Escherichia coli heat-labile enterotoxin promotes protective Th17 responses against infection by driving innate IL-1 and IL-23 production

Escherichia coli heat-labile enterotoxin (LT) is a powerful mucosal adjuvant; however, it is associated with toxic effects when delivered intranasally, and its mechanism of action is poorly understood. In this article, we demonstrate that LT acts as a highly effective adjuvant when administered parenterally, promoting Ag-specific IL-17, as well as IFN-γ, IL-4, and IL-10 production in response to coadministered Ags. We found that the adjuvant activity of LT was mediated in part by inducing dendritic cell (DC) activation; LT promoted CD80 and CD86 expression by DCs and enhanced IL-1α, IL-1β, and IL-23 production. An LT mutant, LTK63, that lacks enzyme activity was less effective than the wild-type toxin in promoting DC maturation and the development of Ag-specific Th17 cells. LT enhanced IL-23 and IL-1α production from DCs via activation of ERK MAPK and IL-1β secretion through activation of caspase-1 and the NLRP3 inflammasome. These cytokines played a major role in promoting Th17 responses by LT and LTK63. The induction of Th17 cells in vivo in response to LT and LTK63 as adjuvants was significantly reduced in IL-1RI-deficient mice. Finally, using a murine respiratory infection model, we demonstrated that LT can act as a highly effective adjuvant for a pertussis vaccine, promoting Ag-specific Th17 cells and protection against Bordetella pertussis challenge, which was significantly reduced in IL-17-defective mice. Our findings provide clear evidence that LT can promote protective immune responses in part through induction of innate IL-1 and, consequently, Th17 cells.     

Developmental response of zygotes exposed to similar mutagens

Exposure of mouse zygotes to ethylene oxide (EtO) or ethyl methanesulfonate (EMS) led to high incidences of fetal death and of certain classes of fetal malformations (Generoso et al., 1987, 1988; Rutledge and Generoso, 1989). These effects were not associated with induced chromosomal aberrations (Katoh et al., 1989) nor are they likely to be caused by gene mutations (Generoso et al., 1990). Nevertheless, the anomalies observed in these studies resemble the large class of stillbirths and sporadic defects in humans that are of unknown etiology, such as cleft palate, omphalocoel, clubfoot, hydrops and stillbirths (Czeizel, 1985; Oakley, 1986). Therefore, we continue to study the possible mechanisms relating to induction of these types of zygote-derived anomalies in mice. Effects of zygote exposure to the compounds methyl methanesulfonate (MMS), dimethyl sulfate (DMS), and diethyl sulfate (DES), which have similar DNA-binding properties as EtO and EMS, were studied. DMS and DES, but not MMS, induced effects that are similar to those induced by EtO and EMS. Thus, no site-specific alkylation product was identifiable as the critical target for these zygote-derived anomalies. We speculate that the developmental anomalies arose as a result of altered programming of gene expression during embryogenesis.     

Induction of metallothionein by superantigenic bacterial exotoxin: probable involvement of the immune system

Metallothionein (MT) can be induced in mouse liver by a bacterial exotoxin, toxic shock syndrome toxin-1 (TSST-1). Hepatic MT was induced by TSST-1 in a dose-dependent manner from 100 μg/kg through 3 mg/kg in CF-1 mice, and by 6 h the induction was almost maximal. The increase of hepatic MT occurred at the mRNA level, also, and both MT-I and II mRNAs increased coordinately. Because TSST-1 is a superantigen, it was investigated whether TSST-1 induces MT through cytokines as a consequences of immunostimulation. In low-cytokine-producing mice (C3H/HeJ), up to a dose of 1 mg/kg of TSST-1, there was only 2- to 3-fold increase of hepatic MT. In contrast, in normal-cytokine-producing mice (C3Heb/FeJ), TSST-1 increased MT in a dose-dependent manner, and at a dose of 1 mg/kg, there was a 25-fold increase in hepatic MT. This suggests that activation of the immune system is probably involved in the induction of MT by TSST-1. Studies on the role of specific hepatic cytokines (IL-1, TNF-α, and IL-6) in TSST-mediated hepatic MT induction showed that TSST-1 did not increase hepatic IL-1 or TNF-α significantly over controls in any of the mouse strains studied. In contrast, TSST-1 induced hepatic IL-6 in all three strains of mice. However, in CF-1 and C3Heb/FeJ mice (normal-cytokine-producing) IL-6 induction preceded MT mRNA induction, but in C3H/HeJ mice (low-cytokine-producing), IL-6 induction did not precede MT and mRNA induction.     

