Transcriptome Analysis Identifies the Crosstalk between Dendritic and Natural Killer Cells in Human Cutaneous Leishmaniasis.

Skin ulcers of cutaneous leishmaniasis (CL) are characterized by a localized inflammatory response mediated by innate and adaptive immune cells, including dendritic cells (DC) and natural killer (NK) cells. Bidirectional interactions between DCs and NK cells contribute to tailor leishmaniasis outcome. Despite advances in the Leishmania biology field in recent decades, the mechanisms involved in DC/NK-mediated control of Leishmania sp. pathogenesis as well as the cellular and molecular players involved in such interaction remain unclear. The present study sought to investigate canonical pathways associated with CL arising from Leishmania braziliensis infection. Initially, two publicly available microarray datasets of skin biopsies from active CL lesions were analyzed, and five pathways were identified using differentially expressed genes. The "Crosstalk between DCs and NK cells" pathway was notable due to a high number of modulated genes. The molecules significantly involved in this pathway were identified, and our findings were validated in newly obtained CL biopsies. We found increased expression of TLR4, TNFRSF1B, IL-15, IL-6, CD40, CCR7, TNF and IFNG, confirming the analysis of publicly available datasets. These findings reveal the "crosstalk between DCs and NK cells" as a potential pathway to be further explored in the pathogenesis of CL, especially the expression of CCR7, which is correlated with lesion development.

Metabolic disorders and post-acute hospitalization in black/mixed-race patients with long COVID in Brazil: A cross-sectional analysis.

BACKGROUND: Although low-middle income countries have been disproportionately affected by the COVID-19 pandemic, there is scarce information about the impact of long COVID on their population. This study aimed to evaluate long COVID symptomatology, complications (hospital readmission and metabolic disorders), and main clinical features that impact Quality of Life (QoL). METHODS: This cross-sectional study provides a detailed clinical and laboratory picture of individuals who presented residual symptoms after mild to severe acute COVID-19. Between Aug-2020 to Sep-2021, long COVID patients were evaluated in a reference center for long COVID in Bahia State, Brazil. The EQ-5D-5L questionnaire accessed QoL. RESULTS: A total of 1164 (52 ±13.4 years, 57% female, 88% black/mixed-race) were evaluated 2.3 [IQR = 1.6-3.7] months after mild (n = 351, 30.2%), moderate (338, 29.0%) or severe (475, 40.8%) acute illness. Dyspnea (790, 67.9%), fatigue (738, 63.5%), and chest pain (525, 42.9%) were the most frequent residual symptoms regardless of acute severity, affecting the QoL of 88.9% of patients (n/N-826/925), mainly the domains of anxiety/depression and pain/discomfort. High levels of HbA1c were detected for 175 out of 664 patients (26.6%), 40% of them without a previous diagnosis of diabetes mellitus. Of note, hospital admission one-to-three months after the acute phase of disease was required for 51 (4.4%) patients. CONCLUSION: In this majority-black/mixed-race population, long COVID was associated with post-acute hospitalization, newly diagnosed diabetes mellitus, and decreased QoL, particularly in women and regardless of disease severity of acute infection, suggesting important implications for health care system.

Prediabetes Induces More Severe Acute COVID-19 Associated With IL-6 Production Without Worsening Long-Term Symptoms.

Aims: Pre-existing conditions, such as age, hypertension, obesity, and diabetes, constitute known risk factors for severe COVID-19. However, the impact of prediabetes mellitus (PDM) on COVID-19 severity is less clear. This study aimed to evaluate the influence of PDM in the acute and long-term phases of COVID-19. Materials and methods: We compared inflammatory mediators, laboratory and clinical parameters and symptoms in COVID-19 patients with prediabetes (PDM) and without diabetes (NDM) during the acute phase of infection and at three months post-hospitalization. Results: Patients with PDM had longer hospital stays and required intensive care unit admission more frequently than NDM. Upon hospitalization, PDM patients exhibited higher serum levels of interleukin 6 (IL-6), which is related to reduced partial pressure of oxygen (PaO2) in arterial blood, oxygen saturation (SpO2) and increased COVID-19 severity. However, at three months after discharge, those with PDM did not exhibit significant alterations in laboratory parameters or residual symptoms; however, PDM was observed to influence the profile of reported symptoms. Conclusions: PDM seems to be associated with increased risk of severe COVID-19, as well as higher serum levels of IL-6, which may constitute a potential biomarker of severe COVID-19 risk in affected patients. Furthermore, while PDM correlated with more severe acute-phase COVID-19, no long-term worsening of sequelae was observed.

