Sing Sing Way

Porphyromonas gingivalis Lipopolysaccharide Contains Multiple Lipid A Species That Functionally Interact with Both Toll-Like Receptors 2 and 4

ABSTRACT The innate host response to lipopolysaccharide (LPS) obtained from Porphyromonas gingivalis is unusual in that different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) as well as an antagonist or agonist for TLR4. In this report it is shown that P. gingivalis LPS is highly heterogeneous, containing more lipid A species than previously described. In addition, purification of LPS can preferentially fractionate these lipid A species. It is shown that an LPS preparation enriched for lipid A species at m/z 1,435 and 1,450 activates human and mouse TLR2, TLR2 plus TLR1, and TLR4 in transiently transfected HEK 293 cells coexpressing membrane-associated CD14. The HEK cell experiments further demonstrated that cofactor MD-2 was required for functional engagement of TLR4 but not of TLR2 nor TLR2 plus TLR1. In addition, serum-soluble CD14 effectively transferred P. gingivalis LPS to TLR2 plus TLR1, but poorly to TLR4. Importantly, bone marrow cells obtained from TLR2−/− and TLR4−/− mice also responded to P. gingivalis LPS in a manor consistent with the HEK results, demonstrating that P. gingivalis LPS can utilize both TLR2 and TLR4. No response was observed from bone marrow cells obtained from TLR2 and TLR4 double-knockout mice, demonstrating that P. gingivalis LPS activation occurred exclusively through either TLR2 or TLR4. Although the biological significance of the different lipid A species found in P. gingivalis LPS preparations is not currently understood, it is proposed that the presence of multiple lipid A species contributes to cell activation through both TLR2 and TLR4.

Characterization of flagellin expression and its role in Listeria monocytogenes infection and immunity

Flagellin is the structural component of flagella produced by many pathogenic bacteria and is a potent proinflammatory molecule that mediates these effects through Toll‐like receptor (TLR) 5. In Listeria monocytogenes (LM), flagellin expression is regulated by temperature and has been described as being shut off at 37°C. In this study, we demonstrate that TLR5‐mediated cell activation and flagellin expression is maintained at 37°C in some laboratory‐adapted strains and in ≈u200a20% of LM clinical isolates. To determine the role of flagellin in LM infection, a targeted mutation in the structural gene for flagellin (flaA) was generated in a parental LM strain that expressed flagellin under all conditions examined. In vitro studies demonstrated that this ΔflaA mutant was (i) non‐motile; (ii) not able to activate TLR5‐transfected HeLa cells; and (iii) induced tumour necrosis factor (TNF)‐α production in ≈u200a50% fewer CD11b+ cells in splenocytes from normal mice compared with the parental strain. However, there was no significant alteration in virulence of the ΔflaA mutant after either intravenous or oral murine infection. Similarly, there was no difference in the generation of LM‐specific CD8 or CD4 T cells after intravenous or oral infection. These data indicate that flagellin is not essential for LM pathogenesis or for the induction of LM‐specific adaptive immune responses in normal mice.

Isolation of Listeria monocytogenes mutants with high‐level in vitro expression of host cytosol‐induced gene products

The facultative intracellular bacterial pathogen Listeria monocytogenes dramatically increases the expression of several key virulence factors upon entry into the host cell cytosol. actA, the protein product of which is required for cell‐to‐cell spread of the bacterium, is expressed at low to undetectable levels in vitro and increases in expression more than 200‐fold after L. monocytogenes escape from the phagosome. To identify bacterial factors that participate in the intracellular induction of actA expression, L. monocytogenes mutants expressing high levels of actA during in vitro growth were selected after chemical mutagenesis. The resulting mutant isolates displayed a wide range of actA expression levels, and many were less sensitive to environmental signals that normally mediate repression of virulence gene expression. Several isolates contained mutations affecting actA gene expression that mapped at least 40 kb outside the PrfA regulon, supporting the existence of additional regulatory factors that contribute to virulence gene expression. Two actA in vitro expression mutants contained novel mutations within PrfA, a key regulator of L. monocytogenes virulence gene expression. PrfA E77K and PrfA G155S mutations resulted in high‐level expression of PrfA‐dependent genes, increased bacterial invasion of epithelial cells and increased virulence in mice. Both prfA mutant strains were significantly less motile than wild‐type L. monocytogenes. These results suggest that, although constitutive activation of PrfA and PrfA‐dependent gene expression may enhance L. monocytogenes virulence, it may conversely hamper the bacteriums ability to compete in environments outside host cells.

