following protocols for the generation of cells used in clinical studies secreted significantly more IL-23 upon CD154 restimulation than following prior activation. Two days after intradermal injection, GA/d-cAMP-activated fluorescence-labeled DCs were detected in the T cell areas of draining lymph nodes. When similarly injected, GA/d-cAMP as well as cytokine-activated protein-loaded DCs induced comparable Th immune responses characterized by secretion of IFN-gamma, TNF, and IL-17, and transiently expanded FOXP3(+) regulatory T cells. Reactivation of primate DCs through CD154 considerably influences their immmunostimulatory properties. This may have a substantial impact on the development of innovative vaccine approaches.

Induction of high expression of CCR7 and high production of IL-12 in human monocyte-derived dendritic cells by a new bacterial component: LCOS 1013

Dendritic cells (DCs) are the most potent antigen presenting cells of the immune system as they can act as initiators, stimulators and regulators of the immune response. Human DCs are most commonly generated for clinical use by in vitro differentiation of monocytes with exogenous cytokines. Here, we investigate the effect of LCOS 1013 on the production of mature Mo-DCs. LCOS 1013 is a new bacterial component from walls of gram + Klebsellia pneumoniae bacteria that contain some OmpA glycoproteins. Purified peripheral blood monocytes were cultured for 6 days with IL-4 and GM-CSF in order to obtain immature dendritic cells (Im-MoDCs). On day six, Im-MoDCs were matured with either LCOS 1013, TNF alpha, LPS or CD40-Ligand. LCOS 1013 matured Mo-DCs (LCO-DCs) showed a higher expression of DC-LAMP, CD80, CD83, CD54 and CD40 than TNF alpha, LPS and CD40L matured Mo-DCs. Interestingly, LCO-DCs exhibited high expression of full competent CCR7 and high secretion of IL-12 during their maturation. Functionally, LCO-DCs have equivalent potency to trigger mixed leukocyte reaction and antigen-specific reaction and polarize immune response towards Th1 way. Moreover, we found that LCOS 1013 activates DCs through TLR2. LCOS 1013 represents an attractive therapeutic maturation agent of DCs allowing the production of Mo-DCs with high capacity to migrate and to induced Th1 immune responses.

Effects of Combined BCG and DHEA Treatment in Preventing the Development of Asthma

Both BCG and dehydroepiandrosterone (DHEA) induce Th1 immune responses and suppress Th2 allergic reactions. To investigate whether the combination of BCG and DHEA has an additive effect on asthma prevention, BALB/c mice (n = 10 per group) were given an intraperitoneal injection of BCG at the beginning of sensitization, and fed mice chow containing DHEA throughout the study. In female mice, the combined administration of 2 × 104 CFUs BCG and 0.01% DHEA effectively suppressed the ovalbumin-induced increase in airway sensitivity to methacholine (56.5 vs. 8.2 mg/mL, p < 0.01), while BCG (13.9 mg/mL) or DHEA (17.9 mg/mL) alone did not. However, the addition of high dose (0.1%) DHEA decreased the efficacy of high dose (2 × 105 CFUs) BCG in suppressing the airway responsiveness and eosinophilia. In male mice, the treatments with BCG and/or DHEA were less effective, and the interferon-γ / interleukin-4 ratio in the splenocyte supernatant was significantly higher and the ovalbumin-specific IgE concentration in the serum was significantly lower as compared to female mice. In conclusion, the combination of low doses of BCG and DHEA had an additive effect in suppressing the development of airway hypersensitivity. Androgens in males and DHEA overdose might reduce the efficacy of BCG.

