Guimaraes-Costa AB, Shannon JP, Waclawiak I, Oliveira J, Meneses C, de Castro W, Wen X, Brzostowski J, Serafim TD, Andersen JF, Hickman HD, Kamhawi S, Valenzuela JG, Oliveira F. A sand fly salivary protein acts as a neutrophil chemoattractant. Nat Commun. 2021 May 28;12(1):3213.
DOI: 10.1038/s41467-021-23002-5
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Apart from bacterial formyl peptides or viral chemokine mimicry, a non-vertebrate or insect protein that directly attracts mammalian innate cells such as neutrophils has not been molecularly characterized. Here, we show that members of sand fly yellow salivary proteins induce in vitro chemotaxis of mouse, canine and human neutrophils in transwell migration or EZ-TAXIScan assays. We demonstrate murine neutrophil recruitment in vivo using flow cytometry and two-photon intravital microscopy in Lysozyme-M-eGFP transgenic mice. We establish that the structure of this ~ 45 kDa neutrophil chemotactic protein does not resemble that of known chemokines. This chemoattractant acts through a G-protein-coupled receptor and is dependent on calcium influx. Of significance, this chemoattractant protein enhances lesion pathology (P < 0.0001) and increases parasite burden (P < 0.001) in mice upon co-injection with Leishmania parasites, underlining the impact of the sand fly salivary yellow proteins on disease outcome. These findings show that some arthropod vector-derived factors, such as this chemotactic salivary protein, activate rather than inhibit the host innate immune response, and that pathogens take advantage of these inflammatory responses to establish in the host.
Borges PA, Waclawiak I, Georgii JL, Fraga-Junior VDS, Barros JF, Lemos FS, Russo-Abrahão T, Saraiva EM, Takiya CM, Coutinho-Silva R, Penido C, Mermelstein C, Meyer-Fernandes JR, Canto FB, Neves JS, Melo PA, Canetti C, Benjamim CF. Adenosine Diphosphate Improves Wound Healing in Diabetic Mice Through P2Y12 Receptor Activation. Front Immunol. 2021 Mar 22;12:651740.
DOI: 10.3389/fimmu.2021.651740
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Chronic wounds are a public health problem worldwide, especially those related to diabetes. Besides being an enormous burden to patients, it challenges wound care professionals and causes a great financial cost to health system. Considering the absence of effective treatments for chronic wounds, our aim was to better understand the pathophysiology of tissue repair in diabetes in order to find alternative strategies to accelerate wound healing. Nucleotides have been described as extracellular signaling molecules in different inflammatory processes, including tissue repair. Adenosine-5'-diphosphate (ADP) plays important roles in vascular and cellular response and is immediately released after tissue injury, mainly from platelets. However, despite the well described effect on platelet aggregation during inflammation and injury, little is known about the role of ADP on the multiple steps of tissue repair, particularly in skin wounds. Therefore, we used the full-thickness excisional wound model to evaluate the effect of local ADP application in wounds of diabetic mice. ADP accelerated cutaneous wound healing, improved new tissue formation, and increased both collagen deposition and transforming growth factor-β (TGF-β) production in the wound. These effects were mediated by P2Y12 receptor activation since they were inhibited by Clopidogrel (Clop) treatment, a P2Y12 receptor antagonist. Furthermore, P2Y1 receptor antagonist also blocked ADP-induced wound closure until day 7, suggesting its involvement early in repair process. Interestingly, ADP treatment increased the expression of P2Y12 and P2Y1 receptors in the wound. In parallel, ADP reduced reactive oxygen species (ROS) formation and tumor necrosis factor-α (TNF-α) levels, while increased IL-13 levels in the skin. Also, ADP increased the counts of neutrophils, eosinophils, mast cells, and gamma delta (γδ) T cells (Vγ4+ and Vγ5+ cells subtypes of γδ+ T cells), although reduced regulatory T (Tregs) cells in the lesion. In accordance, ADP increased fibroblast proliferation and migration, myofibroblast differentiation, and keratinocyte proliferation. In conclusion, we provide strong evidence that ADP acts as a pro-resolution mediator in diabetes-associated skin wounds and is a promising intervention target for this worldwide problem.
Keywords: P2Y12 recepor; adenosine diphosphate (ADP); diabetes; inflammation; mice; purinergic signaling; skin; wound healing.
Silva JBNF, Calcia TBB, Silva CP, Guilherme RF, Almeida-Souza F, Lemos FS, Calabrese KS, Caruso-Neves C, Neves JS, Benjamim CF. ATRvD1 Attenuates Renal Tubulointerstitial Injury Induced by Albumin Overload in Sepsis-Surviving Mice. Int J Mol Sci. 2021 Oct 27;22(21):11634.
