Identification of connective tissue disease autoantibodies and a novel autoantibody anti-annexin A11 in patients with "idiopathic" interstitial lung disease.

BACKGROUND: Autoantibodies are a hallmark feature of Connective Tissue Diseases (CTD). Their presence in patients with idiopathic interstitial lung disease (ILD) may suggest covert CTD. We aimed to determine the prevalence of CTD autoantibodies in patients diagnosed with idiopathic ILD. METHODS: 499 patient sera were analysed: 251 idiopathic pulmonary fibrosis (IPF), 206 idiopathic non-specific interstitial pneumonia (iNSIP) and 42 cryptogenic organising pneumonia (COP). Autoantibody status was determined by immunoprecipitation. RESULTS: 2.4% of IPF sera had a CTD-autoantibody compared to 10.2% of iNSIP and 7.3% of COP. 45% of autoantibodies were anti-synthetases. A novel autoantibody targeting an unknown 56 kDa protein was found in seven IPF patients (2.8%) and two NSIP (1%) patients. This was characterised as anti-annexin A11. CONCLUSION: Specific guidance on autoantibody testing and interpretation in patients with ILD could improve diagnostic accuracy. Further work is required to determine the clinical significance of anti-annexin A11.

Leishmania major-derived lipophosphoglycan influences the host's early immune response by inducing platelet activation and DKK1 production via TLR1/2.

Background: Platelets are rapidly deployed to infection sites and respond to pathogenic molecules via pattern recognition receptors (TLR, NLRP). Dickkopf1 (DKK1) is a quintessential Wnt antagonist produced by a variety of cell types including platelets, endothelial cells, and is known to modulate pro-inflammatory responses in infectious diseases and cancer. Moreover, DKK1 is critical for forming leukocyte-platelet aggregates and induction of type 2 cell-mediated immune responses. Our previous publication showed activated platelets release DKK1 following Leishmania major recognition. Results: Here we probed the role of the key surface virulence glycoconjugate lipophosphoglycan (LPG), on DKK1 production using null mutants deficient in LPG synthesis (Δlpg1- and Δlpg2-). Leishmania-induced DKK1 production was reduced to control levels in the absence of LPG in both mutants and was restored upon re-expression of the cognate LPG1 or LPG2 genes. Furthermore, the formation of leukocyte-platelet aggregates was dependent on LPG. LPG mediated platelet activation and DKK1 production occurs through TLR1/2. Conclusion: Thus, LPG is a key virulence factor that induces DKK1 production from activated platelets, and the circulating DKK1 promotes Th2 cell polarization. This suggests that LPG-activated platelets can drive innate and adaptive immune responses to Leishmania infection.

Profiles of Local and Systemic Inflammation in the Outcome of Treatment of Human Cutaneous Leishmaniasis Caused by Leishmania (Viannia).

The immune mechanisms that contribute to the efficacy of treatment of cutaneous leishmaniasis (CL) are not fully understood. The aim of this study was to define immune correlates of the outcome of treatment of CL caused by Leishmania (Viannia) species during standard of care treatment with pentavalent antimonials. We conducted a comparative expression profiling of immune response genes in peripheral blood mononuclear cells (PBMCs) and lesion biopsy specimens obtained from CL patients before and at the end of treatment (EoT) with meglumine antimoniate. The ex vivo response of PBMCs to L (V) panamensis partially reflected that of lesion microenvironments. Significant downregulation of gene expression profiles consistent with local innate immune responses (monocyte and neutrophil activation and chemoattractant molecules) was observed at EoT in biopsy specimens of patients who cured (n = 8), compared to those from patients with treatment failure (n = 8). Among differentially expressed genes, pretreatment expression of CCL2 was significantly predictive of the therapeutic response (receiver operating characteristic [ROC] curve, area under the curve [AUC] = 0.82, P = 0.02). Polymorphisms in regulatory regions of the CCL2 promoter were analyzed in a pilot cohort of DNA samples from CL patients (cures, n = 20, and treatment failure, n = 20), showing putative association of polymorphisms rs13900(C/T) and rs2857656(G/C) with treatment outcome. Our data indicate that dampening gene expression profiles of monocyte and neutrophil activation characterize clinical cure after treatment of CL, supporting participation of parasite-sustained inflammation or deregulated innate immune responses in treatment failure.

Profiling gene expression of antimony response genes in Leishmania (Viannia) panamensis and infected macrophages and its relationship with drug susceptibility.

