Daniel Claudio Oliveira Gomes

Background: Leishmaniasis, a parasitic disease caused by Leishmania protozoa, has various clinical forms and is endemic in Brazil. Traditional experimental infection methods using intradermal and subcutaneous needles do not resemble natural sand fly transmission and are associated with risks of laboratory accidents owing to the use of low-gauge needles. In this study, we investigated the application of microneedles for transdermal (TD) infections to better replicate the deposition of parasites observed in the natural infection environment while ensuring safe handling in the laboratory. Methods: Initial experiments involved inducing TD infections using 1-, 7- or 12-microneedle cartridges, compared with inducing conventional intradermal (ID) infections in the ears of BALB/c mice. Subsequent tests used a 12-microneedle cartridge at different tissue depths (0.25, 0.5, 0.75, 1.0 and 1.5 mm), followed by challenge with different doses of Leishmania amazonensis . In addition, histology and flow cytometry studies were performed 30 min post-infection. Results: Transdermal infections produced similar lesion development and parasite loads as ID infections. More tissue destruction was induced using the 1- or 7-microneedle cartridge compared to the 12-microneedle cartridge and ID infection. Infection was successfully established at all microneedle depths (0.25, 0.5, 0.75 and 1.0 mm), resulting in comparable lesion thickness across the different experimental groups, with no statistically significant differences observed. The parasite load and lesion thickness were dependent on the inoculum size in the ear infection via the TD route, with efficient lesion progression using 2 × 10 ⁶ and 2 × 10 ⁵ promastigotes. Parasite loads in the ear were similar between the two approaches at the early stages, specifically at 30 min and 12 h post-infection. Thirty minutes post-infection, cytometry analysis indicated recruitment of neutrophils to the lesion site, regardless of the infection model used. The TD model specifically attracted neutrophils, independent of the presence of parasites. Conclusions: This novel microneedle infection model enables efficient challenge and reduces risks during animal experimentation. This technique holds promise for future studies of leishmaniasis therapy and vaccine development. Graphical

Background/Objectives: The total lysate of Leishmania amazonensis (LaAg) is one of the most extensively studied vaccine formulations against leishmaniasis. Despite demonstrating safety and immunogenicity when administered intramuscularly, LaAg has failed to show efficacy in clinical trials and, in some cases, has even been associated with an enhanced susceptibility to infection. Adjuvants, which are molecules or compounds added to antigens to enhance the immunogenicity or modulate the immune response, are frequently employed in vaccine studies. This study aimed to evaluate different adjuvants to improve the protective efficacy of LaAg in L.amazonensis infection using a BALB/c mouse model. Methods: BALB/c mice were immunized with LaAg in combination with various adjuvants. The delayed-type hypersensitivity (DTH) test was assessed by measuring the infected paw and was used to evaluate the immunogenicity and to determine the most effective adjuvant. The immune response was analyzed through flow cytometry, focusing on cytokine production, immune cell recruitment and lesion size, alongside the control of parasite load at the infection site. The expression levels of iNOS and TGF-β were quantified using RT-qPCR, while IgG1, IgG2a and IgE antibody levels were determined via ELISA. Results: Among the adjuvants tested, only saponin (SAP) elicited a significant DTH response following LaAg challenge. SAP enhanced the immunogenicity of LaAg, as evidenced by increased IFN-γ-producing CD4⁺ and CD8⁺ T cells in the draining lymph nodes at 18 h post-challenge. Additionally, SAP facilitated the recruitment of lymphocytes, macrophages, neutrophils and eosinophils to the infection site. Conclusions: The LaAg + SAP combination conferred partial protection, as demonstrated by a reduction in lesion size and the partial control of parasite load. In conclusion, the addition of SAP as an adjuvant to LaAg effectively modulates the immune response, enhancing the vaccine’s protective efficacy. These findings provide valuable insights into the development of improved vaccines against L.amazonensis infection.

Cytotoxic activity is a hallmark of the immunopathogenesis in human cutaneous leishmaniasis (CL). In this study, we identified accumulation of CD4⁺ granzyme B producing T cells with increased cytotoxic capacity in CL lesions. These cells showed enhanced expression of activating NK receptors (NKG2D and NKG2C), diminished expression of inhibitory NKG2A, along with the upregulation of the senescence marker CD57. Notably, CD4⁺ T cells freshly isolated from CL lesions demonstrated remarkable capacity to mediate NL-like bystander cytolysis. Phenotypic analyses revealed that lesional CD4⁺ T cells are mainly composed of late-differentiated effector (CD27-CD45RA-) and terminally differentiated (senescent) TEMRA (CD27-CD45RA+) subsets. Interestingly, the TEMRA CD4⁺ T cells exhibited higher expression of granzyme B and CD107a. Collectively, our results provide the first evidence that senescent cytotoxic CD4⁺ T cells may support the skin pathology of human cutaneous leishmaniasis and, together with our previous findings, support the notion that multiple subsets of cytotoxic senescent cells may be involved in inducing the skin lesions in these patients.

