RANK Ligand Helps Immunity to Leishmania major by Skewing M2-Like Into M1 Macrophages.

Macrophages host Leishmania major infection, which causes cutaneous Leishmaniasis in humans. In the murine model, resistance to infection depends on the host immunity mediated by CD4 T-cell cytokines and macrophages. In association to other stimuli, the Th1 cytokine IFN-γ induces NO-mediated microbial killing by M1/classically-activated macrophages. By contrast, the Th2 cytokine IL-4 promotes M2/alternatively activated macrophages, which express arginase-1 and shelter infection. Other cytokines, such as RANKL, might also participate in the crosstalk between T cells and macrophages to restrict parasite infection. RANKL and its receptor RANK are known to play an essential role in bone remodeling, by inducing osteoclatogenesis. It has also been shown that RANKL stimulates antigen-presenting cells, such as DCs and macrophages, to enhance T cell responses. Here we investigated how RANKL directly modulates the effector macrophage phenotypes and immunity to L. major parasites. We found that inflammatory peritoneal macrophages from B6 mice express RANK and M2 features, such as CD301 (MGL) and CD206 (mannose receptor). Nonetheless, treatment with RANKL or IFN-γ induced macrophage differentiation into more mature F40/80hi macrophages able to produce IL-12 and TNF-α. In parallel, macrophages treated with RANKL, IFN-γ, or RANKL along with IFN-γ progressively downregulated the expression of the M2 hallmarks MGL, arginase-1, and CCL17. Moreover, a synergism between IFN-γ and RANKL enhanced inducible NO synthase (iNOS) expression and NO production by macrophages. These results are consistent with the idea that RANKL helps IFN-γ to induce a M2-like to M1 phenotype shift. Accordingly, concomitant treatment with RANKL and IFN-γ promoted macrophage-mediated immunity to L. major, by inducing NO and ROS-dependent parasite killing. Furthermore, by cooperating with IFN-γ, endogenous RANKL engages CD4 T-cell help toward L. major-infected macrophages to upregulate M1 and Th1 cytokine responses. Therefore, RANKL, in combination with IFN-γ, is a potential local therapeutic tool to improve immune responses in Leishmaniasis, by skewing M2-like into effector M1 macrophages.

Involvement of the capsular GalXM-induced IL-17 cytokine in the control of Cryptococcus neoformans infection.

Cryptococcus neoformans is an opportunistic fungus that can cause lethal brain infections in immunosuppressed individuals. Infection usually occurs via the inhalation of a spore or desiccated yeast which can then disseminate from the lung to the brain and other tissues. Dissemination and disease is largely influence by the production of copious amounts of cryptococcal polysaccharides, both which are secreted to the extracellular environment or assembled into a thick capsule surrounding the cell body. There are two important polysaccharides: glucuronoxylomannan (GXM) and galactoxylomannan, also called as glucuronoxylomanogalactan (GXMGal or GalXM). Although GXM is more abundant, GalXM has a more potent modulatory effect. In the present study, we show that GalXM is a potent activator of murine dendritic cells, and when co-cultured with T cells, induces a Th17 cytokine response. We also demonstrated that treating mice with GalXM prior to infection with C. neoformans protects from infection, and this phenomenon is dependent on IL-6 and IL-17. These findings help us understand the immune biology of capsular polysaccharides in fungal pathogenesis.

Trypanosoma cruzi Infection Induces Cellular Stress Response and Senescence-Like Phenotype in Murine Fibroblasts.

Trypanosoma cruzi infects and replicates within a wide variety of immune and non-immune cells. Here, we investigated early cellular responses induced in NIH-3T3 fibroblasts upon infection with trypomastigote forms of T. cruzi. We show that fibroblasts were susceptible to T. cruzi infection and started to release trypomastigotes to the culture medium after 4 days of infection. Also, we found that T. cruzi infection reduced the number of fibroblasts in 3-day cell cultures, by altering fibroblast proliferation. Infected fibroblasts displayed distinctive phenotypic alterations, including enlarged and flattened morphology with a nuclei accumulation of senescence-associated heterochromatin foci. In addition, infection induced an overexpression of the enzyme senescence-associated ß-galactosidase (SA-ß-gal), an activation marker of the cellular senescence program, as well as the production of cytokines and chemokines involved with the senescence-associated secretory phenotype (SASP) such as IL-6, TNF-α, IL-1ß, and MCP-1. Infected fibroblasts released increased amounts of stress-associated factors nitric oxide (NO) and reactive oxygen species (ROS), and the treatment with antioxidants deferoxamine (DFO) and N-acetylcysteine reduced ROS generation, secretion of SASP-related cytokine IL-6, SA-ß-gal activity, and parasite load by infected fibroblasts. Taken together, our data suggest that T. cruzi infection triggers a rapid cellular stress response followed by induction of a senescent-like phenotype in NIH-3T3 fibroblasts, enabling them to act as reservoirs of parasites during the early stages of the Chagas disease.