The mechanisms of Leishmania resistance to antimonials have been primarily determined in experimentally derived Leishmania strains. However, their participation in the susceptibility phenotype in field isolates has not been conclusively established. Being an intracellular parasite, the activity of antileishmanials is dependent on internalization of drugs into host cells and effective delivery to the intracellular compartments inhabited by the parasite. In this study we quantified and comparatively analyzed the gene expression of nine molecules involved in mechanisms of xenobiotic detoxification and Leishmania resistance to antimonial drugs in resistant and susceptible laboratory derived and clinical L.(Viannia) panamensis strains(n=19). In addition, we explored the impact of Leishmania susceptibility to antimonials on the expression of macrophage gene products having putative functions in transport, accumulation and metabolism of antimonials. As previously shown for other Leishmania species, a trend of increased abcc3 and lower aqp-1 expression was observed in the laboratory derived Sb-resistant L.(V.) panamensis line. However, this was not found in clinical strains, in which the expression of abca2 was significantly higher in resistant strains as both, promastigotes and intracellular amastigotes. The effect of drug susceptibility on host cell gene expression was evaluated on primary human macrophages from patients with cutaneous leishmaniasis (n=17) infected ex-vivo with the matched L.(V.) panamensis strains isolated at diagnosis, and in THP-1 cells infected with clinical strains (n=6) and laboratory adapted L.(V.) panamensis lines. Four molecules, abcb1 (p-gp), abcb6, aqp-9 and mt2a were differentially modulated by drug resistant and susceptible parasites, and among these, a consistent and significantly increased expression of the xenobiotic scavenging molecule mt2a was observed in macrophages infected with Sb-susceptible L. (V.) panamensis. Our results substantiate that different mechanisms of drug resistance operate in laboratory adapted and clinical Leishmania strains, and provide evidence that parasite-mediated modulation of host cell gene expression of molecules involved in drug transport and metabolism could contribute to the mechanisms of drug resistance and susceptibility in Leishmania.

Local Delivery of the Toll-Like Receptor 9 Ligand CpG Downregulates Host Immune and Inflammatory Responses, Ameliorating Established Leishmania (Viannia) panamensis Chronic Infection.

Infection by Leishmania (Viannia) panamensis, the predominant etiologic agent for cutaneous leishmaniasis in Colombia, is characterized by a chronic mixed inflammatory response. Current treatment options are plagued by toxicity, lengthy treatment regimens, and growing evidence of drug resistance. Immunotherapy, modulating the immune system to mount a protective response, may provide an alternate therapeutic approach. We investigated the ability of the Toll-like receptor 9 (TLR9) ligand CpG to modulate established disease in the L (V) panamensis mouse model. Treatment of established infection with a high dose (50 µg) of CpG ameliorated disease and lowered parasite burden. Interestingly, immediately after treatment there was a significant increase in transforming growth factor ß (TGF-ß) and concomitantly an increase in T regulatory cell (Treg) function. Although a general reduction in cell-mediated immune cytokine and chemokine (gamma interferon [IFN-γ], interleukin 10 [IL-10], IL-13, IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF], IL-4, and MIP-1α) responses of the treated mice was observed, certain chemokines (RANTES, monocyte chemoattractant protein 1[MCP-1], and IP-10) were increased. Further, in peripheral blood mononuclear cells (PBMCs) from patients with cutaneous leishmaniasis, CpG treatment similarly exhibited a dose-response effect on the production of IFN-γ, IL-17, IL-10, and IL-13, with reductions observed at higher doses. To further understand the underlying mechanisms and cell populations driving the CpG mediated response, we examined the ex vivo dose effects mediated by the TLR9+ cell populations (dendritic cells, macrophages, and B cells) found to accumulate labeled CpG in vivo Notably, B cells altered the production of IL-17, IL-13, and IFN-γ, supporting a role for B cells functioning as antigen-presenting cells (APCs) and/or regulatory cells during infection. Interestingly, B cells have been previously demonstrated as a primary type of APC in patients infected with L (V) panamensis and thus may be useful targets of immunotherapy. Collectively, our results show that CpG-induced immune regulation leads to a dampening of the host immune response and healing in the mouse model, and it may provide an alternate approach to treatment of cutaneous leishmaniasis caused by L (V) panamensis.

Targeting the HSP60/10 chaperonin systems of Trypanosoma brucei as a strategy for treating African sleeping sickness.

