Listeria Monocytogenes Challenge Research Articles

The Probiotic Bacillus subtilis MB40 Improves Immunity in a Porcine Model of Listeriosis.

Probiotics for humans and direct-fed microbials for livestock are increasingly popular dietary ingredients for supporting immunity. The aim of this study was to determine the effects of dietary supplementation of Bacillus subtilis MB40 (MB40) on immunity in piglets challenged with the foodborne pathogen Listeria monocytogenes (LM). Three-week-old piglets (n = 32) were randomly assigned to four groups: (1) basal diet, (2) basal diet with LM challenge, (3) MB40-supplemented diet, and (4) MB40-supplemented diet with LM challenge. Experimental diets were provided throughout a 14-day (d) period. On d8, piglets in groups 2 and 4 were intraperitoneally inoculated with LM at 108 CFU/mL per piglet. Blood samples were collected at d1, d8, and d15 for biochemical and immune response profiling. Animals were euthanized and necropsied at d15 for liver and spleen bacterial counts and intestinal morphological analysis. At d15, LM challenge was associated with increased spleen weight (p = 0.017), greater circulating populations of neutrophils (p = 0.001) and monocytes (p = 0.008), and reduced ileal villus height to crypt depth ratio (p = 0.009), compared to non-challenged controls. MB40 supplementation reduced LM bacterial counts in the liver and spleen by 67% (p < 0.001) and 49% (p < 0.001), respectively, following the LM challenge, compared to the basal diet. MB40 supplementation was also associated with decreased circulating concentrations of monocytes (p = 0.007). Altogether, these data suggest that MB40 supplementation is a safe and well-tolerated approach to enhance immunity during systemic Listeria infection.

A live attenuated DIVA vaccine affords protection against Listeria monocytogenes challenge in sheep

Listeria monocytogenes (Lm) is a deadly foodborne pathogen that comprises 14 serotypes, among which, serotype 4b Lm is the primary cause of listeriosis outbreaks in humans and animals. Here, we evaluated the safety, immunogenicity, and protective efficacy of a serotype 4b vaccine candidate Lm NTSNΔactA/plcB/orfX in sheep. The infection dynamics, clinical features, and pathological observation verified that the triple genes deletion strain has adequate safety for sheep. Moreover, NTSNΔactA/plcB/orfX significantly stimulated humoral immune response and provided 78% immune protection to sheep against lethal wild-type strain challenge. Notably, the attenuated vaccine candidate could differentiate infected and vaccinated animals (DIVA) via serology determination of the antibody against listeriolysin O (LLO, encoded by hly) and phosphatidylinositol-specific phospholipase C (PI-PLC, encoded by plcB). These data suggest that the serotype 4b vaccine candidate has high efficacy, safety, and DIVA characteristics, and may be used to prevent Lm infection in sheep. Our study provides a theoretical basis for its future application in livestock and poultry breeding.

Natural Microbial Exposure from the Earliest Natural Time Point Enhances Immune Development by Expanding Immune Cell Progenitors and Mature Immune Cells.

Microbial experience fundamentally shapes immunity, particularly during the perinatal period when the immune system is underdeveloped, and novel microbial encounters are common. Most animal models are raised in specific pathogen-free (SPF) conditions with relatively uniform microbial communities. How SPF housing conditions alter early-life immune development relative to natural microbial exposure (NME) has not been thoroughly investigated. In this article, we compare immune development in SPF-raised mice with mice born from immunologically experienced mothers in microbially diverse environments. NME induced broad immune cell expansion, including naive cells, suggesting mechanisms besides activation-induced proliferation contribute to the increase in immune cell numbers. We found NME conditions also expanded immune cell progenitor cell populations in the bone marrow, suggesting microbial experience enhances immune development at the earliest stages of immune cell differentiation. Multiple immune functions characteristically impaired in infants were also enhanced by NME, including T cell memory and Th1 polarization, B cell class switching and Ab production, proinflammatory cytokine expression, and bacterial clearance after Listeria monocytogenes challenge. Collectively, our studies reveal numerous impairments in immune development in SPF conditions relative to natural immune development.

