Optimization of quenched fluorescent peptide substrates of SARS-CoV-2 3CLpro main protease (Mpro) from proteomic identification of P6-P6' active site specificity.

SARS-CoV-2 3C-like main protease (3CLpro) is essential for protein excision from the viral polyprotein. 3CLpro inhibitor drug development to block SARS-CoV-2 replication focuses on the catalytic non-prime (P) side for specificity and potency, but the importance of the prime (P') side in substrate specificity and for drug development remains underappreciated. We determined the P6-P6' specificity for 3CLpro from >800 cleavage sites that we identified using Proteomic Identification of Cleavage site Specificity (PICS). Cleavage occurred after the canonical P1-Gln and non-canonical P1-His and P1-Met residues. Moreover, P3 showed a preference for Arg/Lys and P3' for His. Essential H-bonds between the N-terminal Ser1 of protomer-B in 3CLpro dimers form with P1-His, but not with P1-Met. Nonetheless, cleavage occurs at P1-Met456 in native MAP4K5. Elevated reactive oxygen species in SARS-CoV-2 infection oxidize methionines. Molecular simulations revealed P1-MetOX forms an H-bond with Ser1 and notably, strong positive cooperativity between P1-Met with P3'-His was revealed, which enhanced peptide-cleavage rates. The highly plastic S3' subsite accommodates P3'-His that displays stabilizing backbone H-bonds with Thr25 lying central in a "'threonine trio" (Thr24-Thr25-Thr26) in the P'-binding domain I. Molecular docking simulations unveiled structure-activity relationships impacting 3CLpro-substrate interactions, and the role of these structural determinants was confirmed by MALDI-TOF-MS cleavage assays of P1'- and P3'-positional scanning peptide libraries carrying a 2nd optimal cut-site as an internal positive control. These data informed the design of two new and highly soluble 3CLproquenched-fluorescent peptide substrates for improved FRET monitoring of 3CLpro activity with 15× improved sensitivity over current assays.IMPORTANCEFrom global proteomics identification of >800 cleavage sites, we characterized the P6-P6' active site specificity of SARS-CoV-2 3CLpro using proteome-derived peptide library screens, molecular modeling simulations, and focussed positional peptide libraries. In P1', we show that alanine and serine are cleaved 3× faster than glycine and the hydrophobic small amino acids Leu, Ile, or Val prevent cleavage of otherwise optimal non-prime sequences. In characterizing non-canonical non-prime P1 specificity, we explored the unusual P1-Met specificity, discovering enhanced cleavage when in the oxidized state (P1-MetOX). We unveiled unexpected amino acid cooperativity at P1-Met with P3'-His and noncanonical P1-His with P2-Phe, and the importance of the threonine trio (Thr24-Thr25-Thr26) in the prime side binding domain I in defining prime side binding in SARS-CoV-2 3CLpro. From these analyses, we rationally designed quenched-fluorescence natural amino acid peptide substrates with >15× improved sensitivity and high peptide solubility, facilitating handling and application for screening of new antiviral drugs.

Glucocorticoid modulates oxidative and thermogenic function of rat brown adipose tissue and human brown adipocytes.

Chronic and excessive glucocorticoid (GC) exposure can cause Cushing's syndrome, resulting in fat accumulation in selected body areas. Particularly in the brown adipose tissue (BAT), GC acts negatively, resulting in whitening of the tissue. We hypothesized that dysregulation of microRNAs by GC could be an additional mechanism to explain its negative actions in BAT. Male Wistar rats were divided into two groups: (1) Control sham and (2) GC group that was administered dexamethasone 6.25 mg/200 µL via osmotic pump implantation over 28 days. After this period, the animals were euthanized and BAT tissue was properly stored. Human fat cells treated with dexamethasone were used to translate the experimental results found in animals to human biology. GC-treated rat BAT presented with large lipid droplets, severely impaired thermogenic activation, and reduced glucose uptake measured by 18F-FDG PET/CT. GC exposure induced a reduction in the mitochondrial OXPHOS system and oxygen consumption. MicroRNA profiling of BAT revealed five top-regulated microRNAs and among them miR-21-5p was the most significantly upregulated in GC-treated rats compared to the control group. Although upregulation of miR-21-5p in the tissue, differentiated primary brown adipocytes from GC-treated rats had decreased miR-21-5p levels compared to the control group. To translate these results to the clinic, human brown adipocytes were treated with dexamethasone and miR-21-5p inhibitor. In human brown cells, inhibition of miR-21-5p increased brown adipocyte differentiation and prevented GC-induced glucose uptake, resulting in a lower glycolysis rate. In conclusion, high-dose GC therapy significantly impacts brown adipose tissue function, with a notable association between glucose uptake and miR-21-5p.

