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The larval stage of the cestode Echinococcus granulosus causes cystic echinococcosis in humans and livestock. This larva is protected by the millimeter-thick, mucin-based laminated layer (LL), from which materials have to be shed to allow parasite growth. We previously reported that dendritic cells (DCs) respond to microscopic pieces of the mucin gel of the LL (pLL) with unconventional maturation phenotypes, in the absence or presence of Toll-like receptor (TLR) agonists, including lipopolysaccharide (LPS). We also reported that the presence of pLL inhibited the activating phosphorylation of the phosphatidylinositol 3-kinase (PI3K) effector Akt induced by granulocyte-macrophage colony-stimulating factor or interleukin-4. We now show that the inhibitory effect of pLL extends to LPS as a PI3K activator, and results in diminished phosphorylation of GSK3 downstream from Akt. Functionally, the inhibition of Akt and GSK3 phosphorylation are linked to the blunted upregulation of CD40, a major feature of the unconventional maturation phenotype. Paradoxically, all aspects of unconventional maturation induced by pLL depend on PI3K class I. Additional components of the phagocytic machinery are needed, but phagocytosis of pLL particles is not required. These observations hint at a DC response mechanism related to receptor-independent mechanisms proposed for certain crystalline and synthetic polymer-based particles; this would fit the previously reported lack of detection of molecular-level motifs necessary of the effects of pLL on DCs. Finally, we report that DCs exposed to pLL are able to condition DCs not exposed to the material so that these cannot upregulate CD40 in full in response to LPS., (Copyright © 2019 Pittini et al.)

Toxoplasma gondii is an obligate intracellular parasite that can cause severe neurological disease in infected humans. CD40 is a receptor on macrophages that plays a critical role in controlling T. gondii infection. We examined the regulation of CD40 on the surface of T. gondii-infected bone marrow-derived macrophages (BMdMs). T. gondii induced CD40 expression both at the transcript level and on the cell surface, and interestingly, the effect was parasite strain specific: CD40 levels were dramatically increased in type II T. gondii-infected BMdMs compared to type I- or type III-infected cells. Type II induction of CD40 was specific to cells harboring intracellular parasites and detectable as early as 6 h postinfection (hpi) at the transcript level. CD40 protein expression peaked at 18 hpi. Using forward genetics with progeny from a type II × type III cross, we found that CD40 induction mapped to a region of chromosome X that included the gene encoding the dense granule protein 15 (GRA15). Using type I parasites stably expressing the type II allele of GRA15 (GRA15II), we found that type I GRA15II parasites induced the expression of CD40 on infected cells in an NF-κB-dependent manner. In addition, stable expression of hemagglutinin-tagged GRA15II in THP-1 cells resulted in CD40 upregulation in the absence of infection. Since CD40 signaling contributes to interleukin-12 (IL-12) production, we examined IL-12 from infected macrophages and found that CD40L engagement of CD40 amplified the IL-12 response in type II-infected cells. These data indicate that GRA15II induction of CD40 promotes parasite immunity through the production of IL-12., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

A Salmonella vector vaccine expressing the saliva-binding region (SBR) of the adhesin AgI/II of Streptococcus mutans has been shown to induce a mixed Th1/Th2 anti-SBR immune response in mice and to require Toll-like receptor 2 (TLR2), TLR4, and MyD88 signaling for the induction of mucosal anti-SBR antibody responses. Since dendritic cells (DC) are critical in innate and adaptive immunity, the present study assessed the role of SBR expression by the vector vaccine in DC activation. Bone marrow-derived DC from wild-type and TLR2, TLR4, and MyD88 knockout mice were stimulated with Salmonella vector BRD509, the SBR-expressing Salmonella vector vaccine BRD509(pSBRT7), or SBR protein, and the DC responses to different stimuli were compared by assessing costimulatory molecule expression, cytokine production, and signaling pathways. The DC response to both BRD509(pSBRT7) and BRD509 was dependent mainly on TLR4. BRD509(pSBRT7) and BRD509 induced upregulation of CD80, CD86, CD40, and major histocompatibility complex class II (MHC II) expression. Lower levels of interleukin-10 (IL-10) and IL-12p40 were produced by BRD509(pSBRT7)-stimulated DC than by BRD509-stimulated DC. Furthermore, BRD509(pSBRT7)-stimulated DC showed decreased p38 phosphorylation compared to that induced by DC stimulated with BRD509. However, BRD509(pSBRT7)-treated DC produced a higher level of IL-6 than BRD509-stimulated cells. The low IL-12p40 and high IL-6 cytokine profile expressed by BRD509(pSBRT7)-stimulated DC may represent a shift toward a Th2 response, as suggested by the increased expression in Jagged-1. These results provide novel evidence that a heterologous protein expressed by a Salmonella vector vaccine can differentially affect DC activation.