Trypanosoma brucei are protozoan parasites that cause African sleeping sickness in humans (also known as Human African Trypanosomiasis-HAT). Without treatment, T. brucei infections are fatal. There is an urgent need for new therapeutic strategies as current drugs are toxic, have complex treatment regimens, and are becoming less effective owing to rising antibiotic resistance in parasites. We hypothesize that targeting the HSP60/10 chaperonin systems in T. brucei is a viable anti-trypanosomal strategy as parasites rely on these stress response elements for their development and survival. We recently discovered several hundred inhibitors of the prototypical HSP60/10 chaperonin system from Escherichia coli, termed GroEL/ES. One of the most potent GroEL/ES inhibitors we discovered was compound 1. While examining the PubChem database, we found that a related analog, 2e-p, exhibited cytotoxicity to Leishmania major promastigotes, which are trypanosomatids highly related to Trypanosoma brucei. Through initial counter-screening, we found that compounds 1 and 2e-p were also cytotoxic to Trypanosoma brucei parasites (EC50=7.9 and 3.1µM, respectively). These encouraging initial results prompted us to develop a library of inhibitor analogs and examine their anti-parasitic potential in vitro. Of the 49 new chaperonin inhibitors developed, 39% exhibit greater cytotoxicity to T. brucei parasites than parent compound 1. While many analogs exhibit moderate cytotoxicity to human liver and kidney cells, we identified molecular substructures to pursue for further medicinal chemistry optimization to increase the therapeutic windows of this novel class of chaperonin-targeting anti-parasitic candidates. An intriguing finding from this study is that suramin, the first-line drug for treating early stage T. brucei infections, is also a potent inhibitor of GroEL/ES and HSP60/10 chaperonin systems.

Immunomodulatory nanoparticles ameliorate disease in the Leishmania (Viannia) panamensis mouse model.

Leishmania (Viannia) panamensis (L. (V.) panamensis) is a species of protozoan parasites that causes New World leishmaniasis, which is characterized by a hyper-inflammatory response. Current treatment strategies, mainly chemotherapeutic, are suboptimal due to adverse effects, long treatment regimens, and increasing drug resistance. Recently, immunotherapeutic approaches have shown promise in preclinical studies of leishmaniasis. As NPs may enable broad cellular immunomodulation through internalization in phagocytic and antigen-presenting cells, we tested the therapeutic efficacy of biodegradable NPs encapsulating a pathogen-associated molecular pattern (PAMP), CpG-rich oligonucleotide (CpG; NP-CpG), in mice infected with L. (V.) panamensis. NP-CpG treatment reduced lesion size and parasite burden, while neither free CpG nor empty NP showed therapeutic effects. NP-encapsulation led to CpG persistence at the site of infection along with an unexpected preferential cellular uptake by myeloid derived suppressor cells (MDSCs; CD11b(+)Ly6G(+)Ly6C(-)) as well as CD19(+) dendritic cells. This corresponded with the suppression of the ongoing immune response measured by the reduction of pathogenic cytokines IL-10 and IL-13, as well as IL-17 and IFNγ, in comparison to other treatment groups. As chronic inflammation is generally associated with the accumulation of MDSCs, this study may enable the rational design of cost-effective, safe, and scalable delivery systems for the treatment of inflammation-mediated diseases.

The Src kinases Hck, Fgr and Lyn activate Arg to facilitate IgG-mediated phagocytosis and Leishmania infection.

Leishmaniasis is a devastating disease that disfigures or kills nearly two million people each year. Establishment and persistence of infection by the obligate intracellular parasite Leishmania requires repeated uptake by macrophages and other phagocytes. Therefore, preventing uptake could be a novel therapeutic strategy for leishmaniasis. Amastigotes, the life cycle stage found in the human host, bind Fc receptors and enter macrophages primarily through immunoglobulin-mediated phagocytosis. However, the host machinery that mediates amastigote uptake is poorly understood. We have previously shown that the Arg (also known as Abl2) non-receptor tyrosine kinase facilitates L. amazonensis amastigote uptake by macrophages. Using small-molecule inhibitors and primary macrophages lacking specific Src family kinases, we now demonstrate that the Hck, Fgr and Lyn kinases are also necessary for amastigote uptake by macrophages. Src-mediated Arg activation is required for efficient uptake. Interestingly, the dual Arg and Src kinase inhibitor bosutinib, which is approved to treat cancer, not only decreases amastigote uptake, but also significantly reduces disease severity and parasite burden in Leishmania-infected mice. Our results suggest that leishmaniasis could potentially be treated with host-cell-active agents such as kinase inhibitors.

