The Paradox of a Phagosomal Lifestyle: How Innate Host Cell-Leishmania amazonensis Interactions Lead to a Progressive Chronic Disease.

Intracellular phagosomal pathogens represent a formidable challenge for innate immune cells, as, paradoxically, these phagocytic cells can act as both host cells that support pathogen replication and, when properly activated, are the critical cells that mediate pathogen elimination. Infection by parasites of the Leishmania genus provides an excellent model organism to investigate this complex host-pathogen interaction. In this review we focus on the dynamics of Leishmania amazonensis infection and the host innate immune response, including the impact of the adaptive immune response on phagocytic host cell recruitment and activation. L. amazonensis infection represents an important public health problem in South America where, distinct from other Leishmania parasites, it has been associated with all three clinical forms of leishmaniasis in humans: cutaneous, muco-cutaneous and visceral. Experimental observations demonstrate that most experimental mouse strains are susceptible to L. amazonensis infection, including the C57BL/6 mouse, which is resistant to other species such as Leishmania major, Leishmania braziliensis and Leishmania infantum. In general, the CD4+ T helper (Th)1/Th2 paradigm does not sufficiently explain the progressive chronic disease established by L. amazonensis, as strong cell-mediated Th1 immunity, or a lack of Th2 immunity, does not provide protection as would be predicted. Recent findings in which the balance between Th1/Th2 immunity was found to influence permissive host cell availability via recruitment of inflammatory monocytes has also added to the complexity of the Th1/Th2 paradigm. In this review we discuss the roles played by innate cells starting from parasite recognition through to priming of the adaptive immune response. We highlight the relative importance of neutrophils, monocytes, dendritic cells and resident macrophages for the establishment and progressive nature of disease following L. amazonensis infection.

Phenotypical and genotypical differences among Leishmania (Leishmania) amazonensis isolates that caused different clinical frames in humans and dogs: A systematic review.

Leishmania (Leishmania) amazonensis is an important etiological agent of American cutaneous leishmaniasis (ACL) in Brazil. The species causes a large spectrum of clinical manifestations in humans and dogs, ranging from cutaneous, cutaneous diffuse, mucocutaneous, and visceral involvement, however, the factors that drive the development of different disease forms by the same species are not yet fully known. In the present work, it was systematically reviewed the studies addressing phenotypic and genotypic characteristics of Leishmania (L.) amazonensis isolates causing cutaneous and visceral clinical frames in humans and dogs, comparing the results observed. For this, four research databases were searched for the following keywords: (Leishmania amazonensis AND visceral leishmaniasis) AND (tropism OR virulence OR visceralization OR adaptations OR mutation OR clinical presentation OR resistance OR survival OR wide spectrum). The results revealed that the complexity disease seems to involve the combination of genetic factors of the parasite (as modifications in molecules related to the virulence and metabolism) and also of the host's immune background and status. Nonetheless, the exact mechanism that leads to different clinical manifestations between strains of the same species is still uncertain and future studies must be developed to better elucidate this phenomenon.

Potential antigenic targets used in immunological tests for diagnosis of tegumentary leishmaniasis: A systematic review.

Immunological tests may represent valuable tools for the diagnosis of human tegumentary leishmaniasis (TL) due to their simple execution, less invasive nature and potential use as a point-of-care test. Indeed, several antigenic targets have been used with the aim of improving the restricted scenario for TL-diagnosis. We performed a worldwide systematic review to identify antigenic targets that have been evaluated for the main clinical forms of TL, such as cutaneous (CL) and mucosal (ML) leishmaniasis. Included were original studies evaluating the sensitivity and specificity of immunological tests for human-TL, CL and/or ML diagnosis using purified or recombinant proteins, synthetic peptides or polyclonal or monoclonal antibodies to detect Leishmania-specific antibodies or antigens. The review methodology followed PRISMA guidelines and all selected studies were evaluated in accordance with QUADAS-2. Thirty-eight original studies from four databases fulfilled the selection criteria. A total of 79 antigens were evaluated for the detection of antibodies as a diagnostic for TL, CL and/or ML by ELISA. Furthermore, three antibodies were evaluated for the detection of antigen by immunochromatographic test (ICT) and immunohistochemistry (IHC) for CL-diagnosis. Several antigenic targets showed 100% of sensitivity and specificity, suggesting potential use for TL-diagnosis in its different clinical manifestations. However, a high number of proof-of-concept studies reinforce the need for further analysis aimed at verifying true diagnostic accuracy in clinical practice.

