Differential phagocytosis of Leishmania mexicana promastigotes and amastigotes by monocyte‐derived dendritic cells

Leishmania species are dimorphic protozoan parasites that live and replicate in the gut of sand flies as promastigotes or in mammalian hosts as amastigotes. Different immune cells, including DCs, and receptors differ in their involvement in phagocytosis of promastigotes and amastigotes and in recognition of different Leishmania species. In the case of L. mexicana, differences in phagocytosis of promastigotes and amastigotes by DCs and participation of C‐type lectin receptors (CLRs) have not been established. In the present study, flow cytometry and confocal microscopy were used to investigate the phagocytosis by monocyte‐derived dendritic cells (moDCs) of L. mexicana promastigotes and amastigotes in the presence or absence of immune serum during various periods of time. Blocking antibodies against mannose receptors and DC‐SIGN were used to explore the participation of these receptors in the phagocytosis of L. mexicana by moDC. The major differences in interactions of L. mexicana promastigotes and amastigotes with moDC were found to occur within the first 3 hr, during which phagocytosis of promastigotes predominated as compared with opsonization of promastigotes and amastigotes. However, after 6 hr of incubation, opsonized promastigotes were preferentially phagocytosed as compared with unopsonized promastigotes and amastigotes and after 24 hr of incubation there were no differences in the phagocytosis of promastigotes and amastigotes. Finally, after 3 hr incubation, DC‐SIGN was involved in the phagocytosis of promastigotes, but not of amastigotes.     

Interaction between mannosylated lipoarabinomannan and dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin influences dendritic cells maturation and T cell immunity

The aim of the study was to investigate the interaction between manLAM and DC-SIGN influencing DCs maturation and downstream immune response using small interfering RNA-expressing lentiviral vectors to specifically knockdown DC-SIGN. Our data indicated that DC-SIGN knockdown alone in DCs did not affect the maturation or the immunological function of lipopolysacharide (LPS)-activated DCs. Surface molecules were dramatically down-regulated in DCs primed with manLAM but not in mock control DCs (P < 0.05). Meanwhile, manLAM enhanced the production of the immunosuppressive cytokine IL-10 in DCs (P < 0.05). The level of IFN-γ was significantly down-regulated in the supernatants of naive T cells after co-cultured with DCs primed with manLAM (P < 0.05). We demonstrated that DCs primed with manLAM may partially impair maturation phenotypes and immune response in LPS-activated DCs. However, the alterations of DCs function and downstream immune response caused by manLAM were reversed by the knockdown of DC-SIGN.     

Mycobacterium tuberculosislpdC, Rv0462, induces dendritic cell maturation and Th1 polarization

Mycobacterium tuberculosis, the etiological factor of pulmonary tuberculosis, causes significant morbidity and mortality worldwide. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). In this study, we demonstrated that the gene encoding lipoamide dehydrogenase C (lpdC) from M. tuberculosis, Rv0462, induce maturation and activation of DCs involved in the MAPKs signaling pathway. Moreover, Rv0462-treated DCs activated naïve T cells, polarized CD4⁺ and CD8⁺ T cells to secrete IFN-γ in syngeneic mixed lymphocyte reactions, which would be expected to contribute to Th1 polarization of the immune response. Our results suggest that Rv0462 can contribute to the innate and adaptive immune responses during tuberculosis infection, and thus modulate the clinical course of tuberculosis.     

Fas Signal Promotes the Immunosuppressive Function of Regulatory Dendritic Cells via the ERK/β-Catenin Pathway

