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An intricate network of innate and immune cells and their derived mediators function in unison to protect us from toxic elements and infectious microbial diseases that are encountered in our environment. This vast network operates efficiently by use of a single cell epithelium in, for example, the gastrointestinal (GI) and upper respiratory (UR) tracts, fortified by adjoining cells and lymphoid tissues that protect its integrity. Perturbations certainly occur, sometimes resulting in inflammatory diseases or infections that can be debilitating and life threatening. For example, allergies in the eyes, skin, nose, and the UR or digestive tracts are common. Likewise, genetic background and environmental microbial encounters can lead to inflammatory bowel diseases (IBDs). This mucosal immune system (MIS) in both health and disease is currently under intense investigation worldwide by scientists with diverse expertise and interests. Despite this activity, there are numerous questions remaining that will require detailed answers in order to use the MIS to our advantage. In this issue of PLOS Biology, a research article describes a multi-scale in vivo systems approach to determine precisely how the gut epithelium responds to an inflammatory cytokine, tumor necrosis factor-alpha (TNF-α), given by the intravenous route. This article reveals a previously unknown pathway in which several cell types and their secreted mediators work in unison to prevent epithelial cell death in the mouse small intestine. The results of this interesting study illustrate how in vivo systems biology approaches can be used to unravel the complex mechanisms used to protect the host from its environment.
Previously, we showed that nasal administration of a naked cDNA plasmid expressing Flt3 ligand (FL) cDNA (pFL) enhanced CD4(+) Th2-type, cytokine-mediated mucosal immunity and increased lymphoid-type dendritic cell (DC) numbers. In this study, we investigated whether targeting nasopharyngeal-associated lymphoreticular tissue (NALT) DCs by a different delivery mode of FL, i.e., an adenovirus (Ad) serotype 5 vector expressing FL (Ad-FL), would provide Ag-specific humoral and cell-mediated mucosal immunity. Nasal immunization of mice with OVA plus Ad-FL as mucosal adjuvant elicited high levels of OVA-specific Ab responses in external secretions and plasma as well as significant levels of OVA-specific CD4(+) T cell proliferative responses and OVA-induced IFN-gamma and IL-4 production in NALT, cervical lymph nodes, and spleen. We also observed higher levels of OVA-specific CTL responses in the spleen and cervical lymph nodes of mice given nasal OVA plus Ad-FL than in mice receiving OVA plus control Ad. Notably, the number of CD11b(+)CD11c(+) DCs expressing high levels of costimulatory molecules was preferentially increased. These DCs migrated from the NALT to mucosal effector lymphoid tissues. Taken together, these results suggest that the use of Ad-FL as a nasal adjuvant preferentially induces mature-type NALT CD11b(+)CD11c(+) DCs that migrate to effector sites for subsequent CD4(+) Th1- and Th2-type cytokine-mediated, Ag-specific Ab and CTL responses.
Mucosal tolerance induction generally requires multiple or large Ag doses. Because microfold (M) cells have been implicated as being important for mucosal tolerance induction and because reovirus attachment protein sigma1 (psigma1) is capable of binding M cells, we postulated that targeting a model Ag to M cells via psigma1 could induce a state of unresponsiveness. Accordingly, a genetic fusion between OVA and the M cell ligand, reovirus psigma1, termed OVA-psigma1, was developed to enhance tolerogen uptake. When applied nasally, not parenterally, as little as a single dose of OVA-psigma1 failed to induce OVA-specific Abs even in the presence of adjuvant. Moreover, the mice remained unresponsive to peripheral OVA challenge, unlike mice given multiple nasal OVA doses that rendered them responsive to OVA. The observed unresponsiveness to OVA-psigma1 could be adoptively transferred using cervical lymph node CD4(+) T cells, which failed to undergo proliferative or delayed-type hypersensitivity responses in recipients. To discern the cytokines responsible as a mechanism for this unresponsiveness, restimulation assays revealed increased production of regulatory cytokines, IL-4, IL-10, and TGF-beta1, with greatly reduced IL-17 and IFN-gamma. The induced IL-10 was derived predominantly from FoxP3(+)CD25(+)CD4(+) T cells. No FoxP3(+)CD25(+)CD4(+) T cells were induced in OVA-psigma1-dosed IL-10-deficient (IL-10(-/-)) mice, and despite showing increased TGF-beta1 synthesis, these mice were responsive to OVA. These data demonstrate the feasibility of using psigma1 as a mucosal delivery platform specifically for low-dose tolerance induction.
