Mariarosaria Marinaro

Probiotics Ameliorate Recurrent Th1-Mediated Murine Colitis by Inducing IL-10 and IL-10-Dependent TGF-β-Bearing Regulatory Cells

Recent studies of murine models of mucosal inflammation suggest that, whereas some kinds of bacterial microflora are inducers of disease, others, known as probiotics, prevent disease. In the present study, we analyzed the regulatory cytokine and cell response to probiotic (VSL#3) administration in the context of the Th1 T cell colitis induced by trinitrobenzene sulfonic acid treatment of SJL/J mice. Daily administration of probiotics for 3 wk to mice during a remission period between a first and second course of colitis induced by trinitrobenzene sulfonic acid, resulted in a milder form of recurrent colitis than observed in mice administered PBS during this same period. This protective effect was attributable to effects on the lamina propria mononuclear cell (LPMC) population, because it could be transferred by LPMC from probiotic-treated mice to naive mice. Probiotic administration was associated with an early increase in the production of IL-10 and an increased number of regulatory CD4+ T cells bearing surface TGF-β in the form of latency-associated protein (LAP) (LAP+ T cells). The latter were dependent on the IL-10 production because administration of anti-IL-10R mAb blocked their appearance. Finally, the LAP+ T cells were essential to the protective effect of probiotics because administration of anti-IL-10R or anti-TGF-β at the initiation of recurrent colitis induction or depletion of LAP+ T cells from LPMC abolished the latter’s capacity to transfer protection to naive recipients. These studies show that probiotic (VSL#3) administration during a remission period ameliorates the severity of recurrent colitis by inducing an immunoregulatory response involving TGF-β-bearing regulatory cells.

Cholera toxin induces maturation of human dendritic cells and licences them for Th2 priming

Cholera toxin (CT) is a potent mucosal adjuvant that amplifies B and T cell responses to mucosally co‐administered antigens, stimulating predominant Th2‐type responses. However, little is known about the mechanism of adjuvanticity of CT and on the influence this toxin may have on Th2 cell development during the priming of an immune response. We analyzed the effect of CT on dendritic cells (DC), which are responsible for the priming of immune responses at the systemic as well as at the mucosal level. We found that CT induces phenotypic and functional maturation of blood monocyte‐derived DC. Indeed, CT‐treated DC up‐regulate expression of HLA‐DR molecules, B7.1 and B7.2 co‐stimulatory molecules, and are able to prime naive CD4+CD45RA+ T cells in vitro, driving their polarization towards the Th2 phenotype. Furthermore, CT‐matured DC express functional chemokine receptors CCR7 and CXCR4 which may render them responsive to migratory stimuli towards secondary lymphoid organs. Interestingly, the maturation program induced by CT is unique since CT does not induce but rather inhibits cytokine (IL‐12p70 and TNF‐α) and chemokine (RANTES, MIP‐1α and MIP‐1β) secretion by lipopolysaccharide‐ or CD40 ligand‐activated DC. Our results help to elucidate the mechanism of action of CT as an adjuvant and highlight a new stimulus of bacterial origin that promotes maturation of DC.

Mucosal immunity to infection with implications for vaccine development

The induction of effective mucosal immunity that also provides systemic immunity is a considerable challenge. Over the past two years, efforts to develop novel mucosal vaccine delivery systems to induce mucosal immunity against bacterial and viral diseases, including HIV, have dramatically increased. Here we cite novel vaccines and delivery systems being used to establish effective mucosal immunity.

A Transient Breach in the Epithelial Barrier Leads to Regulatory T-Cell Generation and Resistance to Experimental Colitis

BACKGROUND & AIMS Previous studies have indicated that a defective epithelial barrier leads to inflammation of the underlying lamina propria. Nevertheless, it is likely that physiologic breaks in the barrier must occur for homeostatic regulatory T cells to develop. We determined the effect of agents that disrupt epithelial tight junctions (ethanol and AT1002, a Vibrio cholerae zonula occludens toxin hexapeptide) on regulatory T-cell induction and resistance to induction of colitis by trinitrobenzene sulfonic acid (TNBS). METHODS The effects of ethanol and AT1002 on colon immune function were evaluated by their capacity to induce direct phenotypic or functional changes in effector and regulatory cell populations and their indirect effect on the development of TNBS-induced colitis. The basis of regulatory cell development was evaluated with in vitro studies of isolated dendritic cell populations. The role of innate immunity was evaluated by in vivo gene silencing studies utilizing Toll-like receptor (TLR)-2-specific small interfering RNA (siRNA). RESULTS Both ethanol and AT1002 induced persistent latency-associated peptide-positive CD4(+) regulatory T cells that, as shown in adoptive transfer studies, render mice resistant to the induction of TNBS colitis. The development of these cells requires the presence of an intact microflora and the activity of CD11c(+) dendritic cells. Their induction is also influenced by innate immune factors operating through TLR-2, because attenuation of TLR-2 signaling by in vivo TLR-2 siRNA administration prevents their development. CONCLUSIONS A mild and/or transient breach in epithelial barrier function leads to dominant regulatory T-cell responses that protect the mucosa from inflammation.