Normal microbial experiences accelerate memory T cell compartment maturation in infants

Abstract Memory T cells cause faster and more effective elimination of pathogens during reinfection by the rapid production of effector T cells. Worldwide, the leading cause of death among children and infants is infection, partly due to under-developed T cell memory populations commonly seen in infants. In the context of biomedical research, the specific pathogen-free (SPF) facilities research animals are housed in further the deficit of memory T cells, as it is known that adult mice housed in SPF facilities have few memory T cells, but that exposure to normal microbial experiences (NME) replenishes this population of cells. This phenomenon, though, has not been investigated in infants. Therefore, we aimed to investigate how NME influences infant memory T cell commitment to reveal novel insights into natural mammalian immune development. At various time points, the trajectory of infant immune maturation of age-matched SPF and NME (pups birthed and raised in an environment exposed to NME) mice were examined using flow cytometry. We observed significant increases in both the number and frequency of memory T cell subsets (CD8+ and CD4+ Tcm and Tem cells) at all time points in pups exposed to NME. We also observed increased serum concentrations of cytokines required for memory T cell differentiation and survival (IL-7 and IL-15) in NME pups. These data support the hypothesis that NME accelerates the maturation of the memory T cell compartment in infant mice. We predict this more mature memory T cell compartment may contribute to improved immunity to novel infections. Our ongoing experiments test this hypothesis using a Listeria monocytogenes challenge model.

B cells promote CD8 Tcell primary and memory responses to subunit vaccines.

SUMMARYThe relationship between B cells and CD4 T cells has been carefully studied, revealing a collaborative effort in which B cells promote the activation, differentiation, and expansion of CD4 T cells while the so-called “helper” cells provide signals to B cells, influencing their class switching and fate. Interactions between B cells and CD8 T cells are not as well studied, although CD8 T cells exhibit an accelerated contraction after certain infections in B-cell-deficient mice. Here, we find that B cells significantly enhance primary CD8 T cell responses after vaccination. Moreover, memory CD8 numbers and function are impaired in B-cell-deficient animals, leading to increased susceptibility to bacterial challenge. We also show that interleukin-27 production by B cells contributes to their impact on primary, but not memory, CD8 responses. Better understanding of the interactions between CD8 T cells and B cells may aid in the design of more effective future vaccine strategies.

Microbiological Stability and Overall Quality of Ready-to-Heat Meals Based on Traditional Recipes of the Basilicata Region

The quality of ready meals is affected by several factors that may impair stability and nutritional value. In this work, we evaluated the overall quality of four traditional meals (Basilicata region) prepared according to the cook&chill method, packaged in air or modified atmosphere packaging (MAP; 70% N2 and 30% CO2), and stored at 4 °C for seven days. The shelf-life was determined by Listeria monocytogenes challenge testing and inactivation by microwave (MW) heating was assessed. The counts at the production day were excellent in three meals out of four, whereas one had high levels of spoilage and pathogens both as soon as the preparation and after seven days. MAP was partially effective only against the growth of the aerobic mesophilic species, whereas sensory analysis revealed that MAP may preserve many of sensory attributes. The average shelf-life of the meals ranged from 11 to 13 days, however, the potential shelf-life was undetectable in one out four meals, as L. monocytogenes growth was inhibited two days after the inoculum. In the inoculated meals, MW heating provided a partial inactivation (25%) of the pathogen. The overall quality of type the meals was partially satisfactory; post-cooking contaminations may affect the microbial load and reduce the palatability over the storage period and, above all, may involve biological hazards which consumers’ habits may not be able to eliminate.

Immune enhancing effects of Weissella cibaria JW15 on BALB/c mice immunosuppressed by cyclophosphamide

Our present study investigates the immune enhancing effects of JW15 on cyclophosphamide (CPM)-induced immunosuppression in BALB/c mice. In our previous study, we demonstrated the immune enhancing effects of Weissella cibaria JW15 (JW15) isolated from Kimchi (Korean traditional fermented vegetables) on macrophage cells and Listeria monocytogenes challenge model. The recovery levels of white blood cells and the mean spleen weight per body weight (g/kg) of JW15-treated group was significantly larger than that of the CPM-treated group. Moreover, JW15 administration enhanced the recovery of splenocyte proliferation in CPM-treated mice. In addition, JW15 supplementation stimulated the production of cytokines (TNF- α and IL-6) in splenocytes. Thus, JW15 could promote the ability from CPM-induced immunosuppression. In conclusion, W. cibaria JW15 exhibits the ability to induce immune-enhancing effects and could be a suitable functional food additive for humans and companion animals.