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Novel strategies for the prevention and treatment of sepsis-associated acute kidney injury and its long-term outcomes have been required and remain a challenge in critical care medicine. Therapeutic strategies using lipid mediators, such as aspirin-triggered resolvin D1 (ATRvD1), can contribute to the resolution of acute and chronic inflammation. In this study, we examined the potential effect of ATRvD1 on long-term kidney dysfunction after severe sepsis. Fifteen days after cecal ligation and puncture (CLP), sepsis-surviving BALB/c mice were subjected to a tubulointerstitial injury through intraperitoneal injections of bovine serum albumin (BSA) for 7 days, called the subclinical acute kidney injury (subAKI) animal model. ATRvD1 treatment was performed right before BSA injections. On day 22 after CLP, the urinary protein/creatinine ratio (UPC), histologic parameters, fibrosis, cellular infiltration, apoptosis, inflammatory markers levels, and mRNA expression were determined. ATRvD1 treatment mitigated tubulointerstitial injury by reducing proteinuria excretion, the UPC ratio, the glomerular cell number, and extracellular matrix deposition. Pro-fibrotic markers, such as transforming growth factor β (TGFβ), type 3 collagen, and metalloproteinase (MMP)-3 and -9 were reduced after ATRvD1 administration. Post-septic mice treated with ATRvD1 were protected from the recruitment of IBA1+ cells. The interleukin-1β (IL-1β) levels were increased in the subAKI animal model, being attenuated by ATRvD1. Tumor necrosis factor-α (TNF-α), IL-10, and IL-4 mRNA expression were increased in the kidney of BSA-challenged post-septic mice, and it was also reduced after ATRvD1. These results suggest that ATRvD1 protects the kidney against a second insult such as BSA-induced tubulointerstitial injury and fibrosis by suppressing inflammatory and pro-fibrotic mediators in renal dysfunction after sepsis.
Keywords: sepsis; renal tubulointerstitial injury; resolvin; ATRvD1; inflammation; kidney
Moura Silva H, Kitoko JZ, Queiroz CP, Kroehling L, Matheis F, Yang KL, Reis BS, Ren-Fielding C, Littman DR, Bozza MT, Mucida D, Lafaille JJ. c-MAF-dependent perivascular macrophages regulate diet-induced metabolic syndrome. Sci Immunol. 2021 Oct;6(64):eabg7506.
DOI: 10.1126/sciimmunol.abg7506
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Macrophages are an essential part of tissue development and physiology. Perivascular macrophages have been described in tissues and appear to play a role in development and disease processes, although it remains unclear what the key features of these cells are. Here, we identify a subpopulation of perivascular macrophages in several organs, characterized by their dependence on the transcription factor c-MAF and displaying nonconventional macrophage markers including LYVE1, folate receptor 2, and CD38. Conditional deletion of c-MAF in macrophage lineages caused ablation of perivascular macrophages in the brain and altered muscularis macrophages program in the intestine. In the white adipose tissue (WAT), c-MAF–deficient perivascular macrophages displayed an altered gene expression profile, which was linked to an increased vascular branching. Upon feeding high-fat diet (HFD), mice with c-MAF–deficient macrophages showed improved metabolic parameters compared with wild-type mice, including less weight gain, greater glucose tolerance, and reduced inflammatory cell profile in WAT. These results define c-MAF as a central regulator of the perivascular macrophage transcriptional program in vivo and reveal an important role for this tissue-resident macrophage population in the regulation of metabolic syndrome.
Juliano MA, Costa SM, Alves AMB, Cordeiro MT, Marques ETA, Scharfstein J, Arruda LB. Contact System Activation in Plasma from Dengue Patients Might Harness Endothelial Virus Replication through the Signaling of Bradykinin Receptors. Pharmaceuticals (Basel). 2021 Jan 12;14(1):56.
DOI: 10.3390/ph14010056
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Since exacerbated inflammation and microvascular leakage are hallmarks of dengue virus (DENV) infection, here we interrogated whether systemic activation of the contact/kallikrein-kinin system (KKS) might hamper endothelial function. In vitro assays showed that dextran sulfate, a potent contact activator, failed to generate appreciable levels of activated plasma kallikrein (PKa) in the large majority of samples from a dengue cohort (n = 70), irrespective of severity of clinical symptoms. Impaired formation of PKa in dengue-plasmas correlated with the presence of cleaved Factor XII and high molecular weight kininogen (HK), suggesting that the prothrombogenic contact system is frequently triggered during the course of infection. Using two pathogenic arboviruses, DENV or Zika virus (ZIKV), we then asked whether exogenous BK could influence the outcome of infection of human brain microvascular endothelial cells (HBMECs). Unlike the unresponsive phenotype of Zika-infected HBMECs, we found that BK, acting via B2R, vigorously stimulated DENV-2 replication by reverting nitric oxide-driven apoptosis of endothelial cells. Using the mouse model of cerebral dengue infection, we next demonstrated that B2R targeting by icatibant decreased viral load in brain tissues. In summary, our study suggests that contact/KKS activation followed by BK-induced enhancement of DENV replication in the endothelium may underlie microvascular pathology in dengue.
