The neurotrophic receptor Ntrk2 directs lymphoid tissue neovascularization during Leishmania donovani infection.

The neurotrophic tyrosine kinase receptor type 2 (Ntrk2, also known as TrkB) and its ligands brain derived neurotrophic factor (Bdnf), neurotrophin-4 (NT-4/5), and neurotrophin-3 (NT-3) are known primarily for their multiple effects on neuronal differentiation and survival. Here, we provide evidence that Ntrk2 plays a role in the pathologic remodeling of the spleen that accompanies chronic infection. We show that in Leishmania donovani-infected mice, Ntrk2 is aberrantly expressed on splenic endothelial cells and that new maturing blood vessels within the white pulp are intimately associated with F4/80(hi)CD11b(lo)CD11c(+) macrophages that express Bdnf and NT-4/5 and have pro-angiogenic potential in vitro. Furthermore, administration of the small molecule Ntrk2 antagonist ANA-12 to infected mice significantly inhibited white pulp neovascularization but had no effect on red pulp vascular remodeling. We believe this to be the first evidence of the Ntrk2/neurotrophin pathway driving pathogen-induced vascular remodeling in lymphoid tissue. These studies highlight the therapeutic potential of modulating this pathway to inhibit pathological angiogenesis.

Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.

Kupffer cells (KCs) represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens. Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8(+) T cells. Here, using a combination of approaches including whole mount and thin section confocal microscopy, adoptive cell transfer and intra-vital 2-photon microscopy, we demonstrate that KCs represent the only detectable population of mononuclear phagocytes within granulomas induced by Leishmania donovani infection that are capable of presenting parasite-derived peptide to effector CD8(+) T cells. This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

Dendritic cells matured by inflammation induce CD86-dependent priming of naive CD8+ T cells in the absence of their cognate peptide antigen.

Dendritic cells (DC) licensed by the interaction between pathogen products and pattern recognition receptors can activate naive T cells to undergo Ag-dependent proliferation and cytokine production. In contrast, DC induced to mature by trans-acting inflammatory stimuli are believed to only be capable of supporting Ag-dependent proliferative responses. In this study, we show that uninfected DC matured as a consequence of Leishmania-induced inflammation induce CD8(+) T cells to proliferate in the absence of their cognate Ag. We separated splenic DC from Leishmania donovani-infected mice into those that contained parasites and had been activated to induce IL-12p40, from those that had undergone only partial maturation, measured by increased CD86 expression in the absence of IL-12p40 induction. We then showed that these partially matured DC could induce exogenous peptide-independent proliferation of OT-I and F5 CD8(+) TCR transgenic T cells, as well as polyclonal CD8(+) T cells. Proliferation of OT-I cells was significantly inhibited in vitro and in vivo by anti-CD86 mAb but not by anti-CD80 mAb and could also be inhibited by cyclosporine A. Proliferating OT-I cells did not produce IFN-gamma, even when re-exposed to mature DC. However, these primed OT-I cells subsequently produced effector cytokines, not just on exposure to their cognate peptide but, more importantly, to weak exogenous TCR agonists that otherwise failed to induce IFN-gamma. We further showed that OT-I cells undergoing locally driven proliferation to another pathogen, Streptococcus pneumoniae, rapidly seeded other lymphoid tissues, suggesting that CD8(+) T cells primed in this way may play a role in rapidly countering pathogen dissemination.

Alveolar macrophages transport pathogens to lung draining lymph nodes.

The first step in inducing pulmonary adaptive immunity to allergens and airborne pathogens is Ag acquisition and transport to the lung draining lymph nodes (dLN). Dendritic cells (DC) sample the airways, and active transfer of Ag to the lung dLN is considered an exclusive property of migratory DC. However, alveolar macrophages (AM) are the first phagocytes to contact inhaled particulate matter. Although having well-defined immunoregulatory capabilities, AM are generally considered as restricted to the alveoli. We show that murine AM constitutively migrate from lung to dLN and that following exposure to Streptococcus pneumoniae, AM rapidly transport bacteria to this site. Thus AM, and not DC, appear responsible for the earliest delivery of these bacteria to secondary lymphoid tissue. The identification of this novel transport pathway has important consequences for our understanding of lung immunity and suggests more widespread roles for macrophages in the transport of Ags to lymphoid organs than previously appreciated.

Natural antibodies and complement are endogenous adjuvants for vaccine-induced CD8+ T-cell responses.