Leishmania-encoded orthologs of macrophage migration inhibitory factor regulate host immunity to promote parasite persistence.

Leishmania major encodes 2 orthologs of the cytokine macrophage migration inhibitory factor (MIF), whose functions in parasite growth or in the host-parasite interaction are unknown. To determine the importance of Leishmania-encoded MIF, both LmMIF genes were removed to produce an mif(-/-) strain of L. major This mutant strain replicated normally in vitro but had a 2-fold increased susceptibility to clearance by macrophages. Mice infected with mif(-/-) L. major, when compared to the wild-type strain, also showed a 3-fold reduction in parasite burden. Microarray and functional analyses revealed a reduced ability of mif(-/-) L. major to activate antigen-presenting cells, resulting in a 2-fold reduction in T-cell priming. In addition, there was a reduction in inflammation and effector CD4 T-cell formation in mif(-/-) L. major-infected mice when compared to mice infected with wild-type L. major Notably, effector CD4 T cells that developed during infection with mif(-/-) L. major demonstrated statistically significant differences in markers of functional exhaustion, including increased expression of IFN-γ and IL-7R, reduced expression of programmed death-1, and decreased apoptosis. These data support a role for LmMIF in promoting parasite persistence by manipulating the host response to increase the exhaustion and depletion of protective CD4 T cells.-Holowka, T., Castilho, T. M., Baeza Garcia, A., Sun, T., McMahon-Pratt, D., Bucala, R. Leishmania-encoded orthologs of macrophage migration inhibitory factor regulate host immunity to promote parasite persistence.

The Wnt Antagonist Dickkopf-1 Promotes Pathological Type 2 Cell-Mediated Inflammation.

Exposure to a plethora of environmental challenges commonly triggers pathological type 2 cell-mediated inflammation. Here we report the pathological role of the Wnt antagonist Dickkopf-1 (Dkk-1) upon allergen challenge or non-healing parasitic infection. The increased circulating amounts of Dkk-1 polarized T cells to T helper 2 (Th2) cells, stimulating a marked simultaneous induction of the transcription factors c-Maf and Gata-3, mediated by the kinases p38 MAPK and SGK-1, resulting in Th2 cell cytokine production. Circulating Dkk-1 was primarily from platelets, and the increase of Dkk-1 resulted in formation of leukocyte-platelet aggregates (LPA) that facilitated leukocyte infiltration to the affected tissue. Functional inhibition of Dkk-1 impaired Th2 cell cytokine production and leukocyte infiltration, protecting mice from house dust mite (HDM)-induced asthma or Leishmania major infection. These results highlight that Dkk-1 from thrombocytes is an important regulator of leukocyte infiltration and polarization of immune responses in pathological type 2 cell-mediated inflammation.

Ex vivo host and parasite response to antileishmanial drugs and immunomodulators.

BACKGROUND: Therapeutic response in infectious disease involves host as well as microbial determinants. Because the immune and inflammatory response to Leishmania (Viannia) species defines the outcome of infection and efficacy of treatment, immunomodulation is considered a promising therapeutic strategy. However, since Leishmania infection and antileishmanial drugs can themselves modulate drug transport, metabolism and/or immune responses, immunotherapeutic approaches require integrated assessment of host and parasite responses. METHODOLOGY: To achieve an integrated assessment of current and innovative therapeutic strategies, we determined host and parasite responses to miltefosine and meglumine antimoniate alone and in combination with pentoxifylline or CpG 2006 in peripheral blood mononuclear cells (PBMCs) of cutaneous leishmaniasis patients. Parasite survival and secretion of TNF-α, IFN-γ, IL-10 and IL-13 were evaluated concomitantly in PBMCs infected with Luc-L. (V.) panamensis exposed to meglumine antimoniate (4, 8, 16, 32 and 64 µg SbV/mL) or miltefosine (2, 4, 8, 16 and 32 µM HePC). Concentrations of 4 µM of miltefosine and 8 µg SbV/mL were selected for evaluation in combination with immunomodulators based on the high but partial reduction of parasite burden by these antileishmanial concentrations without affecting cytokine secretion of infected PBMCs. Intracellular parasite survival was determined by luminometry and cytokine secretion measured by ELISA and multiplex assays. PRINCIPAL FINDINGS: Anti- and pro-inflammatory cytokines characteristic of L. (V.) panamensis infection were evaluable concomitantly with viability of Leishmania within monocyte-derived macrophages present in PBMC cultures. Both antileishmanial drugs reduced the parasite load of macrophages; miltefosine also suppressed IL-10 and IL-13 secretion in a dose dependent manner. Pentoxifylline did not affect parasite survival or alter antileishmanial effects of miltefosine or meglumine antimoniate. However, pentoxifylline diminished secretion of TNF-α, IFN-γ and IL-13, cytokines associated with the outcome of infection by species of the Viannia subgenus. Exposure to CpG diminished the leishmanicidal effect of meglumine antimoniate, but not miltefosine, and significantly reduced secretion of IL-10, alone and in combination with either antileishmanial drug. IL-13 increased in response to CpG plus miltefosine. CONCLUSIONS AND SIGNIFICANCE: Human PBMCs allow integrated ex vivo assessment of antileishmanial treatments, providing information on host and parasite determinants of therapeutic response that may be used to tailor therapeutic strategies to optimize clinical resolution.