In situ study of cellular immune response in human cutaneous lesions caused by Leishmania (Viannia) panamensis in Panama.

AIMS: Leishmaniasis is considered a disease with multiple clinical/immunopathological characteristics, depending on the immunity of the host and the species of the parasite. In Panama, the most prevalent species that causes localized cutaneous leishmaniasis (LCL) is Leishmania (Viannia) panamensis, and its immune response is poorly studied. Therefore, we evaluated by immunohistochemistry, the in situ immune response during this infection. METHODS AND RESULTS: Biopsies from Panamanian patients with LCL were collected and processed by histological techniques. Infection by L. (V.) panamensis was demonstrated by isolation in culture and molecular characterization by Hsp70-RFLP. The in situ immune response was assessed by immunohistochemistry. The immune response was characterized by predominance of T cells, mainly CD8 cells that showed positive correlation with IFN-γ and Granzyme B. CD4 cells presented positive correlation with both IFN-γ and IL-13, pointed by mixed cellular immune response. Regulatory response was characterized by FoxP3 cells, which showed positive correlation to IL-10 but not with TGF-ß. CONCLUSIONS: L. (V.) panamensis infection triggers a mixed cellular immune response, characterized by the presence of pro-inflammatory, anti-inflammatory and regulatory elements in the skin lesion of Panamanian patients. These data contribute to a better understanding of the immunopathogenesis of Leishmania Viannia infection in Panama.

Leishmania Viannia guyanensis, LRV1 virus and extracellular vesicles: a dangerous trio influencing the faith of immune response during muco-cutaneous leishmaniasis.

Parasites of Leishmania genus have developed various strategies to overcome host immune response favoring its infection and development toward leishmaniasis. With an array of virulence factors, those parasites modify host macrophage signaling and functions. Depending of the species involved, visceral or cutaneous leishmaniasis will develop. Several years ago, Leishmania Viannia guyanensis that is naturally infected with the endosymbiotic virus Leishmania RNA Virus 1 was found to cause a particularly aggressive form of South-American mucocutaneous leishmaniasis. This virus, when co-transmitted with the parasite was shown to strongly modulate RNA sensors and NLRP3 inflammasome network that could explain in part the exacerbated skin pathology caused by this particular parasite. In this review, we will be discussing how this endosymbiotic virus-infected Leishmania in conjunction with Leishmania exosomes partner together to manipulate host immune response in their favor.

B-cell profile, B-cell activating factor concentration and IgG levels in human cutaneous and mucosal leishmaniasis.

AIMS: The aim of this study was to evaluate characteristics of B cells in human tegumentary leismaniasis (TL) analysing cutaneous leishmaniasis (CL), most prevalent form and mucosal leishmaniasis (ML), aggressive form characterized by the destruction of the oral-nasal-pharyngeal cavities. METHODS AND RESULTS: By flow cytometry analysis, we found decreased percentages of non-class-switched memory B cells in TL with the degree of the loss related to clinical severity. Using commercial ELISA, we reported high levels of B-cell activating factor (BAFF) and IgG preferentially in aggressive CL and markedly in ML together with decreased BAFF receptors in the latter. We also found lower levels of BAFF after clinical recovery suggesting a relation between BAFF and disease activity. Mucosal leishmaniasis history of therapeutic failure presented high levels of BAFF accompanied by detectable concentrations of IFN-γ and IL-6 (assayed by commercial ELISA and cytometric bead arrays respectively), cytokines involved in exaggerated inflammatory responses and tissue damage in TL. CONCLUSION: We demonstrate B-cell disturbances in TL with the degree of the alterations related to clinical severity. We suggest a relation between excess of BAFF and disease activity and point towards a possible implication of BAFF in the inflammatory phenomenon of ML.

Cutaneous and mucocutaneous leishmaniasis: experience of a Mediterranean hospital.