Human Nasopharyngeal-Associated Lymphoreticular Tissues: Functional Analysis of Subepithelial and Intraepithelial B and T Cells from Adenoids and Tonsils

Subepithelial and intraepithelial lymphocytes of human adenoids and tonsils were characterized and directly compared to determine the potential contribution of these tissues to mucosal and systemic immune responses. The distribution of T and B cell subsets, cytokine patterns, and antibody (Ab) isotype profiles were similar for adenoids and tonsils. Both tissues contained predominantly B cells ( approximately 65%), approximately 5% macrophages, and 30% CD3(+) T cells. The T cells were primarily of the CD4(+) subset ( approximately 80%). Tonsillar intraepithelial lymphocytes were also enriched in B cells. The analysis of dispersed cells revealed a higher frequency of cells secreting IgG than IgA and the predominant Ig subclass profiles were IgG1 > IgG3 and IgA1 > IgA2, respectively. In situ analysis also revealed higher numbers of IgG- than IgA-positive cells. These IgG-positive cells were present in the epithelium and in the subepithelial zones of both tonsils and adenoids. Mitogen-triggered T cells from tonsils and adenoids produced both Th1- and Th2-type cytokines, clearly exhibiting their pluripotentiality for support of cell-mediated and Ab responses. Interestingly, antigen-specific T cells produced interferon-gamma and lower levels of interleukin-5. These results suggest that adenoids and tonsils of the nasopharyngeal-associated lymphoreticular tissues represent a distinct component of the mucosal-associated lymphoreticular tissues with features of both systemic and mucosal compartments.

Immunoglobulin A (IgA) responses and IgE-associated inflammation along the respiratory tract after mucosal but not systemic immunization.

ABSTRACT The purpose of the present study was to determine the extent of immunologic responses, particularly immunopathologic responses, within the upper and lower respiratory tracts after intranasal immunization using the mucosal adjuvant cholera toxin (CT). BALB/c mice were nasally immunized with influenza virus vaccine combined with CT. The inclusion of the mucosal adjuvant CT clearly enhanced generation of antibody responses in both the nasal passages and lungs. After nasal immunization, antigen-specific immunoglobulin A (IgA) antibody-forming cells dominated antibody responses throughout the respiratory tract. However, IgG responses were significant in lungs but not in nasal passages. Furthermore, parenteral immunization did not enhance humoral immunity in the upper respiratory tract even after a nasal challenge, whereas extrapulmonary lymphoid responses enhanced responses in the lung. After nasal immunization, inflammatory reactions, characterized by mononuclear cell infiltration, developed within the lungs of mice but not in nasal passages. Lowering dosages of CT reduced, but did not eliminate, these adverse reactions without compromising adjuvancy. Serum IgE responses were also enhanced in a dose-dependent manner by inclusion of CT. In summary, there are differences in the generation of humoral immunity between the upper respiratory tract and the lung. As the upper respiratory tract is in a separate compartment of the immune system from that stimulated by parenteral immunization, nasal immunization is an optimal approach to generate immunity throughout the respiratory tract. Despite the promise of nasal immunization, there is also the potential to develop adverse immunopathologic reactions characterized by pulmonary airway inflammation and IgE production.

Oral QS-21 Requires Early IL-4 Help for Induction of Mucosal and Systemic Immunity