Cell Migration and Cell Adhesion Assays to Investigate Leishmania-Host Cell Interaction.

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-resolving localized cutaneous lesions to a highly fatal visceral form of the disease. An estimated 12 million people worldwide are currently infected, and another 350 million face risk of infection. It is known that host cells infected by Leishmania parasites, such as macrophages or dendritic cells, can migrate to different host tissues, yet how migration contributes to parasite dissemination and homing remains poorly understood. Therefore, assessing these parasites' ability to modulate host cell response, adhesion, and migration will shed light on mechanisms involved in disease dissemination and visceralization. Cellular migration is a complex process in which cells undergo polarization and protrusion, allowing them to migrate. This process, regulated by actin and tubulin-based microtubule dynamics, involves different factors, including the modulation of cellular adhesion to the substrate. Cellular adhesion and migration processes have been investigated using several models. Here, we describe a method to characterize the migratory aspects of host cells during Leishmania infection. This detailed protocol presents the differentiation and infection of dendritic cells, the analysis of host cell motility and migration, and the formation of adhesion complexes and actin dynamics. This in vitro protocol aims to further elucidate mechanisms involved in Leishmania dissemination within vertebrate host tissues and can also be modified and applied to other cell migration studies.

LTB4-Driven Inflammation and Increased Expression of ALOX5/ACE2 During Severe COVID-19 in Individuals With Diabetes.

Diabetes is a known risk factor for severe coronavirus disease 2019 (COVID-19), the disease caused by the new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there is a lack of knowledge about the mechanisms involved in the evolution of COVID-19 in individuals with diabetes. We aimed to evaluate whether the chronic low-grade inflammation of diabetes could play a role in the development of severe COVID-19. We collected clinical data and blood samples of patients with and without diabetes hospitalized for COVID-19. Plasma samples were used to measure inflammatory mediators and peripheral blood mononuclear cells, for gene expression analysis of the SARS-CoV-2 main receptor system (ACE2/TMPRSS2), and for the main molecule of the leukotriene B4 (LTB4) pathway (ALOX5). We found that diabetes activates the LTB4 pathway and that during COVID-19 it increases ACE2/TMPRSS2 as well as ALOX5 expression. Diabetes was also associated with COVID-19-related disorders, such as reduced oxygen saturation as measured by pulse oximetry/fraction of inspired oxygen (FiO2) and arterial partial pressure of oxygen/FiO2 levels, and increased disease duration. In addition, the expressions of ACE2 and ALOX5 are positively correlated, with increased expression in patients with diabetes and COVID-19 requiring intensive care assistance. We confirmed these molecular results at the protein level, where plasma LTB4 is significantly increased in individuals with diabetes. In addition, IL-6 serum levels are increased only in individuals with diabetes requiring intensive care assistance. Together, these results indicate that LTB4 and IL-6 systemic levels, as well as ACE2/ALOX5 blood expression, could be early markers of severe COVID-19 in individuals with diabetes.

Oral Tolerance Induced by Heat Shock Protein 65-Producing Lactococcus lactis Mitigates Inflammation in Leishmania braziliensis Infection.

