Leishmania (V.) braziliensis and L. (L.) amazonensis promote differential expression of dendritic cells and cellular immune response in murine model

The expression of Langerhans cell (LC) and dermal dendritic cell (dDC) as well as T CD4+ and CD8+ immune responses was evaluated in the skin of BALB/c mice experimentally infected by L. (L.) amazonensis (La) and L. (V.) braziliensis (Lb). At 4th and 8th weeks post infection (PI), skin biopsies were collected to determine the parasite load and CD207+, CD11c+, CD4+, CD8+, iNOS+ cellular densities. Cytokine (IFN-?, IL-4 and IL-10) profiles were also analysed in draining lymph node. At 4th week, the densities of CD207+ and CD11c+ were higher in the La infection, while in the Lb infection, these markers revealed a significant increase at 8th week. At 4th week, CD4+ and CD8+ were higher in the La infection, but at 8th week, there was a substantial increase in both markers in the Lb infection. iNOS+ was higher in the Lb infection at 4th and 8th weeks. In contrast, the parasite load was higher in the La infection at 4th and 8th weeks. The concentration of IFN-? was higher in the Lb infection, but IL-4 and IL-10 were higher in the La infection at 4th and 8th weeks. These results confirm the role of the Leishmania species in the BALB/c mice disease characterized by differences in the expression of dendritic cells and cellular immune response.

B Lymphocyte-Induced Maturation Protein-1 Contributes to Intestinal Mucosa Homeostasis by Limiting the Number of IL-17-Producing CD4(+) T Cells

The transcription factor B lymphocyte induced maturation protein-1 (Blimp-1) plays important roles in embryonic development and immunity. Blimp-1 is required for the differentiation of plasma cells, and mice with T cell specific deletion of Blimp-1 (Blimp-1CKO mice) develop a fatal inflammatory response in the colon. Previous work demonstrated that lack of Blimp-1 in CD4(+) and CD8(+) T cells leads to intrinsic functional defects, but little is known about the functional role of Blimp-1 in regulating differentiation of Th cells in vivo and their contribution to the chronic intestinal inflammation observed in the Blimp1CKO mice. In this study, we show that Blimp-1 is required to restrain the production of the inflammatory cytokine IL-17 by Th cells in vivo. Blimp-1CKO mice have greater numbers of IL-17 producing TCR beta(+)CD4(+)cells in lymphoid organs and in the intestinal mucosa. The increase in IL-17 producing cells was not restored to normal levels in wild-type and Blimp-1CKO mixed bone marrow chimeric mice, suggesting an intrinsic role for Blimp-1 in constraining the production of IL-17 in vivo. The observation that Blimp-1 deficient CD4(+) T cells are more prone to differentiate into IL-17(+)/IFN-gamma(+) cells and cause severe colitis when transferred to Rag1-deficient mice provides further evidence that Blimp-1 represses IL-17 production. Analysis of Blimp-1 expression at the single cell level during Th differentiation reveals that Blimp-1 expression is induced in Th1 and Th2 but repressed by TGF-beta in Th17 cells. Collectively, the results described here establish a new role for Blimp-1 in regulating IL-17 production in vivo. The Journal of Immunology, 2012,189: 5682-5693.

Prophylactic and Therapeutic Vaccination Using Dendritic Cells Primed with Peptide 10 Derived from the 43-Kilodalton Glycoprotein of Paracoccidioides brasiliensis