The highly purified saponin derivative, QS-21, from the Quillaja saponaria Molina tree has been proved to be safe for parenteral administration and represents a potential alternative to bacterial enterotoxin derivatives as a mucosal adjuvant. Here we report that p.o. administration of QS-21 with the vaccine protein tetanus toxoid elicited strong serum IgM and IgG Ab responses, which were only slightly enhanced by further oral immunization. The IgG Ab subclass responses were predominantly IgG1 followed by IgG2b for the 50-μg p.o. dose of QS-21, whereas the 250-μg p.o. dose also induced IgG2a and IgG3 Abs. Low oral QS-21 doses induced transient IgE Ab responses 7 days after the primary immunization, whereas no IgE Ab responses were seen in mice given the higher QS-21 dose. Further, low but not high p.o. QS-21 doses triggered Ag-specific secretory IgA (S-IgA) Ab responses. Th cell responses showed higher IFN-γ (Th1-type) and lower IL-5, IL-6, and IL-10 (Th2-type) secretion after the high QS-21 p.o. dose than after low doses. Interestingly, the mucosal adjuvant activity of low oral QS-21 doses was diminished in IL-4−/− mice, suggesting a role for this cytokine in the initiation of mucosal immunity by oral QS-21. In summary, our results show that oral QS-21 enhances immunity to coadministered Ag and that different doses of QS-21 lead to distinct patterns of cytokine and serum Ab responses. We also show that an early IL-4 response is required for the induction of mucosal immunity by oral QS-21 as adjuvant.

A double-blind, placebo-controlled study of Mycobacterium-specific human immune responses induced by intradermal bacille Calmette-Guérin vaccination.

Recent studies have indicated that type 1 T cell responses (potent interferon-gamma and cytolytic responses, with absence of interleukin-4 production) are important for protective immunity against mycobacteria. These observations suggest that assays of type 1 T cell responses may be useful as surrogate markers of protective immunity in the evaluation of new tuberculosis vaccines. To be useful as surrogate markers, immunologic assays must distinguish between vaccine recipients and control subjects in clinical trials. Previous studies have shown that bacille Calmette-Guérin (BCG) vaccination can induce human type 1 T cell responses, but randomized trials have not been done to determine whether measurement of these responses can distinguish between BCG recipients and control subjects. We have conducted a double-blind, placebo-controlled trial of intradermal vaccination with two different BCG strains. We compared the mean lymphoproliferative, cytotoxic, Th1 and Th2 cytokine, and antibody responses detected in BCG and placebo recipients. These studies demonstrated that significant increases in Mycobacterium-specific T cell proliferative responses and type 1 cytokine responses were induced by BCG when compared with results with a placebo. In addition, BCG induced significant increases in Mycobacterium-specific antibody responses with an isotype profile characteristic of a type 1 cytokine bias. T cell and antibody assays involving the use of mycobacterial whole cell lysates or live BCG were able to discriminate between BCG and placebo recipients better than were assays using mycobacterial culture filtrates. These studies provide important information for the development of immunologic assays that might be useful as surrogate markers of protective immunity in future trials of new tuberculosis vaccines.

Effects of the adjuvant cholera toxin on dendritic cells: stimulatory and inhibitory signals that result in the amplification of immune responses

Cholera toxin (CT) is a potent mucosal adjuvant. When administered through the mucosal route CT amplifies B and T lymphocyte responses to co-administered antigens. Since the discovery of CT as a mucosal adjuvant, other bacterial enterotoxins have been found to have this property. These molecules or their detoxified derivatives are all important for the development of mucosal vaccines for human use, and it is thus necessary to understand their mechanism of action. CT has immunomodulatory effects on different cell types, however, the interaction of CT with dendritic cells (DCs), which have a primary role in the priming of immune responses, may be crucial for its adjuvant activity.

IL-4 Promotes the Migration of Circulating B Cells to the Spleen and Increases Splenic B Cell Survival

We report that IL-4 causes a redistribution of B cells and modestly increases B cell life span. Intravenous injection of a long-acting formulation of IL-4 induces increases in both spleen cell number and the percentage of splenic B cells. These effects are observed within 1 day of IL-4 administration and plateau after ∼3 days if IL-4 treatment is continued. The increase in splenic B cell number is IL-4 dose dependent, CD4+ T cell independent, FcγRII/FcγRIII independent, and Stat6 independent. Decreases in the number of B cells in the blood and the percentage of mature B cells in the bone marrow, concomitant with the increase in splenic B cell number, suggest that redistribution of circulating B cells to the spleen is partially responsible for IL-4 induction of splenic B cell hyperplasia. Considerable reduction in the effect of 5 days of IL-4 treatment on splenic B cell number when B lymphopoiesis is blocked with anti-IL-7 mAb suggests that generation of new B cells is also involved in IL-4-induced splenic B cell hyperplasia. 5-Bromo-2′-deoxyuridine labeling experiments demonstrate that IL-4 modestly prolongs the life span of newly generated splenic B cells, and experiments that measure B cell HSA (CD24) expression as an indicator of B cell age suggest that IL-4 may also prolong the life span of mature splenic B cells. Thus, IL-4 increases splenic B cell number through two Stat6-independent effects: increased net migration of circulating B cells to the spleen and increased B cell life span. Both effects may promote Ab responses to a systemic Ag challenge.