Sara Passos

IL-17 Mediates Immunopathology in the Absence of IL-10 Following Leishmania major Infection

Leishmaniasis, resulting from infection with the protozoan parasite Leishmania, consists of a wide spectrum of clinical manifestations, from healing cutaneous lesions to fatal visceral infections. A particularly severe form of cutaneous leishmaniasis, termed mucosal leishmaniasis, exhibits decreased IL-10 levels and an exaggerated inflammatory response that perpetuates the disease. Using a mouse model of leishmaniasis, we investigated what cytokines contribute to increased pathology when IL-10-mediated regulation is absent. Leishmania major infected C57BL/6 mice lacking IL-10 regulation developed larger lesions than controls, but fewer parasites. Both IFN-γ and IL-17 levels were substantially elevated in mice lacking the capacity to respond to IL-10. IFN-γ promoted an increased infiltration of monocytes, while IL-17 contributed to an increase in neutrophils. Surprisingly, however, we found that IFN-γ did not contribute to increased pathology, but instead regulated the IL-17 response. Thus, blocking IFN-γ led to a significant increase in IL-17, neutrophils and disease. Similarly, the production of IL-17 by cells from leishmaniasis patients was also regulated by IL-10 and IFN-γ. Additional studies found that the IL-1 receptor was required for both the IL-17 response and increased pathology. Therefore, we propose that regulating IL-17, possibly by downregulating IL-1β, may be a useful approach for controlling immunopathology in leishmaniasis.

Differential immune regulation of activated T cells between cutaneous and mucosal leishmaniasis as a model for pathogenesis

Cutaneous (CL) and mucosal leishmaniasis (ML) are characterized by a predominant type 1 immune response (IFN‐γ and TNF‐α production) and strong inflammatory response in the lesions with few parasites. This exacerbated type 1 response is more evident in ML as compared to CL. Our main hypothesis is that a differential immune regulation of T cell activation leads to over reactive T cells in ML. In the present study, we investigated immunological factors that could explain the mechanisms behind it by comparing some immune regulatory mechanisms between ML and CL patients: frequency of cells expressing co‐stimulatory molecules, apoptotic markers, T cell activation markers; and ability of neutralizing antibodies to IL‐2, IL‐12 and IL‐15 do down‐regulate IFN‐γ production in leishmania antigen‐stimulated peripheral blood mononuclear cells (PBMC). Interestingly, in CL anti‐IL‐2 and anti‐IL‐15 significantly suppressed antigen‐specific IFN‐γ production, while in ML only anti‐IL‐2 suppressed IFN‐γ production. Finally, higher frequency of CD4+ T cells expressing CD28−, CD69+ and CD62L low were observed in ML as compared to CL. These data indicate that an exacerbated type 1 response in ML is differentially regulated and not appropriately down modulated, with increased frequencies of activated effectors T cells, maintaining the persistent inflammatory response and tissue damage observed in ML.

Protective and pathologic immune responses in human tegumentary leishmaniasis.

Studies in the recent years have advanced the knowledge of how host and parasite factors contribute to the pathogenesis of human tegumentary leishmaniasis. Polymorphism within populations of Leishmania from the same species has been documented; indicating that infection with different strains may lead to distinct clinical pictures and can also interfere in the response to treatment. Moreover, detection of parasite genetic tags for the precise identification of strains will improve diagnostics and therapy against leishmaniasis. On the host side, while a predominant Th1 type immune response is important to control parasite growth, it does not eradicate Leishmania and, in some cases, does not prevent parasite dissemination. Evidence has accumulated showing the participation of CD4+ and CD8+ T cells, as well as macrophages, in the pathology associated with L. braziliensis, L. guayanensis, and L. major infection. The discovery that a large percentage of individuals that are infected with Leishmania do not develop disease will help to understand how the host controls Leishmania infection. As these individuals have a weaker type 1 immune response than patients with cutaneous leishmaniasis, it is possible that control of parasite replication in these individuals is dependent, predominantly, on innate immunity, and studies addressing the ability of neutrophils, macrophages, and NK cells to kill Leishmania should be emphasized.

