Peptidoglycan-associated lipoprotein of Aggregatibacter actinomycetemcomitans induces apoptosis and production of proinflammatory cytokines via TLR2 in murine macrophages RAW 264.7 in vitro

Peptidoglycan-associated lipoprotein (PAL) is a conserved pro-inflammatory outer membrane lipoprotein in Gram-negative bacteria. Compared to systemic pathogens, little is known about the virulence properties of PAL in Aggregatibacter actinomycetemcomitans (AaPAL). The aims of this study were to investigate the cytolethality of AaPAL and its ability to induce pro-inflammatory cytokine production in macrophages. Mouse macrophages were stimulated with AaPAL, and the production of IL-1β, IL-6, TNF-α, and MCP-1 was measured after 6, 24, and 48 h. To investigate which receptor AaPAL employs for its interaction with macrophages, anti-toll-like receptor (TLR)2 and anti-TLR4 antibodies were used to block respective TLRs on macrophages. Metabolic activity and apoptosis of the macrophages were investigated after stimulation with AaPAL. AaPAL induced the production of MCP-1, TNF-α, IL-6, and IL-1β from mouse macrophages in order of decreasing abundance. The pre-treatment of macrophages with an anti-TLR2 antibody significantly diminished cytokine production. Under AaPAL stimulation, the metabolic activity of macrophages decreased in a dose- and time-dependent manner. Furthermore, AaPAL induced apoptosis in 56% of macrophages after 48 h of incubation. Our data suggest that AaPAL can kill macrophages by apoptosis. The results also emphasize the role of AaPAL as a potent pro-inflammatory agent in A. actinomycetemcomitans-associated infections.</p

Kinetics of the serological response up to one year after tularemia

Serological analysis is the predominant method used to diagnose tularemia, a zoonotic disease caused by the highly virulent bacterium F. tularensis. We determined F. tularensis-specific IgM and IgG antibody titers by an LPS-based ELISA assay on five occasions one to twelve months after onset of ulceroglandular tularemia in 19 individuals. Peak IgM antibody titers were observed at the one-month time point and peak IgG antibody titers at the two-month time point. Both IgG and IgM antibody levels declined linearly thereafter with rather similar kinetics. Compared to the average one-month antibody titers, average IgG titers were not significantly lower before the 12-month time point and IgM titers before the 4-month time point. All, but one average titer, were significantly increased compared to the cut-off of the assay. Average IgG and IgM titers were significantly lower for the group = 69 years old compared to the group &lt; 69 years. Collectively, the data demonstrate a persistence of F. tularensis-specific IgM and IgG antibody titers for at least 12 months after ulceroglandular tularemia. Thus, low, but significantly elevated F. tularensis-specific antibody titers are of limited diagnostic value since they are not indicative of ongoing tularemia

Vesicle-independent extracellular release of a proinflammatory outer membrane lipoprotein in free-soluble form

<p>Abstract</p> <p>Background</p> <p><it>Aggregatibacter actinomycetemcomitans </it>is an oral bacterium associated with aggressively progressing periodontitis. Extracellular release of bacterial outer membrane proteins has been suggested to mainly occur via outer membrane vesicles. This study investigated the presence and conservation of peptidoglycan-associated lipoprotein (AaPAL) among <it>A. actinomycetemcomitans </it>strains, the immunostimulatory effect of AaPAL, and whether live cells release this structural outer membrane lipoprotein in free-soluble form independent of vesicles.</p> <p>Results</p> <p>The <it>pal </it>locus and its gene product were confirmed in clinical <it>A. actinomycetemcomitans </it>strains by PCR-restriction fragment length polymorphism and immunoblotting. Culturing under different growth conditions revealed no apparent requirement for the AaPAL expression. Inactivation of <it>pal </it>in a wild-type strain (D7S) and in its spontaneous laboratory variant (D7SS) resulted in pleiotropic cellular effects. In a cell culture insert model (filter pore size 0.02 μm), AaPAL was detected from filtrates when strains D7S and D7SS were incubated in serum or broth in the inserts. Electron microscopy showed that <it>A. actinomycetemcomitans </it>vesicles (0.05–0.2 μm) were larger than the filter pores and that there were no vesicles in the filtrates. The filtrates were immunoblot negative for a cytoplasmic marker, cyclic AMP (cAMP) receptor protein. An ex vivo model indicated cytokine production from human whole blood stimulated by AaPAL.</p> <p>Conclusion</p> <p>Free-soluble AaPAL can be extracellularly released in a process independent of vesicles.</p

Vaccine-mediated mechanisms controlling replication of Francisella tularensis in human peripheral blood mononuclear cells using a co-culture system