The flagellar protein FLAG1/SMP1 is a candidate for Leishmania-sand fly interaction.

Leishmaniasis is a serious problem that affects mostly poor countries. Various species of Leishmania are the agents of the disease, which take different clinical manifestations. The parasite is transmitted by sandflies, predominantly from the Phlebotomus genus in the Old World and Lutzomyia in the New World. During development in the gut, Leishmania must survive various challenges, which include avoiding being expelled with blood remnants after digestion. It is believed that attachment to the gut epithelium is a necessary step for vector infection, and molecules from parasites and sand flies have been implicated in this attachment. In previous work, monoclonal antibodies were produced against Leishmania. Among these an antibody was obtained against Leishmania braziliensis flagella, which blocked the attachment of Leishmania panamensis flagella to Phlebotomus papatasi guts. The protein recognized by this antibody was identified and named FLAG1, and the complete FLAG1 gene sequence was obtained. This protein was later independently identified as a small, myristoylated protein and called SMP1, so from now on it will be denominated FLAG1/SMP1. The FLAG1/SMP1 gene is expressed in all developmental stages of the parasite, but has higher expression in promastigotes. The anti-FLAG1/SMP1 antibody recognized the flagellum of all Leishmania species tested and generated the expected band by western blots. This antibody was used in attachment and infection blocking experiments. Using the New World vector Lutzomyia longipalpis and Leishmania infantum chagasi, no inhibition of attachment ex vivo or infection in vivo was seen. On the other hand, when the Old World vectors P. papatasi and Leishmania major were used, a significant decrease of both attachment and infection were seen in the presence of the antibody. We propose that FLAG1/SMP1 is involved in the attachment/infection of Leishmania in the strict vector P. papatasi and not the permissive vector L. longipalpis.

The immunotherapeutic role of regulatory T cells in Leishmania (Viannia) panamensis infection.

Leishmania (Viannia) parasites are etiological agents of cutaneous leishmaniasis in the New World. Infection is characterized by a mixed Th1/Th2 inflammatory response, which contributes to disease pathology. However, the role of regulatory T cells (Tregs) in Leishmania (Viannia) disease pathogenesis is unclear. Using the mouse model of chronic L. (V.) panamensis infection, we examined the hypothesis that Treg functionality contributes to control of pathogenesis. Upon infection, Tregs (CD4(+)Foxp3(+)) presented with a dysregulated phenotype, in that they produced IFN-γ, expressed Tbet, and had a reduced ability to suppress T cell proliferation in vitro. Targeted ablation of Tregs resulted in enlarged lesions, increased parasite load, and enhanced production of IL-17 and IFN-γ, with no change in IL-10 and IL-13 levels. This indicated that an increased inflammatory response was commensurate with disease exacerbation and that the remaining impaired Tregs were important in regulation of disease pathology. Conversely, adoptive transfer of Tregs from naive mice halted disease progression, lowered parasite burden, and reduced cytokine production (IL-10, IL-13, IL-17, IFN-γ). Because Tregs appeared to be important for controlling infection, we hypothesized that their expansion could be used as an immunotherapeutic treatment approach. As a proof of principle, chronically infected mice were treated with rIL-2/anti-IL-2 Ab complex to expand Tregs. Treatment transitorily increased the numbers and percentage of Tregs (draining lymph node, spleen), which resulted in reduced cytokine responses, ameliorated lesions, and reduced parasite load (10(5)-fold). Thus, immunotherapy targeting Tregs could provide an alternate treatment strategy for leishmaniasis caused by Leishmania (Viannia) parasites.

