TLR 9 Activation in Dendritic Cells Enhances Salmonella Killing and Antigen Presentation via Involvement of the Reactive Oxygen Species

Synthetic CpG containing oligodeoxynucleotide Toll like receptor-9 agonist (CpG DNA) activates innate immunity and can stimulate antigen presentation against numerous intracellular pathogens. It was observed that Salmonella Typhimurium growth can be inhibited by the CpG DNA treatment in the murine dendritic cells. This inhibitory effect was mediated by an increased reactive oxygen species production. In addition, it was noted that CpG DNA treatment of dendritic cells during Salmonella infection leads to an increased antigen presentation. Further this increased antigen presentation was dependent on the enhanced reactive oxygen species production elicited by Toll like receptor-9 activation. With the help of an exogenous antigen it was shown that Salmonella antigen could also be cross-presented in a better way by CpG induction. These data collectively indicate that CpG DNA enhance the ability of murine dendritic cells to contain the growth of virulent Salmonella through reactive oxygen species dependent killing

Poly-Thymidine Oligonucleotides Mediate Activation of Murine Glial Cells Primarily Through TLR7, Not TLR8

The functional role of murine TLR8 in the inflammatory response of the central nervous system (CNS) remains unclear. Murine TLR8 does not appear to respond to human TLR7/8 agonists, due to a five amino acid deletion in the ectodomain. However, recent studies have suggested that murine TLR8 may be stimulated by alternate ligands, which include vaccinia virus DNA, phosphothioate oligodeoxynucleotides (ODNs) or the combination of phosphothioate poly-thymidine oligonucleotides (pT-ODNs) with TLR7/8 agonists. In the current study, we analyzed the ability of pT-ODNs to induce activation of murine glial cells in the presence or absence of TLR7/8 agonists. We found that TLR7/8 agonists induced the expression of glial cell activation markers and induced the production of multiple proinflammatory cytokines and chemokines in mixed glial cultures. In contrast, pT-ODNs alone induced only low level expression of two cytokines, CCL2 and CXCL10. The combination of pT-ODNs along with TLR7/8 agonists induced a synergistic response with substantially higher levels of proinflammatory cytokines and chemokines compared to CL075. This enhancement was not due to cellular uptake of the agonist, indicating that the pT-ODN enhancement of cytokine responses was due to effects on an intracellular process. Interestingly, this response was also not due to synergistic stimulation of both TLR7 and TLR8, as the loss of TLR7 abolished the activation of glial cells and cytokine production. Thus, pT-ODNs act in synergy with TLR7/8 agonists to induce strong TLR7-dependent cytokine production in glial cells, suggesting that the combination of pT-ODNs with TLR7 agonists may be a useful mechanism to induce pronounced glial activation in the CNS

GM-CSF-Producing Th Cells in Rats Sensitive and Resistant to Experimental Autoimmune Encephalomyelitis

Given that granulocyte macrophage colony-stimulating factor (GM-CSF) is identified as the key factor to endow auto-reactive Th cells with the potential to induce neuroinflammation in experimental autoimmune encephalomyelitis (EAE) models, the frequency and phenotype of GM-CSF-producing (GM-CSF+) Th cells in draining lymph nodes (dLNs) and spinal cord (SC) of Albino Oxford (AO) and Dark Agouti (DA) rats immunized for EAE were examined. The generation of neuroantigen-specific GM-CSF+ Th lymphocytes was impaired in dLNs of AO rats (relatively resistant to EAE induction) compared with their DA counterparts (susceptible to EAE) reflecting impaired CD4+ lymphocyte proliferation and less supportive of GM-CSF+ Th cell differentiation dLN cytokine microenvironment. Immunophenotyping of GM-CSF+ Th cells showed their phenotypic heterogeneity in both strains and revealed lower frequency of IL-17+ IFN-gamma+, IL-17+ IFN-gamma-, and IL-17-IFN-gamma+ cells accompanied by higher frequency of IL-17-IFN-gamma- cells among them in AO than in DA rats. Compared with DA, in AO rats was also found (i) slightly lower surface density of CCR2 (drives accumulation of highly pathogenic GM-CSF+ IFN-gamma+ Th17 cells in SC) on GM-CSF+ IFN-gamma+ Th17 lymphocytes from dLNs, and (ii) diminished CCL2 mRNA expression in SC tissue, suggesting their impaired migration into the SC. Moreover, dLN and SC cytokine environments in AO rats were shown to be less supportive of GM-CSF+ IFN-gamma+ Th17 cell differentiation (judging by lower expression of mRNAs for IL-1 beta, IL-6 and IL-23/p19). In accordance with the (i) lower frequency of GM-CSF+ Th cells in dLNs and SC of AO rats and their lower GM-CSF production, and (ii) impaired CCL2 expression in the SC tissue, the proportion of proinflammatory monocytes among peripheral blood cells and their progeny (CD45(hi) cells) among the SC CD11b+ cells were reduced in AO compared with DA rats. Collectively, the results indicate that the strain specificities in efficacy of several mechanisms controlling (auto) reactive CD4+ lymphocyte expansion/differentiation into the cells with pathogenic phenotype and migration of the latter to the SC contribute to AO rat resistance to EAE

