Neutrophils from Both Susceptible and Resistant Mice Efficiently Kill Opsonized \u3cem\u3eListeria monocytogenes\u3c/em\u3e

Inbred mouse strains differ in their susceptibility to infection with the facultative intracellular bacterium Listeria monocytogenes, largely due to delayed or deficient innate immune responses. Previous antibody depletion studies suggested that neutrophils (polymorphonuclear leukocytes [PMN]) were particularly important for clearance in the liver, but the ability of PMN from susceptible and resistant mice to directly kill L. monocytogenes has not been examined. In this study, we showed that PMN infiltrated the livers of BALB/c/By/J (BALB/c) and C57BL/6 (B6) mice in similar numbers and that both cell types readily migrated toward leukotriene B4 in an in vitro chemotaxis assay. However, CFU burdens in the liver were significantly higher in BALB/c mice than in other strains, suggesting that PMN in the BALB/c liver might not be able to clear L. monocytogenes as efficiently as B6 PMN. Unprimed PMN harvested from either BALB/c or B6 bone marrow killed L. monocytogenes directly ex vivo, and pretreatment with autologous serum significantly enhanced killing efficiency for both. L. monocytogenes were internalized within 10 min and rapidly triggered intracellular production of reactive oxygen species in a dose-dependent manner. However, PMN from gp91phox-deficient mice also readily killed L. monocytogenes, which suggested that nonoxidative killing mechanisms may be sufficient for bacterial clearance. Together, these results indicate that there is not an intrinsic defect in the ability of PMN from susceptible BALB/c mice to kill L. monocytogenes and further suggest that if PMN function is impaired in BALB/c mice, it is likely due to locally produced modulating factors present in the liver during infection

Effect of aqueous extract of Tinospora cordifolia on functions of peritoneal macrophages isolated from CCl4 intoxicated male albino mice

<p>Abstract</p> <p>Background</p> <p>The current practice of ingesting phytochemicals for supporting the immune system or fighting infections is based on centuries-old tradition. Macrophages are involved at all the stages of an immune response. The present study focuses on the immunostimulant properties of <it>Tinospora cordifolia </it>extract that are exerted on circulating macrophages isolated from CCl₄(0.5 ml/kg body weight) intoxicated male albino mice.</p> <p>Methods</p> <p>Apart from damaging the liver system, carbon tetrachloride also inhibits macrophage functions thus, creating an immunocompromised state, as is evident from the present study. Such cell functions include cell morphology, adhesion property, phagocytosis, enzyme release (myeloperoxidase or MPO), nitric oxide (NO) release, intracellular survival of ingested bacteria and DNA fragmentation in peritoneal macrophages isolated from these immunocompromised mice. <it>T. cordifolia </it>extract was tested for acute toxicity at the given dose (150 mg/kg body weight) by lactate dehydrogenase (LDH) assay.</p> <p>Results</p> <p>The number of morphologically altered macrophages was increased in mice exposed to CCl₄. Administration of CCl₄(i.p.) also reduced the phagocytosis, cell adhesion, MPO release, NO release properties of circulating macrophages of mice. The DNA fragmentation of peritoneal macrophages was observed to be higher in CCl₄intoxicated mice. The bacterial killing capacity of peritoneal macrophages was also adversely affected by CCl_4.However oral administration of aqueous fraction of <it>Tinospora cordifolia </it>stem parts at a dose of 40 mg/kg body weight (<it>in vivo</it>) in CCl₄exposed mice ameliorated the effect of CCl₄, as the percentage of morphologically altered macrophages, phagocytosis activity, cell adhesion, MPO release, NO release, DNA fragmentation and intracellular killing capacity of CCl₄intoxicated peritoneal macrophages came closer to those of the control group. No acute toxicity was identified in oral administration of the aqueous extract of <it>Tinospora cordifolia </it>at a dose of 150 mg/kg body weight.</p> <p>Conclusion</p> <p>From our findings it can be suggested that, polar fractions of <it>Tinospora cordifolia </it>stem parts contain major bioactive compounds, which directly act on peritoneal macrophages and have been found to boost the non-specific host defenses of the immune system. However, the molecular mechanism of this activity of <it>Tinospora cordifolia </it>on immune functions needs to be elucidated.</p