Dendritic cells (DCs) play important roles in the initiation of immune response and also in the maintenance of immune tolerance. Now, many kinds of regulatory DCs with different phenotypes have been identified to suppress immune response and contribute to the control of autoimmune diseases. However, the mechanisms by which regulatory DCs can be regulated to exert the immunosuppressive function in the immune microenvironment remain to be fully investigated. In addition, how T cells, once activated, can feedback affect the function of regulatory DCs during immune response needs to be further identified. We previously identified a unique subset of CD11b^hiIa^low regulatory DCs, differentiated from mature DCs or hematopoietic stem cells under a stromal microenvironment in spleen and liver, which can negatively regulate immune response in a feedback way. Here, we show that CD11b^hiIa^low regulatory DCs expressed high level of Fas, and endothelial stromal cell-derived TGF-β could induce high expression of Fas on regulatory DCs via ERK activation. Fas ligation could promote regulatory DCs to inhibit CD4⁺ T cell proliferation more significantly. Furthermore, Fas ligation preferentially induced regulatory DCs to produce IL-10 and IP-10 via ERK-mediated inactivation of GSK-3 and subsequent up-regulation of β-catenin. Interestingly, activated T cells could promote regulatory DCs to secrete more IL-10 and IP-10 partially through FasL. Therefore, our results demonstrate that Fas signal, at least from the activated T cells, can promote the immunosuppressive function of Fas-expressing regulatory DCs, providing a new manner for the regulatory DCs to regulate adaptive immunity.     

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen^1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response³.DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment⁴. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens⁵. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma^6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.     

Lewis X oligosaccharides-heparanase complex targeting to DCs enhance antitumor response in mice

Heparanase has been proved as an promising tumor antigen for the therapeutical target. However, the antigen alone cannot fully elicit the immune response in vivo. In this study, Lewis X oligosaccharides–heparanase complex was prepared, which can target to the dendritic cells (DCs) via dendritic cell-specific intercellular-adhesion-molecule-grabbing non-integrin (DC-SIGN). In addition, the DCs were loaded with the complex, and then were utilized to immunize mice to detect the immune response. Our data demonstrated that the modified DCs could enhance the specific IFN-γ production and cytotoxic T cell response. Furthermore, the modified DCs could also significantly suppress the established tumor growth and prolong the life span of tumor-bearing mice. Therefore, the Lewis X oligosaccharides–heparanase complex might be regarded as an ideal vaccine, and represent a novel way for the therapeutical strategy of tumor.     

Leishmania donovani vs immunity: T-cells sensitized from Leishmania of one donor may modulate their cytokines pattern on re-stimulation with Leishmania from different donor in visceral leishmaniasis

Antimony resistance is frequently encountered during treatment of visceral leishmaniasis (VL) and the differences are well characterized by inadequate IFN-γ dominant type-1 protection mechanisms. The part played by Leishmania parasites derived from antimony treated patients in the outcome of an immune response largely remains to be investigated. In the present study we observed that macrophages of BALB/c mice infected with antimony non-responder (SAG-NR) isolates had a greater amastigote burden than antimony responder (SAG-R) isolates. Later it was observed that antigen from SAG-NR and R L. donovani isolates elicit different cytokine responses in peritoneal blood mononuclear cells (PBMCs) from patients with VL. The production of IFN-γ by T-cells in VL patients increased in response to Leishmania derived from responder patients but this response within same T-cells was lower when sensitized from Leishmania from a non-responder VL patient. On the other hand, IL-4 and IL-10 expression was increased when primed with parasites from non-responder VL source. Such a differential pattern of cytokine expression by the same T-cell population produced to Leishmania from different donors, needs further exploration.     

Leishmania donovani infection differentially regulates small G‐proteins

Leishmania is a protozoan parasite that resides and replicates in macrophages and causes leishmaniasis. The parasite alters the signaling cascade in host macrophages and evades the host machinery. Small G‐proteins are GTPases, grouped in 5 different families that play a crucial role in the regulation of cell proliferation, cell survival, apoptosis, intracellular trafficking, and transport. In particular, the Ras family of small G‐proteins has been identified to play a significant role in the cellular functions mentioned before. Here, we studied the differential expression of the most important small G‐proteins during Leishmania infection. We found major changes in the expression of different isoforms of Ras, mainly in N‐Ras. We observed that Leishmania donovani infection led to enhanced N‐Ras expression, whereas it inhibited K‐Ras and H‐Ras expression. Furthermore, an active N‐Ras pull‐down assay showed enhanced N‐Ras activity. L donovani infection also increased extracellular signal–regulated kinase 1/2 phosphorylation and simultaneously decreased p38 phosphorylation. In contrast, pharmacological inhibition of Ras led to reduction in the phosphorylation of extracellular signal–regulated kinase 1/2 and enhanced the phosphorylation of p38 in Leishmania‐infected cells, which could lead to increased interleukin‐12 expression and decreased interleukin‐10 expression. Indeed, farnesylthiosalicyclic acid (a Ras inhibitor), when used at the effective level in L donovani–infected macrophages, reduced amastigotes in the host macrophages. Thus, upregulated N‐Ras expression during L donovani infection could be a novel immune evasion strategy of Leishmania and would be a potential target for antileishmanial immunotherapy.     