Cutting Edge: Protective Cell-Mediated Immunity to Listeria monocytogenes in the Absence of Myeloid Differentiation Factor 88

In addition to their role in triggering innate immune responses, Toll-like receptors are proposed to play a key role in linking the innate and adaptive arms of the immune response. The majority of cellular responses downstream of Toll-like receptors are mediated through the adapter molecule myeloid differentiation factor 88 (MyD88), and mice with a targeted deletion of MyD88 are highly susceptible to bacterial infections, including primary infection with Listeria monocytogenes (LM). In contrast, herein we demonstrate that MyD88-deficient mice have only a modest impairment in their LM-specific CD4 T cell response, and no impairment in their CD8 T cell response following infection with ActA-deficient LM. Furthermore, CD8 T cells from immunized MyD88-deficient mice protected naive recipient mice following adoptive splenocyte transfer, and immunized MyD88-deficient mice were protected from infection with wild-type LM. These results indicate that adaptive immune responses can be generated and provide protective immunity in the absence of MyD88.

IL-12 and Type-I IFN Synergize for IFN-γ Production by CD4 T Cells, Whereas Neither Are Required for IFN-γ Production by CD8 T Cells after Listeria monocytogenes Infection

Differentiation of Ag-specific T cells into IFN-γ producers is essential for protective immunity to intracellular pathogens. In addition to stimulation through the TCR and costimulatory molecules, IFN-γ production is thought to require other inflammatory cytokines. Two such inflammatory cytokines are IL-12 and type I IFN (IFN-I); both can play a role in priming naive T cells to produce IFN-γ in vitro. However, their role in priming Ag-specific T cells for IFN-γ production during experimental infection in vivo is less clear. In this study, we examine the requirements for IL-12 and IFN-I, either individually or in combination, for priming Ag-specific T cell IFN-γ production after Listeria monocytogenes (Lm) infection. Surprisingly, neither individual nor combined defects in IL-12 or IFN-I signaling altered IFN-γ production by Ag-specific CD8 T cells after Lm infection. In contrast, individual defects in either IL-12 or IFN-I signaling conferred partial (∼50%) reductions, whereas combined deficiency in both IL-12 and IFN-I signaling conferred more dramatic (75–95%) reductions in IFN-γ production by Ag-specific CD4 T cells. The additive effects of IL-12 and IFN-I signaling on IFN-γ production by CD4 T cells were further demonstrated by adoptive transfer of transgenic IFN-IR+/+ and IFN-IR−/− CD4 T cells into normal and IL-12-deficient mice, and infection with rLm. These results demonstrate an important dichotomy between the signals required for priming IFN-γ production by CD4 and CD8 T cells in response to bacterial infection.

Cutting Edge: Recombinant Listeria monocytogenes Expressing a Single Immune-Dominant Peptide Confers Protective Immunity to Herpes Simplex Virus-1 Infection

The vast majority of the world’s population is infected with HSV. Although antiviral therapy can reduce the incidence of reactivation and asymptomatic viral shedding, and limit morbidity and mortality from active disease, it cannot cure infection. Therefore, the development of an effective vaccine is an important global health priority. In this study, we demonstrate that recombinant Listeria monocytogenes (Lm) expressing the H-2Kb glycoprotein B (gB)498–505 peptide from HSV-1 triggers a robust CD8 T cell response to this Ag resulting in protective immunity to HSV infection. Following challenge with HSV-1, immune-competent mice primed with recombinant Lm-expressing gB498–505 Ag were protected from HSV-induced paralysis. Protection was associated with dramatic reductions in recoverable virus, and early expansion of HSV-1-specific CD8 T cells in the regional lymph nodes. Thus, recombinant Lm-expressing Ag from HSV represents a promising new class of vaccines against HSV infection.

