Mannose-Coated Reconstituted Lipoprotein Nanoparticles for the Targeting of Tumor-Associated Macrophages: Optimization, Characterization, and In Vitro Evaluation of Effectiveness.

Reconstituted high-density lipoprotein nanoparticles (rHDL NPs) have been utilized as delivery vehicles to a variety of targets, including cancer cells. However, the modification of rHDL NPs for the targeting of the pro-tumoral tumor-associated macrophages (TAMs) remains largely unexplored. The presence of mannose on nanoparticles can facilitate the targeting of TAMs which highly express the mannose receptor at their surface. Here, we optimized and characterized mannose-coated rHDL NPs loaded with 5,6-dimethylxanthenone-4-acetic acid (DMXAA), an immunomodulatory drug. Lipids, recombinant apolipoprotein A-I, DMXAA, and different amounts of DSPE-PEG-mannose (DPM) were combined to assemble rHDL-DPM-DMXAA NPs. The introduction of DPM in the nanoparticle assembly altered the particle size, zeta potential, elution pattern, and DMXAA entrapment efficiency of the rHDL NPs. Collectively, the changes in physicochemical characteristics of rHDL NPs upon the addition of the mannose moiety DPM indicated that the rHDL-DPM-DMXAA NPs were successfully assembled. The rHDL-DPM-DMXAA NPs induced an immunostimulatory phenotype in macrophages pre-exposed to cancer cell-conditioned media. Furthermore, rHDL-DPM NPs delivered their payload more readily to macrophages than cancer cells. Considering the effects of the rHDL-DPM-DMXAA NPs on macrophages, the rHDL-DPM NPs have the potential to serve as a drug delivery platform for the selective targeting of TAMs.

Neutrophils Are More Effective than Monocytes at Phagosomal Containment and Killing of Listeria monocytogenes.

Neutrophils and inflammatory monocytes are innate immune cells essential for protection during Listeria monocytogenes infection. Although certain functions have been generally assigned to each of the cells, similarities and differences in functions necessary for bacterial clearance have not previously been investigated. In the current study, phagocytosis, phagosomal containment, bacterial killing, and cytokine production by neutrophils and monocytes during L. monocytogenes infection were studied. Data obtained via in vitro studies show that neutrophils are more effective at L. monocytogenes uptake, phagosomal containment, and killing than monocytes. However, monocytes were found to be more effective at cytokine production during L. monocytogenes infection, in vivo. Additionally, the data demonstrated that neutrophils and monocytes are also capable of producing IL-1α, a cytokine that does not yet have a clearly defined role during infection with L. monocytogenes Furthermore, we were able to demonstrate a population of monocytes producing both TNF-α and IL-α, concurrently. This study highlights the multifunctional capabilities of neutrophils and monocytes, further adding to our knowledge of these innate immune cells during L. monocytogenes infection.

The Essential Role of Neutrophils during Infection with the Intracellular Bacterial Pathogen Listeria monocytogenes.

Neutrophils have historically been characterized as first responder cells vital to host survival because of their ability to contain and eliminate bacterial and fungal pathogens. However, recent studies have shown that neutrophils participate in both protective and detrimental responses to a diverse array of inflammatory and infectious diseases. Although the contribution of neutrophils to extracellular infections has been investigated for decades, their specific role during intracellular bacterial infections has only recently been appreciated. During infection with the Gram-positive intracellular pathogen Listeria monocytogenes, neutrophils are recruited from the bone marrow to sites of infection where they use novel bacterial-sensing pathways leading to phagocytosis and production of bactericidal factors. This review summarizes the requirement of neutrophils during L. monocytogenes infection by examining both neutrophil trafficking and function during primary and secondary infection.

Interleukin-23 (IL-23) deficiency disrupts Th17 and Th1-related defenses against Streptococcus pneumoniae infection.

Resolution of acute of infection caused by capsular Streptococcus pneumoniae infection in the absence of effective antibiotic therapy requires tight regulation of immune and inflammatory responses. To provide new mechanistic insight of the requirements needed for innate host defenses against acute S. pneumoniae infection, we examined how IL-23 deficiency mediated acute pulmonary resistance. We found that IL-23 deficient mice were more susceptible to bacterial colonization in the lungs corresponding with greater bacterial dissemination. The lack of IL-23 was found to decrease IL-6 and IL-12p70 cytokine levels in bronchiolar lavage within the initial day after infection. Pulmonary leukocytes isolated from infected IL-23 deficient mice demonstrated a dramatic decrease in IL-17A and IFN-γ in response to heat-killed organisms. These findings corresponded with significant abrogation of neutrophilic infiltrate in the lungs compared to IL-23 competent mice. Whereas previous studies have shown opposing influences of IL-12/IL-23 regulation, our findings suggest a concordant dependency of IL-23 expression on Th1 and Th17-related responses.

Inflammatory monocyte recruitment is regulated by interleukin-23 during systemic bacterial infection.