Evaluation of immune responses and protection induced by A2 and nucleoside hydrolase (NH) DNA vaccines against Leishmania chagasi and Leishmania amazonensis experimental infections

Several antigens have been tested as vaccine candidates against Leishmania infections but controversial results have been reported when different antigens are co-administered in combined vaccination protocols. Immunization with A2 or nucleoside hydrolase (NH) antigens was previously shown to induce Th1 immune responses and protection in BALB/c mice against Leishmania donovani and L. amazonensis (A2) or L. donovani and L. mexicana (NH) infections. In this work, we investigated the protective efficacy of A2 and NH DNA vaccines, in BALB/c mice, against L. amazonensis or L. chagasi challenge infection. Immunization with either A2 (A2-pCDNA3) or NH (NH-VR1012) DNA induced an elevated IFN-γ production before infection; however, only A2 DNA immunized mice were protected against both Leishmania species and displayed a sustained IFN-γ production and very low IL-4 and IL-10 levels, after challenge. Mice immunized with NH/A2 DNA produced higher levels of IFN-γ in response to both specific recombinant proteins (rNH or rA2), but displayed higher IL-4 and IL-10 levels and increased edema and parasite loads after L. amazonensis infection, as compared to A2 DNA immunized animals. These data extend the characterization of the immune responses induced by NH and A2 antigens as potential candidates to compose a defined vaccine and indicate that a highly polarized type 1 immune response is required for improvement of protective levels of combined vaccines against both L. amazonensis and L. chagasi infections.

Mycobacterium tuberculosis Antigen 85A induces Th-1 immune responses in systemic sarcoidosis.

Sarcoidosis is a granulomatous disease of unknown etiology, characterized by a Th-1 immunophenotype. Although humoral immune responses by sarcoidosis subjects to mycobacterial proteins have been detected, mycobacterial antigens capable of inducing cellular immune responses in sarcoidosis subjects have not been reported. We used the enzyme-linked immunospot assay to assess for recognition of the Mycobacterium tuberculosis mycolyl transferase, Antigen 85A, by peripheral blood mononuclear cells from 25 sarcoidosis subjects, 22 PPD- (purified protein derivative) healthy volunteers, and 16 PPD+ healthy subjects. Reactivity to Ag85A whole protein was observed in 15 of 25 sarcoidosis subjects compared to 2 of 22 PPD- subjects (p=0.0006, Fisher's exact test) and to 14 of 16 PPD+ subjects (p=0.084, Fisher's exact test). Monoclonal antibody against HLA-DR inhibited recognition. In addition to immune recognition of Ag85A whole protein, peptide-mapping studies identified four immunogenic Ag85A peptides, which induced Th-1 immune responses in individual sarcoidosis subjects, suggesting that multiple epitopes from a mycobacterial protein may have a role in sarcoidosis immunopathogenesis.

Inhibition of Lewis Lung Carcinoma Growth by Toxoplasma gondii through Induction of Th1 Immune Responses and Inhibition of Angiogenesis

Toxoplasma gondii is an obligate intracellular protozoan parasite that induces antitumor activity against certain types of cancers. However, little information is available regarding the immunologic mechanisms that regulate these effects. For this purpose, C57BL/6 mice were administered either the T. gondii Me49 strain orally or Lewis lung carcinoma (LLC) cells intramuscularly. Survival rates, tumor size, histopathology, and immune responses were determined for each group, and angiogenesis was evaluated by in vivo Matrigel plug assay. Toxoplasma-infected (TG-injected) mice survived the entire experimental period, whereas cancer cell-bearing (LLC-injected) mice died within six weeks. Mice injected with both T. gondii and cancer cells (TG/LLC-injected group) showed significantly increased survival rates, CD8+ T-cell percentages, IFN-γ mRNA expression levels, serum IgG2a titers, and CTL responses as compared to the LLC-injected mice. In addition, angiogenesis in the TG/LLC-injected mice was notably inhibited. These effects in TG/LCC-injected mice were similar or were increased by the addition of an adjuvant, Quil-A. However, TG/LLC-injected mice showed decreased percentages of CD4+ and CD8+ T-cells, IFN-γ mRNA expression levels, and serum IgG1 and IgG2a titers as compared to TG-injected mice. Taken together, our results demonstrate that T. gondii infection inhibits tumor growth in the Lewis lung carcinoma mouse model through the induction of Th1 immune responses and antiangiogenic activity.