In this study, we examine whether native cholera toxin (nCT) as a mucosal adjuvant can support trinitrophenyl (TNP)-LPS-specific mucosal immune responses. C57BL/6 mice were given nasal TNP-LPS in the presence or absence of nCT. Five days later, significantly higher levels of TNP-specific mucosal IgA Ab responses were induced in the nasal washes, saliva, and plasma of mice given nCT plus TNP-LPS than in those given TNP-LPS alone. High numbers of TNP-specific IgA Ab-forming cells were also detected in mucosal tissues such as the nasal passages (NPs), the submandibular glands (SMGs), and nasopharyngeal-associated lymphoreticular tissue of mice given nCT. Flow cytometric analysis showed that higher numbers of surface IgA+, CD5+ B cells (B-1a B cells) in SMGs and NPs of mice given nasal TNP-LPS plus nCT than in those given TNP-LPS alone. Furthermore, increased levels of IL-5R alpha-chain were expressed by B-1a B cells in SMGs and NPs of mice given nasal TNP-LPS plus nCT. Thus, CD4+ T cells from these mucosal effector lymphoid tissues produce high levels of IL-5 at both protein and mRNA levels. When mice were treated with anti-IL-5 mAb, significant reductions in TNP-specific mucosal IgA Ab responses were noted in external secretions. These findings show that nasal nCT as an adjuvant enhances mucosal immune responses to a T cell-independent Ag due to the cross-talk between IL-5Ralpha+ B-1a B cells and IL-5-producing CD4+ T cells in the mucosal effector lymphoid tissues.
Absence of suitable mucosal adjuvants for humans prompted us to consider alternative vaccine designs for mucosal immunization. Because adenovirus is adept in binding to the respiratory epithelium, we tested the adenovirus 2 fiber protein (Ad2F) as a potential vaccine-targeting molecule to mediate vaccine uptake. The vaccine component (the host cell-binding domain to botulinum toxin (BoNT) serotype A) was genetically fused to Ad2F to enable epithelial binding. The binding domain for BoNT was selected because it lies within the immunodominant H chain as a beta-trefoil (Hcbetatre) structure; we hypothesize that induced neutralizing Abs should be protective. Mice were nasally immunized with the Hcbetatre or Hcbetatre-Ad2F, with or without cholera toxin (CT). Without CT, mice immunized with Hcbetatre produced weak secretory IgA (sIgA) and plasma IgG Ab response. Hcbetatre-Ad2F-immunized mice produced a sIgA response equivalent to mice coimmunized with CT. With CT, Hcbetatre-Ad2F-immunized mice showed a more rapid onset of sIgA and plasma IgG Ab responses that were supported by a mixed Th1/Th2 cells, as opposed to mostly Th2 cells by Hcbetatre-dosed mice. Mice immunized with adjuvanted Hcbetatre-Ad2F or Hcbetatre were protected against lethal BoNT serotype A challenge. Using a mouse neutralization assay, fecal Abs from Hcbetatre-Ad2F or Hcbetatre plus CT-dosed mice could confer protection. Parenteral immunization showed that the inclusion of Ad2F enhances anti-Hcbetatre Ab titers even in the absence of adjuvant. This study shows that the Hcbetatre structure can confer protective immunity and that use of Hcbetatre-Ad2F gives more rapid and sustained mucosal and plasma Ab responses.