Cutting Edge: Immunity and IFN-γ Production during Listeria monocytogenes Infection in the Absence of T-bet

The T-box transcription factor T-bet is an important regulator of IFN-γ production in all cell types and is considered to be essential for the generation of CD4 Th1 T cells. IFN-γ in turn plays a critical role in immunity to many infectious agents. In this study, we demonstrate that T-bet is not required for host resistance to primary Listeria monocytogenes (LM) infection. In the innate immune phase, control of LM replication, serum IFN-γ, and numbers of IFN-γ-producing NK cells were similar in T-bet-deficient and control mice. In the adaptive immune phase, there was no defect in bacterial clearance or in the numbers of LM-specific IFN-γ-producing CD8 T cells in T-bet-deficient mice and only a modest, although significant, reduction in the numbers of Th1 CD4 T cells and IFN-γ secretion by CD4 T cells. Thus, host resistance and the generation of IFN-γ-producing cells in response to LM infection are not substantially compromised in the absence of T-bet.

Deviation from a Strong Th1-Dominated to a Modest Th17-Dominated CD4 T Cell Response in the Absence of IL-12p40 and Type I IFNs Sustains Protective CD8 T Cells

The differentiation of naive CD4 T cells into specific effector subsets is controlled in large part by the milieu of cytokines present during their initial encounter with Ag. Cytokines that drive differentiation of the newly described Th17 lineage have been characterized in vitro, but the cytokines that prime commitment to this lineage in response to infection in vivo are less clear. Listeria monocytogenes (Lm) induces a strong Th1 response in wild-type mice. By contrast, we demonstrate that in the absence of IL-12p40 (or IFN-γ) and type I IFN receptor signaling, the Th1 Ag-specific CD4 T cell response is virtually abolished and replaced by a relatively low magnitude Th17-dominated response. This Th17 response was dependent on TGF-β and IL-6. Despite this change in CD4 T cell response, neither the kinetics of the CD4 and CD8 T cell responses, the quality of the CD8 T cell response, nor the ability of CD8 T cells to mediate protection were affected. Thus, generation of protective CD8 T cell immunity was resilient to perturbations that replace a strong Th1-dominated to a reduced magnitude Th17-dominated Ag-specific CD4 T cell response.

Induction of Protective Immunity to Listeria monocytogenes in Neonates

Neonates suffer unduly from infections and also respond suboptimally to most commonly used vaccines. However, a CD8 T cell response can be elicited in neonates if the Ag is introduced into the cytoplasm of APCs. Listeria monocytogenes (Lm) targets the cytoplasm of APC and is a strong CD8 and CD4 Th1-promoting vaccine vehicle in adult mice. We hypothesized that an attenuated strain of Lm would be safe and induce long-lasting protective immunity, even in neonates. We found that neonatal mice immunized only once with the attenuated strain ΔactA-Lm developed robust primary and secondary CD8 and CD4 Th1 responses and were fully protected from lethal challenge with virulent wild-type Lm without the need for a booster immunization. Furthermore, ΔactA-Lm expressing a heterologous recombinant Ag induced a strong CD8 and Th1 memory response to that Ag. Based on these data, we propose that ΔactA-Lm or derivatives thereof might serve as a vaccine vehicle for neonatal immunization.

IL-23 Promotes the Production of IL-17 by Antigen-Specific CD8 T Cells in the Absence of IL-12 and Type-I Interferons

In contrast to CD4 T cells, CD8 T cells inherently differentiate into IFN-γ-producing effectors. Accordingly, while generation of IFN-γ-producing Th1 CD4 T cells was profoundly impaired in mice deficient for both type-I IFN and IL-12 signaling in response to infection with Listeria monocytogenes, generation of Ag-specific, IFN-γ-producing CD8 T cells was unimpaired. However, a fraction of these CD8 T cells also produced IL-17 in an IL-23-dependent manner. Furthermore, the addition of IL-23 in vitro was sufficient for some naive CD8 T cells to differentiate into IFN-γ/IL-17 dual-producing cells and was associated with increased expression of ROR-γt and ROR-α. Addition of IL-6 and TGF-β to IL-23 further augmented ROR-γt and ROR-α expression and suppressed Eomes expression, thereby enhancing IL-17 production by CD8 T cells. A loss of cytotoxic function accompanied the production of IL-17, as the addition of IL-6 and TGF-β resulted in a marked reduction of granzyme B and perforin expression. Thus, CD8 T cells retain sufficient plasticity to respond to environmental cues and can acquire additional effector functions in response to their environmental context.