Abstract 589: Activation of Toll-like Receptor Induces Cardiac Remodeling

Introduction: Diastolic heart failure, due to increased ventricular stiffness, is brought about by extracellular matrix (ECM) remodeling. We have previously shown that T-lymphocyte function directly affects cardiac fibroblast function and thereby the ECM composition and diastolic function. Oligodeoxynucleotides (ODN) that selectively stimulate the Toll-like receptor-9 (TLR9) receptor on dendritic cells (DC), induce TH1 immune response. Hypothesis: Selective stimulation of the DC TLR 9 receptor with ODN CPG1668 activates TH1 lymphocytes resulting in ECM remodeling and diastolic dysfunction. Methods: Weight matched C57BL/6J female mice were treated either with PBS (n=8) or with CPG1668 (n=8) at a dose of 50 μg/mouse/week (i.p.) for 4 weeks. After 6 weeks, left ventricular mechanics were determined with pressure-volume loop analysis, ECHO, and ventricular tissue harvested for gene expression, collagen content and crosslinking, and the collagen crosslinking enzymatic activity of lysyl oxidase (LOX). Splenic lymphocytes were harvested for the cytokine determination. Results: CPG1668 administration increased the TH1 cytokine, IFN-γ, from 429±21 to 954±21 (ng/10 6 T-cells), while TH2 cytokine, IL-4, decreased from 614±79 to 51±19 (pg/10 6 T-cells). CPG1668 significantly decreased the end-diastolic volume from 18±1 to 14±1μL (P=0.02), and cardiac output from 6.7±0.6 to 4.9±0.6 mL/min (P=0.05) with no change of ejection fraction 71±3 to 73±4 compared with controls. An increase of ventricular stiffness (β), from 0.16±0.01 to 0.23±0.01 mmHg/μL (P=0.0007), was supported by a decreased E deceleration slope of −8.8±0.6 to −4.1±0.4 (P&lt;0.0001). CPG increased total cardiac collagen two-fold (P=0.0004) and collagen crosslinking by 3-fold (P=0.0007) compared with controls. Correspondingly, CPG administration increased cardiac LOX enzymatic activity by 61% (P=0.02). Real time PCR showed that LOXL3 gene expression increased 9.1-fold (P&lt;0.0001) as well as LOX 1.7-fold (P=0.05) when comparing CPG with controls. Conclusions: This study demonstrates that stimulation of the TH1 lymphocytes via DC TLR 9 receptor induces cardiac ECM remodeling and diastolic dysfunction associated with LOX induced collagen crosslinking.

T Cell-Independent, TLR-Induced IL-12p70 Production in Primary Human Monocytes

IL-12p70 is a key cytokine for the induction of Th1 immune responses. IL-12p70 production in myeloid cells is thought to be strictly controlled by T cell help. In this work we demonstrate that primary human monocytes can produce IL-12p70 in the absence of T cell help. We show that human monocytes express TLR4 and TLR8 but lack TLR3 and TLR7 even after preincubation with type I IFN. Simultaneous stimulation of TLR4 and TLR8 induced IL-12p70 in primary human monocytes. IL-12p70 production in peripheral blood myeloid dendritic cells required combined stimulation of TLR7/8 ligands together with TLR4 or with TLR3 ligands. In the presence of T cell-derived IL-4, but not IFN-gamma, stimulation with TLR7/8 ligands was sufficient to stimulate IL-12p70 production. In monocytes, type I IFN was required but not sufficient to costimulate IL-12p70 induction by TLR8 ligation. Furthermore, TLR8 ligation inhibited LPS-induced IL-10 in monocytes, and LPS alone gained the ability to stimulate IL-12p70 in monocytes when the IL-10 receptor was blocked. Together, these results demonstrate that monocytes are licensed to synthesize IL-12p70 through type I IFN provided via the Toll/IL-1R domain-containing adaptor inducing IFN-beta pathway and the inhibition of IL-10, both provided by combined stimulation with TLR4 and TLR8 ligands, triggering a potent Th1 response before T cell help is established.