The American Tegumentary Leishmaniasis (ATL) is caused by protozoans of the genus Leishmania and varies from mild localized cutaneous leishmaniasis (LCL) form to more severe manifestations such as the diffuse cutaneous leishmaniasis (DCL) form and the mucosal leishmaniasis (ML) form. Previously, we demonstrated the accumulation of senescent cells in skin lesions of patients with LCL. Moreover, lesional transcriptomic analyses revealed a robust co-induction of senescence and pro-inflammatory gene signatures, highlighting the critical role of senescent T cells in orchestrating pathology. In this work we hypothesized that senescent cells might operate differently among the ATL spectrum, potentially influencing immunopathological mechanisms and clinical outcome. We analysed previously published RNA-Seq datasets of skin biopsies of healthy subjects and lesional skin from DCL patients, LCL patients and LCL patients that, after treatment, progressed to mucosal leishmaniasis (MLP). Our findings demonstrate a robust presence of a CD8 T cell signature associated with both LCL and MLP lesions. Moreover, both inflammatory and cytotoxic signatures were significantly upregulated, showing a strong increase in MLP and LCL groups, but not DCL. The senescence signature was elevated between LCL and MLP groups, representing the only distinguishable signature of immunopathology between them. Interestingly, our analyses further revealed the senescence signature's capacity to predict progression from LCL to mucosal forms, which was not observed with other signatures. Both the senescence-signature score and specific senescence-associated genes demonstrated an increased capacity to predict mucosal progression, with correct predictions exceeding 97% of cases. Collectively, our findings contribute to a comprehensive understanding of immunosenescence in ATL and suggest that senescence may represent the latest and most important signature of the immunopathogenisis. This highlights its potential value in predicting disease severity.

Natural killer (NK) cells include different subsets with diverse effector capacities that are poorly understood in the context of parasitic diseases. Here, we investigated inhibitory and activating receptor expression on NK cells in patients with cutaneous leishmaniasis (CL) and explored their phenotypic and functional heterogeneity based on CD57 and NKG2C expression. The expression of CD57 identified NK cells that accumulated in CL patients and exhibited features of senescence. The CD57⁺ cells exhibited heightened levels of the activating receptor NKG2C and diminished expression of the inhibitory receptor NKG2A. RNA sequencing analyses based on NKG2C transcriptome have revealed two distinct profiles among CL patients associated with cytotoxic and functional genes. The CD57⁺NKG2C⁺ subset accumulated in the blood of patients and presented conspicuous features of senescence, including the expression of markers such as p16, yH2ax, and p38, as well as reduced proliferative capacity. In addition, they positively correlated with the number of days until lesion resolution. This study provides a broad understanding of the NK cell biology during Leishmania infection and reinforces the role of senescent cells in the adverse clinical outcomes of CL. (A) NK subsets present opposite immune and senescence features in cutaneous leishmaniasis. (B) Expansion of cytotoxic NKG2C + CD57 + NK cells is associated with increased lesional healing delay. Spearman’s rank correlation (ρ) between cell subsets frequencies and patient stratified by CD57 and NKG2C expression and lesional healing time (days) taken by cutaneous leishmaniasis patients. The graphs show a median with 95% confidence. The P values were calculated by comparing each treatment round set between the NK cell subsets using RM-one-way ANOVA and Benjamin and Hochberg method. *P < 0.05, **P < 0.01, ***P < 0.001.

The intestinal microbiota has an important role in animal health. Therefore, prebiotics have gained interest in the scientific community for their role in manipulating populations of intestinal microorganisms. Among prebiotics, there is Smallanthus sonchifolius Poepp. Endl (yacon) root, which can be ingested in natura or in different forms, such as flours and extracts. This prebiotic has been associated with beneficial effects in different diseases, including metabolic (like type 2 diabetes) and immunological disorders. Thus, mouse models of human diseases caused by immunological factors have been used to better understand the prebiotic effects of yacon. Since prebiotics interfere with animal microbiota, it is important to take into consideration the quality level of mouse facilities. In this way, the beneficial effects of a yacon root pulp were tested in a mouse model of allergic asthma and, considering how animal facility conditions could affect those results, we performed experiments both in conventional facilities and specific pathogen-free (SPF) ones. Our results showed that better prebiotic effects were observed in a SPF facility than in a conventional one and, in some cases, evidence of yacon beneficial effects was observed exclusively in a SPF facility. For example, there were reductions of 63.6% and 58.0% in IgE and eosinophils, respectively, in immunized/yacon-treated animals compared to immunized mice only. Taken together, our results not only showed the beneficial effects of yacon root pulp in an asthma model but also demonstrated the relevance of performing experiments with prebiotics in SPF facilities.