Probing the Role of Protein Surface Charge in the Activation of PrfA, the Central Regulator of Listeria monocytogenes Pathogenesis

Listeria monocytogenes is a food-borne intracellular bacterial pathogen capable of causing serious human disease. L. monocytogenes survival within mammalian cells depends upon the synthesis of a number of secreted virulence factors whose expression is regulated by the transcriptional activator PrfA. PrfA becomes activated following bacterial entry into host cells where it induces the expression of gene products required for bacterial spread to adjacent cells. Activation of PrfA appears to occur via the binding of a small molecule cofactor whose identity remains unknown. Electrostatic modeling of the predicted PrfA cofactor binding pocket revealed a highly positively charged region with two lysine residues, K64 and K122, located at the edge of the pocket and another (K130) located deep within the interior. Mutational analysis of these residues indicated that K64 and K122 contribute to intracellular activation of PrfA, whereas a K130 substitution abolished protein activity. The requirement of K64 and K122 for intracellular PrfA activation could be bypassed via the introduction of the prfA G145S mutation that constitutively activates PrfA in the absence of cofactor binding. Our data indicate that the positive charge of the PrfA binding pocket contributes to intracellular activation of PrfA, presumably by facilitating binding of an anionic cofactor

Inflammation Triggers Emergency Granulopoiesis through a Density-Dependent Feedback Mechanism

Normally, neutrophil pools are maintained by homeostatic mechanisms that require the transcription factor C/EBPα. Inflammation, however, induces neutrophilia through a distinct pathway of “emergency” granulopoiesis that is dependent on C/EBPβ. Here, we show in mice that alum triggers emergency granulopoiesis through the IL-1RI-dependent induction of G-CSF. G-CSF/G-CSF-R neutralization impairs proliferative responses of hematopoietic stem and progenitor cells (HSPC) to alum, but also abrogates the acute mobilization of BM neutrophils, raising the possibility that HSPC responses to inflammation are an indirect result of the exhaustion of BM neutrophil stores. The induction of neutropenia, via depletion with Gr-1 mAb or myeloid-specific ablation of Mcl-1, elicits G-CSF via an IL-1RI-independent pathway, stimulating granulopoietic responses indistinguishable from those induced by adjuvant. Notably, C/EBPβ, thought to be necessary for enhanced generative capacity of BM, is dispensable for increased proliferation of HSPC to alum or neutropenia, but plays a role in terminal neutrophil differentiation during granulopoietic recovery. We conclude that alum elicits a transient increase in G-CSF production via IL-1RI for the mobilization of BM neutrophils, but density-dependent feedback sustains G-CSF for accelerated granulopoiesis

The Role of Neutrophils during Mild and Severe Influenza Virus Infections of Mice

Neutrophils have been implicated in both protective and pathological responses following influenza virus infections. We have used mAb 1A8 (anti-Ly6G) to specifically deplete LyG6high neutrophils and induce neutropenia in mice infected with virus strains known to differ in virulence. Mice were also treated with mAb RB6-8C5 (anti-Ly6C/G or anti-Gr-1), a mAb widely used to investigate the role of neutrophils in mice that has been shown to bind and deplete additional leukocyte subsets. Using mAb 1A8, we confirm the beneficial role of neutrophils in mice infected with virus strains of intermediate (HKx31; H3N2) or high (PR8; H1N1) virulence whereas treatment of mice infected with an avirulent strain (BJx109; H3N2) did not affect disease or virus replication. Treatment of BJx109-infected mice with mAb RB6-8C5 was, however, associated with significant weight loss and enhanced virus replication indicating that other Gr-1+ cells, not neutrophils, limit disease severity during mild influenza infections

Interplay between CD8α+ Dendritic Cells and Monocytes in Response to Listeria monocytogenes Infection Attenuates T Cell Responses

