Aldehyde dehydrogenase-1a1 induces oncogene suppressor genes in B cell populations.

The deregulation of B cell differentiation has been shown to contribute to autoimmune disorders, hematological cancers, and aging. We provide evidence that the retinoic acid-producing enzyme aldehyde dehydrogenase 1a1 (Aldh1a1) is an oncogene suppressor in specific splenic IgG1(+)/CD19(-) and IgG1(+)/CD19(+) B cell populations. Aldh1a1 regulated transcription factors during B cell differentiation in a sequential manner: 1) retinoic acid receptor alpha (Rara) in IgG1(+)/CD19(-) and 2) zinc finger protein Zfp423 and peroxisome proliferator-activated receptor gamma (Pparg) in IgG1(+)/CD19(+) splenocytes. In Aldh1a1(-/-) mice, splenic IgG1(+)/CD19(-) and IgG1(+)/CD19(+) B cells acquired expression of proto-oncogenic genes c-Fos, c-Jun, and Hoxa10 that resulted in splenomegaly. Human multiple myeloma B cell lines also lack Aldh1a1 expression; however, ectopic Aldh1a1 expression rescued Rara and Znf423 expressions in these cells. Our data highlight a mechanism by which an enzyme involved in vitamin A metabolism can improve B cell resistance to oncogenesis.

Oral but not parenteral interleukin (IL)-12 redirects T helper 2 (Th2)-type responses to an oral vaccine without altering mucosal IgA responses.

Our past studies have shown that the mucosal adjuvant cholera toxin (CT) induces T helper type 2 (Th2) responses with systemic IgG1, IgE and mucosal secretory IgA (S-IgA) antibodies (Abs). In this study, recombinant murine IL-12 (rmIL-12) was given either parenterally or orally to mice orally immunized with tetanus toxoid (TT) and CT to determine whether this cytokine could redirect the CT-induced Th2-type responses and what effect this shift would have on S-IgA Ab responses. Intraperitoneal administration of rmIL-12 shifted TT-specific responses toward Th1-type and resulted in CD4+ T cells producing IFN-gamma and IL-2 with markedly reduced levels of Th2-type cytokines. This cytokine profile was accompanied by increased delayed-type hypersensitivity (DTH) and shifts in serum IgG1 to IgG2a and IgG3 anti-TT Ab responses. Further, serum IgE and S-IgA Ab responses were markedly reduced by parenteral IL-12. When IL-12 complexed to liposomes was given orally both shifts to IgG2a and IgG3 and low IgE Abs again occurred concomitant with enhanced serum IFN-gamma and DTH responses. Interestingly, oral rmIL-12 did not result in significant levels of serum IL-12 nor altered S-IgA Ab responses and resulted in higher levels of some Th2-type cytokines both in vitro and in vivo when compared with parenteral IL-12. Our results show that the shifts in systemic immune responses with intact S-IgA Abs which occur after oral delivery of IL-12-liposomes are due to cytokine effects in the Peyer's patches and suggest new strategies for the targeted manipulation of Th1- and Th2-type responses to mucosal vaccines.

Syntaxin 1A is expressed in airway epithelial cells, where it modulates CFTR Cl(-) currents.

The CFTR Cl(-) channel controls salt and water transport across epithelial tissues. Previously, we showed that CFTR-mediated Cl(-) currents in the Xenopus oocyte expression system are inhibited by syntaxin 1A, a component of the membrane trafficking machinery. This negative modulation of CFTR function can be reversed by soluble syntaxin 1A peptides and by the syntaxin 1A binding protein, Munc-18. In the present study, we determined whether syntaxin 1A is expressed in native epithelial tissues that normally express CFTR and whether it modulates CFTR currents in these tissues. Using immunoblotting and immunofluorescence, we observed syntaxin 1A in native gut and airway epithelial tissues and showed that epithelial cells from these tissues express syntaxin 1A at >10-fold molar excess over CFTR. Syntaxin 1A is seen near the apical cell surfaces of human bronchial airway epithelium. Reagents that disrupt the CFTR-syntaxin 1A interaction, including soluble syntaxin 1A cytosolic domain and recombinant Munc-18, augmented cAMP-dependent CFTR Cl(-) currents by more than 2- to 4-fold in mouse tracheal epithelial cells and cells derived from human nasal polyps, but these reagents did not affect CaMK II-activated Cl(-) currents in these cells.