Cutaneous leishmaniasis caused by L. braziliensis induces a pronounced Th1 inflammatory response characterized by IFN-γ production. Even in the absence of parasites, lesions result from a severe inflammatory response in which inflammatory cytokines play an important role. Different approaches have been used to evaluate the therapeutic potential of orally administrated heat shock proteins (Hsp). These proteins are evolutionarily preserved from bacteria to humans, highly expressed under inflammatory conditions and described as immunodominant antigens. Tolerance induced by the oral administration of Hsp65 is capable of suppressing inflammation and inducing differentiation in regulatory cells, and has been successfully demonstrated in several experimental models of autoimmune and inflammatory diseases. We initially administered recombinant Lactococcus lactis (L. lactis) prior to infection as a proof of concept, in order to verify its immunomodulatory potential in the inflammatory response arising from L. braziliensis. Using this experimental approach, we demonstrated that the oral administration of a recombinant L. lactis strain, which produces and secretes Hsp65 from Mycobacterium leprae directly into the gut, mitigated the effects of inflammation caused by L. braziliensis infection in association or not with PAM 3CSK4 (N-α-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine, a TLR2 agonist). This was evidenced by the production of anti-inflammatory cytokines and the expansion of regulatory T cells in the draining lymph nodes of BALB/c mice. Our in vitro experimental results suggest that IL-10, TLR-2 and LAP are important immunomodulators in L. braziliensis infection. In addition, recombinant L. lactis administered 4 weeks after infection was observed to decrease lesion size, as well as the number of parasites, and produced a higher IL-10 production and decrease IFN-γ secretion. Together, these results indicate that Hsp65-producing L. lactis can be considered as an alternative candidate for treatment in both autoimmune diseases, as well as in chronic infections that cause inflammatory disease.

Metformin promotes susceptibility to experimental Leishmania braziliensis infection.

BACKGROUND: Metformin (MET) is a hypoglycemic drug used for the treatment of diabetes, despite interference in host immunity against microorganisms. Cutaneous infection caused by pathogens such as Leishmania braziliensis (Lb), the agent responsible for cutaneous leishmaniasis (CL) in Brazil, represents an interesting model in which to evaluate the effects associated with MET. OBJECTIVE: To evaluate the modulatory effect of MET in Lb infection. MATERIAL AND METHODS: Experimental study of Lb infection and MET treatment in BALB/c mice and Raw 264.7 macrophages. FINDINGS: MET treatment interfered with lesion kinetics, increased parasite load and reduced macrophage proliferation. Low concentrations of MET in Lb culture allow for the maintenance of stationary parasite growth phase. Lb-infected cells treated with MET exhibited increased parasite load. While both MET and Lb infection alone promoted the production of intracellular reactive oxygen species (ROS), reduced levels of ROS were seen in MET-treated Lb-infected macrophages. MAIN CONCLUSION: Experimental treatment with MET interfered with the kinetics of cutaneous ulceration, increased Lb parasite load, altered ROS production and modulated cellular proliferation. Our experimental results indicate that MET interfere with the evolution of CL.

Leukotriene B4 licenses inflammasome activation to enhance skin host defense.

The initial production of inflammatory mediators dictates host defense as well as tissue injury. Inflammasome activation is a constituent of the inflammatory response by recognizing pathogen and host-derived products and eliciting the production of IL-1ß and IL-18 in addition to inducing a type of inflammatory cell death termed "pyroptosis." Leukotriene B4 (LTB4) is a lipid mediator produced quickly (seconds to minutes) by phagocytes and induces chemotaxis, increases cytokine/chemokine production, and enhances antimicrobial effector functions. Whether LTB4 directly activates the inflammasome remains to be determined. Our data show that endogenously produced LTB4 is required for the expression of pro-IL-1ß and enhances inflammasome assembly in vivo and in vitro. Furthermore, LTB4-mediated Bruton's tyrosine kinase (BTK) activation is required for inflammasome assembly in vivo as well for IL-1ß-enhanced skin host defense. Together, these data unveil a new role for LTB4 in enhancing the expression and assembly of inflammasome components and suggest that while blocking LTB4 actions could be a promising therapeutic strategy to prevent inflammasome-mediated diseases, exogenous LTB4 can be used as an adjuvant to boost inflammasome-dependent host defense.

New Role of P. brasiliensis α-Glucan: Differentiation of Non-conventional Dendritic Cells.