Nasal application of native cholera toxin (nCT) as a mucosal adjuvant has potential toxicity for the CNS through binding to GM1 gangliosides in the olfactory nerves. Although mutants of cholera toxin (mCTs) have been developed that show mucosal adjuvant activity without toxicity, it still remains unclear whether these mCTs will induce CNS damage. To help overcome these concerns, in this study we created new double mutant CTs (dmCTs) that have two amino acid substitutions in the ADP-ribosyltransferase active center (E112K) and COOH-terminal KDEL (E112K/KDEV or E112K/KDGL). Confocal microscopic analysis showed that intracellular localization of dmCTs differed from that of mCTs and nCTs in intestinal epithelial T84 cells. Furthermore, both dmCTs exhibited very low toxicity in the Y1 cell assay and mouse ileal loop tests. When mucosal adjuvanticity was examined, both dmCTs induced enhanced OVA-specific immune responses in both mucosal and systemic lymphoid tissues. Interestingly, although both dmCT E112K/KDEV and dmCT E112K/KDGL showed high Th2-type and significant Th1-type cytokine responses by OVA-specific CD4+ T cells, dmCT E112K/KDEV exhibited significantly lower Th1-type cytokine responses than did nCT and dmCT E112K/KDGL. These results show that newly developed dmCTs retain strong biological adjuvant activity without CNS toxicity.
Anthrax edema toxin (EdTx) is an AB-type toxin that binds to anthrax toxin receptors on target cells via the binding subunit, protective Ag (PA). Edema factor, the enzymatic A subunit of EdTx, is an adenylate cyclase. We found that nasal delivery of EdTx enhanced systemic immunity to nasally coadministered OVA and resulted in high OVA-specific plasma IgA and IgG (mainly IgG1 and IgG2b). The edema factor also enhanced immunity to the binding PA subunit itself and promoted high levels of plasma IgG and IgA responses as well as neutralizing PA Abs. Mice given OVA and EdTx also exhibited both PA- and OVA-specific IgA and IgG Ab responses in saliva as well as IgA Ab responses in vaginal washes. EdTx as adjuvant triggered OVA- and PA-specific + T cells which secreted IFN-gamma and selected Th2-type cytokines. The EdTx up-regulated costimulatory molecule expression by APCs but was less effective than cholera toxin for inducing IL-6 responses either by APCs in vitro or in nasal washes in vivo. Finally, nasally administered EdTx did not target CNS tissues and did not induce IL-1 mRNA responses in the nasopharyngeal-associated lymphoepithelial tissue or in the olfactory bulb epithelium. Thus, EdTx derivatives could represent an alternative to the ganglioside-binding enterotoxin adjuvants and provide new tools for inducing protective immunity to PA-based anthrax vaccines.
The safety of nasal vaccines containing enterotoxin-based mucosal adjuvants has not been studied in detail. Previous studies have indicated that native cholera toxin (nCT) can alter antigen trafficking when applied nasally. In this study, we determined the enterotoxin-based variables that alter antigen trafficking. To measure the influence of enterotoxin-based mucosal adjuvants on antigen trafficking in the nasal tract, native and mutant enterotoxins were coadministered with radiolabeled tetanus toxoid (TT). The nCT and heat-labile enterotoxin type 1 (LTh-1) redirected TT into the olfactory neuroepithelium (ON/E). Antigen redirection occurred mainly across the nasal epithelium without subsequent transport along olfactory neurons into the olfactory bulbs (OB). Thus, no significant accumulation of the vaccine antigen TT was observed in the OB when coadministered with nCT. In contrast, neither mutant CT nor mutant LTh-1, which lack ADP-ribosyltransferase activity, redirected TT antigen into the ON/E. Thus, ADP-ribosyltransferase activity was essential for antigen trafficking across the olfactory epithelium. Accumulation of TT in the ON/E was also due to B-subunit binding to GM1 gangliosides, as was demonstrated (i) by redirection of TT by LTh-1 in a dose-dependent manner, (ii) by ganglioside inhibition of the antigen redirection by LTh-1 and nCT, and (iii) by the use of LT-IIb, a toxin that binds to gangliosides other than GM1. Redirection of TT into the ON/E coincided with elevated production of interleukin 6 (IL-6) but not IL-1beta or tumor necrosis factor alpha in the nasal mucosa. Thus, redirection of TT is dependent on ADP-ribosyltransferase activity and GM1 binding and is associated with production of the inflammatory cytokine IL-6.