Vaccination with peptide 10 (P10), derived from the Paracoccidioides brasiliensis glycoprotein 43 (gp43), induces a Th1 response that protects mice in an intratracheal P. brasiliensis infection model. Combining P10 with complete Freund's adjuvant (CFA) or other adjuvants further increases the peptide's antifungal effect. Since dendritic cells (DCs) are up to 1,000-fold more efficient at activating T cells than CFA, we examined the impact of P10-primed bone-marrow-derived DC vaccination in mice. Splenocytes from mice immunized with P10 were stimulated in vitro with P10 or P10-primed DCs. T cell proliferation was significantly increased in the presence of P10-primed DCs compared to the peptide. The protective efficacy of P10-primed DCs was studied in an intratracheal P. brasiliensis model in BALB/c mice. Administration of P10-primed DCs prior to (via subcutaneous vaccination) or weeks after (via either subcutaneous or intravenous injection) P. brasiliensis infection decreased pulmonary damage and significantly reduced fungal burdens. The protective response mediated by the injection of primed DCs was characterized mainly by an increased production of gamma interferon (IFN-gamma) and interleukin 12 (IL-12) and a reduction in IL-10 and IL-4 compared to those of infected mice that received saline or unprimed DCs. Hence, our data demonstrate the potential of P10-primed DCs as a vaccine capable of both the rapid protection against the development of serious paracoccidioidomycosis or the treatment of established P. brasiliensis disease.

Maternal immunization with ovalbumin prevents neonatal allergy development and up-regulates inhibitory receptor FcγRIIB expression on B cells

Abstract Background Preconception allergen immunization prevents neonatal allergen sensitization in mice by a complex interaction between regulatory cells/factors and antibodies. The present study assessed the influence of maternal immunization with ovalbumin (OVA) on the immune response of 3 day-old and 3 week-old offspring immunized or non-immunized with OVA and evaluated the effect of IgG treatment during fetal development or neonatal period. Results Maternal immunization with OVA showed increased levels of FcγRIIb expression in splenic B cells of neonates, which were maintained for up to 3 weeks and not affected by additional postnatal OVA immunization. Maternal immunization also exerted a down-modulatory effect on both IL-4 and IFN-γ-secreting T cells and IL-4 and IL-12- secreting B cells. Furthermore, immunized neonates from immunized mothers showed a marked inhibition of antigen-specifc IgE Ab production and lowered Th2/Th1 cytokine levels, whereas displaying enhanced FcγRIIb expression on B cells. These offspring also showed reduced antigen-specific proliferative response and lowered B cell responsiveness. Moreover, in vitro evaluation revealed an impairment of B cell activation upon engagement of B cell antigen receptor by IgG from OVA-immunized mice. Finally, in vivo IgG transference during pregnancy or breastfeeding revealed that maternal Ab transference was able to increase regulatory cytokines, such as IL-10, in the prenatal stage; yet only the postnatal treatment prevented neonatal sensitization. None of the IgG treatments induced immunological changes in the offspring, as it was observed for those from OVA-immunized mothers. Conclusion Maternal immunization upregulates the inhibitory FcγRIIb expression on offspring B cells, avoiding skewed Th2 response and development of allergy. These findings contribute to the advancement of prophylactic strategies to prevent allergic diseases in early life.

Estrogen Receptor α Signaling in T Lymphocytes Is Required for Estradiol-Mediated Inhibition of Th1 and Th17 Cell Differentiation and Protection against Experimental Autoimmune Encephalomyelitis

Estrogen treatment exerts a protective effect on experimental autoimmune encephalomyelitis (EAE) and is under clinical trial for multiple sclerosis therapy. Estrogens have been suspected to protect from CNS autoimmunity through their capacity to exert anti-inflammatory as well as neuroprotective effects. Despite the obvious impacts of estrogens on the pathophysiology of multiple sclerosis and EAE, the dominant cellular target that orchestrates the anti-inflammatory effect of 17β-estradiol (E2) in EAE is still ill defined. Using conditional estrogen receptor (ER) α-deficient mice and bone marrow chimera experiments, we show that expression of ERα is critical in hematopoietic cells but not in endothelial ones to mediate the E2 inhibitory effect on Th1 and Th17 cell priming, resulting in EAE protection. Furthermore, using newly created cell type-specific ERα-deficient mice, we demonstrate that ERα is required in T lymphocytes, but neither in macrophages nor dendritic cells, for E2-mediated inhibition of Th1/Th17 cell differentiation and protection from EAE. Lastly, in absence of ERα in host nonhematopoietic tissues, we further show that ERα signaling in T cells is necessary and sufficient to mediate the inhibitory effect of E2 on EAE development. These data uncover T lymphocytes as a major and nonredundant cellular target responsible for the anti-inflammatory effects of E2 in Th17 cell-driven CNS autoimmunity.