Antibody Repertoires Identify ß-Tubulin as a Host Protective Parasite Antigen in Mice Infected With Trypanosoma cruzi.

Few studies investigate the major protein antigens targeted by the antibody diversity of infected mice with Trypanosoma cruzi. To detect global IgG antibody specificities, sera from infected mice were immunoblotted against whole T. cruzi extracts. By proteomic analysis, we were able to identify the most immunogenic T. cruzi proteins. We identified three major antigens as pyruvate phosphate dikinase, Hsp-85, and ß-tubulin. The major protein band recognized by host IgG was T. cruzi ß-tubulin. The T. cruzi ß-tubulin gene was cloned, expressed in E. coli, and recombinant T. cruzi ß-tubulin was obtained. Infection increased IgG reactivity against recombinant T. cruzi ß-tubulin. A single immunization of mice with recombinant T. cruzi ß-tubulin increased specific IgG reactivity and induced protection against T. cruzi infection. These results indicate that repertoire analysis is a valid approach to identify antigens for vaccines against Chagas disease.

All-Trans Retinoic Acid Promotes an M1- to M2-Phenotype Shift and Inhibits Macrophage-Mediated Immunity to Leishmania major.

As key cells, able to host and kill Leishmania parasites, inflammatory monocytes/macrophages are potential vaccine and therapeutic targets to improve immune responses in Leishmaniasis. Macrophage phenotypes range from M1, which express NO-mediated microbial killing, to M2 macrophages that might help infection. Resistance to Leishmaniasis depends on Leishmania species, mouse strain, and both innate and adaptive immunity. C57BL/6 (B6) mice are resistant and control infection, whereas Leishmania parasites thrive in BALB/c mice, which are susceptible to develop cutaneous lesions in the course of infection with Leishmania major, but not upon infection with Leishmania braziliensis. Here, we investigated whether a deficit in early maturation of inflammatory monocytes into macrophages in BALB/c mice underlies increased susceptibility to L. major versus L. braziliensis parasites. We show that, after infection with L. braziliensis, monocytes are recruited to peritoneum, differentiate into macrophages, and develop an M1 phenotype able to produce proinflammatory cytokines in both B6 and BALB/c mice. Nonetheless, more mature macrophages from B6 mice expressed inducible NO synthase (iNOS) and higher NO production in response to L. braziliensis parasites, whereas BALB/c mice developed macrophages expressing an incomplete M1 phenotype. By contrast, monocytes recruited upon L. major infection gave rise to immature macrophages that failed to induce an M1 response in BALB/c mice. Overall, these results are consistent with the idea that resistance to Leishmania infection correlates with improved maturation of macrophages in a mouse-strain and Leishmania-species dependent manner. All-trans retinoic acid (ATRA) has been proposed as a therapy to differentiate immature myeloid cells into macrophages and help immunity to tumors. To prompt monocyte to macrophage maturation upon L. major infection, we treated B6 and BALB/c mice with ATRA. Unexpectedly, treatment with ATRA reduced proinflammatory cytokines, iNOS expression, and parasite killing by macrophages. Moreover, ATRA promoted an M1 to M2 transition in bone marrow-derived macrophages from both strains. Therefore, ATRA uncouples macrophage maturation and development of M1 phenotype and downmodulates macrophage-mediated immunity to L. major parasites. Cautions should be taken for the therapeutic use of ATRA, by considering direct effects on innate immunity to intracellular pathogens.

Degranulating Neutrophils Promote Leukotriene B4 Production by Infected Macrophages To Kill Leishmania amazonensis Parasites.

Neutrophils mediate early responses against pathogens, and they become activated during endothelial transmigration toward the inflammatory site. In the current study, human neutrophils were activated in vitro with immobilized extracellular matrix proteins, such as fibronectin (FN), collagen, and laminin. Neutrophil activation by FN, but not other extracellular matrix proteins, induces the release of the granules' contents, measured as matrix metalloproteinase 9 and neutrophil elastase activity in culture supernatant, as well as reactive oxygen species production. Upon contact with Leishmania amazonensis-infected macrophages, these FN-activated neutrophils reduce the parasite burden through a mechanism independent of cell contact. The release of granule proteases, such as myeloperoxidase, neutrophil elastase, and matrix metalloproteinase 9, activates macrophages through TLRs, leading to the production of inflammatory mediators, TNF-α and leukotriene B4 (LTB4), which are involved in parasite killing by infected macrophages. The pharmacological inhibition of degranulation reverted this effect, abolishing LTB4 and TNF production. Together, these results suggest that FN-driven degranulation of neutrophils induces the production of LTB4 and TNF by infected macrophages, leading to the control of Leishmania infection.

The PGE2/IL-10 Axis Determines Susceptibility of B-1 Cell-Derived Phagocytes (B-1CDP) to Leishmania major Infection.