BACKGROUND: Leishmaniasis, considered by the World Health Organization as one of the most important tropical diseases, is endemic in the Mediterranean Basin. The aim of this study was to evaluate epidemiological and clinical characteristics of cutaneous (CL) and mucocutaneous leishmaniasis (MCL) in La Fe University Hospital, Valencia, Spain. The particular focus was on diagnosis techniques and clinical differences according to the immunological status of the patients. METHODS: An eleven-year retrospective observational study of CL and MCL episodes at the hospital was performed. Epidemiological, clinical and therapeutic variables of each case, together with the microbiological and anatomopathological diagnosis, were analyzed. RESULTS: A total of 42 patients were included, 30 of them were male and 28 were immunocompetent. Most of the cases (36/42) were diagnosed in the last 5 years (2013-2017). The incidence of CL and MCL increased from 3.6/100,000 (2006-2012) to 13.58/100,000 (2013-2017). The majority of the patients (37/42) exhibited CL, in 30 cases as single lesions (30/37). Ulcerative lesions were more common in immunosuppressed patients (13/14) than in immunocompetent patients (20/28), (P = 0.2302). The length of lesion presence before diagnosis was 7.36 ± 6.72 months in immunocompetent patients and 8.79 ± 6.9 months in immunosuppressed patients (P = 0.1863). Leishmania DNA detection (92.3%) was the most sensitive diagnostic technique followed by Giemsa stain (65%) and histopathological examination (53.8%). Twelve patients (12/42) had close contact with dogs or were living near to kennels, and 10 of them did not present underlying conditions. Intralesional glucantime (21/42) and liposomal amphotericin B (7/42) were the most common treatments administered in monotherapy. All patients evolved successfully and no relapse was reported. CONCLUSIONS: Some interesting clinical and epidemiological differences were found in our series between immunocompetent and immunosuppressed patients. Future studies can take these results further especially by studying patients with biological therapy. Skin biopsies combining NAAT with histological techniques are the most productive techniques for CL or MCL diagnosis.

The dichotomy of the systemic and local cytokine responses of mucosal leishmaniasis patients in Sudan: A pilot study.

Sudanese mucosal leishmaniasis (ML) is a rare clinical form of leishmaniasis and characterized by persistent ulcer of the oral and/or the nasal mucous membranes caused by Leishmania donovani. No data is available about the systemic and local immune responses in mucosal leishmaniasis. This study aimed to measure the systemic and the local cytokines responses of Sudanese ML patients compared to cured cutaneous leishmaniasis patients (Leishmanin skin test positive, LST+ve) and unexposed healthy controls (Leishmanin skin test negative, LST-ve). Six parasitological confirmed ML patients, 7 LST+ve, and 6 LST-ve were enrolled. Systemic Th-1 (IFN-γ and TNF-α), Th-2 (IL-10 and IL-13), Treg (TGF-ß1), and inflammatory cytokines IL-6 and IL-8 concentration were measured in the supernatant of whole blood samples following stimulation with live L. donovani promastigotes using ELISA. Local intralesion IL-10, IFN-γ, and IL-13 expression was measured using Real Time PCR. A significant high concentrations of IFN-γ, TNFα, IL-10, TGFß, IL-6, and IL-8 were detected in the supernatant of stimulated whole blood samples of ML patients compared with the LST+ve and LST-ve controls. Using Real Time-PCR and primers for various cytokines, a significant high expression of TH2 cytokines IL-10 and IL-13 mRNA was detected in contrast to a low TH1 cytokine IFN-γ mRNA in the mucosal lesion. There is a clear dichotomy in the cytokine response during Mucosal leishmaniasis. A significantly high TH1, inflammatory and Treg cytokines response is produced systemically, in contrast to a significant high TH2 cytokines response in the mucosal lesion.

Leishmania RNA virus exacerbates Leishmaniasis by subverting innate immunity via TLR3-mediated NLRP3 inflammasome inhibition.

Leishmania RNA virus (LRV) is an important virulence factor associated with the development of mucocutaneous Leishmaniasis, a severe form of the disease. LRV-mediated disease exacerbation relies on TLR3 activation, but downstream mechanisms remain largely unexplored. Here, we combine human and mouse data to demonstrate that LRV triggers TLR3 and TRIF to induce type I IFN production, which induces autophagy. This process results in ATG5-mediated degradation of NLRP3 and ASC, thereby limiting NLRP3 inflammasome activation in macrophages. Consistent with the known restricting role of NLRP3 for Leishmania replication, the signaling pathway triggered by LRV results in increased parasite survival and disease progression. In support of this data, we find that lesions in patients infected with LRV+ Leishmania are associated with reduced inflammasome activation and the development of mucocutaneous disease. Our findings reveal the mechanisms triggered by LRV that contribute to the development of the debilitating mucocutaneous form of Leishmaniasis.