Targeting Leishmania major Antigens to Dendritic Cells In Vivo Induces Protective Immunity

Efficient vaccination against the parasite Leishmania major, the causative agent of human cutaneous leishmaniasis, requires development of type 1 T-helper (Th1) CD4⁺ T cell immunity. Because of their unique capacity to initiate and modulate immune responses, dendritic cells (DCs) are attractive targets for development of novel vaccines. In this study, for the first time, we investigated the capacity of a DC-targeted vaccine to induce protective responses against L. major. To this end, we genetically engineered the N-terminal portion of the stress-inducible 1 protein of L. major (LmSTI1a) into anti-DEC205/CD205 (DEC) monoclonal antibody (mAb) and thereby delivered the conjugated protein to DEC⁺ DCs in situ in the intact animal. Delivery of LmSTI1a to adjuvant-matured DCs increased the frequency of antigen-specific CD4⁺ T cells producing IFN-γ⁺, IL-2⁺, and TNF-α⁺ in two different strains of mice (C57BL/6 and Balb/c), while such responses were not observed with the same doses of a control Ig-LmSTI1a mAb without receptor affinity or with non-targeted LmSTI1a protein. Using a peptide library for LmSTI1a, we identified at least two distinct CD4⁺ T cell mimetopes in each MHC class II haplotype, consistent with the induction of broad immunity. When we compared T cell immune responses generated after targeting DCs with LmSTI1a or other L. major antigens, including LACK (Leishmania receptor for activated C kinase) and LeIF (Leishmania eukaryotic ribosomal elongation and initiation factor 4a), we found that LmSTI1a was superior for generation of IFN-γ-producing CD4⁺ T cells, which correlated with higher protection of susceptible Balb/c mice to a challenge with L. major. For the first time, this study demonstrates the potential of a DC-targeted vaccine as a novel approach for cutaneous leishmaniasis, an increasing public health concern that has no currently available effective treatment.     

Impact of Neutrophil-Secreted Myeloid Related Proteins 8 and 14 (MRP 8/14) on Leishmaniasis Progression

The myeloid-related proteins (MRPs) 8/14 are small proteins mainly produced by neutrophils, which have been reported to induce NO production in macrophages. On the other hand, Leishmania survives and multiplies within phagocytes by inactivating several of their microbicidal functions. Whereas MRPs are rapidly released during the innate immune response, their role in the regulation of Leishmaniasis is still unknown. In vitro experiments revealed that Leishmania infection alters MRP-induced signaling, leading to inhibition of macrophage functions (NO, TNF-α). In contrast, MRP-primed cells showed normal signaling activation and NO production in response to Leishmania infection. Using a murine air-pouch model, we observed that infection with L. major induced leukocyte recruitment and MRP secretion comparable to LPS-treated mice. Depletion of MRPs significantly reduced these inflammatory events and augmented both parasite load and footpad swelling during the first 8 weeks post-infection, as also observed in MRP KO mice. On the contrary, mouse treatment with recombinant MRPs (rMRPs) had the opposite effect. Collectively, our results suggest that rapid secretion of MRPs by neutrophils at the site of infection may protect uninfected macrophages and favor a more efficient innate inflammatory response against Leishmania infection. In summary, our study reveals the critical role played by MRPs in the regulation of Leishmania infection and how this pathogen can subvert its action.     

Snapin promotes HIV‐1 transmission from dendritic cells by dampening TLR8 signaling

HIV‐1 traffics through dendritic cells (DCs) en route to establishing a productive infection in T lymphocytes but fails to induce an innate immune response. Within DC endosomes, HIV‐1 somehow evades detection by the pattern‐recognition receptor (PRR) Toll‐like receptor 8 (TLR8). Using a phosphoproteomic approach, we identified a robust and diverse signaling cascade triggered by HIV‐1 upon entry into human DCs. A secondary siRNA screen of the identified signaling factors revealed several new mediators of HIV‐1 trans‐infection of CD4⁺ T cells in DCs, including the dynein motor protein Snapin. Inhibition of Snapin enhanced localization of HIV‐1 with TLR8⁺ early endosomes, triggered a pro‐inflammatory response, and inhibited trans‐infection of CD4⁺ T cells. Snapin inhibited TLR8 signaling in the absence of HIV‐1 and is a general regulator of endosomal maturation. Thus, we identify a new mechanism of innate immune sensing by TLR8 in DCs, which is exploited by HIV‐1 to promote transmission.     