Chronicity of dermal leishmaniasis caused by Leishmania panamensis is associated with parasite-mediated induction of chemokine gene expression.

Chronic tegumentary leishmaniasis is characterized by a scarcity of parasites in lesions and a heightened inflammatory response. Deregulated and hyperactive inflammation contributes to tissue damage and parasite persistence. The mechanisms by which immune cells are recruited to the lesion and their relationship to clinical outcomes remain elusive. We examined the expression levels of chemokines and their receptors in relation to clinical outcome in dermal leishmaniasis caused by Leishmania (Viannia) panamensis. Primary macrophages from healthy donors were infected with L. panamensis strains isolated from self-healing patients (n = 4) and those presenting chronic disease (n = 5). A consistent pattern of upregulation of neutrophil (cxcl1, cxcl2, cxcl5, and cxcl8/il-8) and monocyte (ccl2, ccl7, ccl8, cxcl3, and cxcl10) chemotactic chemokines and ccr1 and ccr5 receptor genes, evaluated by reverse transcription-quantitative PCR (qRT-PCR), was observed upon infection with strains from patients with chronic dermal leishmaniasis; induction of CXCL5 and CCL8 was corroborated at the protein level. No apparent upregulation was elicited in macrophages infected with strains from self-healing patients. Expression levels of ccl8, cxcl2, cxcl3, and cxcl5 in lesion biopsy specimens from patients with chronic cutaneous leishmaniasis (CL) were compared to those in biopsy specimens from Montenegro skin tests of individuals with asymptomatic infection. Increased expression levels of cxcl5 (P < 0.05), ccl8, and cxcl3 were corroborated in chronic CL lesions. Our study revealed a dichotomy in macrophage chemokine gene expression elicited by L. panamensis strains from patients with self-healing disease and those presenting chronic disease, consistent with parasite-mediated hyperactivation of the inflammatory response driving chronicity. The predominant upregulation of neutrophil and monocyte chemoattractants indicates novel mechanisms of sustained inflammatory activation and may provide new therapeutic targets against chronic dermal leishmaniasis.

CD4 T cell activation by B cells in human Leishmania (Viannia) infection.

BACKGROUND: An effective adaptive immune response requires activation of specific CD4 T cells. The capacity of B cells to activate CD4 T cells in human cutaneous leishmaniasis caused by Leishmania (Viannia) has not been evaluated. METHODS: CD4 T cell activation by B cells of cutaneous leishmaniasis patients was evaluated by culture of PBMCs or purified B cells and CD4 T cells with Leishmania panamensis antigens. CD4 T cell and B cell activation markers were evaluated by flow cytometry and 13 cytokines were measured in supernatants with a bead-based capture assay. The effect of Leishmania antigens on BCR-mediated endocytosis of ovalbumin was evaluated in the Ramos human B cell line by targeting the antigen with anti-IgM-biotin and anti-biotin-ovalbumin-FITC. RESULTS: Culture of PBMCs from cutaneous leishmaniasis patients with Leishmania antigens resulted in upregulation of the activation markers CD25 and CD69 as well as increased frequency of CD25hiCD127- cells among CD4 T cells. Concomitantly, B cells upregulated the costimulatory molecule CD86. These changes were not observed in PBMCs from healthy subjects, indicating participation of Leishmania-specific lymphocytes expanded in vivo. Purified B cells from these patients, when interacting with purified CD4 T cells and Leishmania antigens, were capable of inducing significant increases in CD25 and CD69 expression and CD25hiCD127- frequency in CD4 T cells. These changes were associated with upregulation of CD86 in B cells. Comparison of changes in CD4 T cell activation parameters between PBMC and B cell/CD4 T cell cultures showed no statistically significant differences; further, significant secretion of IFN-γ, TNF-α, IL-6 and IL-13 was induced in both types of cultures. Additionally, culture with Leishmania antigens enhanced BCR-mediated endocytosis of ovalbumin in Ramos human B cells. CONCLUSIONS: The capacity of B cells specific for Leishmania antigens in peripheral blood of cutaneous leishmaniasis patients to activate CD4 T cells and induce cytokine secretion is similar to that of all cell populations present in PBMCs. This capacity implicates B cells as a plausible target for modulation of the immune response to Leishmania infection as a therapeutic strategy.