Listeria monocytogenes is a gram-positive intracellular pathogen that causes meningitis and septicemia in immunocompromised individuals and spontaneous abortion in pregnant women. The innate immune response against L. monocytogenes is primarily mediated by neutrophils and monocytes. Interleukin-23 (IL-23) is an important proinflammatory cytokine well known for its role in neutrophil recruitment in various infectious and autoimmune diseases. We have previously shown that IL-23 is required for host resistance against L. monocytogenes and for neutrophil recruitment to the liver, but not the spleen, during infection. Despite efficient neutrophil recruitment to the spleen, IL-23p19 knockout (KO) mice have an increased bacterial burden in this organ, suggesting that IL-23 may regulate the recruitment/function of another cell type to the spleen. In this study, we show that specific depletion of neutrophils abrogated the differences in bacterial burdens in the livers but not the spleens of C57BL/6 (B6) and IL-23p19 KO mice. Interestingly, L. monocytogenes-infected IL-23p19 KO mice had fewer monocytes in the spleen than B6 mice, as well as a reduction in the monocyte-recruiting chemokines CCL2 and CCL7. Additionally, the overall concentrations of tumor necrosis factor alpha (TNF-α) and nitric oxide (NO(•)), as well as the percentages and total numbers of monocytes producing TNF-α and NO(•), were reduced in IL-23p19 KO mice compared to levels in B6 mice, leading to increased bacterial burdens in the spleens of L. monocytogenes-infected IL-23p19 KO mice. Collectively, our data establish that IL-23 is required for the optimal recruitment of TNF-α- and NO(•)-producing inflammatory monocytes, thus revealing a novel mechanism by which this proinflammatory cytokine provides protection against bacterial infection.

Extracellular superoxide dismutase inhibits innate immune responses and clearance of an intracellular bacterial infection.

Reactive oxygen species and reactive nitrogen species play important roles during immune responses to bacterial pathogens. Extracellular superoxide dismutase (ecSOD) regulates extracellular concentrations of reactive oxygen species and reactive nitrogen species and contributes to tissue protection during inflammatory insults. The participation of ecSOD in immune responses seems therefore intuitive, yet is poorly understood. In the current study, we used mice with varying levels of ecSOD activity to investigate the involvement of this enzyme in immune responses against Listeria monocytogenes. Surprisingly, our data demonstrate that despite enhanced neutrophil recruitment to the liver, ecSOD activity negatively affected host survival and bacterial clearance. Increased ecSOD activity was accompanied by decreased colocalization of neutrophils with bacteria, as well as increased neutrophil apoptosis, which reduced overall and neutrophil-specific TNF-α production. Liver leukocytes from mice lacking ecSOD produced equivalent NO· compared with liver leukocytes from mice expressing ecSOD. However, during infection, there were higher levels of peroxynitrite (NO(3)·(-)) in livers from mice lacking ecSOD compared with livers from mice expressing ecSOD. Neutrophil depletion studies revealed that high levels of ecSOD activity resulted in neutrophils with limited protective capacity, whereas neutrophils from mice lacking ecSOD provided superior protection compared with neutrophils from wild-type mice. Taken together, our data demonstrate that ecSOD activity reduces innate immune responses during bacterial infection and provides a potential target for therapeutic intervention.

Specific depletion reveals a novel role for neutrophil-mediated protection in the liver during Listeria monocytogenes infection.

Previous studies have suggested that neutrophils are required for resistance during infection with multiple pathogenic microorganisms. However, the depleting antibody used in those studies binds to both Ly6G and Ly6C (anti-Gr-1; clone RB6-8C5). This antibody has been shown to deplete not only neutrophils but also monocytes and a subset of CD8(+) T cells. Recently, an antibody against Ly6G, which specifically depletes neutrophils, was characterized. In the present study, neutrophils are depleted using the antibody against Ly6G during infection with the intracellular bacterium Listeria monocytogenes (LM). Our data show that neutrophil-depleted mice are much less susceptible to infection than mice depleted with anti-Gr-1. Although neutrophils are required for clearance of LM, their importance is more pronounced in the liver and during a high-dose bacterial challenge. Furthermore, we demonstrate that the protection mediated by neutrophils is due to the production of TNF-α, but not IFN-γ. Additionally, neutrophils are not required for the recruitment of monocytes or the generation of adaptive T-cell responses during LM infection. This study highlights the importance of neutrophils during LM infection, and indicate that depletion of neutrophils is less detrimental to the host than depletion of all Gr-1-expressing cell populations.

A novel immunoregulatory function for IL-23: Inhibition of IL-12-dependent IFN-γ production.

Most studies investigating the function of IL-23 have concluded that it promotes IL-17-secreting T cells. Although some reports have also characterized IL-23 as having redundant pro-inflammatory effects with IL-12, we have instead found that IL-23 antagonizes IL-12-induced secretion of IFN-γ. When splenocytes or purified populations of T cells were cultured with IL-23, IFN-γ secretion in response to IL-12 was dramatically reduced. The impact of IL-23 was most prominent in CD8(+) T cells, but was also observed in NK and CD4(+) T cells. Mechanistically, the IL-23 receptor was not required for this phenomenon, and IL-23 inhibited signaling through the IL-12 receptor by reducing IL-12-induced signal transducer and activator of transcription 4 (STAT4) phosphorylation. IL-23 was also able to reduce IFN-γ secretion by antagonizing endogenously produced IL-12 from Listeria monocytogenes (LM)-infected macrophages. In vivo, LM infection induced higher serum IFN-γ levels and a greater percentage of IFN-γ(+)CD8(+) T cells in IL-23p19-deficient mice as compared with WT mice. This increase in IFN-γ production coincided with increased LM clearance at days 2 and 3 post-infection. Our data suggest that IL-23 may be a key factor in determining the responsiveness of lymphocytes to IL-12 and their subsequent secretion of IFN-γ.