Immune Evasion by Yersinia enterocolitica: Differential Targeting of Dendritic Cell Subpopulations In Vivo

CD4+ T cells are essential for the control of Yersinia enterocolitica (Ye) infection in mice. Ye can inhibit dendritic cell (DC) antigen uptake and degradation, maturation and subsequently T-cell activation in vitro. Here we investigated the effects of Ye infection on splenic DCs and T-cell proliferation in an experimental mouse infection model. We found that OVA-specific CD4+ T cells had a reduced potential to proliferate when stimulated with OVA after infection with Ye compared to control mice. Additionally, proliferation of OVA-specific CD4+ T cells was markedly reduced when cultured with splenic CD8α+ DCs from Ye infected mice in the presence of OVA. In contrast, T-cell proliferation was not impaired in cultures with CD4+ or CD4−CD8α− DCs isolated from Ye infected mice. However, OVA uptake and degradation as well as cytokine production were impaired in CD8α+ DCs, but not in CD4+ and CD4−CD8α− DCs after Ye infection. Pathogenicity factors (Yops) from Ye were most frequently injected into CD8α+ DCs, resulting in less MHC class II and CD86 expression than on non-injected CD8α+ DCs. Three days post infection with Ye the number of splenic CD8α+ and CD4+ DCs was reduced by 50% and 90%, respectively. The decreased number of DC subsets, which was dependent on TLR4 and TRIF signaling, was the result of a faster proliferation and suppressed de novo DC generation. Together, we show that Ye infection negatively regulates the stimulatory capacity of some but not all splenic DC subpopulations in vivo. This leads to differential antigen uptake and degradation, cytokine production, cell loss, and cell death rates in various DC subpopulations. The data suggest that these effects might be caused directly by injection of Yops into DCs and indirectly by affecting the homeostasis of CD4+ and CD8α+ DCs. These events may contribute to reduced T-cell proliferation and immune evasion of Ye

HIV/SIV Infection Primes Monocytes and Dendritic Cells for Apoptosis

Subversion or exacerbation of antigen-presenting cells (APC) death modulates host/pathogen equilibrium. We demonstrated during in vitro differentiation of monocyte-derived macrophages and monocyte-derived dendritic cells (DCs) that HIV sensitizes the cells to undergo apoptosis in response to TRAIL and FasL, respectively. In addition, we found that HIV-1 increased the levels of pro-apoptotic Bax and Bak molecules and decreased the levels of anti-apoptotic Mcl-1 and FLIP proteins. To assess the relevance of these observations in the context of an experimental model of HIV infection, we investigated the death of APC during pathogenic SIV-infection in rhesus macaques (RMs). We demonstrated increased apoptosis, during the acute phase, of both peripheral blood DCs and monocytes (CD14+) from SIV+RMs, associated with a dysregulation in the balance of pro- and anti-apoptotic molecules. Caspase-inhibitor and death receptors antagonists prevented apoptosis of APCs from SIV+RMs. Furthermore, increased levels of FasL in the sera of pathogenic SIV+RMs were detected, compared to non-pathogenic SIV infection of African green monkey. We suggest that inappropriate apoptosis of antigen-presenting cells may contribute to dysregulation of cellular immunity early in the process of HIV/SIV infection