Human macrophage response to L. (Viannia) panamensis: microarray evidence for an early inflammatory response.

BACKGROUND: Previous findings indicate that susceptibility to Leishmania (Viannia) panamensis infection of monocyte-derived macrophages from patients and asymptomatically infected individuals were associated with the adaptive immune response and clinical outcome. METHODOLOGY/PRINCIPAL FINDINGS: To understand the basis for this difference we examined differential gene expression of human monocyte-derived macrophages following exposure to L. (V.) panamensis. Gene activation profiles were determined using macrophages from healthy volunteers cultured with or without stationary phase promastigotes of L. (V.) panamensis. Significant changes in expression (>1.5-fold change; p<0.05; up- or down-regulated) were identified at 0.5, 4 and 24 hours. mRNA abundance profiles varied over time, with the highest level of activation occurring at earlier time points (0.5 and 4 hrs). In contrast to observations for other Leishmania species, most significantly changed mRNAs were up- rather than down-regulated, especially at early time points. Up-regulated transcripts over the first 24 hours belonged to pathways involving eicosanoid metabolism, oxidative stress, activation of PKC through G protein coupled receptors, or mechanism of gene regulation by peroxisome proliferators via PPARα. Additionally, a marked activation of Toll-receptor mediated pathways was observed. Comparison with published microarray data from macrophages infected with L. (Leishmania) chagasi indicate differences in the regulation of genes involved in signaling, motility and the immune response. CONCLUSIONS: Results show that the early (0.5 to 24 hours) human monocyte-derived macrophage response to L. (Viannia) panamensis is not quiescent, in contrast to published reports examining later response times (48-96 hours). Early macrophage responses are important for the developing cellular response at the site of infection. The kinetics and the mRNA abundance profiles induced by L. (Viannia) panamensis illustrate the dynamics of these interactions and the distinct biologic responses to different Leishmania species from the outset of infection within their primary host cell.

The Abl and Arg kinases mediate distinct modes of phagocytosis and are required for maximal Leishmania infection.

Leishmania, an obligate intracellular parasite, binds several receptors to trigger engulfment by phagocytes, leading to cutaneous or visceral disease. These receptors include complement receptor 3 (CR3), used by promastigotes, and the Fc receptor (FcR), used by amastigotes. The mechanisms mediating uptake are not well understood. Here we show that Abl family kinases mediate both phagocytosis and the uptake of Leishmania amazonensis by macrophages (Ms). Imatinib, an Abl/Arg kinase inhibitor, decreases opsonized polystyrene bead phagocytosis and Leishmania uptake. Interestingly, phagocytosis of IgG-coated beads is decreased in Arg-deficient Ms, while that of C3bi-coated beads is unaffected. Conversely, uptake of C3bi-coated beads is decreased in Abl-deficient Ms, but that of IgG-coated beads is unaffected. Consistent with these results, Abl-deficient Ms are inefficient at C3bi-opsonized promastigote uptake, and Arg-deficient Ms are defective in IgG1-opsonized amastigote uptake. Finally, genetic loss of Abl or Arg reduces infection severity in murine cutaneous leishmaniasis, and imatinib treatment results in smaller lesions with fewer parasites than in controls. Our studies are the first to demonstrate that efficient phagocytosis and maximal Leishmania infection require Abl family kinases. These results highlight Abl family kinase-mediated signaling pathways as potential therapeutic targets for leishmaniasis.

TLR1/2 activation during heterologous prime-boost vaccination (DNA-MVA) enhances CD8+ T Cell responses providing protection against Leishmania (Viannia).