Effects of Th2 cytokines on chemokine expression in the lung: IL-13 potently induces eotaxin expression by airway epithelial cells

Airway inflammation associated with asthma is characterized by massive infiltration of eosinophils, mediated in part by specific chemoattractant factors produced in the lung. Allergen-specific Th2 cells appear to play a central role in asthma; for example, adoptively transferred Th2 cells induced lung eosinophilia associated with induction of specific chemokines. Interestingly, Th2 supernatant alone administered intranasally to naive mice induced eotaxin, RANTES, monocyte-chemotactic protein-1, and KC expression along with lung eosinophilia. We tested the major cytokines individually and found that IL-4 and IL-5 induced higher levels of macrophage-inflammatory protein-1alpha and KC; IL-4 also increased the production of monocyte-chemotactic protein-1; IL-13 and IL-4 induced eotaxin. IL-13 was by far the most potent inducer of eotaxin; indeed, a neutralizing anti-IL-13 Ab removed most of the eotaxin-inducing activity from Th2 supernatants, although it did not entirely block the recruitment of eosinophils. While TNF-alpha did not stimulate eotaxin production by itself, it markedly augmented eotaxin induction by IL-13. IL-13 was able to induce eotaxin in the lung of JAK3-deficient mice, suggesting that JAK3 is not required for IL-13 signaling in airway epithelial cells; however, eosinophilia was not induced in this situation, suggesting that JAK3 transduces other IL-13-mediated mechanisms critical for eosinophil recruitment. Our study suggests that IL-13 is an important mediator in the pathogenesis of asthma and therefore a potential target for asthma therapy.     

Rapid vascular responses to anthrax lethal toxin in mice containing a segment of chromosome 11 from the CAST/Ei strain on a C57BL/6 genetic background

Host allelic variation controls the response to B. anthracis and the disease course of anthrax. Mouse strains with macrophages that are responsive to anthrax lethal toxin (LT) show resistance to infection while mouse strains with LT non-responsive macrophages succumb more readily. B6.CAST.11M mice have a region of chromosome 11 from the CAST/Ei strain (a LT responsive strain) introgressed onto a LT non-responsive C57BL/6J genetic background. Previously, B6.CAST.11M mice were found to exhibit a rapid inflammatory reaction to LT termed the early response phenotype (ERP), and displayed greater resistance to B. anthracis infection compared to C57BL/6J mice. Several ERP features (e.g., bloat, hypothermia, labored breathing, dilated pinnae vessels) suggested vascular involvement. To test this, Evan's blue was used to assess vessel leakage and intravital microscopy was used to monitor microvascular blood flow. Increased vascular leakage was observed in lungs of B6.CAST.11M mice compared to C57BL/6J mice 1 hour after systemic administration of LT. Capillary blood flow was reduced in the small intestine mesentery without concomitant leukocyte emigration following systemic or topical application of LT, the latter suggesting a localized tissue mechanism in this response. Since LT activates the Nlrp1b inflammasome in B6.CAST.11M mice, the roles of inflammasome products, IL-1β and IL-18, were examined. Topical application to the mesentery of IL-1β but not IL-18 revealed pronounced slowing of blood flow in B6.CAST.11M mice that was not present in C57BL/6J mice. A neutralizing anti-IL-1β antibody suppressed the slowing of blood flow induced by LT, indicating a role for IL-1β in the response. Besides allelic differences controlling Nlrp1b inflammasome activation by LT observed previously, evidence presented here suggests that an additional genetic determinant(s) could regulate the vascular response to IL-1β. These results demonstrate that vessel leakage and alterations to blood flow are part of the rapid response in mice resistant to B. anthracis infection.     

