Innovative Pre-Clinical Data Using Peptides to Intervene in the Evolution of Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, relentless, and deadly disease. Little is known about its pathogenetic mechanisms; therefore, developing efficient pharmacological therapies is challenging. This work aimed to apply a therapeutic alternative using immunomodulatory peptides in a chronic pulmonary fibrosis murine model. BALB/c mice were intratracheally instilled with bleomycin (BLM) and followed for 30 days. The mice were treated with the immune modulatory peptides ToAP3 and ToAP4 every three days, starting on the 5th day post-BLM instillation. ELISA, qPCR, morphology, and respiratory function analyses were performed. The treatment with both peptides delayed the inflammatory process observed in the non-treated group, which showed a fibrotic process with alterations in the production of collagen I, III, and IV that were associated with significant alterations in their ventilatory mechanics. The ToAP3 and ToAP4 treatments, by lung gene modulation patterns, indicated that distinct mechanisms determine the action of peptides. Both peptides controlled the experimental IPF, maintaining the tissue characteristics and standard function properties and regulating fibrotic-associated cytokine production. Data obtained in this work show that the immune response regulation by ToAP3 and ToAP4 can control the alterations that cause the fibrotic process after BLM instillation, making both peptides potential therapeutic alternatives and/or adjuvants for IPF.

Pathogenicity and Growth Conditions Modulate Fonsecaea Extracellular Vesicles' Ability to Interact With Macrophages.

Chromoblastomycosis (CBM) is a chronic cutaneous and subcutaneous mycosis caused by black, dimorphic, and filamentous fungi of the Herpothrichiellaceae family, such as species of the genus Fonsecaea. These fungi can switch between the saprophytic forms (conidia and hyphae) and the pathogenic form, the muriform cells (MCs), which is considered an essential mechanism for fungal virulence. Nearly all types of cells can produce membranous structures formed by a lipid bilayer that communicate extracellularly with other cells, known as "extracellular vesicles" (EVs), which may act as virulence factors, as observed for several species of pathogenic fungi. Our findings demonstrated for the first time that F. pedrosoi, F. nubica, and F. erecta produce EVs in response to nutritional conditions. The EVs varied in sterol and protein contents, size, and morphology. Moreover, the EVs induced different cytokine and nitric oxide release patterns by bone marrow-derived macrophages (BMDMs). The EVs activated IL-1ß production, possibly acting as the first signal in inflammasome activation. Unlike the pathogenic species, the EVs isolated from F. erecta did not significantly stimulate TNF and IL-10 production in general. Overall, these results demonstrated that different species of Fonsecaea produce EVs capable of modulating pro- and anti-inflammatory cytokine and nitric oxide production by BMDMs and that growth conditions affected the immunomodulatory capacities of the EVs as well as their size, content, and morphology.

Fish Oil Nanoemulsion Supplementation Attenuates Bleomycin-Induced Pulmonary Fibrosis BALB/c Mice.

Diets rich in omega-3 or -6 fatty acids will produce different profiles for cell membranes phospholipid constitutions. Omegas 3 and 6 are part of the diet and can modulate the inflammatory profile. We evaluated the effects of the oral absorption of fish oil, when associated with a lipid nanoemulsion in an experimental pulmonary inflammatory model. Pulmonary fibrosis is a disease associated with excessive extracellular matrix deposition. We determined to investigate the morphophysiological mechanisms in mice that were pretreated after induction with bleomycin (BLM). The pretreatment was for 21 days with saline solution, sunflower oil (SO), fish oil (FO), and fish oil nanoemulsion (NEW3). The animals received a daily dose of 50 mg/Kg of docosahexaenoic acid DHA and 10 mg/Kg eicosapentaenoic (EPA) (100 mg/Kg), represented by a daily dose of 40 µL of NEW3. The blank group was treated with the same amount daily (40 µL) during the 21 days of pretreatment. The animals were treated with SO and FO, 100 mg/Kg (containing 58 mg/Kg of polyunsaturated fats/higher% linoleic acid) and 100 mg/Kg (50 mg/Kg of DHA and 10 mg/Kg EPA), respectively. A single dose of 5 mg/mL (50 µL) bleomycin sulfate, by the intratracheal surgical method in BALB/cAnNTac (BALB/c). NEW3 significantly reduced fibrotic progression, which can be evidenced by the protection from loss of body mass, increase in respiratory incursions per minute, decreased spacing of alveolar septa, decreased severity of fibrosis, and changes in the respiratory system. NEW3 attenuated the inflammatory changes developed in the experimental model of pulmonary fibrosis, while group SO showed a significant increase in inflammatory changes. This concluded that the presented results demonstrated that is possible to positively modulate the immune and inflamamtory response to an external agressor, by changing the nutitional intake of specific fatty acids, such as omega-3 placed in fish oil. Moreover, these benefits can be improved by the nanoencapsulation of fish oil in lipid nanoemulsions.

Peptides ToAP3 and ToAP4 decrease release of inflammatory cytokines through TLR-4 blocking.

Antimicrobial peptides (AMPs) are small molecules with microbicidal and immunoregulatory activities. In this study we evaluated the anti-inflammatory and antimicrobial activities of peptides ToAP3 and ToAP4, AMPs from the venom of the Brazilian scorpion Tityus obscurus. To test the peptides' activity, murine bone marrow-derived macrophages (BMDMs) or dendritic cells (BMDCs) were stimulated with peptides plus LPS to analyze their ability to modulate cytokine release as well as phenotypic markers. For antimicrobial analysis, we evaluated the indirect activity against macrophage-internalized Cryptococcus neoformans and direct activity against Mycobacterium massiliense. Our data demonstrate that they were able to reduce TNF-α and IL-1ß transcript levels and protein levels for BMDM and BMDC. Furthermore, the reduction of TNF-α secretion, before LPS- inflammatory stimuli, is associated with peptide interaction with TLR-4. ToAP4 increased MHC-II expression in BMDC, while ToAP3 decreased co-stimulatory molecules such as CD80 and CD86. Although these peptides were able to modulate the production of cytokines and molecules associated with antigen presentation, they did not increase the ability of clearance of C. neoformans by macrophages. In antimicrobial analysis, only ToAP3 showed potent action against bacteria. Altogether, these results demonstrate a promising target for the development of new immunomodulatory and anti-bacterial therapies.