Toll-like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

Toll-like receptors (TLRs) are best known for their ability to recognize microbial or viral components and initiate innate immune responses. We showed here that TLRs and their coreceptors were expressed by multipotential hematopoietic stem cells, whose cell cycle entry was triggered by TLR ligation. TLR expression also extended to some of the early hematopoietic progenitors, although not the progenitor cells dedicated to megakaryocyte and erythroid differentiation. TLR signaling via the Myd88 adaptor protein drove differentiation of myeloid progenitors, bypassing some normal growth and differentiation requirements, and also drove lymphoid progenitors to become dendritic cells. CD14 contributed to the efficiency of lipopolysaccharide (LPS) recognition by stem and progenitor cells, and LPS interacted directly with the TLR4/MD-2 complex on these cells in bone marrow. Thus, the preferential pathogen-mediated stimulation of myeloid differentiation pathways may provide a means for rapid replenishment of the innate immune system during infection.

Sympathetic nervous system plays an important role in the relationship between immune mediated diseases

T helper (Th) lymphocytes have been classified into distinct subsets, Th1 and Th2 on the basis of the cytokines they produce. According to the cross-regulatory properties of Th1 and Th2 cells, one would assume that to be affected by a Th1 type disease increases susceptibility to a Th1 type disease and inhibits a Th2 type disease and vice versa about being affected by a Th2 type disease. However, the pattern of related diseases does not necessarily follow the conventional pattern of inhibitory effects of Th1 and Th2 immune responses on each other. For example, Mycobacteria including BCG, that induce Th1 immune responses; can modulate some Th1 type autoimmune diseases including MS, experimental autoimmune encephalomyelitis (EAE; an animal model for Multiple Sclerosis) and insulin-dependent diabetes mellitus (IDDM) thereby leading to an alleviation of their symptoms. Also BCG precipitates a syndrome similar to systemic lupus erythematosus (SLE), a Th2 type disease; in NOD mice. The coexistence of the major Th2-mediated atopic diseases such as asthma, eczema and allergic rhinitis with the Th1-mediated autoimmune conditions including; coeliac disease (CD), IDDM, rheumatoid arthritis (RA) and psoriasis is another example that is in apparent disagreement with counter-regulatory effects of Th1/Th2 phenotypes. SNS can be stimulated by pro-inflammatory cytokines, production of which is induced by mycobacteria including BCG. Although these cytokines can inhibit SNS activity in the site of inflammation and secondary lymphoid organs, they increase sympathetic tone in other places. Increased sympathetic tone can induce an anti-inflammatory and Th2 type milieu. This milieu can inhibit MS and IDDM and provide a susceptible environment for starting of SLE. Atopic diseases are Th2 type immune mediated diseases; therefore, they increase the production of Th2 type cytokine and decrease production of pro-inflammatory cytokines in the site of allergic reaction and also in secondary lymphoid organs. Therefore, atopic diseases decrease sympathetic tone in all tissues except in the sites of allergic reaction and secondary lymphoid organs. Decreased sympathetic tone results in a pro-inflammatory milieu and in such an environment, Th1 type autoimmune diseases can affect tissues.

Therapeutic effect of CpG-enriched plasmid administration on the tight-skin mouse model of scleroderma

Immunostimulatory CpG motifs can preferentially induce Th1 immune responses and have been applied to treat Th2-dominant disease. In this study, we investigated whether a plasmid with the addition of 20 copies of an immunostimulatory CpG motif (pB-CpG20) might prevent the development of scleroderma-like syndrome in tight-skin (Tsk/+) mice. Administration of pB-CpG20 to Tsk/+mice every 3 weeks starting at the age of 1 week reduced skin thickness and collagen content compared to that of pB or saline. The reduction was long lasting even after halting the treatment. Furthermore, this treatment partially reduced the production of anti-nuclear antibodies although it did not decrease the incidence of lung emphysema. pB-CpG20 increased the number of spleen cells secreting IFN-γ and reduced that of the cells secreting IL-4 in vivo and in vitro compared to saline. These results suggest that repeated administration of a CpG-enriched plasmid can ameliorate scleroderma-like syndrome by biasing Th1 immunity in young Tsk/+mice.