Role of 5-lipoxygenase in IL-13-induced pulmonary inflammation and remodeling

Exaggerated levels of IL-13 and leukotriene (LT) pathway activation frequently coexist at sites of Th2 inflammation and in tissue fibrotic responses. However, the relationship(s) between the IL-13 and LTs in these responses have not been defined. We hypothesized that the 5-lipoxygenase (5-LO) pathway of LT metabolism plays an important role in the pathogenesis of IL-13-induced chronic inflammation and remodeling. To test this hypothesis, we evaluated the effects of IL-13 on components of the 5-LO metabolic and activation pathways. We also compared the effects of transgenic IL-13 in C57BL/6 mice with wild-type and null 5-LO genetic loci. These studies demonstrate that IL-13 increases the levels of mRNA encoding cytosolic phospholipase A(2), LTA(4) hydrolase, and 5-LO-activating protein without altering the expression of 5-LO, LTC(4) synthase, LTB(4) receptors 1 and 2, and cysteinyl-LT receptors 1 and 2. They also demonstrate that this activation is associated with the enhanced accumulation of LTB(4) but not of cysteinyl-LTs. Furthermore, they demonstrate that this stimulation plays a critical role in the pathogenesis of IL-13-induced inflammation, tissue fibrosis, and respiratory failure-induced death while inhibiting alveolar remodeling. Lastly, mechanistic insights are provided by demonstrating that IL-13-induced 5-LO activation is required for optimal stimulation and activation of TGF-beta(1) and the inhibition of matrix metalloproteinase-12. When viewed in combination, these studies demonstrate that 5-LO plays an important role in IL-13-induced inflammation and remodeling.     

Mildly oxidized low-density lipoprotein inhibits the in vitro induction of the specific antibody response to Candida albicans

Oxidized low-density lipoprotein plays a critical role in the pathogenesis of atherosclerosis and exerts pleiotropic effects on various cellular functions. The present study was designed to evaluate the effects of mildly oxidized LDL (mLDL) on the induction and regulation of an in vitro specific antibody response. We found that mLDL significantly inhibited the induction of the anti-Candida albicans antibody response by human peripheral blood mononuclear cells (PBMC). mLDL-induced down-regulation of antibody production was abrogated by blocking the major receptors that bind and internalize modified LDL. In the mLDL-treated C. albicans-stimulated PBMC cultures an early increase in IL-1beta production was observed and the addition of anti-IL-1beta antibody abrogated the mLDL-induced inhibitory effect. Moreover, the addition of IL-1beta to the cultures inhibited the induction of the specific antibody response, similar to mLDL. On the other hand, mLDL up-regulated PWM-induced polyclonal immunoglobulin (Ig) production. In the same cultures IgM anti-mLDL was found. These results indicate that the up-regulation of IL-1beta production induced by mLDL may be involved in the hindering of B cell function, i.e., specific antibody production. This could be relevant in the pathogenesis of inflammatory diseases such as atherosclerosis.     

Rotavirus 2/6 virus-like particles administered intranasally in mice, with or without the mucosal adjuvants cholera toxin and Escherichia coli heat-labile toxin, induce a Th1/Th2-like immune response

We investigated the rotavirus-specific lymphocyte responses induced by intranasal immunization of adult BALB/c mice with rotavirus 2/6 virus-like particles (2/6-VLPs) of the bovine RF strain, by assessing the profile of cytokines produced after in vitro restimulation and serum and fecal antibody responses. The cytokines produced by splenic cells were first evaluated. Intranasal immunization with 50 microg of 2/6-VLPs induced a high serum antibody response, including immunoglobulin G1 (IgG1) and IgG2a, a weak fecal antibody response, and a mixed Th1/Th2-like profile of cytokines characterized by gamma interferon and interleukin 10 (IL-10) production and very low levels of IL-2, IL-4, and IL-5. Intranasal immunization with 10 microg of 2/6-VLPs coadministered with the mucosal adjuvants cholera toxin and Escherichia coli heat-labile toxin (LT) considerably enhanced the Th1/Th2-like response; notably, significant levels of IL-2, IL-4, and IL-5 were observed. Since rotavirus is an enteric pathogen, we next investigated the production of IL-2 and IL-5, as being representative of Th1 and Th2 responses, by Peyer's patch and mesenteric lymph node cells from mice immunized intranasally with 2/6-VLPs and LT. The results were compared to those obtained from splenic and cervical lymph node cells. We found that both cytokines were produced by cells from each of these lymphoid tissues. These results confirm the Th1/Th2-like response observed at the systemic level and show, on the assumption that T cells are the primary cells producing the cytokines after in vitro restimulation, that rotavirus-specific T lymphocytes are present in the intestine after intranasal immunization with 2/6-VLPs and LT.     