Cell-mediated immunity (CMI) is normally required for efficient protection against intracellular infections, however, identification of correlates is challenging and they are generally lacking. Francisella tularensis is a highly virulent, facultative intracellular bacterium and CMI is critically required for protection against the pathogen, but how this is effectuated in humans is poorly understood. To understand the protective mechanisms, we established an in vitro co-culture assay to identify how control of infection of F. tularensis is accomplished by human cells and hypothesized that the model will mimic in vivo immune mechanisms. Non-adherent peripheral blood mononuclear cells (PBMCs) were expanded with antigen and added to cultures with adherent PBMC infected with the human vaccine strain, LVS, or the highly virulent SCHU S4 strain. Intracellular numbers of F. tularensis was followed for 72 h and secreted and intracellular cytokines were analyzed. Addition of PBMC expanded from naïve individuals, i.e., those with no record of immunization to F. tularensis, generally resulted in little or no control of intracellular bacterial growth, whereas addition of PBMC from a majority of F. tularensis-immune individuals executed static and sometimes cidal effects on intracellular bacteria. Regardless of infecting strain, statistical differences between the two groups were significant, P &lt; 0.05. Secretion of 11 cytokines was analyzed after 72 h of infection and significant differences with regard to secretion of IFN-γ, TNF, and MIP-1β was observed between immune and naïve individuals for LVS-infected cultures. Also, in LVS-infected cultures, CD4 T cells from vaccinees, but not CD8 T cells, showed significantly higher expression of IFN-γ, MIP-1β, TNF, and CD107a than cells from naïve individuals. The co-culture system appears to identify correlates of immunity that are relevant for the understanding of mechanisms of the protective host immunity to F. tularensis

Analyses of human immune responses to Francisella tularensis identify correlates of protection

Francisella tularensis is the etiological agent of the potentially severe infection tularemia. An existing F: tularensis vaccine, the live vaccine strain (LVS), has been used to protect at-risk personnel, but it is not licensed in any country and it has limited efficacy. Therefore, there is a need of a new, efficacious vaccine. The aim of the study was to perform a detailed analysis of the characteristics of the human immune response to F. tularensis, since this will generate crucial knowledge required to develop new vaccine candidates. Nine individuals were administered the LVS vaccine and peripheral blood mononuclear cells (PBMC) were collected before and at four time points up to one year after vaccination. The properties of the PBMC were characterized by flow cytometry analysis of surface markers and intracellular cytokine staining. In addition, the cytokine content of supernatants from F. tularensis-infected PBMC cultures was determined and the protective properties of the supernatants investigated by adding them to cultures with infected monocyte-derived macrophages (MDM). Unlike before vaccination, PBMC collected at all four time points after vaccination demonstrated F. tularensis-specific cell proliferation, cytokine secretion and cytokine-expressing memory cells. A majority of 17 cytokines were secreted at higher levels by PBMC collected at all time points after vaccination than before vaccination. A discriminative analysis based on IFN-γ and IL-13 secretion correctly classified samples obtained before and after vaccination. Increased expression of IFN-γ, IL-2, and MIP-1β were observed at all time points after vaccination vs. before vaccination and the most significant changes occurred among the CD4 transient memory, CD8 effector memory, and CD8 transient memory T-cell populations. Growth restriction of the highly virulent F. tularensis strain SCHU S4 in MDM was conferred by supernatants and protection correlated to levels of IFN-γ, IL-2, TNF, and IL-17. The findings demonstrate that F. tularensis vaccination induces long-term T-cell reactivity, including TEM and TTM cell populations. Individual cytokine levels correlated with the degree of protection conferred by the supernatants. Identification of such memory T cells and effector mechanisms provide an improved understanding of the protective mechanisms against F. tularensis. mechanisms against F. tularensis

Vaccine-Mediated Mechanisms Controlling Francisella tularensis SCHU S4 Growth in a Rat Co-Culture System

Francisella tularensis causes the severe disease tularemia. In the present study, the aim was to identify correlates of protection in the rat co-culture model by investigating the immune responses using two vaccine candidates conferring distinct degrees of protection in rat and mouse models. The immune responses were characterized by use of splenocytes from naive or Live vaccine strain- (LVS) or clpB/wbtC-immunized Fischer 344 rats as effectors and bone marrow-derived macrophages infected with the highly virulent strain SCHU S4. A complex immune response was elicited, resulting in cytokine secretion, nitric oxide production, and efficient control of the intracellular bacterial growth. Addition of LVS-immune splenocytes elicited a significantly better control of bacterial growth than clpB/wbtC splenocytes. This mirrored the efficacy of the vaccine candidates in the rat model. Lower levels of IFN-gamma, TNF, fractalkine, IL-2, and nitrite were present in the co-cultures with clpB/wbtC splenocytes than in those with splenocytes from LVS-immunized rats. Nitric oxide was found to be a correlate of protection, since the levels inversely correlated to the degree of protection and inhibition of nitric oxide production completely reversed the growth inhibition of SCHU S4. Overall, the results demonstrate that the co-culture assay with rat-derived cells is a suitable model to identify correlates of protection against highly virulent strains of F. tularensi