Cell death induced by NLRP3-palmitate axis impairs pulmonary damage tolerance and aggravates immunopathology during obesity-tuberculosis comorbidity.

A low-grade and persistent inflammation, which is the hallmark of obesity, requires the participation of NLRP3 and cell death. During Mycobacterium tuberculosis infection, NLRP3 signaling is important for bacterial killing by macrophages in vitro but was shown to be dispensable for host protection in vivo. We hypothesized that during obesity-tuberculosis (TB) comorbidity, NLRP3 signaling might play a detrimental role by inducing excessive inflammation. We employed a model of high-fat-diet-induced obesity, followed by M. tuberculosis infection in C57BL/6 mice. Obese mice presented increased susceptibility to infection and pulmonary immunopathology compared to lean mice. Using treatment with NLRP3 antagonist and Nlrp3-/- mice, we showed that NLRP3 signaling promoted cell death, with no effect in bacterial loads. The levels of palmitate were higher in the lungs of obese infected mice compared to lean counterparts, and we observed that this lipid increased M. tuberculosis-induced macrophage death in vitro, which was dependent on NLRP3 and caspase-1. At the chronic phase, although lungs of obese Nlrp3-/- mice showed an indication of granuloma formation compared to obese wild-type mice, there was no difference in the bacterial load. Our findings indicate that NLRP3 may be a potential target for host-directed therapy to reduce initial and severe inflammation-mediated disease and to treat comorbidity-associated TB. © 2022 The Pathological Society of Great Britain and Ireland.

Acetylcholine, Fatty Acids, and Lipid Mediators Are Linked to COVID-19 Severity.

Lipid and cholinergic mediators are inflammatory regulators, but their role in the immunopathology of COVID-19 is still unclear. Here, we used human blood and tracheal aspirate (TA) to investigate whether acetylcholine (Ach), fatty acids (FAs), and their derived lipid mediators (LMs) are associated with COVID-19 severity. First, we analyzed the perturbation profile induced by SARS-CoV-2 infection in the transcriptional profile of genes related to the ACh and FA/LM pathways. Blood and TA were used for metabolomic and lipidomic analyses and for quantification of leukocytes, cytokines, and ACh. Differential expression and coexpression gene network data revealed a unique transcriptional profile associated with ACh and FA/LM production, release, and cellular signaling. Transcriptomic data were corroborated by laboratory findings: SARS-CoV-2 infection increased plasma and TA levels of arachidonic acid, 5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid, 11-hydroxy-5Z,8Z,12E,14Z-eicosatetraenoic acid, and ACh. TA samples also exhibited high levels of PGE2, thromboxane B2, 12-oxo-5Z,8Z,10E,14Z-eicosatetraenoic acid, and 6-trans-leukotriene B4 Bioinformatics and experimental approaches demonstrated robust correlation between transcriptional profile in Ach and FA/LM pathways and parameters of severe COVID-19. As expected, the increased neutrophil-to-lymphocyte ratio, neutrophil counts, and cytokine levels (IL-6, IL-10, IL-1ß, and IL-8) correlated with worse clinical scores. Glucocorticoids protected severe and critical patients and correlated with reduced Ach levels in plasma and TA samples. We demonstrated that pulmonary and systemic hyperinflammation in severe COVID-19 are associated with high levels of Ach and FA/LM. Glucocorticoids favored the survival of patients with severe/critical disease, and this effect was associated with a reduction in ACh levels.

Investigation of the involvement of platelet-activating factor in the control of hypertension by aerobic training. A randomized controlled trial.