The sphingosine kinase 1 and S1P1 axis specifically counteracts LPS-induced IL-12p70 production in immune cells of the spleen

Sphingosine-1-phosphate (S1P) has been implicated in angiogenesis, inflammation, cancerogenesis, neurological excitability and immune regulation and is synthesized by two different sphingosine kinases (SphK). It was suggested that mice lacking the gene for SphK1 exhibit no obvious phenotype, because SphK2 compensates for its absence. However, recent investigations revealed that under challenge SphK1 contributed to pro-inflammatory processes favoring Th2 and Th17 rather than Th1-type reactions. To investigate the immune modulatory role of SphK1 as opposed to SphK2 specifically for the Th1 propagating IL-12p70 we compared WT and SphK1(-/-) splenocytes and Flt3-ligand differentiated BMCs of WT and SphK1(-/-), representing dendritic cells as major producers of IL-12p70, incubated with LPS. We determined the impact on IL-12p70 in comparison to other inflammatory cytokines, and on DC and macrophage surface marker expression, SphK mRNA, protein expression and enzymatic activity in splenocytes. Our data demonstrated that SphK1 deficiency enhanced LPS-induced IL-12p70 production although SphK2 was present. To further characterize SphK1-dependent IL-12p70 regulation we exogenously applied S1P, SEW2871 and the new potent S1P1 agonist CYM5442. Both S1P and S1P1-specific analogs fully compensated the increase of IL-12p70 production in SphK1-deficient splenocytes. The use of pertussis toxin, to block G(i)-coupled signaling downstream of S1P1, again increased IL-12p70 and neglected the compensation achieved by addition of S1P and S1P1 agonists pointing on the importance of this specific S1P-receptor. Given that, in parallel to a prominent IL-12p35 increase following LPS stimulation, LPS also enhanced SphK expression and total SphK activity, we concluded that SphK1-derived S1P acting via S1P1 is a major mechanism of this negative IL-12p70 feedback loop, which did not affect other cytokines. Moreover, our data showed that SphK2 activity failed to compensate for SphK1 deficiency. These findings clearly point to a divergent and cytokine-specific impact of immune cell SphK1 and SphK2 in chronic inflammation and cancer.

Skin-infiltrating T cells and cytokine expression in Icelandic horses affected with insect bite hypersensitivity: a possible role for regulatory T cells

Equine insect bite hypersensitivity (IBH) is a seasonally recurrent, pruritic skin disorder caused by an IgE-mediated reaction to salivary proteins of biting flies, predominantly of the genus Culicoides. The aim of this study was to define T cell subsets and cytokine profile in the skin of IBH-affected Icelandic horses with particular focus on the balance between T helper (Th) 1, Th2 and T regulatory (Treg) cells. Distribution and number of CD4+, CD8+ and Forkhead box P3 (FoxP3)+ T cells were characterized by immunohistochemical staining in lesional and non-lesional skin of moderately and severely IBH-affected horses (n=14) and in the skin of healthy control horses (n=10). Using real-time quantitative reverse transcription-polymerase chain reaction, mRNA expression levels of Th2 cytokines (Interleukin (IL)-4, IL-5, IL-13), Th1 cytokines (Interferon-gamma), regulatory cytokines (Transforming Growth Factor beta1, IL-10) and the Treg transcription factor FoxP3 were measured in skin and blood samples. Furthermore, Culicoides nubeculosus specific serum IgE levels were assessed. Lesions of IBH-affected horses contained significantly higher numbers of CD4+ cells than skin of healthy control horses. Furthermore, the total number of T cells (CD4+ and CD8+) was significantly increased in lesional compared to non-lesional skin and there was a tendency (p=0.07) for higher numbers of CD4+ cells in lesional compared to non-lesional skin. While the number of FoxP3+ T cells did not differ significantly between the groups, the ratio of Foxp3 to CD4+ cells was significantly lower in lesions of severely IBH-affected horses than in moderately affected or control horses. Interestingly, differences in FoxP3 expression were more striking at the mRNA level. FoxP3 mRNA levels were significantly reduced in lesional skin, compared both to non-lesional and to healthy skin and were also significantly lower in non-lesional compared to healthy skin. Expression levels of IL-13, but not IL-4 or IL-5, were significantly elevated in lesional and non-lesional skin of IBH-affected horses. IL-10 levels were lower in lesional compared to non-lesional skin (p=0.06) and also lower (p=0.06) in the blood of IBH-affected than of healthy horses. No significant changes were observed regarding blood expression levels of Th1 and Th2 cytokines or FoxP3. Finally, IBH-affected horses had significantly higher Culicoides nubeculosus specific serum IgE levels than control horses. The presented data suggest that an imbalance between Th2 and Treg cells is a characteristic feature in IBH. Treatment strategies for IBH should thus aim at restoring the balance between Th2 and Treg cells.