MARVELD1 promotes survival during septic shock in mice

Abstract MARVELD1 is integral to the inflammatory response occurring during septic shock, although its precise function has yet to be determined. Here we show that compared with wildtype mice, listeria monocytogenes (LM)-induced septic shock in MARVELD1 knockout mice resulted in a heightened pro-inflammatory response and increased mortality. This observation was associated with impaired bacterial clearance related to marked inhibition of the macrophage phagocytic acitivity in MARVELD1 knockout mice. Consistently, the MARVELD1 knockout mice were more sensitive to a lethal endotoxin or LM challenge, while wildtype mice in the bacterial peritonitis model produced a significant survival benefit. These data highlight the crucial role of the MARVELD1 pathway in inducing an adequate macrophage response to polymicrobial sepsis, and both the therapeutic promise and potential risks of its modulation.

Adenovirus Serotype 5 Vectors Trigger IL-27-Dependent Inhibitory CD4+ T Cell Responses

Abstract Viral vectors are attractive vaccine platforms that elicit robust innate and adaptive immune responses, but they vary greatly in their ability to induce protective immunity. Adenovirus serotype 5 (Ad5) vectors elicit robust CD8+ T cell responses but are typically characterized by an exhausted phenotype. The mechanisms by which Ad5 vectors induce dysfunctional CD8+ T cells have not been fully elucidated. Here we demonstrate that Ad5 vectors, but not Ad26 vectors, elicit exhausted antigen-specific IL-10+PD1+ CD4+ T cells with a dysfunctional transcriptional profile, and these cells effectively suppress CD8+ T cell responses in vivo. The blockade of IL-10 increased the frequency of antigen-specific CD8+ T cells with enhanced cytokine polyfunctionality and protective capacity against a Listeria monocytogenes challenge. Induction of inhibitory CD4+ T cells by Ad5 vectors is associated with increased IL-27 expression, and IL-27 blockade improves CD4+ T cell cytokine polyfunctionality. Together our data highlight a novel role for IL-27 induced inhibitory CD4+ T cells in regulating responses to viral vector vaccines.

Rescue of CD8+ T cell vaccine memory following sublethal γ irradiation

Sublethal γ irradiation eliminates CD8+ T cell mediated memory responses. In this work, we explored how these memory responses could be rescued in the aftermath of such exposure. We utilized two models of CD8+ T cell mediated immunity: a mouse model of Listeria monocytogenes (LM) infection in which CD8+ T cells specific for LM expressed antigens (Listeriolysin O, LLO) can be tracked, and a murine skin graft model in which CD8+ T cells mediate rejection across a MHC class I (Dd) disparity. In the LM immunized mice, LL0 specific CD8+ T memory cells were lost on irradiation, preserved with rapid revaccination with an attenuated strain 1–3 days post-irradiation (PI), and these mice survived a subsequent wild type LM challenge. A genetic “signature of rescue” identified a group of immune-associated mRNA maintained or upregulated following irradiation and rescue. A number of these factors, including IL-36γ, dectin-2 (Clec4n), and mir101c are upregulated rapidly after exposure of mice to sublethal γ radiation alone and are sustained by early, but not later rescue. Such factors will be evaluated as potential therapeutics to replace individual vaccines for global rescue of CD8+ T memory cell responses following sublethal γ irradiation. The skin allograft model mirrored that of the LM model in that the accelerated Dd skin allograft rejection response was lost in mice exposed to sublethal γ radiation, but infusion of allogeneic Dd expressing bone marrow cells 1–4 days PI preserved the CD8+ T memory mediated accelerated rejection response, further suggesting that innate immune responses may not always be essential to rescue of CD8+ memory T cells following γ irradiation.

Longitudinal requirement for CD4+ T cell help for adenovirus vector-elicited CD8+ T cell responses.