Human Classical Monocytes Control the Intracellular Stage of Leishmania braziliensis by Reactive Oxygen Species

Leishmania braziliensis are intracellular parasites that cause unique clinical forms of cutaneous leishmaniasis. Previous studies with other leishmania species demonstrated that reactive oxygen species (ROS) control promastigotes, the infective stage of the parasite, but not the amastigote form that exists in the mammalian host. Here we show that ROS inhibits growth of L. braziliensis amastigotes in resting monocytes, and that classical monocytes are primarily responsible for this control. ROS, but not nitric oxide, also contributed to killing of L. braziliensis by IFN-γ activated monocytes. Furthermore, by gene expression profiling of human lesions we found greater expression of genes associated with ROS, but not nitric oxide, compared to normal skin. This study shows that ROS are important for control of L. braziliensis both at the initial stages of infection, as well as at later time points, and highlights that monocyte subsets may play different roles during leishmaniasis.

Influence of Helminth Infections on the Clinical Course of and Immune Response to Leishmania braziliensis Cutaneous Leishmaniasis

BACKGROUND Helminth infections influence the clinical outcome of and immune response to certain immune-mediated diseases. METHODS We conducted a cohort study of 120 patients to examine the role that intestinal helminth infection plays in the clinical course of and immune response to cutaneous leishmaniasis (CL) treated with pentavalent antimony. RESULTS Patients coinfected with Leishmania braziliensis and helminths took longer to heal (relative hazard for healing, 0.47 [95% confidence interval, 0.26-0.85]; P=.01) than patients with CL without helminths, with 70% of coinfected patients being cured at 90 days, compared with 92% of helminth-free patients. Coinfected patients had an immune response shifted toward the T helper 2 type, with increased total immunoglobulin E levels (P<.06) and a tendency toward increased interleukin-5 levels, compared with helminth-free patients with CL. CONCLUSIONS Helminths influence both the clinical outcome and the immune response of patients with CL. These results may have clinical implications for the care of patients with CL caused by Leishmania braziliensis, because screening for and treatment of helminths may improve responses to treatment and possibly reduce the risk of progression to mucosal disease.

IFN-γ production to leishmania antigen supplements the leishmania skin test in identifying exposure to L. braziliensis infection.

Background Cutaneous leishmaniasis due to L. braziliensis (CL) is characterized by a positive delayed type hypersensitivity test (DTH) leishmania skin test (LST) and high IFN-γ production to soluble leishmania antigen (SLA). The LST is used for diagnosis of CL and for identification of individuals exposed to leishmania infection but without disease. The main aim of the present study was to identify markers of exposure to L. braziliensis infection. Methodolgy/Principal Findings This cohort study enrolled 308 household contacts (HC) of 76 CL index cases. HC had no active or past history of leishmaniasis. For the present cross-sectional study cytokines and chemokines were determined in supernatants of whole blood culture stimulated with SLA. Of the 308 HC, 36 (11.7%) had a positive LST but in these IFN-γ was only detected in 22 (61.1%). Moreover of the 40 HC with evidence of IFN-γ production only 22 (55%) had a positive LST. A total of 54 (17.5%) of 308 HC had specific immune response to SLA. Only a moderate agreement (Kappa = 0.52; 95% CI: 0.36–0.66) was found between LST and IFN-γ production. Moreover while enhancement of CXCL10 in cultures stimulated with SLA was observed in HC with DTH+ and IFN-γ+ and in patients with IFN-γ+ and DTH−, no enhancement of this chemokine was observed in supernatants of cells of HC with DTH+ and IFN-γ−. Conclusions/Significance This study shows that in addition of LST, the evaluation of antigen specific IFN-γ production should be performed to determine evidence of exposure to leishmania infection. Moreover it suggests that in some HC production of IFN-γ and CXCL10 are performed by cells not involved with DTH reaction.

Effect of LACK and KMP11 on IFN-gamma production by peripheral blood mononuclear cells from cutaneous and mucosal leishmaniasis patients.