Modulation of allergy incidence in icelandic horses is associated with a change in IL-4-producing T cells

BACKGROUND: Equine insect bite hypersensitivity (IBH) is an immediate-type hypersensitivity reaction provoked by insect-derived allergens. Icelandic horses living in Iceland do not have IBH due to absence of relevant insects, but acquire it at high frequency after being imported to mainland Europe. In contrast, their offspring born in mainland Europe has reduced IBH incidence. T helper 1 (Th1) and Th2 cells and cytokines were determined in Icelandic horses born in Iceland and on the continent and which either have IBH or are healthy. METHODS: Peripheral blood mononuclear cells (PBMC) from these horses were stimulated for 18 h during summer and winter with polyclonal T cell stimuli, IBH allergen(s) or irrelevant allergen(s). Cells were analysed by flow cytometry for interferon-gamma (IFN-gamma) and interleukin-4 (IL-4); RNA was analysed for IFN-gamma, IL-4, IL-5 and IL-13 mRNA. RESULTS: During summer, but not during winter, IBH PBMC stimulated polyclonally showed reduced IFN-gamma mRNA and IFN-gamma-producing cells when compared with those of healthy horses, regardless of origin. PBMC stimulated polyclonally or with IBH allergen showed increased IL-4 mRNA levels and higher numbers of IL-4-producing cells when born in Iceland or showing IBH symptoms. IL-5 and IL-13 mRNA were modulated neither by disease nor by origin. Abrogation of IL-4 production in healthy horses born in mainland Europe may be due, at least in part, to IL-10. There was an increased level of IL-10 in supernatants from PBMC of healthy horses born in mainland Europe and stimulated polyclonally or with IBH allergen. CONCLUSIONS: Modulation of IBH incidence is governed by altered Th1/Th2 ratio, which might be influenced by IL-10.

Using distinct molecular signatures of human monocytes and dendritic cells to predict adjuvant activity and pyrogenicity of TLR agonists

We present a systematic study that defines molecular profiles of adjuvanticity and pyrogenicity induced by agonists of human Toll-like receptor molecules in vitro. Using P(3)CSK(4), Lipid A and Poly I:C as model adjuvants we show that all three molecules enhance the expansion of IFNgamma(+)/CD4(+) T cells from their naïve precursors following priming with allogeneic DC in vitro. In contrast, co-culture of naive CD4(+) T cells with allogeneic monocytes and TLR2/TLR4 agonists only resulted in enhanced T cell proliferation. Distinct APC molecular signatures in response to each TLR agonist underline the dual effect observed on T cell responses. Using protein and gene expression assays, we show that TNF-alpha and CXCL10 represent DC-restricted molecular signatures of TLR2/TLR4 and TLR3 activation, respectively, in sharp contrast to IL-6 produced by monocytes upon stimulation with P(3)CSK(4) and Lipid A. Furthermore, although all TLR agonists are able to up-regulate proIL-1beta specific gene in both cell types, only monocyte activation with Lipid A results in detectable IL-1beta release. These molecular profiles, provide a simple screen to select new immune enhancers of human Th1 responses suitable for clinical application.