Despite the widespread use of replication-incompetent recombinant adenovirus (Ad) vectors as candidate vaccine platforms, the mechanism by which these vectors elicit CD8(+) T cell responses remains poorly understood. Our data demonstrate that induction and maintenance of CD8(+) T cell responses by Ad vector immunization is longitudinally dependent on CD4(+) T cell help for a prolonged period. Depletion of CD4(+) T cells in wild type mice within the first 8 d following Ad immunization resulted in dramatically reduced induction of Ag-specific CD8(+) T cells, decreased T-bet and eomesodermin expression, impaired KLRG1(+) effector differentiation, and atypical expression of the memory markers CD127, CD27, and CD62L. Moreover, these CD8(+) T cells failed to protect against a lethal recombinant Listeria monocytogenes challenge. Depletion of CD4(+) T cells between weeks 1 and 4 following immunization resulted in increased contraction of memory CD8(+) T cells. These data demonstrate a prolonged temporal requirement for CD4(+) T cell help for vaccine-elicited CD8(+) T cell responses in mice. These findings have important implications in the design of vaccines aimed at eliciting CD8(+) T cell responses and may provide insight into the impaired immunogenicity of vaccines in the context of AIDS and other CD4(+) T cell immune deficiencies.

Rescue of vaccine T cell memory following sublethal gamma irradiation (VAC3P.952)

Abstract Subsequent to sublethal γ radiation exposure, survivors reacquire immune competence, but lose vaccine immunity. Our goal is to rescue CD8+ T cell-mediated memory responses following γ radiation exposure. In a mouse model of Listeria monocytogenes (LM) infection, we found that rapid revaccination (rescue) of immunized mice following irradiation preserves CD8+ T cell mediated immunity. Rescued mice are able to survive a wild type LM challenge infection when the rescue vaccine strain, LM-deltaActA, is given 0-3 days following radiation exposure. A signature of rescue was revealed in gene profiling studies in which a group of cytokines and chemokines were uniquely up regulated in the spleens of LM-rescued animals as compared to mice irradiated without rescue. These included factors important in resistance to LM, such as proinflammatory factors CCL2, TNFα, and IL-6 and bacterocidal factors CXCL9, 10, and 11 as well as anti-apoptotic factors including IL-15 and IER3, which may have a role in rescue of memory CTL. RAW264.7, a macrophage cell line, produced a similar cytokine profile when irradiated and infected with LM, suggesting a probable myeloid source for the majority of these factors. Monocyte activation by the combination of irradiation and LM infection may thus create conditions of rescue by producing cytokines and chemokines that enhance clearance of LM and rescue CD8+ CTL mediating vaccine immunity.

Nanoparticle Adjuvant Sensing by TLR7 Enhances CD8+ T Cell–Mediated Protection from Listeria Monocytogenes Infection

Developing new adjuvants and vaccination strategies is of paramount importance to successfully fight against many life-threatening infectious diseases and cancer. Very few adjuvants are currently authorized for human use, and these mainly stimulate a humoral response. However, specific Abs are not sufficient to confer protection against persisting infections or cancer. Therefore, development of adjuvants and immunomodulators able to enhance cell-mediated immune responses represents a major medical need. We recently showed that papaya mosaic virus nanoparticles (PapMV), self-assembled from the coat protein of a plant virus and a noncoding ssRNA molecule, are highly immunogenic in mice. PapMV can be used either as a vaccine delivery platform, through fusion of various epitopes to the coat protein or as adjuvant to enhance humoral immune responses against coadministered Ags or vaccines. However, the mechanisms that confer these immunomodulatory properties to PapMV and its ability to enhance T cell vaccines remain unknown. Using immunization studies in mice, we demonstrate in this paper that PapMV represents a novel TLR7 agonist with strong immunostimulatory properties. More importantly, pretreatment with PapMV significantly improves effector and memory CD8(+) T cell responses generated through dendritic cell vaccination increasing protection against a Listeria monocytogenes challenge.