Leishmania braziliensis: Strain-Specific Modulation of Phagosome Maturation.

Leishmania (Viannia) braziliensis is responsible for the largest number of American tegumentary leishmaniasis (ATL) in Brazil. ATL can present several clinical forms including typical (TL) and atypical (AL) cutaneous and mucocutaneous (ML) lesions. To identify parasite and host factors potentially associated with these diverse clinical manifestations, we first surveyed the expression of two virulence-associated glycoconjugates, lipophosphoglycan (LPG) and the metalloprotease GP63 by a panel of promastigotes of Leishmania braziliensis (L. braziliensis) strains isolated from patients with different clinical manifestations of ATL and from the sand fly vector. We observed a diversity of expression patterns for both LPG and GP63, which may be related to strain-specific polymorphisms. Interestingly, we noted that GP63 activity varies from strain to strain, including the ability to cleave host cell molecules. We next evaluated the ability of promastigotes from these L. braziliensis strains to modulate phagolysosome biogenesis in bone marrow-derived macrophages (BMM), by assessing phagosomal recruitment of the lysosome-associated membrane protein 1 (LAMP-1) and intraphagosomal acidification. Whereas, three out of six L. braziliensis strains impaired the phagosomal recruitment of LAMP-1, only the ML strain inhibited phagosome acidification to the same extent as the L. donovani strain that was used as a positive control. While decreased phagosomal recruitment of LAMP-1 correlated with higher LPG levels, decreased phagosomal acidification correlated with higher GP63 levels. Finally, we observed that the ability to infect and replicate within host cells did not fully correlate with the inhibition of phagosome maturation. Collectively, our results revealed a diversity of strain-specific phenotypes among L. braziliensis isolates, consistent with the high genetic diversity within Leishmania populations.

In Situ Cellular Response Underlying Successful Treatment of Mucosal Leishmaniasis with a Combination of Pentavalent Antimonial and Pentoxifylline.

Mucosal leishmaniasis (ML) is characterized by high production of inflammatory cytokines. Administration of pentoxifylline (PTX), an inhibitor of TNF-alpha, with pentavalent antimony (Sbv), has been successfully used as alternative treatment for refractory ML. Our study aims to investigate the in situ cellular response underlying the effectiveness of this therapy, by evaluating the intensity of the inflammatory infiltrate, cellular composition, and expression of cytokines and granzyme A in lesions from ML before and after treatment with Sbv alone or in combination with PTX. Our data showed no differences in the intensity of inflammatory infiltrate comparing before and after treatment, and comparing between different treatments. However, although the number and frequency of CD4+ and CD8+ cells were not different before and after treatments or comparing different treatments, frequency of CD68+ cells decreased after treatment with Sbv + PTX, but not with Sbv. This was due to a reduction in CD68+ TNF-alpha+ and not in CD68+ IL-10+ cells. The frequency of TNF-alpha+ cells was correlated with the intensity of the inflammatory infiltrate before treatment, but this correlation was lost after treatment with Sbv + PTX. Although the total expression of granzyme A did not significantly change after treatments, a clear trend of decrease was observed after treatment with Sbv + PTX. Interestingly, patients who took longer to heal, regardless of the treatment, displayed a higher frequency of granzyme A+ cells. Our data suggest that treatment with Sbv + PTX acts in CD68+ cells reducing the expression of TNF-alpha but not IL-10, resulting in more efficient modulation of the inflammatory response, accelerating the healing process.

Promastigote parasites cultured from the lesions of patients with mucosal leishmaniasis are more resistant to oxidative stress than promastigotes from a cutaneous lesion.