Although studies have demonstrated the effectiveness of exercise in controlling systemic arterial hypertension (SAH), the mechanisms involved in this effect are still poorly understood. Thus, this study investigated the impact of aerobic training on the relationship between platelet-activating factor (PAF) circulating levels and blood pressure in hypertensives. Seventy-seven hypertensive subjects were enrolled in this randomized controlled trial (age 66.51 ± 7.53 years, body mass 76.17 ± 14.19 kg). Participants were randomized to two groups: the intervention group (IG, n = 36), composed of hypertensive individuals submitted to an aerobic training protocol, and the control group (CG, n = 41), composed of non-exercised hypertensives. Body mass index, arterial blood pressure, quality of life, respiratory muscle strength, and functional capacity were assessed before and after 12 weeks. PAF and plasma cytokine levels were also evaluated respectively by liquid chromatography coupled with mass spectrometry and enzyme-linked immunosorbent assay. Aerobic training promoted a significant reduction in blood pressure while functional capacity, expiratory muscle strength, and quality of life, PAFC16:0 and PAFC18:1 plasma levels were increased in comparison to the CG (p < 0.05). In addition, multiple correlation analysis indicated a positive correlation [F (3.19) = 6.322; p = 0.001; R2adjusted = 0.499] between PAFC16:0 levels and expiratory muscle strength after aerobic training. Taken together, our findings indicate that PAF may be involved in the indirect mechanisms that control SAH, being mainly associated with increased respiratory muscle strength in hypertensive subjects undergoing aerobic training.

Oral Squamous Cell Carcinoma Lipid Evaluation in Gingiva Tissue Stored in TRIzol via Shotgun Lipidomics and MALDI Mass Spectrometry Imaging.

Oral squamous cell carcinoma (OSCC) is the prevalent type of oral cavity cancer, requiring precise, accurate, and affordable diagnosis to identify the disease in early stages, Comprehending the differences in lipid profiles between healthy and cancerous tissues encompasses great relevance in identifying biomarker candidates and enhancing the odds of successful cancer treatment. Therefore, the present study evaluates the analytical performance of simultaneous mRNA and lipid extraction in gingiva tissue from healthy patients and patients diagnosed with OSCC preserved in TRIzol reagent. The data was analyzed by partial least-squares discriminant analysis (PLS-DA) and confirmed via matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). The lipid extraction in TRIzol solution was linear in a range from 330 to 2000 ng mL-1, r2 > 0.99, intra and interday precision and accuracy <15%, and absolute recovery values ranging from 90 to 110%. The most important lipids for tumor classification were evaluated by MALDI-MSI, revealing that the lipids responsible for distinguishing the OSCC group are more prevalent in the cancerous tissue in contrast to the healthy group. The results exhibit the possibilities to do transcriptomic and lipidomic analyses in the same sample and point out important candidates related to the presence of OSCC.

The turning point of COVID-19 severity is associated with a unique circulating neutrophil gene signature.

COVID-19 has a broad spectrum of clinical manifestations associated with the host immune response heterogeneity. Despite the advances in COVID-19 research, it is still crucial to seek a panel of molecular markers that enable accurate stratification of COVID-19 patients. Here, we performed a study that combined analysis of blood transcriptome, demographic data, clinical aspects and laboratory findings from 66 participants classified into different degrees of COVID-19 severity and healthy subjects. We identified a perturbation in blood-leukocyte transcriptional profile associated with COVID-19 aggravation, which was mainly related to processes that disfavoured lymphocyte activation and favoured neutrophil activation. This transcriptional profile stratified patients according to COVID-19 severity. Hence, it enabled identification of a turning point in transcriptional dynamics that distinguished disease outcomes and non-hospitalized from hospitalized moderate patients. Central genes of this unique neutrophil signature were S100A9, ANXA3, CEACAM6, VNN1, OLFM4, IL1R2, TCN1 and CD177. Our study indicates the molecular changes that are linked with the differing clinical aspects presented by humans when suffering from COVID-19, which involve neutrophil activation.

Divergent androgenic modulation of SARS-CoV-2 infection cooperates with dysregulated immune response to dictate worse COVID-19 outcomes in men.