Immunomodulatory properties of Enterococcus faecium JWS 833 isolated from duck intestinal tract and suppression of Listeria monocytogenes infection

The aim of this study was to evaluate the immunomodulatory properties of Enterococcus faecium JWS 833 (JWS 833) isolated from duck intestine and compare them to those of Lactobacillus rhamnosus GG (LGG), a proven immunity-enhancing probiotic. To investigate the immune-enhancing properties of JWS 833, production of nitric oxide (NO) and cytokines was measured in mouse peritoneal macrophages. In addition, a Listeria monocytogenes challenge model was used in the assessment. It was found that heat-killed JWS 833 stimulates mouse peritoneal macrophages to produce NO, interleukin-1 β (IL-1β) and tumor necrosis factor-α (TNF-α) and that oral administration of viable JWS833 enhances NO, IL-1β and TNF-α synthesis upon L. monocytogenes challenge. Moreover, mice fed with JWS 833 were partially protected against lethal challenge with L. monocytogenes. JWS 833 strain has significantly greater immunostimulatory properties than LGG. Moreover, JWS 833 strain partially protects mice against lethal challenge with L. monocytogenes. JWS 833, a novel strain of E. faecium isolated from duck intestine, is potentially a useful feed supplement for controlling pathogens and enhancing host immune responses.

TLR11-dependent and -independent recognition of Toxoplasma gondii enhances innate immunity to bacterial infection (53.3)

Abstract Previous studies have shown that mice infected with the obligate intracellular parasite Toxoplasma gondii are resistant to infection with a variety of bacterial, viral, and fungal pathogens, but the factors and mechanisms mediating these phenomena are unknown. This study identifies the TLR11 ligand profilin (TgPRF) as one T. gondii factor sufficient to confer resistance to bacterial infection using a lethal Listeria monocytogenes challenge model. Mice stimulated with a single injection of purified TgPRF had a 3- to 4-log reduction in bacterial burden in the spleen and liver compared to control animals at day 3 post infection, did not develop clinical signs of disease, and survived for over 30 days. TgPRF stimulation was associated with a rapid increase in the number of neutrophils in the spleen and with production of CCL2, TNF-α, IL-12 and IFN-γ. TgPRF stimulation reduced bacterial burden in mice deficient in T cells, NK cells, IL-12 or IFN-γ, but did not provide any benefit in animals treated with neutrophil depleting antibody, suggesting its protective effect was primarily dependent on neutrophil recruitment. T. gondii-infected TLR11-deficient animals showed similar resistance to L. monocytogenes challenge, indicating that other T. gondii factors may function in similar, but TLR11-independent manners. Together these data show that TgPRF and other T. gondii factors may be used as therapeutics to stimulate innate immunity against bacterial infections.

Immuno-enhancing Effects of Lactobacillus salivarius JWS 58 and Lactobacillus plantarum JWS 1354 isolated from duck

Lactobacillus salivarius JWS 58 (JWS 58) and Lactobacillus plantarum JWS 1354 (JWS 1354) are isolated from duck intestine and have ability to produce bacteriocin. The objective of this study was to evaluate the immunomodulatory effects of JWS 58 and JWS 1354. The nitric oxide (NO) and cytokines (IL- and TNF-) were measured in C57BL/6 mouse peritoneal macrophages to determine immune enhancing effects of JWS 58 and JWS 1354. A Listeria (L.) monocytogenes challenge mice model was used to evaluate immune enhancement ability of JWS 58 and JWS 1354 in vivo. The results showed that JWS 58 and JWS 1354 increased the production of NO or cytokines by peritoneal macrophages and that oral administration of viable probiotic strains in mice elicited the immuno-modulatory effect upon L. monocytogenes challenge. JWS 1354 showed stronger immune enhancing effects than JWS 58. Collectively, this study demonstrated that Lactobacillus strain JWS 58 and JWS 1354 possess immune enhancing effect. Furthermore, two stains are expected to use feed supplement to prevent diseases by pathogenic bacteria through releasing bacteriocin and enhancing host immune responses in animal.

Immuno-Modulatory Effects of Bacteriocin-Producing Pediococcus pentosaceus JWS 939 in Mice

Pediococcus pentosaceus JWS 939 (JWS 939) is a nonpathogenic bacteriocin-producing probiotic isolated from the duck intestine. This study assessed the immunomodulatory effects of JWS 939 and compared them with those of Lactobacillus rhamnosus GG (LGG), a well-known immune enhancer. The immune-enhancing effects of JWS 939 were measured by measuring the production of nitric oxide (NO) and cytokines in C57BL/6 mouse peritoneal macrophages. In addition, to assess the immune enhancement abilities of JWS 939, in vivo, a Listeria monocytogenes challenge mice model was used. The results showed that heat-killed JWS 939 induced more NO and interleukin (IL)-<TEX>$1{\beta}$</TEX> production in mouse peritoneal macrophages than in LGG, and that oral administration of viable JWS 939 in mice increased more NO, IL-<TEX>$1{\beta}$</TEX>, and tumor necrosis factor (TNF)-<TEX>${\alpha}$</TEX> level than in LGG in serum upon L. monocytogenes challenge. In addition, mice fed with JWS 939 had a longer survival time after lethal challenge with L. monocytogenes, and these effects were stronger than those induced by LGG. Collectively, P. pentosaceus JWS 939 is a remarkable strain that, by releasing bacteriocin and enhancing host immune responses, may have potential as a duck feed additive to suppress pathogens.