Human leishmaniasis caused by Leishmania (Viannia) braziliensis can be presented as localized cutaneous leishmaniasis (LCL) or mucosal leishmaniasis (ML). Macrophages kill parasites using nitric oxide (NO) and reactive oxygen species (ROS). The aim of this study was to evaluate the ability of parasites obtained from patients with LCL or ML to produce and resist NO or ROS. Promastigotes and amastigotes from LCL or ML isolates produced similar amounts of NO in culture. Promastigotes from ML isolates were more resistant to NO and H2O2 than LCL parasites in a stationary phase, whereas amastigotes from LCL isolates were more resistant to NO. In addition, in the stationary phase, promastigote isolates from patients with ML expressed more thiol-specific antioxidant protein (TSA) than LCL isolates. Therefore it is suggested that infective promastigotes from ML isolates are more resistant to microbicidal mechanisms in the initial phase of infection. Subsequently, amastigotes lose this resistance. This behavior of ML parasites can decrease the number of parasites capable of stimulating the host immune response shortly after the infection establishment.

Leprosy and American cutaneous leishmaniasis coinfection.

Brazil is a country with a high prevalence of infectious diseases such as leprosy and leishmaniasis. However, coinfection of these diseases is still poorly understood. We report a case of a patient who presented with lepromatous leprosy and cutaneous-mucosal leishmaniasis at the same period. After clinical, laboratory, and histopathological diagnosis, the treatment was introduced and the patient showed important clinical improvement. He was followed in our outpatient clinic. Both pathologies play an important role in the immune system. Depending on the immune response profile of the host, diseases may present themselves in different ways. In this case, the patient showed a divergent immune response for each disease. We hypothesized that this response is specific for each pathogen.

Leishmaniasis mucocutánea asociada a infección diseminada por paracoccidioides brasiliensis / MUCOCUTANEOUS LEISHMANIASIS ASSOCIATED TO DISEMINATED INFECTION BY PARACOCCIDIOIDES BRASILIENSIS

Leishmaniasis, a parasitic disease produced by a protozoan of the genus Leishmania, is triggered by the bite of an infected sandfly. It is endemic in tropical and subtropical areas of the Americas, places of poor socioeconomic health conditions and malnutrition. These conditions favor the entry of other pathogens such as the dimorphic fungus Paracoccidioides brasiliensis, responsible for Paracoccidioidomycosis (PCM), deep mycosis of inhalatory entry that initially affects the lungs; then skin, mucous membranes, lymph nodes and adrenal glands. The association of reported cases of Leishmaniasis and Paracoccidiodoimicosis in the same patient is infrequent. We point out the importance of the multidisciplinary approach for the correct diagnosis and treatment.

Leishmaniasis mucocutánea en pacientes inmunocomprometidos: reporte de 4 casos autóctonos / Mucocutaneous Leishmaniasis in Immunocompromised Patients: Report of 4 Cases in Spain

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Regulatory T-Cell Dynamics in Cutaneous and Mucocutaneous Leishmaniasis due to Leishmania braziliensis.

To evaluate the dynamics of regulatory T cells (Tregs) during tegumentary leishmaniasis, we assessed peripheral blood and biopsies from 54 patients. Patients with cutaneous leishmaniasis (CL) had a decreased proportion of Tregs in the peripheral blood, but the proportion was higher in the biopsies of lesions. During treatment of CL, circulating Tregs increased reaching normal proportions, whereas antigen-specific interferon-γ responses diminished. By contrast, circulating Tregs from mucosal leishmaniasis patients failed to normalize during treatment. C-C chemokine receptor type 5 was expressed on a large proportion of Tregs at the site of infection. These results demonstrate increased Tregs at the site of infection, possibly homing from the peripheral circulation.

Leprosy and American cutaneous leishmaniasis coinfection

Abstract: Brazil is a country with a high prevalence of infectious diseases such as leprosy and leishmaniasis. However, coinfection of these diseases is still poorly understood. We report a case of a patient who presented with lepromatous leprosy and cutaneous-mucosal leishmaniasis at the same period. After clinical, laboratory, and histopathological diagnosis, the treatment was introduced and the patient showed important clinical improvement. He was followed in our outpatient clinic. Both pathologies play an important role in the immune system. Depending on the immune response profile of the host, diseases may present themselves in different ways. In this case, the patient showed a divergent immune response for each disease. We hypothesized that this response is specific for each pathogen.

Type I interferons induced by endogenous or exogenous viral infections promote metastasis and relapse of leishmaniasis.