BACKGROUND: Sex-determined differences are rarely addressed in the management of diseases, despite well-known contrasting outcomes between female and male patients. In COVID-19 there is a remarkable disparity, with higher rates of mortality and more severe acute disease in men compared to women, who are mostly affected by long COVID-19. Furthermore, whether androgens play a protective or detrimental role in COVID-19 is still a matter of debate. Hence, the adequate management of the disease, especially regarding men presenting acute disease aggravation, still needs important data to elucidate the interplay between sex hormones and host immune responses that drive the worse evolution in male patients. METHODS: A cohort of 92 controls and 198 non-severe and severe COVID-19 patients, from both sexes, was assessed for clinical outcomes, plasma steroids, gonadotropins, sex hormone binding globulin (SHBG) and immune mediators, before vaccination. These data were correlated with the global gene expression of blood leukocytes. The androgen receptor (AR) signaling pathway was investigated by transcriptomics and tracheal aspirate was obtained from severe patients for SARS-COV-2 quantification in the respiratory tract. The interplay among clinical, endocrine and immunological data deciphered the sex differences in COVID-19. Importantly, statistical analyses, using 95% confidence interval, considered confounding factors such as age and comorbidities, to definitely parse the role of androgens in the disease outcome. RESULTS: There were notable contrasting levels of testosterone and dihydrotestosterone (DHT) throughout the disease course in male but not female patients. Inflammatory mediators presented significant negative correlations with testosterone, which was partially dependent on age and diabetes in men. Male subjects with severe COVID-19 had a significant up regulation of the AR signaling pathway, including modulation of TMPRSS2 and SRD5A1 genes, which are related to the viral infection and DHT production. Indeed, men had a higher viral load in the tracheal aspirate and levels of DHT were associated with increased relative risk of death. In contrast, the testosterone hormone, which was notably reduced in severe disease, was significantly related with susceptibility to COVID-19 worsening in male patients. Secondary hypogonadism was ruled out in the male severe COVID-19 subjects, as FSH, LH, and SHBG levels were not significantly altered. Instead, these subjects tended to have increased gonadotropin levels. Most interestingly, in this study we identified, for the first time, combined sets of clinical and immunoendocrine parameters that together predicted progression from non-severe to severe COVID-19 in men. One of the limitations of our study was the low or undetectable levels of DHT in many patients. Then, the evaluation of enzymes related to biosynthesis and signaling by androgens was mandatory and reiterated our findings. CONCLUSIONS: These original results unraveled the disease immunoendocrine regulation, despite vaccination or comorbidities and pointed to the fundamental divergent role of the androgens testosterone and DHT in the determination of COVID-19 outcomes in men. Therefore, sex-specific management of the dysregulated responses, treatments or public health measures should be considered for the control of COVID-19 pandemic.

Polyunsaturated fatty acids alter the formation of lipid droplets and eicosanoid production in Leishmania promastigotes.

BACKGROUND: The knowledge about eicosanoid metabolism and lipid droplet (LD) formation in the Leishmania is very limited and new approaches are needed to identify which bioactive molecules are produced of them. OBJECTIVES: Herein, we compared LDs and eicosanoids biogenesis in distinct Leishmania species which are etiologic agents of different clinical forms of leishmaniasis. METHODS: For this, promastigotes of Leishmania amazonensis, L. braziliensis and L. infantum were stimulated with polyunsaturated fatty acids (PUFA) and LD and eicosanoid production was evaluated. We also compared mutations in structural models of human-like cyclooxygenase-2 (GP63) and prostaglandin F synthase (PGFS) proteins, as well as the levels of these enzymes in parasite cell extracts. FINDINGS: PUFAs modulate the LD formation in L. braziliensis and L. infantum. Leishmania spp with equivalent tissue tropism had same protein mutations in GP63 and PGFS. No differences in GP63 production were observed among Leishmania spp, however PGFS production increased during the parasite differentiation. Stimulation with arachidonic acid resulted in elevated production of hydroxyeicosatetraenoic acids compared to prostaglandins. MAIN CONCLUSIONS: Our data suggest LD formation and eicosanoid production are distinctly modulated by PUFAS dependent of Leishmania species. In addition, eicosanoid-enzyme mutations are more similar between Leishmania species with same host tropism.