Coordinated Regulation of Virulence during Systemic Infection of Salmonella enterica Serovar Typhimurium

To cause a systemic infection, Salmonella must respond to many environmental cues during mouse infection and express specific subsets of genes in a temporal and spatial manner, but the regulatory pathways are poorly established. To unravel how micro-environmental signals are processed and integrated into coordinated action, we constructed in-frame non-polar deletions of 83 regulators inferred to play a role in Salmonella enteriditis Typhimurium (STM) virulence and tested them in three virulence assays (intraperitoneal [i.p.], and intragastric [i.g.] infection in BALB/c mice, and persistence in 129X1/SvJ mice). Overall, 35 regulators were identified whose absence attenuated virulence in at least one assay, and of those, 14 regulators were required for systemic mouse infection, the most stringent virulence assay. As a first step towards understanding the interplay between a pathogen and its host from a systems biology standpoint, we focused on these 14 genes. Transcriptional profiles were obtained for deletions of each of these 14 regulators grown under four different environmental conditions. These results, as well as publicly available transcriptional profiles, were analyzed using both network inference and cluster analysis algorithms. The analysis predicts a regulatory network in which all 14 regulators control the same set of genes necessary for Salmonella to cause systemic infection. We tested the regulatory model by expressing a subset of the regulators in trans and monitoring transcription of 7 known virulence factors located within Salmonella pathogenicity island 2 (SPI-2). These experiments validated the regulatory model and showed that the response regulator SsrB and the MarR type regulator, SlyA, are the terminal regulators in a cascade that integrates multiple signals. Furthermore, experiments to demonstrate epistatic relationships showed that SsrB can replace SlyA and, in some cases, SlyA can replace SsrB for expression of SPI-2 encoded virulence factors

Lymph-borne CD8α+ dendritic cells are uniquely able to cross-prime CD8+ T cells with antigen acquired from intestinal epithelial cells

Cross-presentation of cellular antigens is crucial for priming CD8<sup>+</sup> T cells, and generating immunity to intracellular pathogens—particularly viruses. It is unclear which intestinal phagocytes perform this function in vivo. To address this, we examined dendritic cells (DCs) from the intestinal lymph of IFABP-tOVA 232-4 mice, which express ovalbumin in small intestinal epithelial cells (IECs). Among lymph DCs (LDCs) only CD103<sup>+</sup> CD11b<sup>−</sup> CD8α<sup>+</sup> DCs cross-present IEC-derived ovalbumin to CD8<sup>+</sup> OT-I T cells. Similarly, in the mesenteric lymph nodes (MLNs), cross-presentation of IEC–ovalbumin was limited to the CD11c<sup>+</sup> MHCII<sup>hi</sup> CD8α<sup>+</sup> migratory DCs, but absent from all other subsets, including the resident CD8α<sup>hi</sup> DCs. Crucially, delivery of purified CD8α<sup>+</sup> LDCs, but not other LDC subsets, into the MLN subcapsular lymphatic sinus induced proliferation of ovalbumin-specific, gut-tropic CD8<sup>+</sup> T cells <i>in vivo</i>. Finally, in 232-4 mice treated with R848, CD8α<sup>+</sup> LDCs were uniquely able to cross-prime interferon γ-producing CD8<sup>+</sup>T cells and drive their migration to the intestine. Our results clearly demonstrate that migrating CD8α<sup>+</sup> intestinal DCs are indispensable for cross-presentation of cellular antigens and, in conditions of inflammation, for the initial differentiation of effector CD8<sup>+</sup> T cells. They may therefore represent an important target for the development of antiviral vaccinations

Subsets of intestinal dendritic cells and their role in orally-induced immune responses