B-1 cells can be differentiated from B-2 cells because they are predominantly located in the peritoneal and pleural cavities and have distinct phenotypic patterns and activation properties. A mononuclear phagocyte derived from B-1 cells (B-1CDP) has been described. As the B-1CDP cells migrate to inflammatory/infectious sites and exhibit phagocytic capacity, the microbicidal ability of these cells was investigated using the Leishmania major infection model in vitro. The data obtained in this study demonstrate that B-1CDP cells are more susceptible to infection than peritoneal macrophages, since B-1CDP cells have a higher number of intracellular amastigotes forms and consequently release a larger number of promastigotes. Exacerbated infection by L. major required lipid bodies/PGE2 and IL-10 by B-1CDP cells. Both infection and the production of IL-10 were decreased when PGE2 production was blocked by NSAIDs. The involvement of IL-10 in this mechanism was confirmed, since B-1CDP cells from IL-10 KO mice are more competent to control L. major infection than cells from wild type mice. These findings further characterize the B-1CDP cells as an important mononuclear phagocyte that plays a previously unrecognized role in host responses to L. major infection, most likely via PGE2-driven production of IL-10.

Capsular polysaccharides from Cryptococcus neoformans modulate production of neutrophil extracellular traps (NETs) by human neutrophils.

In the present study, we characterized the in vitro modulation of NETs (neutrophil extracellular traps) induced in human neutrophils by the opportunistic fungus Cryptococcus neoformans, evaluating the participation of capsular polysaccharides glucuronoxylomanan (GXM) and glucuronoxylomannogalactan (GXMGal) in this phenomenon. The mutant acapsular strain CAP67 and the capsular polysaccharide GXMGal induced NET production. In contrast, the wild-type strain and the major polysaccharide GXM did not induce NET release. In addition, C. neoformans and the capsular polysaccharide GXM inhibited PMA-induced NET release. Additionally, we observed that the NET-enriched supernatants induced through CAP67 yeasts showed fungicidal activity on the capsular strain, and neutrophil elastase, myeloperoxidase, collagenase and histones were the key components for the induction of NET fungicidal activity. The signaling pathways associated with NET induction through the CAP67 strain were dependent on reactive oxygen species (ROS) and peptidylarginine deiminase-4 (PAD-4). Neither polysaccharide induced ROS production however both molecules blocked the production of ROS through PMA-activated neutrophils. Taken together, the results demonstrate that C. neoformans and the capsular component GXM inhibit the production of NETs in human neutrophils. This mechanism indicates a potentially new and important modulation factor for this fungal pathogen.

Neutrophils increase or reduce parasite burden in Trypanosoma cruzi-infected macrophages, depending on host strain: role of neutrophil elastase.

Neutrophils are involved in the initial steps of most responses to pathogens and are essential components of the innate immune response. Due to the ability to produce and release various soluble mediators, neutrophils may participate in the regulation of the inflammatory response. Little is known about the role of neutrophils during protozoan infections including infection by Trypanosoma cruzi. In the present study we investigated the importance of inflammatory neutrophils on macrophage activation and T. cruzi replication in vitro, in cells obtained from BALB/c mice and C57Bl/6 mice. Co-cultures of BALB/c apoptotic or live neutrophils with infected peritoneal macrophages resulted in increased replication of the parasites and in the production of TGF-ß and PGE2. The treatment with anti-TGF-ß neutralizing antibody and COX inhibitor blocked the parasite replication in vitro. On the other hand, co-cultures of T. cruzi infected macrophages with live neutrophils isolated from C57BL/6 mice resulted in decreased number of trypomastigotes in culture and increased production of TNF-α and NO. The addition of anti-TNF-α neutralizing antibody or elastase inhibitor resulted in the abolishment of macrophage microbicidal effect and increased parasite replication. Addition of elastase to infected macrophages reduced the replication of the parasites, and on the other hand, addition of a selective inhibitor of iNOS increased parasite growth, suggesting the role of NO in this system. Our findings reveal that neutrophils may regulate T. cruzi experimental infection and determine susceptibility and resistance to infection.

Infection with Leishmania major induces a cellular stress response in macrophages.

We investigated early cellular responses induced by infection with Leishmania major in macrophages from resistant C57/BL6 mice. Infection increased production of reactive oxygen species by resident, but not inflammatory peritoneal macrophages. In addition, infection increased activation of stress-activated protein kinases/c-Jun N-terminal kinases (SAPK/JNK) in resident, but not in inflammatory peritoneal macrophages. Infection also increased expression of membrane and soluble FasL, but infected macrophages remained viable after 48 h. Infection increased secretion of cytokines/chemokines TNF-α, IL-6, TIMP-1, IL-1RA, G-CSF, TREM, KC, MIP-1α, MIP-1ß, MCP-1, and MIP-2 in resident macrophages. Addition of antioxidants deferoxamine and N-acetylcysteine reduced ROS generation and JNK activation. Addition of antioxidants or JNK inhibitor SP600125 reduced secretion of KC. Furthermore, treatment with antioxidants or JNK inhibitor also reduced intracellular parasite replication. These results indicated that infection triggers a rapid cellular stress response in resident macrophages which induces proinflammatory signals, but is also involved in parasite survival and replication in host macrophages.