Th17 cell-linked mechanisms mediate vascular dysfunction induced by testosterone in a mouse model of gender-affirming hormone therapy.

Clinical data point to adverse cardiovascular events elicited by testosterone replacement therapy. Testosterone is the main hormone used in gender-affirming hormone therapy (GAHT) by transmasculine people. However, the cardiovascular impact of testosterone in experimental models of GAHT remains unknown. Sex hormones modulate T-cell activation, and immune mechanisms contribute to cardiovascular risk. The present study evaluated whether testosterone negatively impacts female cardiovascular function by enhancing Th17 cell-linked effector mechanisms. Female (8 wk old) C57BL/6J mice received testosterone (48 mg/kg/wk) for 8 wk. Male mice were used for phenotypical comparisons. The hormone treatment in female mice increased circulating testosterone to levels observed in male mice. Testosterone increased lean body mass and body mass index, and decreased perigonadal fat mass, mimicking clinical findings. After 8 wk, testosterone decreased endothelium-dependent vasodilation and increased peripheral Th17 cells. After 24 wk, testosterone increased blood pressure in female mice. Ovariectomy did not intensify phenotypical or cardiovascular effects by testosterone. Female mice lacking T and B cells [Rag1 knockout (-/-)], as well as female mice lacking IL-17 receptor (IL-17Ra-/-), did not exhibit vascular dysfunction induced by testosterone. Testosterone impaired endothelium-dependent vasodilation in female mice lacking γδ T cells, similarly to the observed in wild-type female mice. Adoptive transfer of CD4+ T cells restored testosterone-induced vascular dysfunction in Rag1-/- female mice. Together, these data suggest that CD4+ T cells, most likely Th17 cells, are central to vascular dysfunction induced by testosterone in female mice, indicating that changes in immune-cell balance are important in the GAHT in transmasculine people.NEW & NOTEWORTHY Sex hormone-induced cardiovascular events are important undesirable effects in transgender people under GAHT. Studies addressing the cardiovascular impact of GAHT will certainly contribute to improve healthcare services offered to this population. Our study showing that vascular dysfunction, via Th17 cell-related mechanisms, precedes increased blood pressure induced by testosterone in a GAHT mouse model, reveals potential mechanisms involved in GAHT-related cardiovascular events and may provide new markers/targets for clinical practices in transmasculine people.

The Severity of COVID-19 Affects the Plasma Soluble Levels of the Immune Checkpoint HLA-G Molecule.

The non-classical histocompatibility antigen G (HLA-G) is an immune checkpoint molecule that has been implicated in viral disorders. We evaluated the plasma soluble HLA-G (sHLA-G) in 239 individuals, arranged in COVID-19 patients (n = 189) followed up at home or in a hospital, and in healthy controls (n = 50). Increased levels of sHLA-G were observed in COVID-19 patients irrespective of the facility care, gender, age, and the presence of comorbidities. Compared with controls, the sHLA-G levels increased as far as disease severity progressed; however, the levels decreased in critically ill patients, suggesting an immune exhaustion phenomenon. Notably, sHLA-G exhibited a positive correlation with other mediators currently observed in the acute phase of the disease, including IL-6, IL-8 and IL-10. Although sHLA-G levels may be associated with an acute biomarker of COVID-19, the increased levels alone were not associated with disease severity or mortality due to COVID-19. Whether the SARS-CoV-2 per se or the innate/adaptive immune response against the virus is responsible for the increased levels of sHLA-G are questions that need to be further addressed.

Multifaceted effect of caffeic acid against Streptococcus mutans infection: microbicidal and immunomodulatory agent in macrophages.