Vaccination is the most effective means of preventing infectious diseases and improving global health. However, few vaccines have successfully been developed for protection at mucosal surfaces where most infectious pathogens enter our body. One major reason for this is the lack of adjuvants, immune enhancing agents, that can be administered together with the vaccine. The enterotoxin cholera toxin (CT) is a potent mucosal adjuvant but the toxicity precludes its use in humans. Derivatives of enterotoxins with reduced toxicity are today the most promising candidates for safe and efficient oral adjuvants. However, the underlying mechanisms for the adjuvant activity of enterotoxins are still not fully known. Dendritic cells (DCs) are immune cells that sense the microenvironment and confer T cells with ability to help B cells differentiate into antibody-producing plasma cells, necessary for vaccine-induced protection. Intestinal DCs are important both for immunity and tolerance. However, intestinal DCs constitute a heterogeneous population of cells. The function of intestinal DC subsets therefore needs to be defined further to understand how these contribute to tolerance under steady state and to induce immunity during infection or following oral immunization. In this thesis the role of intestinal DC subsets, in the induction of immune responses following oral administration of antigen, with or without CT as adjuvant, was elucidated. This was done after developing a microsurgical technique in mice that by cannulation of lymphatic vessels allows the direct collection of DCs that exit the intestine under steady state and following vaccination. This technique was combined with the use of genetically modified mice 1) in which DCs can be ablated; 2) that lack specific DC subsets; 3) that are deficient in intracellular signaling pathways in DCs or in other immune cells or 4) that lack CD47, a surface receptor known to influence cell migration. In the thesis we demonstrate the requirement of cDCs for the activation of antigen-specific T cells and the generation of antigen-specific antibodies following oral immunization when using limiting doses of antigen and CT as an adjuvant. In addition, we show in vivo that intact signaling through Gsα specifically in cDCs is essential for the oral adjuvant activity of CT. Using the cannulation technique we show that four subsets of DCs migrate from the intestine under steady state and following oral immunization. Selectively the CD11b-CD8+ subset does not show signs of activation after oral CT and this subset was also found to be dispensable for the generation of antigen-specific intestinal antibodies using this adjuvant. The necessity for CD11b+CD8- cDCs could not be establish in CD47 deficient mice, although these mice display significant reduction of this subset in intestinal tissues. Rather, expression of CD47 by non-hematopoietic cells is pivotal for intestinal antibody generation after oral immunization. Finally, signaling pathways involved in CT’s adjuvanticity were addressed and shown to be independent of classical TLR-signaling. Moreover, caspase 1/11 activity was not necessary for the generation of antigen-specific serum IgG but for intestinal IgA following oral immunization with CT. In conclusion, we have shown a requirement for cDCs and an intact signaling specifically in these cells for the oral adjuvant activity of CT. Furthermore we have identified that the generation of intestinal and systemic antibodies following oral immunization with CT are differentially regulated. These results may therefore have important implications for the development of improved oral vaccines

Relationships between distinct blood monocyte subsets and migrating intestinal lymph dendritic cells in vivo under steady-state conditions.

The origins of dendritic cells (DCs) are poorly understood. In inflammation, DCs can arise from blood monocytes (M(O)s), but their steady-state origin may differ, as shown for Langerhans cells. Two main subsets of M(O)s, defined by expression of different chemokine receptors, CCR2 and CX(3)CR1, have been described in mice and humans. Recent studies have identified the inflammatory function of CCR2(high)CX(3)CR1(low) M(O)s but have not defined unambiguously the origin and fate of CCR2(low)CX(3)CR1(high) cells. In this study, we show that rat M(O)s can also be divided into CCR2(high)CX(3)CR1(low)(CD43(low)) and CCR2(low)CX(3)CR1(high)(CD43(high)) subsets with distinct migratory properties in vivo. Using whole body perfusion to obtain M(O)s, including the marginating pool, we show by adoptive transfer that CD43(low) M(O)s can differentiate into CD43(high) M(O)s in blood without cell division. By adoptive transfer of blood M(O)s followed by collection of pseudoafferent lymph, we show for the first time that a small proportion of intestinal lymph DCs are derived from CCR2(low)CX(3)CR1(high)(CD43(high)) blood M(O)s in vivo under steady-state conditions. This study confirms one of the possible origins of CCR2(low)CX(3)CR1(high) blood M(O)s and indicate that they may contribute to migratory intestinal DCs in vivo in the absence of inflammatory stimuli