During the course of a microbial infection, different antigen presenting cells (APCs) are exposed and contribute to the ensuing immune response. CD8α+ dendritic cells (DCs) are an important coordinator of early immune responses to the intracellular bacteria Listeria monocytogenes (Lm) and are crucial for CD8+ T cell immunity. In this study, we examine the contribution of different primary APCs to inducing immune responses against Lm. We find that CD8α+ DCs are the most susceptible to infection while plasmacytoid DCs are not infected. Moreover, CD8α+ DCs are the only DC subset capable of priming an immune response to Lm in vitro and are also the only APC studied that do so when transferred into β2 microglobulin deficient mice which lack endogenous cross-presentation. Upon infection, CD11b+ DCs primarily secrete low levels of TNFα while CD8α+ DCs secrete IL-12 p70. Infected monocytes secrete high levels of TNFα and IL-12p70, cytokines associated with activated inflammatory macrophages. Furthermore, co-culture of infected CD8α+ DCs and CD11b+ DCs with monocytes enhances production of IL-12 p70 and TNFα. However, the presence of monocytes in DC/T cell co-cultures attenuates T cell priming against Lm-derived antigens in vitro and in vivo. This suppressive activity of spleen-derived monocytes is mediated in part by both TNFα and inducible nitric oxide synthase (iNOS). Thus these monocytes enhance IL-12 production to Lm infection, but concurrently abrogate DC-mediated T cell priming

Utility of In Vivo Transcription Profiling for Identifying Pseudomonas aeruginosa Genes Needed for Gastrointestinal Colonization and Dissemination

Microarray analysis of Pseudomonas aeruginosa mRNA transcripts expressed in vivo during animal infection has not been previously used to investigate potential virulence factors needed in this setting. We compared mRNA expression in bacterial cells recovered from the gastrointestinal (GI) tracts of P. aeruginosa-colonized mice to that of P. aeruginosa in the drinking water used to colonize the mice. Genes associated with biofilm formation and type III secretion (T3SS) had markedly increased expression in the GI tract. A non-redundant transposon library in P. aeruginosa strain PA14 was used to test mutants in genes identified as having increased transcription during in vivo colonization. All of the Tn-library mutants in biofilm-associated genes had an attenuated ability to form biofilms in vitro, but there were no significant differences in GI colonization and dissemination between these mutants and WT P. aeruginosa PA14. To evaluate T3SS factors, we tested GI colonization and neutropenia-induced dissemination of both deletional (PAO1 and PAK) and insertional (PA14) mutants in four genes in the P. aeruginosa T3SS, exoS or exoU, exoT, and popB. There were no significant differences in GI colonization among these mutant strains and their WT counterparts, whereas rates of survival following dissemination were significantly decreased in mice infected by the T3SS mutant strains. However, there was a variable, strain-dependent effect on overall survival between parental and T3SS mutants. Thus, increased transcription of genes during in vivo murine GI colonization is not predictive of an essential role for the gene product in either colonization or overall survival following induction of neutropenia

Dynamic Imaging of the Effector Immune Response to Listeria Infection In Vivo

Host defense against the intracellular pathogen Listeria monocytogenes (Lm) requires innate and adaptive immunity. Here, we directly imaged immune cell dynamics at Lm foci established by dendritic cells in the subcapsular red pulp (scDC) using intravital microscopy. Blood borne Lm rapidly associated with scDC. Myelomonocytic cells (MMC) swarmed around non-motile scDC forming foci from which blood flow was excluded. The depletion of scDC after foci were established resulted in a 10-fold reduction in viable Lm, while graded depletion of MMC resulted in 30–1000 fold increase in viable Lm in foci with enhanced blood flow. Effector CD8+ [CD8 superscript +] T cells at sites of infection displayed a two-tiered reduction in motility with antigen independent and antigen dependent components, including stable interactions with infected and non-infected scDC. Thus, swarming MMC contribute to control of Lm prior to development of T cell immunity by direct killing and sequestration from blood flow, while scDC appear to promote Lm survival while preferentially interacting with CD8+ [CD8 superscript +] T cells in effector sites.National Institutes of Health (U.S.) (Grant P01AI-071195

The interaction of bacterial pathogens with platelets.

In recent years, the frequency of serious cardiovascular infections such as endocarditis has increased, particularly in association with nosocomially acquired antibiotic-resistant pathogens. Growing evidence suggests a crucial role for the interaction of bacteria with human platelets in the pathogenesis of cardiovascular infections. Here, we review the nature of the interactions between platelets and bacteria, and the role of these interactions in the pathogenesis of endocarditis and other cardiovascular diseases