Temporal Regulation of Rapamycin on Memory CTL Programming by IL-12

Mammalian target of rapamycin (mTOR) is a master regulator of cell growth. Recent reports have defined its important role in memory cytotoxic T lymphocyte (CTL) differentiation in infections and memory programming. We report that rapamycin regulated memory CTL programming by IL-12 to a similar level in a wide range of concentrations, and the enhanced memory CTLs by rapamycin were functional and provided similar protection against Listeria Monocytogenes challenge compared to the control. In addition, rapamycin-experienced CTLs went through substantially enhanced proliferation after transfer into recipients. Furthermore, the regulatory function of rapamycin on CD62L expression in memory CTLs was mainly contributed by the presence of rapamycin in the first 24-hr of stimulation in vitro, whereas the effective window of rapamycin on the size of memory CTLs was determined between 24 to 72 hrs. In conclusion, rapamycin regulates IL-12-driven programming of CTLs to a similar level in a wide range of concentrations, and regulates the phenotype and the size of memory CTLs in different temporal windows.

TCR Revision Generates Functional CD4+ T Cells

CD4(+)Vβ5(+) peripheral T cells in C57BL/6 mice respond to encounter with a peripherally expressed endogenous superantigen by undergoing either deletion or TCR revision. In this latter process, cells lose surface Vβ5 expression and undergo RAG-dependent rearrangement of endogenous TCRβ genes, driving surface expression of novel TCRs. Although postrevision CD4(+)Vβ5(-)TCRβ(+) T cells accumulate with age in Vβ5 transgenic mice and bear a diverse TCR Vβ repertoire, it is unknown whether they respond to homeostatic and antigenic stimuli and thus may benefit the host. We demonstrate in this study that postrevision cells are functional. These cells have a high rate of steady-state homeostatic proliferation in situ, and they undergo extensive MHC class II-dependent lymphopenia-induced proliferation. Importantly, postrevision cells do not proliferate in response to the tolerizing superantigen, implicating TCR revision as a mechanism of tolerance induction and demonstrating that TCR-dependent activation of postrevision cells is not driven by the transgene-encoded receptor. Postrevision cells proliferate extensively to commensal bacterial Ags and can generate I-A(b)-restricted responses to Ag by producing IFN-γ following Listeria monocytogenes challenge. These data show that rescued postrevision T cells are responsive to homeostatic signals and recognize self- and foreign peptides in the context of self-MHC and are thus useful to the host.

Platelet-derived CD154 enables T-cell priming and protection against Listeria monocytogenes challenge

AbstractCollagen exposure in tissue activates platelets, initiates wound healing, and modulates adaptive immunity. In this report, data are presented to demonstrate a requirement for platelet-derived CD154 for both collagen-induced augmentation of T-cell immunity and induction of pro-tective immunity to Listeria challenge. Specifically, we demonstrate that Ad5 encoding the membrane-bound form of ovalbumin (Ad5-mOVA) delivered in collagen induces higher ovalbumin-specific cytotoxic T lymphocyte (CTL) activity in a dose-dependent manner compared with Ad5-mOVA delivered in PBS. Increased CTL activity was dependent on the ability of platelets to respond to collagen and to express CD154. Furthermore, mice immunized with low-dose Ad5-mOVA in collagen were able to control a challenge of Listeria monocytogenes recombinant for ovalbumin expression (Lm-OVA), whereas mice immunized with low-dose Ad5-mOVA in PBS were not. These data indicate that in a physiologic setting that mimics wounding, platelets perform a sentinel function when antigen dose is too low to provoke an efficient immune response, and can enhance the generation of antigen-specific CD8 T cells that are functionally relevant to the host.