Medroxyprogesterone acetate and levonorgestrel increase genital mucosal permeability and enhance susceptibility to genital herpes simplex virus type 2 infection.

Depot-medroxyprogesterone acetate (DMPA) is a hormonal contraceptive especially popular in areas with high prevalence of HIV and other sexually transmitted infections (STI). Although observational studies identify DMPA as an important STI risk factor, mechanisms underlying this connection are undefined. Levonorgestrel (LNG) is another progestin used for hormonal contraception, but its effect on STI susceptibility is much less explored. Using a mouse model of genital herpes simplex virus type 2 (HSV-2) infection, we herein found that DMPA and LNG similarly reduced genital expression of the desmosomal cadherin desmoglein-1α (DSG1α), enhanced access of inflammatory cells to genital tissue by increasing mucosal epithelial permeability, and increased susceptibility to viral infection. Additional studies with uninfected mice revealed that DMPA-mediated increases in mucosal permeability promoted tissue inflammation by facilitating endogenous vaginal microbiota invasion. Conversely, concomitant treatment of mice with DMPA and intravaginal estrogen restored mucosal barrier function and prevented HSV-2 infection. Evaluating ectocervical biopsy tissue from women before and 1 month after initiating DMPA remarkably revealed that inflammation and barrier protection were altered by treatment identically to changes seen in progestin-treated mice. Together, our work reveals DMPA and LNG diminish the genital mucosal barrier; a first-line defense against all STI, but may offer foundation for new contraceptive strategies less compromising of barrier protection.

Neutrophils negatively regulate induction of mucosal IgA responses after sublingual immunization.

Induction of mucosal immunoglobulin-A (IgA) capable of providing a first line of defense against bacterial and viral pathogens remains a major goal of needle-free vaccines given via mucosal routes. Innate immune cells are known to play a central role in induction of IgA responses by mucosal vaccines, but the relative contribution of myeloid cell subsets to these responses has not firmly been established. Using an in vivo model of sublingual vaccination with Bacillus anthracis edema toxin (EdTx) as adjuvant, we examined the role of myeloid cell subsets for mucosal secretory IgA responses. Sublingual immunization of wild-type mice resulted in a transient increase of neutrophils in sublingual tissues and cervical lymph nodes. These mice later developed Ag-specific serum IgG responses, but not serum or mucosal IgA. Interestingly, EdTx failed to increase neutrophils in sublingual tissues and cervical lymph nodes of IKKß(ΔMye) mice, and these mice developed IgA responses. Partial depletion of neutrophils before immunization of wild-type mice allowed the development of both mucosal and serum IgA responses. Finally, co-culture of B cells with neutrophils from either wild-type or IKKß(ΔMye) mice suppressed secretion of IgA, but not IgM or IgG. These results identify a new role for neutrophils as negative regulators of IgA responses.

Cytokines as adjuvants for the induction of mucosal immunity.