The innate immune system of humans recognizes the human pathogenic fungus Candida albicans via sugar polymers present in the cell wall, such as mannan and beta-glucan. Here, we examined whether nucleic acids from C. albicans activate dendritic cells. C. albicans DNA induced interleukin-12p40 (IL-12p40) production and CD40 expression by murine bone marrow-derived myeloid dendritic cells (BM-DCs) in a dose-dependent manner. BM-DCs that lacked Toll-like receptor 4 (TLR4), TLR2, and dectin-1, which are pattern recognition receptors for fungal cell wall components, produced IL-12p40 at levels comparable to the levels produced by BM-DCs from wild-type mice, and DNA from a C. albicans pmr1Delta null mutant, which has a gross defect in mannosylation, retained the ability to activate BM-DCs. This stimulatory effect disappeared completely after DNase treatment. In contrast, RNase treatment increased production of the cytokine. A similar reduction in cytokine production was observed when BM-DCs from TLR9(-/-) and MyD88(-/-) mice were used. In a luciferase reporter assay, NF-kappaB activation was detected in TLR9-expressing HEK293T cells stimulated with C. albicans DNA. Confocal microscopic analysis showed similar localization of C. albicans DNA and CpG-oligodeoxynucleotide (CpG-ODN) in BM-DCs. Treatment of C. albicans DNA with methylase did not affect its ability to induce IL-12p40 synthesis, whereas the same treatment completely eliminated the ability of CpG-ODN to induce IL-12p40 synthesis. Finally, impaired clearance of this fungal pathogen was not found in the kidneys of TLR9(-/-) mice. These results suggested that C. albicans DNA activated BM-DCs through a TLR9-mediated signaling pathway using a mechanism independent of the unmethylated CpG motif.

Immunization of mice with Plasmodium berghei or Plasmodium yoelii synthetic linear peptide chimeras (LPCs) based on the circumsporozoite protein protects against experimental challenge with viable sporozoites. The immunogenicity of LPCs is significantly enhanced by spontaneous polymerization. To better understand the antigenic properties of polymeric antimalarial peptides, we studied the immune responses elicited in mice immunized with a polymer or a monomer of a linear peptide construct specific for P. yoelii and compared the responses of antigen-presenting cells following incubation with both peptide species. Efficient uptake of the polymeric peptide in vitro resulted in higher expression of the coactivation markers CD80, CD40, and CD70 on dendritic cells and higher proinflammatory cytokine production than with the monomeric peptide. Macropinocytosis seems to be the main route used by polymeric peptides internalized by antigen-presenting cells. Spontaneous polymerization of synthetic antimalarial-peptide constructs to target professional antigen-presenting cells shows promise for simple delivery of subunit malaria vaccines.

The innate and the adaptive arms of the mucosal immune system must be coordinated to facilitate the control of pathogenic invasion while maintaining immune homeostasis. Toll-like receptors, able to activate the cell to produce bactericidal and inflammatory cytokines but also able to upregulate antigen (Ag)-presenting and costimulatory molecules, are particularly important in this regard. We have previously shown that the chronically infected oral mucosa is in a state of endotoxin tolerance, as evidenced by the downregulation of Toll-like receptors 2 and 4 and of inflammatory cytokines and the upregulation of SH2-containing inositol phosphatase, an inhibitor of NF-kappaB signaling. In the present study, we hypothesized that endotoxin tolerance would influence the ability of human macrophages to engage in Ag capture and killing of the oral pathogen Porphyromonas gingivalis and to upregulate costimulatory molecules and stimulate autologous T-cell proliferation. We show that uptake, but not killing, of P. gingivalis 381 is enhanced by endotoxin tolerance. Reduced killing is possibly due to a reduction of the intracellular lysosomes. We further show that the expression of the Ag-presenting molecule HLA-DR and costimulatory molecules CD40 and CD86 is dampened by endotoxin tolerance to the constitutive level. This, along with our previous evidence for reduction in immunostimulatory cytokines, is consistent with the observed decrease in the induction of autologous CD4(+) T-cell proliferation by endotoxin-tolerized macrophages. Overall, these studies suggest that endotoxin tolerance, as observed in the inflamed oral mucosa, potentiates the innate Ag capture activity of macrophages but diminishes the potential of human macrophages to initiate the adaptive immune response. In conclusion, endotoxin tolerance, while helpful in bacterial clearance and in surmounting excessive inflammatory tissue damage, could potentially reduce the (protective) adaptive immune response during chronic infections such as periodontitis.