The presence of the endogenous Leishmania RNA virus 1 (LRV1) replicating stably within some parasite species has been associated with the development of more severe forms of leishmaniasis and relapses after drug treatment in humans. Here, we show that the disease-exacerbatory role of LRV1 relies on type I IFN (type I IFNs) production by macrophages and signaling in vivo. Moreover, infecting mice with the LRV1-cured Leishmania guyanensis (LgyLRV1- ) strain of parasites followed by type I IFN treatment increased lesion size and parasite burden, quantitatively reproducing the LRV1-bearing (LgyLRV1+ ) infection phenotype. This finding suggested the possibility that exogenous viral infections could likewise increase pathogenicity, which was tested by coinfecting mice with L. guyanensis and lymphocytic choriomeningitis virus (LCMV), or the sand fly-transmitted arbovirus Toscana virus (TOSV). The type I IFN antiviral response increased the pathology of L. guyanensis infection, accompanied by down-regulation of the IFN-γ receptor normally required for antileishmanial control. Further, LCMV coinfection of IFN-γ-deficient mice promoted parasite dissemination to secondary sites, reproducing the LgyLRV1+ metastatic phenotype. Remarkably, LCMV coinfection of mice that had healed from L. guyanensis infection induced reactivation of disease pathology, overriding the protective adaptive immune response. Our findings establish that type I IFN-dependent responses, arising from endogenous viral elements (dsRNA/LRV1), or exogenous coinfection with IFN-inducing viruses, are able to synergize with New World Leishmania parasites in both primary and relapse infections. Thus, viral infections likely represent a significant risk factor along with parasite and host factors, thereby contributing to the pathological spectrum of human leishmaniasis.

AIM2 inflammasome is associated with disease severity in tegumentary leishmaniasis caused by Leishmania (V.) braziliensis.

The inflammasome is a multiprotein signalling platform involved in the pathogenesis of various inflammatory skin diseases. Herein, we investigated gene and protein expression of the inflammasome molecules AIM2 and NLRP3 in active lesions from patients with L. (V.) braziliensis-associated tegumentary leishmaniasis (TL) and correlated these findings with the clinical presentations and responses to therapy. Real-time PCR assays showed a significantly higher AIM2 gene expression in mucosal leishmaniasis (ML) compared with that in cutaneous leishmaniasis (CL). Additionally, AIM2 mRNA expression was significantly higher in lesions from poor responders than in lesions from good responders. In situ protein quantification analyses revealed greater AIM2 expression in ML lesions than in CL lesions. The percentage of AIM2-producing cells was higher in poor responders than in good responders. Although not quite significant, IL-1ß+ cells were slightly more prominent in poor responders than in good responders. Similar results were observed when patients were evaluated according to clinical form. GP63 immunostaining was identified in all samples, but no significant variation between mucosal and cutaneous lesions was observed. GP63 could be associated with reduced NLRP3 inflammasome expression in CL and ML patients. Taken together, these data demonstrate that AIM2 is an important component of the inflammasome in TL patients and is directly associated with the severity of lesions.

Reactivation of cutaneous and mucocutaneous tegumentary leishmaniasis in rheumatoid arthritis patients: an emerging problem?

Rheumatoid arthritis (RA) is a chronic condition that is frequent in patients living in tropical areas exposed to leishmaniasis. RA therapy involves immunosuppressant drugs such as methotrexate (MTX), monoclonal antibodies (mAbs) and prednisone. We report an unusual presentation of cutaneous (CL) or mucocutaneous leishmaniasis (ML) in RA patients from an endemic area of leishmaniasis. A 51-year-old woman noted a cutaneous ulcer on her left ankle during MTX and prednisone RA therapy. Initially diagnosed as a venous stasis ulcer, the aspirate of the injury revealed the presence of Leishmania DNA. A 73-year-old woman presenting non-ulcerated, infiltrated and painful erythematous nodules inside her nostrils while receiving MTX, anti-TNF mAb, and prednisone for RA, had also the aspirate of injuries showing the presence of Leishmania DNA. Both patients healed after the therapy with liposomal amphotericin. The RA therapy has changed to low-dose prednisone, without further reactivation episodes. Both cases suggest that CL or ML can reactivate after administration of an immunosuppressant for RA treatment. Therefore, immunosuppressive treatments for RA should be carefully prescribed in patients from endemic areas or with a history of CL and ML.