Keywords: dengue; bradykinin; endothelial cells; kallikrein-kinin system; contact pathway; bradykinin receptor B2
Silva RCMC, Tan L, Rodrigues DA, Prestes EB, Gomes CP, Gama AM, Oliveira PL, Paiva CN, Manoury B, Bozza MT. Chloroquine inhibits pro-inflammatory effects of heme on macrophages and invivo. Free Radic Biol Med. 2021 Sep;173:104-116.
DOI: 10.1016/j.freeradbiomed.2021.07.028
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Background: Chloroquine has been used successfully to treat Malaria, including by chloroquine-resistant Plasmodium sp., indicating that it has effects on disease itself. Since heme has inflammatory effects and contributes to the pathogenesis of hemolytic diseases, we hypothesize that the anti-inflammatory effect of chloroquine is partially due to its inhibitory effect on heme-induced macrophage activation and on inflammatory tissue damage.
Methods: Bone marrow derived macrophages (BMDMs) were incubated with chloroquine before stimulation with heme, in different conditions, to evaluate cytokines secretion, ROS production, mitogen activated protein kinases (MAPK) or spleen tyrosine kinase (Syk) activation, alone or combined with LPS. The effects of chloroquine upon heme inflammation were also evaluated in vivo, through simultaneous i.p. injection of LPS and heme, intratracheal instillation of Poly-IC followed by heme injection, and in a rhabdomyolysis model.
Results: Chloroquine inhibited TNF secretion, mitochondrial ROS production, MAPK, and Syk activation induced by heme. Inhibition of TNF production could be mimicked by zinc ionophore quercetin, but not by primaquine, a chloroquine analog with low affinity for heme. IL-6 and IL-1β secretions induced by heme in the presence of PRRs agonists were inhibited by chloroquine, but not by calcium chelator BAPTA or inhibitor of endosomal acidification concamycin B. Chloroquine also protected mice from heme inflammatory effects in vivo, inhibiting lethal synergism with PRR agonists, lung pathology caused by heme injection after intratracheal instillation of Poly-IC, and delaying death after rhabdomyolisis.
Keywords: Chloroquine; Cytokines; Heme; Inflammation; ROS
Jennings-Almeida B, Castelpoggi JP, Ramos-Junior ES, Ferreira EO, Domingues RMCP, Echevarria-Lima J, Coutinho-Silva R, Moreira-Souza ACA, Mariño E, Mackay CR, Zamboni DS, Bellio M, Scharfstein J, Lobo LA, Oliveira AC. Dietary Fiber Drives IL-1β-Dependent Peritonitis Induced by Bacteroides fragilis via Activation of the NLRP3 Inflammasome. J Immunol. 2021 May 15;206(10):2441-2452.
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Intestinal barrier is essential for dietary products and microbiota compartmentalization and therefore gut homeostasis. When this barrier is broken, cecal content overflows into the peritoneal cavity, leading to local and systemic robust inflammatory response, characterizing peritonitis and sepsis. It has been shown that IL-1β contributes with inflammatory storm during peritonitis and sepsis and its inhibition has beneficial effects to the host. Therefore, we investigated the mechanisms underlying IL-1β secretion using a widely adopted murine model of experimental peritonitis. The combined injection of sterile cecal content (SCC) and the gut commensal bacteria Bacteroides fragilis leads to IL-1β–dependent peritonitis, which was mitigated in mice deficient in NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome components. Typically acting as a damage signal, SCC, but not B. fragilis, activates canonical pathway of NLRP3 promoting IL-1β secretion in vitro and in vivo. Strikingly, absence of fiber in the SCC drastically reduces IL-1β production, whereas high-fiber SCC conversely increases this response in an NLRP3-dependent manner. In addition, NLRP3 was also required for IL-1β production induced by purified dietary fiber in primed macrophages. Extending to the in vivo context, IL-1β–dependent peritonitis was worsened in mice injected with B. fragilis and high-fiber SCC, whereas zero-fiber SCC ameliorates the pathology. Corroborating with the proinflammatory role of dietary fiber, IL-1R–deficient mice were protected from peritonitis induced by B. fragilis and particulate bran. Overall, our study highlights a function, previously unknown, for dietary fibers in fueling peritonitis through NLRP3 activation and IL-1β secretion outside the gut.