The aim of the present study was to investigate the effects of caffeic acid in the interface between the antimicrobial and anti-inflammatory function in macrophage response against S. mutans. S. mutans (108 cfu/mL) were incubated with caffeic acid to determinate the half-maximal inhibitory concentration (IC50) and macrophage cells were incubated with caffeic acid to determinate cell viability and toxicity. Anti-inflammatory effects were measured by nitrite accumulation, TNF-α and PGE2 production, and NF-kB phosphorylation, and S. mutans survival following internalization by macrophages was investigated. We found that caffeic acid presented antimicrobial activity against S. mutans (IC50 = 2.938 ± 0.1225 mM) without exerting cytotoxicity. Caffeic acid inhibited nitrite, TNF-α and PGE2 production by the NF-kB dependent pathway, indicating an immunomodulatory property. Caffeic acid also contributed to macrophage bacteria clearance activity. In summary, caffeic acid presented antimicrobial activity against S. mutans and anti-inflammatory effects in macrophages.

Photosensitizers attenuate LPS-induced inflammation: implications in dentistry and general health.

Antimicrobial photodynamic therapy (aPDT) is a complementary therapeutic modality for periodontal and endodontic diseases, in which Gram-negative bacteria are directly involved. Currently, there are few evidences regarding the effects of aPDT on bacterial components such as lipopolysaccharide (LPS) and it would represent a major step forward in the clinical use of this therapy. In this context, this study aimed to evaluate the efficacy of different photosensitizers (PSs) used in aPDT in LPS inhibition. Four PSs were used in this study: methylene blue (MB), toluidine blue (TBO), new methylene blue (NMB), and curcumin (CUR). Different approaches to evaluate LPS interaction with PSs were used, such as spectrophotometry, Limulus amebocyte lysate (LAL) test, functional assays using mouse macrophages, and an in vivo model of LPS injection. Spectrophotometry showed that LPS decreased the absorbance of all PSs used, indicating interactions between the two species. LAL assay revealed significant differences in LPS concentrations upon pre-incubation with the different PSs. Interestingly, the inflammatory potential of LPS decreased after previous treatment with the four PSs, resulting in decreased secretion of inflammatory cytokines by macrophages. In vivo, pre-incubating curcumin with LPS prevented animals from undergoing septic shock within the established time. Using relevant models to study the inflammatory activity of LPS, we found that all PSs used in this work decreased LPS-induced inflammation, with a more striking effect observed for NMB and curcumin. These data advance the understanding of the mechanisms of LPS inhibition by PSs.

Immunomodulatory activity of hyaluronidase is associated with metabolic adaptations during acute inflammation.

OBJECTIVE AND DESIGN: Investigate survival outcomes, and immunological and metabolomic effects of hyaluronidase (Hz) treatment during mouse models of acute inflammation and sepsis. METHODS: Survival of C57Bl/6 mice was monitored after lethal challenge with lipopolysaccharide (LPS) or cecal and ligation puncture (CLP)-induced sepsis and treated with Hz or saline. Mice were also challenged with LPS and treated with Hz for leukocyte counting, cytokine quantification and determination of metabolomic profiles in the peritoneal fluid. RESULTS: Hz treatment improved survival outcomes after lethal challenge with LPS or CLP-induced sepsis. LPS challenge promoted acute neutrophil accumulation and production of interleukin-1ß (IL-1ß) and IL-6 in the peritoneum, whereas Hz treatment suppressed neutrophil infiltration and cytokine production. We further characterized the metabolomic alterations caused by LPS challenge, which predicted activity of metabolic pathways related to fatty acids and eicosanoids. Hz treatment had a profound effect over the metabolic response, reflected by reductions of the relative levels of fatty acids. CONCLUSION: Collectively, these data demonstrate that Hz treatment is associated with metabolic reprogramming of pathways that sustain the inflammatory response.

Phospholipids modifications in human hepatoma cell lines (HepG2) exposed to silver and iron oxide nanoparticles.