The cell wall has a critical role in the host immune response to fungal pathogens. In this study, we investigated the influence of two cell wall fractions of the dimorphic fungi Paracoccidioides brasiliensis (Pb) in the in vitro generation of monocyte-derived dendritic cells (MoDCs). Monocytes were purified from the peripheral blood of healthy donors and cultivated for 7 days in medium supplemented with IL-4 and GM-CSF in the presence of Pb cell wall fractions: the alkali-insoluble F1, constituted by ß-1,3-glucans, chitin and proteins, and the alkali-soluble F2, mainly constituted by α-glucan. MoDCs phenotypes were evaluated regarding cell surface expression of CD1a, DC-SIGN, HLA-DR, CD80, and CD83 and production of cytokines. The α-glucan-rich cell wall fraction downregulated the differentiation of CD1a+ MoDCs, a dendritic cell subset that stimulate Th1 responses. The presence of both cell fractions inhibited DC-SIGN and HLA-DR expression, while the expression of maturation markers was differentially induced in CD1a- MoDCs. Differentiation upon F1 and F2 stimulation induced mixed profile of inflammatory cytokines. Altogether, these data demonstrate that Pb cell wall fractions differentially induce a dysregulation in DCs differentiation. Moreover, our results suggest that cell wall α-glucan promote the differentiation of CD1a- DCs, potentially favoring Th2 polarization and contributing to pathogen persistence.

Integrated Analysis Reveals That miR-193b, miR-671, and TREM-1 Correlate With a Good Response to Treatment of Human Localized Cutaneous Leishmaniasis Caused by Leishmania braziliensis.

Localized cutaneous leishmaniasis (LCL) is a chronic disease characterized by ulcerated skin lesion(s) and uncontrolled inflammation. The mechanisms underlying the pathogenesis of LCL are not completely understood, and little is known about posttranscriptional regulation during LCL. MicroRNAs (miRNAs) are non-coding small RNAs that regulate gene expression and can be implicated in the pathogenesis of LCL. We investigated the involvement of miRNAs and their targets genes in human LCL using publicly available transcriptome data sets followed by ex vivo validation. Initial analysis highlighted that miRNA expression is altered during LCL, as patients clustered separately from controls. Joint analysis identified eight high confidence miRNAs that had altered expression (-1.5 ≤ fold change ≥ 1.5; p < 0.05) between cutaneous ulcers and uninfected skin. We found that the expression of miR-193b and miR-671 are greatly associated with their target genes, CD40 and TNFR, indicating the important role of these miRNAs in the expression of genes related to the inflammatory response observed in LCL. In addition, network analysis revealed that miR-193b, miR-671, and TREM1 correlate only in patients who show faster wound healing (up to 59 days) and not in patients who require longer cure times (more than 60 days). Given that these miRNAs are associated with control of inflammation and healing time, our findings reveal that they might influence the pathogenesis and prognosis of LCL.

Understanding the mechanisms controlling Leishmania amazonensis infection in vitro: the role of LTB4 derived from human neutrophils.

Neutrophils are rapidly recruited to the site of Leishmania infection and play an active role in capturing and killing parasites. They are the main source of leukotriene B4 (LTB4), a potent proinflammatory lipid mediator. However, the role of LTB4 in neutrophil infection by Leishmania amazonensis is not clear. In this study, we show that L. amazonensis or its lipophosphoglycan can induce neutrophil activation, degranulation, and LTB4 production. Using pharmacological inhibitors of leukotriene synthesis, our findings reveal an LTB4-driven autocrine/paracrine regulatory effect. In particular, neutrophil-derived LTB4 controls L. amazonensis killing, degranulation, and reactive oxygen species production. In addition, L. amazonensis infection induces an early increase in Toll-like receptor 2 expression, which facilitates parasite internalization. Nuclear factor kappa B (NFkB) pathway activation represents a required upstream event for L. amazonensis-induced LTB4 synthesis. These leishmanicidal mechanisms mediated by neutrophil-derived LTB4 act through activation of its receptor, B leukotriene receptor 1 (BLT1).