The immune modulatory properties of recombinant antigens Kinetoplasmid membrane protein‐11 (KMP11) and Leishmania homologue of receptors for activated C kinase (LACK) in cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) patients were evaluated. The mean levels of interferon‐γ (IFN‐γ) in soluble leishmania antigen (SLA) stimulated peripheral blood mononuclear cells (PBMC) of ML and CL patients were 5625 ± 2333 pg/ml and 4422 ± 3665 pg/ml, respectively. IFN‐γ was not detected in cultures stimulated with KMP11 or LACK. Interleukin‐10 (IL‐10) concentration in SLA, KMP11 and LACK‐stimulated PBMC of ML patients was 13 ± 12 pg/ml, 285 ± 388 pg/ml and 802 ± 483 pg/ml, respectively. Addition of KMP11 or LACK to SLA‐stimulated PBMC of CL and ML patients enhanced IL‐10 production (P < 0.05). Addition of KMP11 decreased IFN‐γ levels by 52% in CL patients and by 19% in ML patients. Addition of LACK to SLA‐stimulated cultures decreased IFN‐γ levels by 58% in CL patients and by 30% in ML patients. Neutralization of IL‐10 abrogated the downregulatory effect of LACK and KMP11. The modulatory properties of LACK and KMP11 are due to induction of IL‐10 production and may be helpful for attenuating chronic inflammatory diseases. However, in some clinical conditions, as demonstrated for ML, these molecules are not able to suppress the IFN‐γ response, even inducing IL‐10 production.

Intermediate Monocytes Contribute to Pathologic Immune Response in Leishmania braziliensis Infections

Ulcer development in patients with cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is associated with high levels of tumor necrosis factor (TNF). We found that early after infection, before ulcer development, the frequency of CD16(+) (both intermediate [CD14(+)CD16(+)] and nonclassical [CD14(dim)CD16(+)]) monocytes was increased in the peripheral blood of patients with L. braziliensis, compared with uninfected controls. These results suggest that CD16(+) monocytes might promote disease. Also, we found that intermediate monocytes expressed CCR2 and that increased levels of CCL2 protein were present in lesions from patients, suggesting that intermediate monocytes are more likely than nonclassical monocytes to migrate to the lesion site. Finally, we found that the intermediate monocytes produced TNF. Our results show that intermediate monocytes are increased in frequency soon after infection; express CCR2, which would promote their migration into the lesions; and, owing to their production of TNF, can enhance the inflammatory response.

Clinical and Immunological Outcome in Cutaneous Leishmaniasis Patients Treated with Pentoxifylline

Pentoxifylline is a tumor necrosis factor-α (TNF-α) inhibitor that also attenuates the immune response and decreases tissue inflammation. The association of pentoxifylline with antimony improves the cure rate of mucosal and cutaneous leishmaniasis. In this randomized and double blind pilot trial, cure rate was higher, although not significant, in patients who received antimony plus pentoxifylline than in those patients receiving antimony plus placebo. A significant decrease in TNF-α and interferon-γ (IFN-γ) levels during therapy was more pronounced in the antimony plus pentoxifylline group, whereas CCL-3 (Chemokine [C-C motif] ligand 3) decreased similarly in both groups. The increased levels of CXCL-9 (Chemokine [C-X-C motif] ligand 9) during therapy were lower in the antimony plus pentoxifylline group. Therapy with pentoxifylline modifies cytokines and chemokines production, which may be associated with therapeutic outcome.

Comparative analysis of the tissue inflammatory response in human cutaneous and disseminated leishmaniasis

Cutaneous leishmaniasis (CL) is the most frequent clinical form of tegumentary leishmaniasis and is characterised by a single or a few ulcerated skin lesions that may disseminate into multiple ulcers and papules, which characterise disseminated leishmaniasis (DL). In this study, cells were quantified using immunohistochemistry and haematoxylin and eosin staining (CD4+, CD68+, CD20+, plasma cells and neutrophils) and histopathology was used to determine the level of inflammation in biopsies from patients with early CL, late CL and DL (ulcers and papules). The histopathology showed differences in the epidermis between the papules and ulcers from DL. An analysis of the cells present in the tissues showed similarities between the ulcers from localised CL (LCL) and DL. The papules had fewer CD4+ T cells than the DL ulcers. Although both CD4+ cells and macrophages contribute to inflammation in early CL, macrophages are the primary cell type associated with inflammation intensity in late ulcers. The higher frequency of CD20+ cells and plasma cells in lesions demonstrates the importance of B cells in the pathogenesis of leishmaniasis. The number of neutrophils was the same in all of the analysed groups. A comparison between the ulcers from LCL and DL and the early ulcers and papules shows that few differences between these two clinical forms can be distinguished by observing only the tissue.