A novel IL-17-dependent mechanism of cross protection: respiratory infection with mycoplasma protects against a secondary listeria infection.

Immune responses to pathogens occur within the context of current and previous infections. Cross protection refers to the phenomena where infection with a particular pathogen provides enhanced resistance to a subsequent unrelated pathogen in an antigen-independent manner. Proposed mechanisms of antigen-independent cross protection have involved the secretion of IFN-gamma, which activates macrophages, thus providing enhanced innate immunity against the secondary viral or bacterial pathogen. Here we provide evidence that a primary infection with the chronic respiratory pathogen, Mycoplasma pulmonis, provides a novel form of cross protection against a secondary infection with Listeria monocytogenes that is not mediated by IFN-gamma, but instead relies upon IL-17 and mobilization of neutrophils. Mice infected with M. pulmonis have enhanced clearance of L. monocytogenes from the spleen and liver, which is associated with increased numbers of Gr-1(+)CD11b(+) cells and higher levels of IL-17. This enhanced clearance of L. monocytogenes was absent in mice depleted of Gr-1(+) cells or in mice deficient in the IL-17 receptor. Additionally, both the IL-17 receptor and neutrophils were essential for optimal clearance of M. pulmonis. Thus, a natural component of the immune response directed against M. pulmonis was able to enhance clearance of L. monocytogenes.

Imatinib mesylate inhibits antigen-specific memory CD8 T cell responses in vivo.

Imatinib mesylate (IM) is effective at inducing complete cytogenetic remission in patients with chronic myelogenous leukemia. Because its influence on CD8 T cell responsiveness in vivo is unknown, we investigated the effects of IM by analyzing the response of OT-1 CD8 T cells to Listeria monocytogenes (LM) that express the cognate epitope OVA(257-264) (LM-OVA). In vitro, IM had no effect on Ag-specific expansion, cell division, cell cycle progression, or IFN-gamma expression in naive or memory OT-1 T cells. However, IM induced apoptosis of naive and memory OT-1 T cells at doses of >5 microM. At 15 microM IM, OT-1 T cells did not survive in in vitro cultures. The primary response of OT-1 T cells in vivo to LM-OVA infection was unaltered. In contrast, continuous IM treatment resulted in a diminished memory OT-1 response. The expression of IL-7Ralpha, a receptor required for memory cell survival, was lower (on OT-1 cells) in animals receiving IM. These results indicate that IM treatment affects the ability of the CD8 memory pool to respond to Ag and has the potential to increase susceptibility to infection.

The role of CD8 T cells in innate immunity and in antigen non-specific protection.

The role of CD8 T cells in adaptive immune responses is well understood. These lymphocytes respond through their T cell receptors to diverse antigens presented by MHC class I molecules by proliferating, secreting cytokines and chemokines, and directly lysing infected cells. Recently, a role for CD8 T cells in the innate immune response has become apparent. Independent of T cell receptor ligation, CD8 T cells can mount a response against pathogens by secreting cytokines and can defend against tumors by directly killing transformed cells. This innate response has been shown to be beneficial in controlling several types of bacterial infections. However, a subset of CD8 T cells that have innate non-antigen-specific capabilities has been implicated in self-reactivity, which could lead to autoimmunity.

Relative contributions of NK and CD8 T cells to IFN-gamma mediated innate immune protection against Listeria monocytogenes.

During the innate immune response to Listeria monocytogenes (LM), the secretion of IFN-gamma is crucial in controlling bacterial numbers. We have shown recently that CD8 T cells have the ability to rapidly secrete IFN-gamma independent of Ag, in response to IL-12 and IL-18, during a LM infection. In the current study, we compared the relative abilities of NK and CD8 T cells to provide innate immune protection. Upon transfer of either NK or memory OT-I T cells (specific for the OVA protein) into IFN-gamma-deficient hosts that were infected subsequently with wild-type LM, both cell types were found in the spleen and had the ability to secrete IFN-gamma. However, the OT-I T cells were more effective at providing innate immune protection as determined by spleen and liver LM burdens. We used immunocytochemistry to demonstrate that upon infection with LM, marginal zone macrophages were localized to the T cell area of the splenic follicle. Transferred memory OT-I T cells were also found in the T cell area of the spleen, co-localizing with the LM and macrophages. In sharp contrast, NK cells were found predominantly in the red pulp region of the spleen. In addition, memory OT-I T cells were also found to be associated with LM lesions in the liver. These results highlight the importance of CD8 T cells in innate immune responses to LM and suggest that their increased protective ability compared with NK cells is the result of their colocalization with LM and macrophages.