Human bronchial epithelium controls TH2 responses by TH1-induced, nitric oxide-mediated STAT5 dephosphorylation: implications for the pathogenesis of asthma

Increased levels of NO in exhaled air in association with increased NO synthetase (NOS)2 expression in bronchial epithelial are hallmark features of asthma. It has been suggested that NO contributes to asthma pathogenesis by selective down-regulation of TH1 responses. We demonstrate, however, that NO can reversibly limit in vitro expansion of both human TH1 and TH2 CD4+ T cells. Mechanistically, NO induces cGMP-mediated reversible STAT5 dephosphorylation and therefore interferes with the IL-2R activation cascade. Human bronchial epithelial cells (HBEC) up-regulate NOS2 after stimulation with IFN-gamma secreted by TH1 CD4+ T cells and release NO, which inhibits both TH1 and TH2 cell proliferation. This reversible T cell growth arrest depends on NO because T cell proliferation is completely restored after in vitro blocking of NOS2 on HBEC. HBEC thus drive the effector end of a TH1-controlled feedback loop, which protects airway mucosal tissues at the potential lesional site in asthma from overwhelming CD4+ TH2 (and potentially TH1) responses following allergen exposure. Variations in the efficiency of this feedback loop provides a plausible mechanism to explain why only a subset of atopics sensitized to ubiquitous aeroallergens progress to expression of clinically relevant levels of airways inflammation.

Neospora caninum and bone marrow-derived dendritic cells: parasite survival, proliferation, and induction of cytokine expression

Dendritic cells (DCs) represent the first line defence of the innate immune system following infection with pathogens. We exploratively addressed invasion and survival ability of Neospora caninum, a parasite causing abortion in cattle, in mouse bone marrow DCs (BMDCs), and respective cytokine expression patterns. Immature BMDCs were exposed to viable (untreated) and nonviable parasites that had been inactivated by different means. Invasion and/or internalization, as well as intracellular survival and proliferation of tachyzoites were determined by NcGRA2-RT-PCR and transmission electron microscopy (TEM). Cytokine expression was evaluated by reverse transcription (RT)-PCR and cytokine ELISA. Transmission electron microscopy of DCs stimulated with untreated viable parasites revealed that N. caninum was able to invade and proliferate within BMDCs. This was confirmed by NcGRA2-RT-PCR. On the other hand, no viable parasite organisms were revealed by TEM when exposing BMDCs to inactivated parasites (nonviability demonstrated by NcGRA2-RT-PCR). Cytokine expression analysis (as assessed by both RT-PCR and ELISA) demonstrated that both viable and nonviable parasites stimulated mBMDCs to express IL-12p40, IL-10 and TNF-alpha, whereas IL-4 RNA expression was not detected. Thus, exposure of mBMDCs to both viable and nonviable parasites results in the expression of cytokines that are relevant for a mixed Th1/Th2 immune response.

TH2 cytokines from malignant cells suppress TH1 responses and enforce a global TH2 bias in leukemic cutaneous T-cell lymphoma

PURPOSE In leukemic cutaneous T-cell lymphoma (L-CTCL), malignant T cells accumulate in the blood and give rise to widespread skin inflammation. Patients have intense pruritus, increased immunoglobulin E (IgE), and decreased T-helper (TH)-1 responses, and most die from infection. Depleting malignant T cells while preserving normal immunity is a clinical challenge. L-CTCL has been variably described as a malignancy of regulatory, TH2 and TH17 cells. EXPERIMENTAL DESIGN We analyzed phenotype and cytokine production in malignant and benign L-CTCL T cells, characterized the effects of malignant T cells on healthy T cells, and studied the immunomodulatory effects of treatment modalities in patients with L-CTCL. RESULTS Twelve out of 12 patients with L-CTCL overproduced TH2 cytokines. Remaining benign T cells were also strongly TH2 biased, suggesting a global TH2 skewing of the T-cell repertoire. Culture of benign T cells away from the malignant clone reduced TH2 and enhanced TH1 responses, but separate culture had no effect on malignant T cells. Coculture of healthy T cells with L-CTCL T cells reduced IFNγ production and neutralizing antibodies to interleukin (IL)-4 and IL-13 restored TH1 responses. In patients, enhanced TH1 responses were observed following a variety of treatment modalities that reduced malignant T-cell burden. CONCLUSIONS A global TH2 bias exists in both benign and malignant T cells in L-CTCL and may underlie the infectious susceptibility of patients. TH2 cytokines from malignant cells strongly inhibited TH1 responses. Our results suggest that therapies that inhibit TH2 cytokine activity, by virtue of their ability to improve TH1 responses, may have the potential to enhance both anticancer and antipathogen responses.