The structure and function LT-IIa, a type II heat-labile enterotoxin of Escherichia coli, are closely related to the structures and functions of cholera toxin and LT-I, the type I heat-labile enterotoxins of Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. While LT-IIa is a potent systemic and mucosal adjuvant, recent studies demonstrated that mutant LT-IIa(T34I), which exhibits no detectable binding activity as determined by an enzyme-linked immunosorbent assay, with gangliosides GD1b, GD1a, and GM1 is a very poor adjuvant. To evaluate whether other mutant LT-IIa enterotoxins that also exhibit diminished ganglioside-binding activities have greater adjuvant activities, BALB/c mice were immunized by the intranasal route with the surface adhesin protein AgI/II of Streptococcus mutans alone or in combination with LT-IIa, LT-IIa(T14S), LT-IIa(T14I), or LT-IIa(T14D). All three mutant enterotoxins potentiated strong mucosal immune responses that were equivalent to the response promulgated by wt LT-IIa. All three mutant enterotoxins augmented the systemic immune responses that correlated with their ganglioside-binding activities. Only LT-IIa and LT-IIa(T14S), however, enhanced expression of major histocompatibility complex class II and the costimulatory molecules CD40, CD80, and CD86 on splenic dendritic cells. LT-IIa(T14I) and LT-IIa(T14D) had extremely diminished toxicities in a mouse Y1 adrenal cell bioassay and reduced abilities to induce the accumulation of intracellular cyclic AMP in a macrophage cell line.

Helicobacter pylori causes chronic gastric infection that affects the majority of the world's population. Despite generating an inflammatory response, the immune system usually fails to clear the infection. Since dendritic cells (DCs) play a pivotal role in shaping the immune response, we investigated the effects of H. pylori on DC function. We have demonstrated that H. pylori increased the expression of activation markers on DCs while upregulating the inhibitory B7 family molecule, PD-L1. Functionally, H. pylori-treated DCs resulted in the production of interleukin-10 (IL-10) and IL-23 but not of alpha interferon (IFN-alpha). While very little or no IL-12 was produced to H. pylori alone, simultaneous ligation of CD40 on DCs induced IL-12 release. We also demonstrated that DCs treated with H. pylori-induced IFN-gamma production by allogeneic naive T cells. However, stimulation of DCs with H. pylori for an extended period of time impaired their ability to produce cytokines after CD40 ligation and limited their ability to promote IFN-gamma release, suggesting that the DCs had become exhausted by the prolonged stimulation. The effect of chronic infection with H. pylori on DC function was further investigated by focusing on DC development. Demonstrating that monocytes differentiated into DCs in the presence of H. pylori exhibited an exhausted phenotype with an impaired ability to produce IL-12 and a downregulation of CD1a. Our results raise the possibility that in chronic H. pylori infection DCs become exhausted after prolonged antigen exposure leading to suboptimal Th1 development. This effect may contribute to persistence of H. pylori infection.

Human monocytes displayed increased expression of CD40 following infection with virulent Mycobacterium tuberculosis. Nevertheless, soluble CD40 ligand (CD40L; also designated CD154) had no effect on the intracellular growth of the organism. Restriction of the intracellular growth of M. tuberculosis by peripheral blood lymphocytes and antigen-specific CD4+ T-cell lines likewise was not reduced by blocking anti-CD40L monoclonal antibody 5c8.