CD8(+) T cells are essential for long-term, vaccine-induced resistance against intracellular pathogens. Here we show that natural antibodies, acting in concert with complement, are endogenous adjuvants for the generation of protective CD8(+) T cells after vaccination against visceral leishmaniasis. IL-4 was crucial for the priming of vaccine-specific CD8(+) T cells, and we defined the primary source of IL-4 as a CD11b(+)CD11c(lo) phagocyte. IL-4 secretion was not observed in antibody-deficient mice and could be reconstituted with serum from normal, but not Btk immune-deficient, mice. Similarly, no IL-4 response or CD8(+) T-cell priming was seen in C1qa(-/-) mice. These results identify a new pathway by which immune complex-mediated complement activation can regulate T-cell-mediated immunity. We propose that this function of natural antibodies could be exploited when developing new vaccines for infectious diseases.

SIGNR1-negative red pulp macrophages protect against acute streptococcal sepsis after Leishmania donovani-induced loss of marginal zone macrophages.

Marginal zone macrophages in the murine spleen play an important role in the capture of blood-borne pathogens and are viewed as an essential component of host defense against the development of pneumococcal sepsis. However, we and others have previously described the loss of marginal zone macrophages associated with the splenomegaly that follows a variety of viral and protozoal infections; this finding raises the question of whether these infected mice would become more susceptible to secondary pneumococcal infection. Contrary to expectations, we found that mice lacking marginal zone macrophages resulting from Leishmania donovani infection have increased resistance to Streptococcus pneumoniae type 3 and do not develop sepsis. Using biophotonic imaging, we observed that pneumococci are rapidly trapped in the spleens of L. donovani-infected mice. By selective depletion studies using clodronate liposomes, depleting monoclonal antibodies specific for Ly6C/G and Ly6G, and CD11c-DTR mice, we show that the enhanced early resistance in L. donovani-infected mice is entirely due to the activity of SIGNR1(-) red pulp macrophages. Our data demonstrate, therefore, that the normal requirement for SIGNR1(+) marginal zone macrophages to protect against a primary pneumococcal infection can, under conditions of splenomegaly, be readily compensated for by activated red pulp macrophages.

Pneumolysin binds to the mannose receptor C type 1 (MRC-1) leading to anti-inflammatory responses and enhanced pneumococcal survival.

Streptococcus pneumoniae (the pneumococcus) is a major cause of mortality and morbidity globally, and the leading cause of death in children under 5 years old. The pneumococcal cytolysin pneumolysin (PLY) is a major virulence determinant known to induce pore-dependent pro-inflammatory responses. These inflammatory responses are driven by PLY-host cell membrane cholesterol interactions, but binding to a host cell receptor has not been previously demonstrated. Here, we discovered a receptor for PLY, whereby pro-inflammatory cytokine responses and Toll-like receptor signalling are inhibited following PLY binding to the mannose receptor C type 1 (MRC-1) in human dendritic cells and mouse alveolar macrophages. The cytokine suppressor SOCS1 is also upregulated. Moreover, PLY-MRC-1 interactions mediate pneumococcal internalization into non-lysosomal compartments and polarize naive T cells into an interferon-γlow, interleukin-4high and FoxP3+ immunoregulatory phenotype. In mice, PLY-expressing pneumococci colocalize with MRC-1 in alveolar macrophages, induce lower pro-inflammatory cytokine responses and reduce neutrophil infiltration compared with a PLY mutant. In vivo, reduced bacterial loads occur in the airways of MRC-1-deficient mice and in mice in which MRC-1 is inhibited using blocking antibodies. In conclusion, we show that pneumococci use PLY-MRC-1 interactions to downregulate inflammation and enhance bacterial survival in the airways. These findings have important implications for future vaccine design.

Changes in soluble adhesion molecules in gingival crevicular fluid following periodontal surgery.