The threat posed by botulism, classically a food- and waterborne disease with a high morbidity and mortality, has increased exponentially in an age of bioterrorism. Because botulinum neurotoxin (BoNT) could be easily disseminated by terrorists using an aerosol or could be used to contaminate the food or water supply, the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases has classified it as a category A agent. Although clearly the development of a safe and effective mucosal vaccine against this toxin should be a high priority, essentially no studies to date have assessed mucosal immune responses to this disease. To bridge this gap in our knowledge, we immunized mice weekly for 4 wk with nasal doses of BoNT type A toxoid and a mutant of cholera toxin termed E112K. We found elevated levels of BoNT-specific IgG Abs in plasma and of secretory IgA Abs in external secretions (nasal washes, saliva, and fecal extracts). When mice given nasal BoNT vaccine were challenged with 4 x 10(3) LD50 of BoNT type A (BoNT/A) via the i.p. route, complete protection was seen, while naive mice given the same dosage died within 2 h. To further confirm the efficacy of this nasal BoNT vaccine, an oral LD50 was determined. When mice were given an oral challenge of 5 microg (2 x oral LD50) of progenitor BoNT/A, all immunized mice survived beyond 5 days, while nonimmunized mice did not. The fecal extract samples from nasally vaccinated mice were found to contain neutralizing secretory IgA Abs. Taken together, these results show that nasal BoNT/A vaccine effectively prevents mucosal BoNT intoxication.
Studies using the enterotoxin cholera toxin and the related heat-labile toxin I of Escherichia coli as adjuvants have established that effective immunity can be provided in mucosal tissues and the general blood stream by vaccines administered by the oral or nasal route. Despite this knowledge and other recognized advantages, needleless mucosal vaccines rarely appear in the health care market and classic injected vaccines remain the norm. Safety issues related to the adjuvants needed for oral and nasal vaccines are the chief factor that hampers their generalized use in humans. Initial strategies to overcome the toxicity of enterotoxin adjuvants consisted of removing the enzymatic activity of these molecules. The binding of these toxins to their ganglioside receptors was later found to be a major source of safety concern regarding nasal vaccines. Non-ganglioside-binding bacterial toxins and their derivatives are being evaluated as alternatives for the development of effective and safe mucosal vaccines. The CTA1-DD adjuvant, which acts by B-cell targeting of the enzymatically active cholera toxin A subunit, represents one of the strategies to induce immunity by nasal immunization without targeting central nervous system tissues. More recently, we found that the edema toxin of Bacillus anthracis, which binds to target cells via the anthrax toxin receptors and delivers its adenylate cyclase subunit, is an effective adjuvant for nasal vaccines. Interestingly, the unique receptors of anthrax toxin derivatives appear to prevent their accumulation in the central nervous system.
Nasal antigen plus mucosal adjuvant delivery has emerged as perhaps the most effective route for induction of both mucosal and peripheral immune response. It has been shown that dendritic cells (DCs) played essential roles for the induction of both immunity and tolerance. Thus, nasal application of CpG ODN targeting plasmacytoid DCs effectively induced antigen-specific immunity. Our recent studies showed that nasal or oral immunization with protein antigen, ovalbumin plus plasmid encoding Flt3 ligand (pFL) cDNA as adjuvant effectively elicited OVA-specific immune responses in both mucosal and systemic lymphoid tissues. The numbers of (DCs) in mice given pFL as mucosal adjuvant were significantly increased. Further, these expanded DCs were activated and expressed MHC II and co-stimulatory molecules (CD40, CD80 and CD86). These results show that mucosal delivery of pFL is DCs targeting adjuvant system that effectively induce both mucosal and systemic antigen-specific immune responses.
Preclinical study reveals mutant cholera toxin promotes mucosal HIV-1 immunity without neuronal toxicity in macaques, indicating potential for safe needle-free vaccines.