Arginine-specific cysteine proteinases (gingipains-R) from periodontopathic Porphyromonas gingivalis cleaved CD14, a bacterial pattern recognition receptor, on human gingival fibroblasts (HGF). Consequently, gingipains-R reduced lipopolysaccharide-induced interleukin-8 production by HGF, indicating that gingipains-R inhibited CD14-dependent HGF activation and are involved in immune evasion by the bacterium in periodontal tissues.

Interleukin-10 (IL-10) is associated with inhibition of cell-mediated immunity and downregulation of the expression of costimulatory molecules required for T-cell activation. When IL-10-deficient (IL-10KO) mice are infected with Toxoplasma gondii, they succumb to a T-cell-mediated shock-like reaction characterized by the overproduction of IL-12 and gamma interferon (IFN-gamma) associated with widespread necrosis of the liver. Since costimulation is critical for T-cell activation, we investigated the role of the CD28-B7 and CD40-CD40 ligand (CD40L) interactions in this infection-induced immunopathology. Our studies show that infection of mice with T. gondii resulted in increased expression of B7 and CD40 that was similar in wild-type and IL-10KO mice. In vivo blockade of the CD28-B7 or CD40-CD40L interactions following infection of IL-10KO mice with T. gondii did not affect serum levels of IFN-gamma or IL-12, nor did it prevent death in these mice. However, when both pathways were blocked, the IL-10KO mice survived the acute phase of infection and had reduced serum levels of IFN-gamma and alanine transaminase as well as decreased expression of inducible nitric oxide synthase in the liver and spleen. Analysis of parasite-specific recall responses from infected IL-10KO mice revealed that blockade of the CD40-CD40L interaction had minimal effects on cytokine production, whereas blockade of the CD28-B7 interaction resulted in decreased production of IFN-gamma but not IL-12. Further reduction of IFN-gamma production was observed when both costimulatory pathways were blocked. Together, these results demonstrate that the CD28-B7 and CD40-CD40L interactions are involved in the development of infection-induced immunopathology in the absence of IL-10.

Expression of CD40 ligand (CD40L) correlated directly with Mycobacterium tuberculosis-stimulated gamma interferon (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) from tuberculosis patients and healthy tuberculin reactors. The CD40L agonist increased M. tuberculosis-induced IFN-gamma production by PBMC, and anti-CD40 or anti-CD40L antibodies reduced IFN-gamma production. CD40L expression on PBMC was reduced by exposure to B cells and to soluble factors from M. tuberculosis-infected monocytes. These findings suggest that CD40L dysregulation contributes to reduced IFN-gamma production in human tuberculosis.

Optimal protective immunity against babesial infection is postulated to require both complement-fixing and opsonizing antibodies in addition to gamma interferon (IFN-gamma)-mediated macrophage activation. The rhoptry-associated protein 1 (RAP-1) of Babesia bigemina induces partial protective immunity and is a candidate vaccine antigen. Previous studies demonstrated that cattle immunized with native protein that were subsequently protected against challenge had a strong IFN-gamma and weaker interleukin-4 (IL-4) response in immune lymph node lymphocytes that reflected the cytokine profile of the majority of CD4(+) T-cell clones obtained from peripheral blood. RAP-1-specific T helper (Th) cell clones that coexpress IFN-gamma and IL-4 are typical of numerous parasite-specific clones examined. However, the function of such cells as helper cells to enhance immunoglobulin secretion by bovine B cells has not been reported. In cattle, both immunoglobulin G1 (IgG1) and IgG2 can fix complement, but IgG2 is the superior opsonizing subclass. Therefore, studies were undertaken to ascertain the functional relevance of RAP-1-specific, CD4(+) Th0 cells as helper cells to enhance IgG1 and/or IgG2 production by autologous B lymphocytes. For comparison, Th0 clones specific for the metazoan parasite Fasciola hepatica that expressed relatively more IL-4 than the B. bigemina-specific Th cells were similarly assayed. B. bigemina RAP-1-specific clones could enhance production of both IgG1 and IgG2 by autologous B cells, whereas Th cell clones specific for F. hepatica enhanced predominantly IgG1 production. The capacity to enhance IgG2 production was associated with production of IFN-gamma by Th cells cocultured with B cells, antigen, and IL-2. The in vitro helper T-cell activity of these T-cell clones was representative of the in vivo serologic responses, which were composed of a mixed IgG1-IgG2 response in B. bigemina RAP-1 immune cattle and a biased IgG1 response in F. hepatica-immune cattle.