BACKGROUND: Inflammation of periodontal tissues during postoperative wound healing is mediated by cell surface adhesion molecules. Soluble forms of these antigens have also been identified and shown to be important in immunoregulatory processes, but have previously not been investigated during periodontal repair and regeneration. The present study has examined the presence and possible changes in soluble intercellular adhesion molecule-1 (sICAM-1; CD54) and lymphocyte function-associated antigen-3 (sLFA-3; CD58) in gingival crevical fluid (GCF) following periodontal surgery. METHODS: GCF samples were collected from four groups: 1) a guided tissue regeneration (GTR) test; 2) a GTR control, at least one complete tooth unit away from the periodontal defect; 3) a conventional flap (CF) surgery; and 4) a crown lengthening (CL). Sandwich enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of sICAM-1 and sLFA-3 in the GCF samples. RESULTS: A marked increase in GCF volumes was found in all sites after surgery, although a persistent increase was associated only with the period of membrane retention at the GTR test sites. In addition, sICAM-1 and sLFA-3 were found in the GCF of healthy as well as diseased sites prior to treatment and the total amounts of both increased transiently following surgical intervention, especially sLFA-3. However, the concentrations of these GCF components, particularly sICAM-1, tended to decrease. CONCLUSIONS: The temporal decrease in the concentration of sICAM-1 and sLFA-3 in GCF may serve to enhance inflammatory reactions at surgically-treated periodontal sites, thereby limiting repair and regeneration in the periodontium. These soluble adhesion molecules may thereby be of potential therapeutic value and might also be useful markers for monitoring periodontal wound healing.

c-di-GMP is an effective immunomodulator and vaccine adjuvant against pneumococcal infection.

Cyclic diguanylate (c-di-GMP) is a unique bacterial intracellular signaling molecule capable of stimulating enhanced protective innate immunity against various bacterial infections. The effects of intranasal pretreatment with c-di-GMP, or intraperitoneal coadministration of c-di-GMP with the pneumolysin toxoid (PdB) or pneumococcal surface protein A (PspA) before pneumococcal challenge, were investigated in mice. We found that c-di-GMP had no significant direct short-term effect on the growth rate of Streptococcus pneumoniae either in vitro or in vivo. However, intranasal pretreatment of mice with c-di-GMP resulted in a significant decrease in bacterial load in lungs and blood after serotypes 2 and 3 challenge, and a significant decrease in lung titers after serotype 4 challenge. Potential cellular mediators of these enhanced protective responses were identified in lungs and draining lymph nodes. Intraperitoneal coadministration of c-di-GMP with PdB or PspA before challenge resulted in significantly higher antigen-specific antibody titers and increased survival of mice, compared to that obtained with alum adjuvant. These findings demonstrate that local or systemic c-di-GMP administration stimulates innate and adaptive immunity against invasive pneumococcal disease. We propose that c-di-GMP can be used as an effective broad spectrum immunomodulator and vaccine adjuvant to prevent infectious diseases.

Evidence for the involvement of lung-specific gammadelta T cell subsets in local responses to Streptococcus pneumoniae infection.

Although gammadelta T cells are involved in the response to many pathogens, the dynamics and heterogeneity of the local gammadelta T cell response remains poorly defined. We recently identified gammadelta T cells as regulators of macrophages and dendritic cells during the resolution of Streptococcus pneumoniae-mediated lung inflammation. Here, using PCR, spectratype analysis and flow cytometry, we show that multiple gammadelta T cell subsets, including those bearing Vgamma1, Vgamma4 and Vgamma6 TCR, increase in number in the lungs of infected mice, but not in associated lymphoid tissue. These gammadelta T cells displayed signs of activation, as defined by CD69 and CD25 expression. In vivo BrdU incorporation suggested that local expansion, rather than recruitment, was the principal mechanism underlying this increase in gammadelta T cells. This conclusion was supported by the finding that pulmonary gammadelta T cells, but not alphabeta T cells, isolated from mice that had resolved infection exhibited lung-homing capacity in both naive and infected recipients. Together, these data provide novel insights into the origins of the heterogeneous gammadelta T cell response that accompanies lung infection, and the first evidence that inflammation-associated gammadelta T cells may exhibit distinct tissue-homing potential.

CD11b regulates recruitment of alveolar macrophages but not pulmonary dendritic cells after pneumococcal challenge.

Despite their close physical and functional relationships, alveolar macrophages (AMs) and pulmonary dendritic cells (pulDCs) have rarely been examined together in the context of infection. Using a nonlethal, resolving model of pneumonia caused by intranasal injection of Streptococcus pneumoniae, we demonstrate that AMs and pulDCs exhibit distinct characteristics during pulmonary inflammation. Recruitment of AMs and pulDCs occurred with different kinetics, and increased numbers of AMs resulted mainly from the appearance of a distinct subset of CD11b(High) AMs. Increased numbers of CD11b(High) and CD11b(Low) AMs, but not pulDCs, were recoverable from bronchoalveolar lavage fluid. CD11b expression on AMs was significantly increased by granulocyte-macrophage colony-stimulating factor but not by interleukin-10 or pathogen-associated stimuli. Finally, antibody blockade demonstrated that CD11b was critical for the recruitment of AMs, but not pulDCs, into the lung after pneumococcal challenge. These data demonstrate that there are significant differences between AM and pulDC responses to inflammatory pathogenic stimuli in vivo.