BACKGROUND: Leishmania (Viannia) parasites present particular challenges, as human and murine immune responses to infection are distinct from other Leishmania species, indicating a unique interaction with the host. Further, vaccination studies utilizing small animal models indicate that modalities and antigens that prevent infection by other Leishmania species are generally not protective. METHODOLOGY: Using a newly developed mouse model of chronic L. (Viannia) panamensis infection and the heterologous DNA prime - modified vaccinia virus Ankara (MVA) boost vaccination modality, we examined whether the conserved vaccine candidate antigen tryparedoxin peroxidase (TRYP) could provide protection against infection/disease. RESULTS: Heterologous prime - boost (DNA/MVA) vaccination utilizing TRYP antigen can provide protection against disease caused by L. (V.) panamensis. However, protection is dependent on modulating the innate immune response using the TLR1/2 agonist Pam3CSK4 during DNA priming. Prime-boost vaccination using DNA alone fails to protect. Prior to infection protectively vaccinated mice exhibit augmented CD4 and CD8 IFNγ and memory responses as well as decreased IL-10 and IL-13 responses. IL-13 and IL-10 have been shown to be independently critical for disease in this model. CD8 T cells have an essential role in mediating host defense, as CD8 depletion reversed protection in the vaccinated mice; vaccinated mice depleted of CD4 T cells remained protected. Hence, vaccine-induced protection is dependent upon TLR1/2 activation instructing the generation of antigen specific CD8 cells and restricting IL-13 and IL-10 responses. CONCLUSIONS: Given the general effectiveness of prime-boost vaccination, the recalcitrance of Leishmania (Viannia) to vaccine approaches effective against other species of Leishmania is again evident. However, prime-boost vaccination modality can with modulation induce protective responses, indicating that the delivery system is critical. Moreover, these results suggest that CD8 T cells should be targeted for the development of a vaccine against infection caused by Leishmania (Viannia) parasites. Further, TLR1/2 modulation may be useful in vaccines where CD8 T cell responses are critical.

Murine model of chronic L. (Viannia) panamensis infection: role of IL-13 in disease.

Leishmania (Viannia) organisms are the most prevalent etiologic agents of human cutaneous leishmaniasis in the Americas. Nevertheless, our knowledge of the immunological mechanisms exploited by L. (Viannia) organisms remains limited and the mechanisms underlying disease are not well understood. Here, we report the development of a BALB/c mouse model of L. (V.) panamensis infection that is able to reproduce chronic disease, with persistent infection and clinically evident lesions for over 1 year. The immune response of the mouse resembles that found for L. (V.) panamensis-infected patients with chronic and recurrent lesions, presenting a mixed Th1/Th2 response with the presence of TNF-α, IFN-γ, IL-10 and IL-13. Using immunodeficient mice, the critical role for IL-13 and/or IL-4Rα in determining susceptibility to chronic infection was evident. With the induction of healing in the immunodeficient mice, increases in IFN-γ and IL-17 were found, concomitant with parasite control and elimination. Specifically, increases in CD4(+) (but not CD8(+)) T cells producing IFN-γ were observed. These results suggest that IL-13 represents an important target for disease control of L. (V.) panamensis infection. This murine model should be useful to further understand the pathology associated with chronic disease and to develop methods for the treatment and prevention of leishmaniasis caused by L. (Viannia) parasites.

Murine visceral leishmaniasis: IgM and polyclonal B-cell activation lead to disease exacerbation.

In visceral leishmaniasis, the draining LN (DLN) is the initial site for colonization and establishment of infection after intradermal transmission by the sand fly vector; however, little is known about the developing immune response within this site. Using an intradermal infection model, which allows for parasite visceralization, we have examined the ongoing immune responses in the DLN of BALB/c mice infected with Leishmania infantum. Although not unexpected, at early times post-infection there is a marked B-cell expansion in the DLN, which persists throughout infection. However, the characteristics of this response were of interest; as early as day 7 post-infection, polyclonal antibodies (TNP, OVA, chromatin) were observed and the levels appeared comparable to the specific anti-leishmania response. Although B-cell-deficient JhD BALB/c mice are relatively resistant to infection, neither B-cell-derived IL-10 nor B-cell antigen presentation appear to be primarily responsible for the elevated parasitemia. However, passive transfer and reconstitution of JhD BALB/c with secretory immunoglobulins, (IgM or IgG; specific or non-specific immune complexes) results in increased susceptibility to L. infantum infection. Further, JhD BALB/c mice transgenetically reconstituted to secrete IgM demonstrated exacerbated disease in comparison to WT BALB/c mice as early as 2 days post-infection. Evidence suggests that complement activation (generation of C5a) and signaling via the C5a receptor (CD88) is related to the disease exacerbation caused by IgM rather than cytokine levels (IL-10 or IFN-gamma). Overall these studies indicate that polyclonal B-cell activation, which is known to be associated with human visceral leishmaniasis, is an early and intrinsic characteristic of disease and may represent a target for therapeutic intervention.