Chemokines constitute a large family of structurally related proteins that play a role in leukocyte migration and differentiation. Indeed, the early expression of human CXC chemokine receptor 1 (hCXCR1) and hCXCR2 [homologous to mouse interleukin (IL)-8Rbeta] ligands by the epithelium is a hallmark of the mucosal host defense. Mice lack IL-8; however, granulocyte chemotactic protein-2 (GCP-2)/lipopolysaccharide-induced CXC chemokine, a murine homologue of human GCP-2, has 32% and 61% sequence identity to human IL-8 and GCP-2, respectively, and binds hCXCR1, hCXCR2, and mouse IL-8Rbeta. To better understand the role of GCP-2 in adaptive immunity and as a nasal adjuvant, we characterized the exogenous effects of this CXC chemokine on cellular and humoral mucosal immune responses. GCP-2 significantly enhanced serum immunoglobulin G (IgG) and mucosal IgA antibodies through increased cytokine secretion by CD4+ T cells. These alterations in humoral and cellular responses were preceded by an increase in the number of B cells in the nasal tract, a decrease in the number of CD4+ T cells in the nasal tract as well as cervical lymph nodes, and an increase in the number of neutrophils in the nasal tract 12 h after GCP-2 immunization. This chemokine also modulated CD28 expression by CD4+ T cells during CD3epsilon stimulation of wild-type mice. GCP-2 increased CD80 and CD86 expression on B cells during in vitro stimulation in a dose-dependent manner. In contrast, cytokine and costimulatory molecule enhancement by GCP-2 was not induced by lymphocytes from IL-8Rbeta-/- mice, suggesting that GCP-2 modulates cellular immunity in part through IL-8Rbeta interactions.
This study assessed the roles of the postnatal lymphotoxin-beta receptor (LTbetaR)-mediated signals in the gut-associated lymphoreticular tissues of mice for subsequent regulation of Ag-specific intestinal IgA responses. Blockade of LTbetaR-dependent events by postnatal administration of the fusion protein of LTbetaR and IgG Fc (LTbetaR-Ig) reduced both the size and numbers of Peyer's patches (PP) without influencing the PP microarchitecture. Interestingly, inhibition of LTbetaR-dependent signaling revealed significant reductions in the formation of follicular dendritic cell clusters in mesenteric lymph nodes (MLN). Furthermore, these postnatal signaling events controlled the development of isolated lymphoid follicles (ILF) because treatment with LTbetaR-Ig eliminated the formation of ILF. LTbetaR-Ig-treated mice with altered microarchitecture of MLN and lacking ILF were still able to produce significant Ag-specific mucosal IgA responses after oral immunization; however, the levels were significantly lower than those seen in control mice. These results imply the importance of ILF for Ag-specific intestinal immunity. However, mice treated with both TNFR55-Ig and LTbetaR-Ig in utero, which lack PP and MLN, but retain intact ILF, failed to induce Ag-specific IgA responses after oral immunization. These findings demonstrate that ILF are not essential for induction of intestinal IgA Ab responses to orally administered Ag. Furthermore, the induction of intestinal IgA Ab responses requires the proper maintenance of the MLN microarchitecture, including a follicular dendritic cell network.
Nasal immunization is an effective way to induce both mucosal and systemic immune responses. In this study, we assessed a cDNA vector for Flt3 ligand (FL) for its potential to enhance mucosal immunity or tolerance. Interestingly, tolerance was avoided and elevated levels of OVA-specific Ab responses were induced in nasal washes, fecal extracts, and saliva as well as in plasma when compared with mice given nasal OVA plus DNA plasmid without the FL gene. In addition, significant levels of OVA-specific CD4+ T cell proliferative responses and OVA-induced IL-4 and IL-2 production were noted in spleen and cervical lymph nodes. Further, marked increases in FL protein occurred in the nasal lamina propria and submandibular glands and the frequencies of CD11c+CD8+ dendritic cells (DCs) significantly increased in the mucosal tissues. Moreover, these DCs expressed high levels of CD40, CD80, CD86, and MHC class II molecules. Nasal delivery of plasmid FL with OVA resulted in FL expression in both mucosal inductive and effector sites and resulted in expanded activated lymphoid DCs. Thus, nasal plasmid FL prevents mucosal tolerance and enhances active immunity when given by a mucosal route.