The role played by tumor necrosis factor during localized and systemic infection with Streptococcus pneumoniae.

Tumor necrosis factor (TNF) has been proposed as a major mediator of host resistance in murine models of Streptococcus pneumoniae infection; in humans, anti-TNF therapies have been implicated in increased susceptibility to pneumococcal infection. Here, we use nonlethal (serotype 6B) and lethal (serotype 3) S. pneumoniae, neutralizing monoclonal antibodies to TNF, and TNF gene-deficient mice to reexamine the role played by TNF in antistreptococcal responses. After nonlethal challenge, primary resistance and all examined parameters of the cellular inflammatory response occurred independently of TNF activity. After lethal challenge, TNF deficiency resulted in more-rapid death but did not affect lung inflammation. However, the livers of the TNF gene-deficient mice, but not of the control mice, exhibited extensive signs of systemic disease. TNF, therefore, is dispensable for a complete cellular pulmonary inflammatory response to S. pneumoniae infection but enhances survival from disseminated lethal infection, at least in part by delaying systemic organ damage.

The innate immune response differs in primary and secondary Salmonella infection.

This study examines innate immunity to oral Salmonella during primary infection and after secondary challenge of immune mice. Splenic NK and NKT cells plummeted early after primary infection, while neutrophils and macrophages (Mphi) increased 10- and 3-fold, respectively. In contrast, immune animals had only a modest reduction in NK cells, no loss of NKT cells, and a slight increase in phagocytes following secondary challenge. During primary infection, the dominant sources of IFN-gamma were, unexpectedly, neutrophils and Mphi, the former having intracellular stores of IFN-gamma that were released during infection. IFN-gamma-producing phagocytes greatly outnumbered IFN-gamma-producing NK cells, NKT cells, and T cells during the primary response. TNF-alpha production was also dominated by neutrophils and Mphi, which vastly outnumbered NKT cells producing this cytokine. Neither T cells nor NK cells produced TNF-alpha early during primary infection. The TNF-alpha response was reduced in a secondary response, but remained dominated by neutrophils and Mphi. Moreover, no significant IFN-gamma production by Mphi was associated with the secondary response. Indeed, only NK1.1(+) cells and T cells produced IFN-gamma in these mice. These studies provide a coherent view of innate immunity to oral Salmonella infection, reveal novel sources of IFN-gamma, and demonstrate that immune status influences the nature of the innate response.

In vivo compartmentalization of functionally distinct, rapidly responsive antigen-specific T-cell populations in DNA-immunized or Salmonella enterica serovar Typhimurium-infected mice.

The location and functional properties of antigen-specific memory T-cell populations in lymphoid and nonlymphoid compartments following DNA immunization or infection with Salmonella were investigated. Epitope-specific CD8+ -T-cell expansion and retention during the memory phase were analyzed for DNA-immunized mice by use of a 5-h peptide restimulation assay. These data revealed that epitope-specific gamma interferon (IFN-gamma)-positive CD8+ T cells occur at higher frequencies in the spleen, liver, and blood than in draining or peripheral lymph nodes during the expansion phase. Moreover, this distribution is maintained into long-term memory. The location and function of both CD4+ and CD8+ Salmonella-specific memory T cells in mice who were given a single dose of Salmonella enterica serovar Typhimurium was also quantitated by an ex vivo restimulation with bacterial lysate to detect the total Salmonella-specific memory pool. Mice immunized up to 6 months previously with S. enterica serovar Typhimurium had bacterium-specific CD4+ T cells that were capable of producing IFN-gamma or tumor necrosis factor alpha (TNF-alpha) at each site analyzed. Similar findings were observed for CD8+ T cells that were capable of producing IFN-gamma, while a much lower frequency and more restricted distribution were associated with TNF-alpha-producing CD8+ T cells. This study is the first to assess the frequencies, locations, and functions of both CD4+ and CD8+ memory T-cell populations in the same Salmonella-infected individuals and demonstrates the organ-specific functional compartmentalization of memory T cells after Salmonella infection.