Divergent regulation of the murine CC chemokine C10 by Th(1) and Th(2) cytokines

Chemokines are typically found as products of acute stimulation of host defence cells. In contrast, the mouse CC chemokine C10 was previously shown to be a delayed, stably induced product of macrophages treated with interleukin 3 (IL-3), IL-4 or GM-CSF. We investigated the possibility that C10 is differentially regulated by cytokines associated with Th(1)and Th(2)cells. Northern blot analysis of bone marrow-derived macrophages showed that, in addition to IL-4, the Th(2)-specific cytokines IL-10 and IL-13 upregulated C10 over a 48-h period in a dose-dependent manner. In contrast, MIP-1alpha and MCP-1/JE were induced by IL-3 or GM-CSF at 48 h and this induction was inhibited by IL-4. Interferon gamma, a Th(1)-specific product, abolished the induction of C10 mRNA and protein by either IL-3 or granulocyte-macrophage colony-stimulating factor (GM-CSF) in either bone marrow-derived or peritoneal macrophages. The inhibition of C10 production by interferon gamma was not NO dependent. Finally the GM-CSF-mediated induction of C10 in peritoneal macrophages was eliminated when these cells presented antigen to established T cells of Th(1)phenotype. The findings are consistent with a potential role for C10 in the modulation of immune reactions of Th(2)type.     

The network of hemopoietic regulatory proteins in myeloid cell differentiation.

There are clones of myeloid leukemic cells that can be induced to undergo terminal cell differentiation to macrophages by normal hemopoietic regulatory proteins. Induction of differentiation in two different clones of myeloid leukemic cells with interleukin 6 (IL-6) or granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in induction of mRNA for the hemopoietic regulatory proteins IL-6, GM-CSF, interleukin 1 alpha and interleukin 1 beta, tumor necrosis factor, and transforming growth factor beta 1. In one of these clones, induction of differentiation with GM-CSF was also associated with induction of mRNA for macrophage colony-stimulating factor (M-CSF) but not for the receptor for M-CSF (c-fms), whereas in the other clone, induction of differentiation with IL-6 was associated with induction of mRNA for both c-fms and M-CSF. The clones also differed in their responsiveness to these regulators. There was no induction of mRNA for granulocyte colony-stimulating factor or interleukin 3 during differentiation of either clone. The results indicate that the genes for a nearly normal network of positive and negative hemopoietic regulatory proteins are induced during differentiation of these myeloid leukemic cells and that there are leukemic clones with specific defects in this network.     

Interplay of cytokines and adjuvants in the regulation of mucosal and systemic HIV-specific CTL

We examined the interplay between cytokines and adjuvants to optimize the induction of CTL by a mucosal HIV peptide vaccine. We show synergy between IL-12 and GM-CSF when administered together with the HIV peptide PCLUS3-18IIIB and cholera toxin (CT) in the induction of CTL activity and protection against mucosal viral transmission. Further, we examine the efficacy of mutant Escherichia coli labile toxin, LT(R192G), as a less toxic adjuvant than CT. LT(R192G) was as effective as or more effective than CT at inducing a mucosal CTL response. Moreover, LT(R192G) was as effective without IL-12 as CT was when combined with IL-12, and the response elicited by LT(R192G) with the vaccine was not further enhanced by the addition of IL-12. GM-CSF synergized with LT(R192G) without exogenous IL-12. Therefore, LT(R192G) may induce a more favorable cytokine response by not inhibiting IL-12 production. In particular, less IL-4 is made after LT(R192G) than CT immunization, and the response is less susceptible to anti-IL-12 inhibition. Thus, the choice of mucosal adjuvant affects the cytokine environment, and the mucosal response and protection can be enhanced by manipulating the cytokine environment with synergistic cytokine combinations incorporated in the vaccine.     