Streptococcus pneumoniae cause considerable morbidity and mortality, with persistent neurological sequelae, particularly in young children and the elderly. It is widely assumed that carriage occurs through direct mucosal colonization from the environment whereas meningitis results from invasion from the blood. However, the results of published studies can be interpreted that pneumococci may enter the brain directly from the nasal cavity by axonal transport through olfactory nerves. This hypothesis is based on findings that (i) teichoic acid of the pneumococcal cell wall interact with gangliosides (GLS), (ii) the interaction of GLS with cholera toxin leads to axonal transport through the olfactory nerves into the brain, and (iii) viruses enter the brain through axonal transport into olfactory nerves. After nasal inoculation, we observe high numbers of pneumococci in nasal washes and the olfactory nerves and epithelium. Significant numbers of pneumococci also infected the olfactory bulbs, brain, and the trigeminal ganglia. The absence of bacteremia in this model makes it unlikely that the bacteria entered the brain from the blood stream. Recovery of colony-forming units from the brain, lungs, olfactory nerves, and epithelium and nasal washes was inhibited by incubating pneumococci with GLS before nasal inoculation. These findings, confirmed by PCR and immunohistochemistry, support a GLS-mediated process of infection and are consistent with pneumococci reaching the brain through retrograde axonal transport.
Cholera toxin (CT) is a potent adjuvant; however, the mechanism for its ability to enhance mucosal immunity has not been fully elucidated. We report here that CT exerts its adjuvant properties by signaling through the GM1 ganglioside receptor. When ganglioside-defective mice were given the antigen (Ag) ovalbumin (OVA) with CT by the oral route, CT failed to support either OVA-specific antibody or CD4+ T cell responses. In vitro treatment of murine bone marrow-derived dendritic cells (DC) with CT induced full maturation as evidenced by up-regulation of the costimulatory molecules, as well as by an enhanced ability to effectively present OVA for Ag-specific T cell responses. On the other hand, ganglioside-defective DC failed to differentiate to full function as Ag-presenting cells in response to CT. Since ganglioside-defective DC showed a mature phenotype after stimulation with lipopolysaccharide (LPS), the effects of CT on DC was independent of signal transduction through adjuvant receptor for LPS, the Toll-like receptor 4. Furthermore, CT also induced nuclear translocation of nuclear factor (NF)-kappaB in DC in a GM1-dependent fashion. These results highlight gangliosides expressed by DC for recognition of the non-self protein bacterial enterotoxin, which employ a unique signaling pathway to induce both innate and adaptive immunity.
T helper type 1 (Th1) cells secreting interferon-gamma (IFN-gamma) have been closely associated with Crohn's disease (CD). Monokine-induced by IFN-gamma (MIG), IFN-gamma-inducible T cell alpha chemoattractant (I-TAC), and IFN-gamma-inducible protein-10 (IP-10), are chemokines that bind CXCR3 and mediate the chemotaxis of leukocytes. IP-10, MIG, and CXCR3 have been shown to be expressed at sites of CD. The current study stems from our recent findings that IP-10, MIG, and I-TAC significantly contribute to the development of Th1-mediated inflammatory responses. To better understand the role of CXCR3 interactions during CD, we characterized the effects of IP-10, MIG, I-TAC, and CXCR3+ T cells on mucosal immune responses. IP-10, MIG, and I-TAC significantly enhanced antigen-specific serum and mucosal antibodies through Th1-mediated events and CD28 modulation. Additionally, the adoptive transfer of naive CXCR3+ T cells and CD4+CD45RB(HI) to T cell receptor beta (TCRbeta) x delta(-/-) mice resulted in the onset of murine colitis. Taken together, these studies suggest that IP-10, MIG, I-TAC, and CXCR3 interactions are involved in mucosal immune responses required for the induction of CD.