Metallic nanoparticles such as silver (Ag NPs) and iron oxide (Fe3O4 NPs) nanoparticles are high production volume materials due to their applications in various consumer products, and in nanomedicine. However, their inherent toxicities to human cells remain a challenge. The present study was aimed at combining lipidomics data with common phenotypically-based toxicological assays to gain better understanding into cellular response to Ag NPs and Fe3O4 NPs exposure. HepG2 cells were exposed to different concentrations (3.125, 6.25, 12.5, 25, 50 and 100 µg/ml) of the nanoparticles for 24 h, after which they were assayed for toxic effects using toxicological assays like cytotoxicity, mutagenicity, apoptosis and oxidative stress. The cell membrane phospholipid profile of the cells was also performed using shotgun tandem mass spectrometry. The results showed that nanoparticles exposure resulted in concentration-dependent cytotoxicity as well as reduced cytokinesis-block proliferation index (CBPI). Also, there was an increase in the production of ROS and superoxide anions in exposed cells compared to the negative control. The lipidomics data revealed that nanoparticles exposure caused a modulation of the phospholipidome of the cells. A total of 155 lipid species were identified, out of which the fold changes of 23 were significant. The high number of differentially changed phosphatidylcholine species could be an indication that inflammation is one of the major mechanisms of toxicity of the nanoparticles to the cells.

Prostaglandins D2 and E2 have opposite effects on alveolar macrophages infected with Histoplasma capsulatum.

Prostaglandin E2 (PGE2) suppresses macrophage effector mechanisms; however, little is known about the function of PGD2 in infected alveolar macrophages (AMs). Using serum-opsonized Histoplasma capsulatum (Ops-H. capsulatum) in vitro, we demonstrated that AMs produced PGE2 and PGD2 in a time-dependent manner, with PGE2 levels exceeding those of PGD2 by 48 h postinfection. Comparison of the effects of both exogenous PGs on AMs revealed that PGD2 increased phagocytosis and killing through the chemoattractant receptor-homologous molecule expressed on Th2 lymphocytes receptor, whereas PGE2 had opposite effects, through E prostanoid (EP) receptor 2 (EP2)/EP4-dependent mechanisms. Moreover, PGD2 inhibited phospholipase C-γ (PLC-γ) phosphorylation, reduced IL-10 production, and increased leukotriene B4 receptor expression. In contrast, exogenous PGE2 treatment reduced PLC-γ phosphorylation, p38 and nuclear factor κB activation, TNF-α, H2O2, and leukotriene B4, but increased IL-1ß production. Using specific compounds to inhibit the synthesis of each PG in vitro and in vivo, we found that endogenous PGD2 contributed to fungicidal mechanisms and controlled inflammation, whereas endogenous PGE2 decreased phagocytosis and killing of the fungus and induced inflammation. These findings demonstrate that, although PGD2 acts as an immunostimulatory mediator to control H. capsulatum infection, PGE2 has immunosuppressive effects, and the balance between these two PGs may limit collateral immune damage at the expense of microbial containment.

The inhibition of 5-Lipoxygenase (5-LO) products leukotriene B4 (LTB4) and cysteinyl leukotrienes (cysLTs) modulates the inflammatory response and improves cutaneous wound healing.

To analyze the participation of the enzyme 5-lipoxygenase (5-LO) in skin repair, WT wounds were compared to those in 5-LO deficient mice (5-LO-/-), which presented faster closure and reduced inflammatory infiltrate in the skin, together with increased CD4 regulatory T cells markers in the draining lymph nodes. The 5-LO-/- wounds also had diminished TNF-α, CCL11, CCL7, CCL2, CXCL9, CCR1 and CXCR2 mRNA expression in the lesions, besides differential extracellular matrix remodeling. Furthermore, when cysteinyl leukotriene (cysLT) and leukotriene (LTB4) receptors were antagonized in WT mice, there was a remarkable reduction in TNF-α expression and faster skin healing, similarly to the findings in 5-LO-/- animals. Finally, our results suggested that 5-LO products, in special cysLT and LTB4, underline skin inflammation that follows skin injury and their neutralization may be an important strategy to improve cutaneous healing.

Hyaluronidase decreases neutrophils infiltration to the inflammatory site.