Safe nasal vaccines capable of promoting both mucosal and systemic immunity are needed for effective protection against bacterial and viral pathogens. While parenteral cytokine treatment could lead to unwanted toxicity, the nasal delivery route results in low but biologically active serum cytokine levels. Interleukin (IL)-6, IL-1 and IL-12, which promote either Th2- or Th1-type responses, respectively, also enhance systemic immunity to co-administered antigens. The chemoattractants lymphotactin (Lptn), RANTES and defensins also exerted adjuvant activity for systemic immunity when nasally administered with antigens. However, each cytokine or innate factor promoted a distinct pattern of T helper cell responses and corresponding IgG subclass response. Interleukin-12, IL-1, and the chemokines Lptn and RANTES promote mucosal immunity. In contrast, nasal IL-6 and defensins failed to induce mucosal S-IgA Ab responses, suggesting that mechanisms more complex than T cell activation and chemotaxis are required for the development of mucosal immunity after nasal delivery of cytokines.

Interleukin 12 and innate molecules for enhanced mucosal immunity.

Recent strategies for understanding the mechanisms underlying mucosal immune responses and subsequent development of mucosal vaccines for induction of targeted immunity now include cytokines and molecules of innate immunity. These studies have shown that cytokines influencing the development of T helper (Th) cells differentially affect the outcome of mucosal vs. systemic immune responses to mucosal vaccines. Serum antigen-specific antibody (Ab) responses were enhanced when either IL-6 or IL-12 was mucosally administered with a protein antigen, while only IL-12 induced antigen-specific mucosal IgA Ab responses. Mucosal IL-6 and IL-12 also affected the type of Th cell responses induced by CD4+ T cells from mice that received IL-12 secreted larger amounts of IFN-gamma and IL-6 when compared with mice nasally treated with IL-6. Discrepancies in the ability to enhance mucosal or systemic immune responses were also observed when human neutrophil peptide (HNP) defensins or lymphotactin were nasally coadministered with protein antigens. Only lymphotactin promoted mucosal secretory IgA (S-IgA) Ab responses while both lymphotactin and defensins enhanced systemic immunity to mucosally co-administered protein antigens. Mixed antigen-specific Th1 -and Th2-type CD4+ T cell responses were induced in the systemic compartment by both lymphotactin and the mixture of HNP-1, HNP-2, and HNP-3 defensins. However, HNPs failed to significantly enhance cytokine secretion by mucosally derived, antigen-specific CD4+ T cells relative to those isolated from the systemic compartment. In summary, these studies clearly show that IL-12 and lymphotactin are able to trigger S-IgA Ab responses and provide new avenues for the design of safe and targeted mucosal vaccines.

Interleukin-12 alters helper T-cell subsets and antibody profiles induced by the mucosal adjuvant cholera toxin.

We have shown that systemic administration of rmIL-12 could trigger Th1-type responses to a protein antigen delivered orally with CT as mucosal adjuvant. The most striking finding was that IL-12 could retain its regulatory effects when orally administered and could redirect the immune response to the oral vaccine toward a Th1-type. However, regulation by orally administered IL-12 differed from parenteral treatment with IL-12 since only the latter treatment affected mucosal S-IgA responses. These findings have important implications for the development of mucosal vaccines that induce the desired immune response.

Strategies for mucosal vaccine development.

Vaccines able to induce both secretory IgA for protection of mucosal surfaces and systemic immunity to pathogens invading the host are of great interest in the war against infectious diseases. Mucosal vaccines trigger immune cells in mucosal inductive sites and thus can induce immunity in both the mucosal and systemic compartments. This review presents a critical survey of adjuvants and delivery systems currently being tested for mucosal immunization. A better understanding of cellular and molecular factors involved in the regulation of mucosal immunity will help in the design of safer mucosal vaccines to elicit the appropriate protective immune response to a given pathogen.

IKKß in intestinal epithelial cells regulates allergen-specific IgA and allergic inflammation at distant mucosal sites.