Adoptive transfer of dendritic cells modulates immunogenesis and tolerogenesis in a neonatal model of murine cutaneous leishmaniasis

We evaluated the adoptive transfer of DCs on Leishmania (L.) mexicana-infected neonatal BALB/c mice. DCs were isolated and purified from the spleens of the following donor groups: a) Adult BALB/c mice infected during adulthood with L. (L) mexicana; b) Adult BALB/c mice infected during neonatal life; c) Healthy neonatal BALB/c mice; d) Healthy adult BALB/c mice. A neonatal model of infection, generated after inoculation with 5 × 10⁵ promastigotes of L. (L) mexicana, was used as the infection control group. Sixteen hours after intraperitoneal transfer of DCs (1 × 10³, 1 × 10⁵, or 1 × 10⁶ cells/ml), neonatal recipient BALB/c mice were infected. The adoptive transfer of DCs diminished disease progression in neonatal mice. This reduction depends on the quantity and provenance of transferred DCs, since the effect was more evident with high numbers of DCs from adult mice infected during adulthood and healthy neonatal mice. Protection was significantly reduced in animals receiving DCs from healthy adult mice but it was absent in mice receiving DCs from adult mice infected during neonatal life. These results suggest that genetic susceptibility to Leishmania infection can be modified during neonatal life, and that the period of life when antigens are encountered is crucial in influencing the capacity of DCs to induce resistance or tolerance.     

Identification of Th1/Th2 regulatory switch to promote healing response during leishmaniasis: a computational approach

Leishmania devices its survival strategy by suppressing the host’s immune functions. The antigen molecules produced by Leishmania interferes with the host’s cell signaling cascades and consequently changes the protein expression pattern of the antigen-presenting cell (APC). This creates an environment suitable for the switching of the T-cell responses from a healing Th1 response to a non-healing Th2 response that is favorable for the continued survival of the parasite inside the host APC. Using a reconstructed signaling network of the intracellular and intercellular reactions between a Leishmania infected APC and T-cell, we propose a computational model to predict the inhibitory effect of the Leishmania infected APC on the T-cell and to identify the regulators of this Th1-/Th2-switching behavior as observed during Leishmania infection. In this work, we hypothesize that a complete removal of the parasite could only be achieved with a simultaneous up-regulation of the healing Th1 response and stimulation of nitric oxide (NO) production from the APCs, and downregulation of the non-healing Th2 response and thereby propose several unique combinations of protein molecules that could elicit this anti-Leishmania immune response. Our results indicate that TLR3 may play a positive role in eliciting NO synthesis, while TLR2 may be responsible for inhibiting an anti-Leishmania immune response. Also, TLR3 overexpression (in the APC), when combined with SHP2 inhibition (in the T cell), produces an anti-Leishmania response that is better than the conventional IFN-gamma or IL12 treatment. A similar anti-Leishmania response is also obtained in another combination where TLR3 (in APC) is overexpressed, and SHC and MKP (of T cell) are inhibited and activated, respectively. Through our study, we also observe that Leishmania infection may induce an upregulation of IFN-beta production from the APC that may lead to an upregulation of the RAP1 and SOCS3 proteins inside the T cell, the potential inhibitors of MAPK and JAK-STAT signaling pathways, respectively, via the TYK2-mediated pathway. This study not only enhances our knowledge in understanding the Th1/Th2 regulatory switch to promote healing response during leishmaniasis but also helps to identify novel combinations of proteins as potential immunomodulators.     