OBJECTIVE: To evaluate the in vivo anti-inflammatory potential of bovine hyaluronidase (HYAL) using two different models of acute inflammation. METHODS: Air pouches were produced in the dorsal subcutaneous of mice and injected with phosphate saline solution or HYAL. The antiinflammatory action of HYAL was evaluated in carrageenan (Cg)-inflamed air pouches. After 4 and 24 h the cellular influx, protein exudation, cytokines and lipid mediators were evaluated. The action of HYAL on the rolling and adhesion of leukocytes was investigated in the LPS-stimulated mesenteric microcirculation by intravital microscopic. RESULTS: Treatment with HYAL reduced the cellular influx and protein exudation in non-inflamed and inflamed air pouches. HYAL treatment of Cg-inflamed air pouch reduced the production of tumor necrosis factor-alpha (TNF-α), interleukin-8 (IL-8), leukotriene B4 (LTB4) and LTC4, whereas prostaglandins E2 (PGE2) and D2 (PGD2) concentrations were unchanged. Histological analyses showed that HYAL administration diminished cell infiltration in the air-pouch lining. In LPS-stimulated mesenteric microcirculation, HYAL usage decreased rolling and adhesion of leukocytes, but did not affect the blood vessels diameters. CONCLUSION: The results demonstrate that HYAL inhibited cellular recruitment, edema formation and pro-inflammatory mediators production, resulting in decreased adherence of leukocytes to blood vessels and tissue infiltration. Our data suggest that HYAL may be considered an effective candidate to ameliorate acute inflammation.

Moojenactivase, a novel pro-coagulant PIIId metalloprotease isolated from Bothrops moojeni snake venom, activates coagulation factors II and X and induces tissue factor up-regulation in leukocytes.

Coagulopathies following snakebite are triggered by pro-coagulant venom toxins, in which metalloproteases play a major role in envenomation-induced coagulation disorders by acting on coagulation cascade, platelet function and fibrinolysis. Considering this relevance, here we describe the isolation and biochemical characterization of moojenactivase (MooA), a metalloprotease from Bothrops moojeni snake venom, and investigate its involvement in hemostasis in vitro. MooA is a glycoprotein of 85,746.22 Da, member of the PIIId group of snake venom metalloproteases, composed of three linked disulfide-bonded chains: an N-glycosylated heavy chain, and two light chains. The venom protease induced human plasma clotting in vitro by activating on both blood coagulation factors II (prothrombin) and X, which in turn generated α-thrombin and factor Xa, respectively. Additionally, MooA induced expression of tissue factor (TF) on the membrane surface of peripheral blood mononuclear cells (PBMC), which led these cells to adopt pro-coagulant characteristics. MooA was also shown to be involved with production of the inflammatory mediators TNF-α, IL-8 and MCP-1, suggesting an association between MooA pro-inflammatory stimulation of PBMC and TF up-regulation. We also observed aggregation of washed platelets when in presence of MooA; however, the protease had no effect on fibrinolysis. Our findings show that MooA is a novel hemostatically active metalloprotease, which may lead to the development of coagulopathies during B. moojeni envenomation. Moreover, the metalloprotease may contribute to the development of new diagnostic tools and pharmacological approaches applied to hemostatic disorders.

Protective Effect of Galectin-1 during Histoplasma capsulatum Infection Is Associated with Prostaglandin E2 and Nitric Oxide Modulation.

Histoplasma capsulatum is a dimorphic fungus that develops a yeast-like morphology in host's tissue, responsible for the pulmonary disease histoplasmosis. The recent increase in the incidence of histoplasmosis in immunocompromised patients highlights the need of understanding immunological controls of fungal infections. Here, we describe our discovery of the role of endogenous galectin-1 (Gal-1) in the immune pathophysiology of experimental histoplasmosis. All infected wild-type (WT) mice survived while only 1/3 of Lgals1-/- mice genetically deficient in Gal-1 survived 30 days after infection. Although infected Lgals1-/- mice had increased proinflammatory cytokines, nitric oxide (NO), and elevations in neutrophil pulmonary infiltration, they presented higher fungal load in lungs and spleen. Infected lung and infected macrophages from Lgals1-/- mice exhibited elevated levels of prostaglandin E2 (PGE2, a prostanoid regulator of macrophage activation) and prostaglandin E synthase 2 (Ptgs2) mRNA. Gal-1 did not bind to cell surface of yeast phase of H. capsulatum, in vitro, suggesting that Gal-1 contributed to phagocytes response to infection rather than directly killing the yeast. The data provides the first demonstration of endogenous Gal-1 in the protective immune response against H. capsulatum associated with NO and PGE2 as an important lipid mediator in the pathogenesis of histoplasmosis.