Activation of cultured human endothelial cells by recombinant lymphotoxin: comparison with tumor necrosis factor and interleukin 1 species

Recombinant human lymphotoxin (LT) was compared with recombinant human tumor necrosis factor (TNF) for direct actions on cultured human endothelial cells (HEC). At equivalent half-maximal concentrations (based on L929 cytotoxicity units) LT and TNF each caused rapid and transient induction (peak 4 to 6 hr) of an antigen associated with leukocyte adhesion (detected by monoclonal antibody H4/18), a rapid but sustained increased expression (plateau 24 hr) of a lymphocyte adhesion structure (ICAM-1), a gradual (plateau 4 to 6 days) increase in expression of HLA-A,B antigens, and gradual (4 to 6 days) conversion of HEC culture morphology from epithelioid to fibroblastoid, an effect enhanced by immune interferon (IFN-gamma). Induction of H4/18 binding by maximal concentrations of LT or TNF could not be augmented by addition of the other cytokine, and 24 hr pretreatment with LT or TNF produced hyporesponsiveness to both mediators for reinduction. H4/18 binding can be transiently induced by tumor-promoting phorbol esters. Pretreatment with either LT or TNF also fully inhibited induction of H4/18 binding by phorbol ester, whereas phorbol ester pretreatment only variably and partially inhibited reinduction by LT or TNF. These actions of LT on endothelium shared with TNF may serve in vivo to promote lymphocyte and inflammatory leukocyte adhesion and transendothelial migration. Recombinant human interleukin 1 species (IL 1 alpha and IL 1 beta) shared many of the actions of LT and TNF and were indistinguishable from each other. However, IL 1 species could be distinguished from LT/TNF by their relative inability to enhance HLA-A,B expression, by their ability to augment H4/18 binding caused by maximally effective concentrations of LT or TNF, and by their inability to inhibit reinduction of H4/18 binding by LT or TNF. In contrast to the actions of LT or TNF, pretreatment with IL 1 alpha or IL 1 beta only partially inhibited induction of H4/18 binding by phorbol ester, and phorbol ester pretreatment consistently, albeit partially, inhibited induction by IL 1 species. These studies suggest that activated T cells through the secretion of LT can in turn activate the local endothelial lining so as to promote homing and extravasation of inflammatory cells. Furthermore, these LT actions can be augmented or complemented by other locally produced mediators such as IFN-gamma or IL 1.     

IL-6 induces NF-kappa B activation in the intestinal epithelia

IL-6 is a potent proinflammatory cytokine that has been shown to play an important role in the pathogenesis of inflammatory bowel disease (IBD). It is classically known to activate gene expression via the STAT-3 pathway. Given the crucial role of IL-6 in the pathogenesis of chronic intestinal inflammation, it is not known whether IL-6 activates NF-kappaB, a central mediator of intestinal inflammation. The model intestinal epithelial cell line, Caco2-BBE, was used to study IL-6 signaling and to analyze whether suppressor of cytokine signaling 3 (SOCS-3) proteins play a role in the negative regulation of IL-6 signaling. We show that IL-6 receptors are present in intestinal epithelia in a polarized fashion. Basolateral IL-6 and, to a lesser extent, apical IL-6 induces the activation of the NF-kappaB pathway. Basolateral IL-6 stimulation results in a maximal induction of NF-kappaB activation and NF-kappaB nuclear translocation at 2 h. IL-6 induces polarized expression of ICAM-1, an adhesion molecule shown to be important in the neutrophil-epithelial interactions in IBD. Using various deletion constructs of ICAM-1 promoter, we show that ICAM-1 induction by IL-6 requires the activation of NF-kappaB. We also demonstrate that overexpression of SOCS-3, a protein known to inhibit STAT activation in response to IL-6, down-regulates IL-6-induced NF-kappaB activation and ICAM-1 expression. In summary, we demonstrate the activation of NF-kappaB by IL-6 in intestinal epithelia and the down-regulation of NF-kappaB induction by SOCS-3. These data may have mechanistic and therapeutic implications in diseases such as IBD and rheumatoid arthritis in which IL-6 plays an important role in the pathogenesis.