Avaliação dos mecanismos de controle da infecção por leishmania amazonensis em neutrófilos humanos: o papel do LTB4 / Evaluation of mechanisms controlling the infection by Leishmania amazonensis in human neutrophils: the role of LTB4

Transmitidas por diferentes espécies de Flebótomos, as Leishmanioses apresentam uma grande variedade de manifestações clínicas. Tem sido demonstrada, a possibilidade de imunização contra a infecção por Leishmania utilizando-se a saliva do vetor Flebotomíneo Lutzomyia Longipalpis. O proncipal vetor da Leishmania Braziliensis é o Flebótomo Lutzomyia Intermedia. O objetivo deste estudo foi avaliar se a imunização de hamsters com a saliva de L. Longipalpis confere proteção contra a infecção por L. Braziliensis na presença da saliva de L. Intermedia. Hamsters machos forma imunizados com o Sonicado de Glândula Salivar (SGS) por três vezes com intervalo de quinze dias. O desafio foi feito na orelha contra-lateral quinze dias após a última imunização 105(com dez elevado à quinta potência) formas promastigotas de L. Braziliensis na presença do SGS de L. Intermedia. O desenvolvimento da lesão foi acompanhado semanalmente e 3,5 e oito semanas, após o desafio, as orelhas e lifondos drenantes foram retirados para a avaliação da carga parasitária, bem como da produção de citocinas durante a infecção. Quarenta e oito horas após o desafio com SGS, observa-se um infiltrado inflamatório composto predominantemente de células mononucleares nas orelhas dos animais imunizados. Estes animais apresentam redução significativa na carga parasitária dos lifondos drenantes, bem como das orelhas desafiadas. Além disso, produzem significativamente mais anticorpos anti-saliva quando comparados aos não-imunizados e menos anticorpos anti-Leishmania. Os possíveis mecanismos envolvidos nesta proteção são os níveis reduzidos da expressão de IL-10 e TGF- ao longo da infecção. Estes resultados sugerem que a imunização com o SGS de L. Longipalpis confere proteção contra a infecção por L. Braziliensis de modo inespecífico à presença da saliva no momento do desafio. Dessa forma, a possibilidade de vacinação contra diferentes espécies de Leishmania, utilizando proteínas salivares de uma espécie de vetor.

Avaliação da imunidade protetora conferida pela imunização de hamsters com saliva do flebotomíneo Lutzomyia Longipalpis contra desafio com Leishmania Brasiliensis na presença da saliva de diferentes vetores / Assessment of protective immunity conferred by immunization of hamsters with the saliva of Lutzomyia longipalpis phlebotomine against challenge with Leishmania brasiliensis in the presence of saliva of different vectors

Transmitidas por diferentes espécies de Flebótomos, as Leishmanioses apresentam uma grande variedade de manifestações clínicas. Tem sido demonstrada, a possibilidade de imunização contra a infecção por Leishmania utilizando-se a saliva do vetor Flebotomíneo Lutzomyia Longipalpis. O principal vetor da Leishmania Braziliensis é o Flebótomo Lutzomyia Intermedia. O objetivo deste estudo foi avaliar se a imunização de hamsters com a saliva de L. Longipalpis confere proteção contra a infecção por L. Braziliensis na presença da saliva de L. Intermedia. Hamsters machos forma imunizados com o Sonicado de Glândula Salivar (SGS) por três vezes com intervalo de quinze dias. O desafio foi feito na orelha contra-lateral quinze dias após a última imunização 105 (com dez elevado à quinta potência) formas promastigotas de L. Braziliensis na presença do SGS de L. Intermedia. O desenvolvimento da lesão foi acompanhado semanalmente e 3,5 e oito semanas, após o desafio, as orelhas e lifondos drenantes foram retirados para a avaliação da carga parasitária, bem como da produção de citocinas durante a infecção. Quarenta e oito horas após o desafio com SGS, observa-se um infiltrado inflamatório composto predominantemente de células mononucleares nas orelhas dos animais imunizados. Estes animais apresentam redução significativa na carga parasitária dos lifondos drenantes, bem como das orelhas desafiadas. Além disso, produzem significativamente mais anticorpos anti-saliva quando comparados aos não-imunizados e menos anticorpos anti-Leishmania. Os possíveis mecanismos envolvidos nesta proteção são os níveis reduzidos da expressão de IL-10 e TGF- ao longo da infecção. Estes resultados sugerem que a imunização com o SGS de L. Longipalpis confere proteção contra a infecção por L. Braziliensis de modo inespecífico à presença da saliva no momento do desafio. Dessa forma, a possibilidade de vacinação contra diferentes espécies de Leishmania, utilizando proteínas salivares de uma espécie de vetor...