Regulation of allergic responses by intestinal epithelial cells (IECs) remains poorly understood. Using a model of oral allergen sensitization in the presence of cholera toxin as adjuvant and mice with cell-specific deletion of inhibitor-κB kinase (IKKß) in IECs (IKKß(ΔIEC)), we addressed the contribution of IECs to allergic sensitization to ingested antigens and allergic manifestations at distant mucosal site of the airways. Cholera toxin induced higher pro-inflammatory responses and altered the profile of the gut microbiota in IKKß(ΔIEC) mice. Antigen-specific immunoglobulin E (IgE) responses were unaltered in IKKß(ΔIEC) mice, but their IgA antibodies (Abs), T helper type 1 (Th1) and Th17 responses were enhanced. Upon nasal antigen challenge, these mice developed lower levels of allergic lung inflammation, which correlated with higher levels of IgA Abs in the airways. The IKKß(ΔIEC) mice also recruited a higher number of gut-sensitized T cells in the airways after nasal antigen challenge and developed airway hyper-responsiveness, which were suppressed by treatment with anti-interleukin-17A. Fecal microbiota transplant during allergic sensitization reduced Th17 responses in IKKß(ΔIEC) mice, but did not affect IgA Ab responses. In summary, we show that IKKß in IECs shapes the gut microbiota and immune responses to ingested antigens and influences allergic responses in the airways via regulation of IgA Ab responses.

RANTES potentiates antigen-specific mucosal immune responses.

RANTES is produced by lymphoid and epithelial cells of the mucosa in response to various external stimuli and is chemotactic for lymphocytes. The role of RANTES in adaptive mucosal immunity has not been studied. To better elucidate the role of this chemokine, we have characterized the effects of RANTES on mucosal and systemic immune responses to nasally coadministered OVA. RANTES enhanced Ag-specific serum Ab responses, inducing predominately anti-OVA IgG2a and IgG3 followed by IgG1 and IgG2b subclass Ab responses. RANTES also increased Ag-specific Ab titers in mucosal secretions and these Ab responses were associated with increased numbers of Ab-forming cells, derived from mucosal and systemic compartments. Splenic and mucosally derived CD4(+) T cells of RANTES-treated mice displayed higher Ag-specific proliferative responses and IFN-gamma, IL-2, IL-5, and IL-6 production than control groups receiving OVA alone. In vitro, RANTES up-regulated the expression of CD28, CD40 ligand, and IL-12R by Ag-activated primary T cells from DO11.10 (OVA-specific TCR-transgenic) mice and by resting T cells in a dose-dependent fashion. These studies suggest that RANTES can enhance mucosal and systemic humoral Ab responses through help provided by Th1- and select Th2-type cytokines as well as through the induction of costimulatory molecule and cytokine receptor expression on T lymphocytes. These effects could serve as a link between the initial innate signals of the host and the adaptive immune system.

Salmonella-mediated mucosal cell-mediated immunity.

Oral immunization with the recombinant Salmonella typhimurium strain (BRD 847) expressing the C fragment of tetanus toxin (TT) induces brisk Ag-specific mucosal S-IgA and serum Ab responses characterized by strong IgG2a Abs to the encoded antigen. We have constructed an attenuated Salmonella typhimurium (aroA- aroD-) strain that expresses chicken egg albumin (OVA) to further elucidate the role of Salmonella-induced Th1 cell phenotype on mucosal cell-mediated immunity (CMI). Peyer's patches and spleen lymphocytes from mice that received the oral Salmonella-OVA vaccine showed dramatic increases in the percent cell lysis of the H-2b restricted EG7.OVA tumor cell line. These results indicate that a single dose of rSalmonella vaccine antigen vector is required to illicit systemic and mucosal Th1-type responses and CTLs. These results also support the existence of a highly regulated relationship between specific cell-mediated immunity and a branch of the humoral immune system, i.e. mucosal IgA responses.

Spontaneously hypertensive rat: cholera toxin converts suppression to immunity through a Th2 cell-IL-4 pathway.