MOLECULAR MECHANISMS UNDERLYING THE TRANSCRIPTIONAL REGULATION OF T HELPER 17 AND REGULATORY T CELLS

CD4+ T helper (Th) lymphocytes are vital for integrating immune responses by orchestrating the function of other immune cell types. Naïve Th cells can differentiate into different effector subsets that are characterized by their cytokine profile and immune regulatory functions. These subsets include Th1, Th2, Th17, natural and inducible regulatory T cells (nTreg and iTreg respectively), among others. We focused our investigation on two Th lineages, Th17 and regulatory T cells, with opposing functions in the immune system. These subsets have been suggested to be reciprocally regulated since they both require TGF-b for their development. We investigated the role of the Treg-associated master transcription factor Foxp3, and found that Foxp3 inhibits Th17 cell generation by preventing the transcriptional activity of the two main Th17-specific transcription factors, nuclear orphan receptors RORa and RORgt. At the molecular level, we identified two different functional domains in Foxp3 required for such inhibition: the LQALL sequence in exon 2 and the TIP60/HDAC7 binding domain. These domains could be crucial to either prevent the association of the nuclear receptors to coactivators or to recruit histone deacetylases to RORa- or RORgt-target genes. Since TGF-b is a common cytokine required for the commitment towards both Th lineages, we determined the role of the TGF-b-dependent signaling pathway in the generation of each subset. By using mice with deficiencies in signaling molecules downstream of TGF-b, we found that while Smad2, Smad3 and Smad4 are required for the generation of iTreg cells, only Smad2 is indispensable for the induction of IL-17-producing cells, suggesting that TGF-b induces these T helper lineages through differential signaling pathways. Thus, our findings describe novel transcriptional regulatory mechanisms that control the generation of two T helper lineages with opposing functions. These findings could provide novel therapeutic targets to treat diseases where the balance of these T cells is dysregulated, such as in autoimmunity, chronic infectious diseases and cancer.

Interferon-gamma and interleukin-4 mRNA expression by peripheral blood mononuclear cells from pregnant and non-pregnant cattle seropositive for bovine viral diarrhea virus

The acceptance of the fetal allograft by pregnant women and mice seems to be associated with a shift from a Th 1 dominated to a Th 2 dominated immune response to certain infectious agents. The goal of this study was to examine cytokine expression in peripheral blood mononuclear cells (PBMCs) from cattle immune to bovine viral diarrhea virus (BVDV) to determine whether pregnancy also has an influence on the type of immune response in this species. Forty-six heifers and cows between 14 months and 13 years of age were included in this study. Twenty-four were seropositive and 22 seronegative for BVDV. Eleven of the seropositive animals and 11 of the seronegative animals were in the eighth month of gestation, the remaining animals were virgin heifers. PBMC from these animals were analyzed for Interferon (IFN)-gamma and Interleukin (IL)-4 mRNA expression by real-time RT-PCR after stimulation with a non-cytopathic strain of BVDV. Additionally, an ELISA was performed to measure IFN-gamma in the supernatants of stimulated cell cultures. In BVDV seropositive animals, IFN-gamma mRNA levels were significantly higher than in BVDV seronegative animals and there was a significant positive correlation between the changes in IFN-gamma and IL-4 mRNA expression. There was, however, no significant difference in IFN-gamma and IL-4 mRNA levels between pregnant and non-pregnant animals. These results are inconsistent with BVDV inducing a Th1 or Th2 biased immune response. Furthermore, a shift in the cytokine pattern during bovine pregnancy was not evident.