An In Vitro Co-culture Mouse Model Demonstrates Efficient Vaccine-Mediated Control of Francisella tularensis SCHU S4 and Identifies Nitric Oxide as a Predictor of Efficacy

Francisella tularensis is a highly virulent intracellular bacterium and cell-mediated immunity is critical for protection, but mechanisms of protection against highly virulent variants, such as the prototypic strain F. tularensis strain SCHU S4, are poorly understood. To this end, we established a co-culture system, based on splenocytes from naive, or immunized mice and in vitro infected bone marrow-derived macrophages that allowed assessment of mechanisms controlling infection with F. tularensis. We utilized the system to understand why the clpB gene deletion mutant, Delta clpB, of SCHU S4 shows superior efficacy as a vaccine in the mouse model as compared to the existing human vaccine, the live vaccine strain (LVS). Compared to naive splenocytes, Delta clpB-, or LVS-immune splenocytes conferred very significant control of a SCHU S4 infection and the Delta clpB-immune splenocytes were superior to the LVS-immune splenocytes. Cultures with the Delta clpB-immune splenocytes also contained higher levels of IFN-gamma, IL-17, and GM-CSF and nitric oxide, and T cells expressing combinations of IFN-gamma, TNF-alpha, and IL-17, than did cultures with LVS-immune splenocytes. There was strong inverse correlation between bacterial replication and levels of nitrite, an end product of nitric oxide, and essentially no control was observed when BMDM from iNOS(-/-) mice were infected. Collectively, the co-culture model identified a critical role of nitric oxide for protection against a highly virulent strain of F. tularensis

Guanylate-Binding Proteins Are Critical for Effective Control of Francisella tularensis Strains in a Mouse Co-Culture System of Adaptive Immunity

Francisella tularensis is a Select Agent that causes the severe disease tularemia in humans and many animal species. The bacterium demonstrates rapid intracellular replication, however, macrophages can control its replication if primed and activation with IFN-γ is known to be essential, although alone not sufficient, to mediate such control. To further investigate the mechanisms that control intracellular F. tularensis replication, an in vitro co-culture system was utilized containing splenocytes obtained from naïve or immunized C57BL/6 mice as effectors and infected bone marrow-derived wild-type or chromosome-3-deficient guanylate-binding protein (GBP)-deficient macrophages. Cells were infected either with the F. tularensis live vaccine strain (LVS), the highly virulent SCHU S4 strain, or the surrogate for F. tularensis, F. novicida. Regardless of strain, significant control of the bacterial replication was observed in co-cultures with wild-type macrophages and immune splenocytes, but not in cultures with immune splenocytes and GBPchr3-deficient macrophages. Supernatants demonstrated very distinct, infectious agent-dependent patterns of 23 cytokines, whereas the cytokine patterns were only marginally affected by the presence or absence of GBPs. Levels of a majority of cytokines were inversely correlated to the degree of control of the SCHU S4 and LVS infections, but this was not the case for the F. novicida infection. Collectively, the co-culture assay based on immune mouse-derived splenocytes identified a dominant role of GBPs for the control of intracellular replication of various F. tularensis strains, regardless of their virulence, whereas the cytokine patterns markedly were dependent on the infectious agents, but less so on GBPs

Levels of cytokines secreted by human PBMC after recall stimulation with ffLVS or ffSchu S4 for five days.

<p>Cytokine concentrations were measured in cell culture supernatants using multiplex analysis. Median values ± SEM from PBMC samples of 14–16 individuals per donor group are shown (black bars indicate convalescent patients; grey bars indicate LVS vaccinees; white bars indicate naïve donors). Statistically significant differences between immune and naïve donors are marked by asterisks (<i>P</i><0.05). For IL-7, 36 out of 40 values were below the detection limit and therefore not included in the data analysis.</p

Histograms indicating the frequency of mono-, bi- and tri-functional cell subsets in CD45RO⁺ and CD45RA⁺ T-cell populations of convalescent patients.

<p>Mean values are illustrated throughout. The black axis indicates percentages for mono-functional IFN-γ-positive and all bi- and tri-functional T-cell subsets. The red axis indicates percentages for MIP-1β- and CD107a-positive mono-functional T cell subsets.</p