The spontaneously hypertensive rat (SHR) exhibits a number of T cell dysfunctions that develop concurrently with elevated blood pressure. Studies have shown a mitogen-induced lymphocyte suppression mediated in part by the production of interferon-gamma (IFN-gamma), which stimulated NO production by macrophages. To assess whether this immune suppression is reversible, SHR were immunized with diphtheria toxoid (DT) with or without cholera toxin (CT) as adjuvant. SHR immunized with DT only displayed weak serum immunoglobulin G (IgG) anti-DT titers, tenfold less than similarly treated normotensive Wistar-Kyoto rats (WKYR). SHR CD4+ T cells failed to proliferate upon in vitro stimulation with DT. In contrast, SHR coimmunized with DT and CT showed serum IgG antibody titers similar to WKYR and Brown Norway rats. Coimmunization with CT rescued SHR CD4+ T cells from suppression and supported DT- or B subunit of CT-specific proliferative responses, and these cells produced more interleukin-4 (IL-4) than IFN-gamma, and anti-IFN-gamma antibody treatment enhanced IL-4 production. Exogenous IL-4 increased the proliferation of antigen-specific CD4+ T cells, whereas IFN-gamma was inhibitory. This study shows that the adjuvant CT induces T helper 2-type responses, reversing the T cell dysfunction in the SHR.

Novel approaches for the induction of T helper 1 (Th1)- or Th2-type mucosal and parenteral immune responses.

Mucosal surfaces are constantly challenged by micro-organisms and are protected by an integrated component of the immune system called mucosa-associated lymphoreticular tissue (MALT). The immune responses elicited at the mucosal level are regulated by T-helper (Th) cells and involve secretory IgA (S-IgA) antibodies (Abs) and cytotoxic T-lymphocytes (CTLs). Mucosal immunisation has the advantage over parenteral immunisation, of inducing S-IgA Abs and of conferring protection at both the mucosal and parenteral levels; however, administration of soluble antigens through a mucosal route very seldom results in significant mucosal and systemic immune responses. Therefore, appropriate mucosal adjuvants, recombinant bacterial and viral vectors and delivery systems have been developed to increase the immunogenicity of vaccine antigens and to preferentially induce antigen-specific T-helper (Th)1- or Th2-type responses, which in turn result in polarised effector immune responses. Understanding the mechanisms underlying Th1- and Th2-type developmental pathways and the ability of novel mucosal adjuvants and delivery systems to target the desired Th1- or Th2-type immune response would help to design effective mucosal vaccines, inducing predominant cell-mediated or humoral responses.

Lymphotactin acts as an innate mucosal adjuvant.

Lymphotactin (Lptn) is a C chemokine produced predominantly by NK and CD8-positive (CD8+) T cells including gammadelta TCR-positive (TCR+) intraepithelial lymphocytes. Lptn is chemotactic for NK and T cells and likely plays an important role in maintaining the integrity of the epithelium and in mucosal immune responses. In this study, we characterized the immune responses to OVA given intranasally with Lptn to mice. This regimen enhanced OVA-specific serum Ab responses and Ab titers in mucosal secretions. Lptn also enhanced OVA-specific Ab-forming cells in mucosal and systemic compartments. CD4-positive (CD4+) T cells isolated from mucosal compartments and spleens of mice intranasally immunized with OVA plus Lptn displayed higher OVA-specific proliferative responses and greater synthesis of IFN-gamma, IL-2, IL-4, IL-5, IL-6, and IL-10 than did CD4+ T cells from mice given OVA without Lptn. These studies indicate that Lptn has adjuvant properties and suggest that Lptn present in the mucosa has the potential to enhance mucosal and systemic Ab responses through help provided by Th1- and Th2-type cells to link the initial innate signals of the mucosa with the acquired immune system.

Mechanisms for induction of acquired host immunity by neutrophil peptide defensins.