Hizikia fusiforme extract enhances dendritic cell maturation in vitro and in vivo

ABSTRACT

Dendritic cells (DCs) are play critical roles in the priming and regulation of immune responses. DCs rapidly process and convey these antigens to prime antigen-specific T cells. Therefore, regulation of DCs functions is important for immunity and immunotherapies. Immune adjuvants for DCs activation are needed to improve the efficacy of vaccines against tumors and many infectious diseases. Therefore, we demonstrate that H. fusiformis extract can regulate DCs maturation and activation. H. fusiformis extract induced costimulatory molecules (CD 80 and CD86), antigen-presenting molecules (major histocompatibility complex (MHC) I and II), CCR7 expression, and interleukin (IL)-12 production in DCs. These effects are associated with upregulation of mitogen-activated protein kinase (MAPK) signaling pathway. In addition, H. fusiformis extract induces costimulatory molecules on splenic DCs and activated CD8⁺ T cells in vivo. Taken together, these findings suggest that H. fusiformis extract may be a potential efficient immune therapeutic compound in DCs-mediated immunotherapies.     

Evaluation of DNA/DNA and prime-boost vaccination using LPG3 against Leishmania major infection in susceptible BALB/c mice and its antigenic properties in human leishmaniasis

One of the main issues in vaccine development is implementation of new adjuvants to improve the antigen presentation and eliciting the protective immune response. Heat shock protein (HSP) molecules are known as natural adjuvants. They can stimulate the innate and adaptive immune response against infectious diseases and cancer. Lipophosphoglycan 3 (LPG3), the Leishmania homologous with GRP94 (glucose regulated protein 94), a member of HSP90 family, is involved in assembly of LPG as the most abundant macromolecule on the surface of Leishmania promastigotes. In the present study as a primary step, we tested LPG3 as a vaccine candidate in two regimens, DNA/DNA and prime-boost (DNA/Protein), against Leishmania major infection in BALB/c mice model. Our results showed that LPG3 and its fragment (rNT-LPG3) are highly immunogenic in BALB/c mice and can stimulate the production of both IgG1 and IgG2a. In prime-boost immunization strategy, the level of antibody response was higher compared with DNA/DNA immunization. The levels of IFN-γ in the supernatant of splenocytes from mice immunized with DNA/DNA and prime-boost regimens were significantly higher when compared to control groups. In fact, immunization with prime-boost vaccination has higher ratio of IFN-γ/IL-5, suggesting a shift towards a Th1 response.In addition, sera reactivity against LPG3 in visceral leishmaniasis (VL) patients was significantly higher in comparison with cutaneous leishmaniasis (CL) patients. Therefore, we recommend further investigations on the usage of LPG3 co-delivery with candidate antigens for vaccine development against leishmaniasis.     

Innate immunity against Leishmania infections

Leishmaniasis is a major health problem that affects more than 300 million people throughout the world. The morbidity associated with the disease causes serious economic burden in Leishmania endemic regions. Despite the morbidity and economic burden associated with Leishmaniasis, this disease rarely gets noticed and is still categorized under neglected tropical diseases. The lack of research combined with the ability of Leishmania to evade immune recognition has rendered our efforts to design therapeutic treatments or vaccines challenging. Herein, we review the literature on Leishmania from innate immune perspective and discuss potential problems as well as solutions and future directions that could aid in identifying novel therapeutic targets to eliminate this parasite.     

Mucosal immunity against Eimeria acervulina infection in broiler chickens following oral immunization with profilin in Montanide™ adjuvants

Leishmaniasis is a disease caused by the unicellular Leishmania parasite. World wide millions of people are affected by this vector born disease. The disease presents itself in different clinical manifestations which are caused by specific Leishmania species. The therapeutic strategy depends on the Leishmania species involved. It is important to detect Leishmania and subsequently type the infecting species in a sensitive way using PCR. Various targets have been proposed but two seem to be best suited, the ITS1 region and the mini-exon. There is, however, no consensus as to which of these two is best. The aim of this study was to compare both targets with our current method, a PCR on the 18S ribosomal RNA gene. The ITS1 PCR proved to be slightly more sensitive and more practical than the mini-exon. Nevertheless, the mini-exon is more polymorphic and is needed in subtyping Leishmania species belonging to the L. Viannia subgenus. The ITS1 method was adapted to use as a real-time PCR for diagnostic purposes. In addition, designing and testing a new primer set improved sensitivity of the PCR on the mini-exon.