Human neutrophil peptide (HNP) defensins were studied to determine their potential effects on adaptive mucosal immunity. Intranasal delivery of HNPs plus ovalbumin (OVA) enhanced OVA-specific serum IgG antibody (Ab) responses. However, OVA-specific IgA Abs were not induced in mucosal secretions or in serum. CD4(+) T cells of intranasally immunized mice displayed higher OVA-specific proliferative responses and elevated production of interferon gamma, interleukin (IL) 5, IL-6, and IL-10 when compared with control groups receiving OVA alone. In vitro, HNPs also enhanced both proliferative responses and T helper (Th) cytokine secretion profiles of CD3epsilon-stimulated spleen- and Peyer's patch-derived naive CD4(+) T cells. HNPs modulated the expression of costimulatory molecules by lipopolysaccharide- or CD3epsilon-stimulated splenic and Peyer's patch B or T cell populations, respectively. These studies show that defensins enhance systemic IgG, but not IgA, Ab responses through help provided by CD4(+) Th1- and Th2-type cytokines and foster B and T cell interactions to link innate immunity with the adaptive immune system.

A novel alkaline phosphatase-based isolation method allows characterization of intraepithelial lymphocytes from villi tip and crypt regions of murine small intestine.

The isolation of intestinal intraepithelial lymphocytes (IEL) is a major prerequisite for the investigation of cellular and molecular cross-talk in the intestinal mucosa. Since intestinal epithelial cells exhibit distinct functional features at the villi tip and crypt levels, such differences could extend to IEL. We developed a mechanical procedure for isolation of IEL from these distinct epithelial sites to test our hypothesis. Cells isolated from the intestinal epithelium by sequential incubations under stirring were segregated based upon their alkaline phosphatase (AP) activity since villi tip and crypt fractions expressed high and low AP activity, respectively. IEL preparations obtained after a further purification step in Percoll gradient contained > 90% Integrin alpha IEL chain+, CD3+ T cells, and no Ig+ cells. Villi tip IEL preparations possessed increased numbers of low density IEL when compared to crypt IEL, suggesting that distinct IEL-epithelial cell interactions occur at the intestinal villi tip and crypt levels.

Intraepithelial lymphocytes from villus tip and crypt portions of the murine small intestine show distinct characteristics.

BACKGROUND & AIMS: Intraepithelial lymphocytes (IELs) are located between epithelial cells that are thought to display unique features and functions at the small intestinal villus tip and crypt levels. We have addressed whether the spatial differences in the intestinal epithelium extend to IELs and subsequent cross-talk between IELs and epithelial cells. METHODS: IELs were isolated from villus tip and crypt portions of mouse small intestine and then compared for spontaneous cytokine production and responsiveness to interleukin (IL)-2 and/or IL-7. RESULTS: No difference was observed between number of beta IELs in villus tips and crypts, whereas a trend toward increased frequencies of IELs bearing the gamma delta form of T-cell receptor was noted in villus tips. Interestingly, the number of beta IELs producing interferon gamma and IL-5 was significantly reduced in the cells from crypts compared with villus tips. Furthermore, villus tip beta IELs exhibited higher responses to stimulation signals provided by IL-2 and/or IL-7 than their crypt counterpart. Such functional differences were not observed with gamma delta IELs from the two intestinal sites. CONCLUSIONS: Distinct molecular cross-talk between IELs and epithelial cells occurs in intestinal villus tips and crypts.

Influenza virus-infected epithelial cells present viral antigens to antigen-specific CD8+ cytotoxic T lymphocytes.

We have investigated the mechanisms involved in the clearance of viral infection at the epithelium level by analyzing the activity of influenza virus-specific cytotoxic T lymphocytes (CTL) against virus-infected CMT-93 intestinal epithelial cells. Epithelial cells infected with live influenza virus effectively present viral antigens and were lysed by both homotypic and heterotypic influenza virus-specific CD8+ T cells. These results shed new light on the control of viral infection through the elimination of virus-infected epithelial cells by virus-specific CTL and demonstrate that CMT-93 cells furnish an appropriate model for in vitro evaluation of CTL activity against virus-infected epithelial cells.