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.

Hyaluronidase modulates inflammatory response and accelerates the cutaneous wound healing.

Hyaluronidases are enzymes that degrade hyaluronan an important constituent of the extracellular matrix. They have been used as a spreading agent, improving the absorption of drugs and facilitating the subcutaneous infusion of fluids. Here, we investigated the influence of bovine testes hyaluronidase (HYAL) during cutaneous wound healing in in vitro and in vivo assays. We demonstrated in the wound scratch assay that HYAL increased the migration and proliferation of fibroblasts in vitro at low concentration, e.g. 0.1 U HYAL enhanced the cell number by 20%. HYAL presented faster and higher reepithelialization in in vivo full-thickness excisional wounds generated on adult Wistar rats back skin already in the early phase at 2nd day post operatory compared to vehicle-control group. Wound closured area observed in the 16 U and 32 U HYAL treated rats reached 38% and 46% compared to 19% in the controls, respectively. Histological and biochemical analyses supported the clinical observations and showed that HYAL treated wounds exhibited increased granulation tissue, diminished edema formation and regulated the inflammatory response by modulating the release of pro and anti-inflammatory cytokines, growth factor and eicosanoids mediators. Moreover, HYAL increased gene expression of peroxisome proliferator-activated receptors (PPAR) γ and PPAR ß/δ, the collagen content in the early stages of healing processes as well as angiogenesis. Altogether these data revealed that HYAL accelerates wound healing processes and might be beneficial for treating wound disorders.

Cooperative role of endogenous leucotrienes and platelet-activating factor in ischaemia-reperfusion-mediated tissue injury.

Insufficient oxygen delivery to organs leads to tissue dysfunction and cell death. Reperfusion, although vital to organ survival, initiates an inflammatory response that may both aggravate local tissue injury and elicit remote organ damage. Polymorphonuclear neutrophil (PMN) trafficking to remote organs following ischaemia/reperfusion (I/R) is associated with the release of lipid mediators, including leucotriene (LT) B4 , cysteinyl-LTs (CysLTs) and platelet-activating factor (PAF). Yet, their potentially cooperative role in regulating I/R-mediated inflammation has not been thoroughly assessed. The present study aimed to determine the cooperative role of lipid mediators in regulating PMN migration, tissue oedema and injury using selective receptor antagonists in selected models of I/R and dermal inflammation. Our results show that rabbits, pre-treated orally with BIIL 284 and/or WEB 2086 and MK-0571, were protected from remote tissue injury following I/R or dermal inflammation in an additive or synergistic manner when the animals were pre-treated with two drugs concomitantly. The functional selectivity of the antagonists towards their respective agonists was assessed in vitro, showing that neither BIIL 284 nor WEB 2086 prevented the inflammatory response to IL-8, C5a and zymosan-activated plasma stimulation. However, these agonists elicited LTB4 biosynthesis in isolated rabbit PMNs. Similarly, a cardioprotective effect of PAF and LTB4 receptor antagonists was shown following myocardial I/R in mice. Taken together, these results underscore the intricate involvement of LTB4 and PAF in each other's responses and provide further evidence that targeting both LTs and PAF receptors provides a much stronger anti-inflammatory effect, regulating PMN migration and oedema formation.

Biodegradable microspheres containing leukotriene B(4) and cell-free antigens from Histoplasma capsulatum activate murine bone marrow-derived macrophages.

Because of the potential protective role of leukotrienes (LTs) in histoplasmosis and the therapeutic and prophylactic effects of cell-free antigens from Histoplasmacapsulatum (CFAgs), the aim of this study was to develop and characterise biodegradable LTB(4)/CFAgs-loaded microspheres (MS) that could promote cellular activation for future immunisation purposes. LTB(4)/CFAgs-loaded MS that were developed through a double emulsion/extraction process were characterised according to their size, zeta potential, morphology, entrapment efficiency and in vitro release kinetics. We evaluated the uptake of LTB(4)/CFAgs-loaded MS by bone marrow derived-macrophages (BMDM). The TNF-α and chemokines, and nitrite production, in the supernatant of BMDM cultures were analysed by enzyme-linked immunosorbent assay (ELISA) and Griess reaction, respectively. We found an instantaneous release of CFAgs and a prolonged release of LTB(4) from the poly-(d,l-lactide-co-glycolide) (PLGA) MS. The microencapsulation process did not alter the zeta potential nor the spherical morphology of the MS. The appropriate size of the LTB(4)/CFAgs-loaded MS (smaller than 10µm) enabled the efficient uptake by BMDM and also induced TNF-α, CXCL1/KC, CCL2/MCP-1, CCL5/RANTES and nitrite oxide release by these cells. In conclusion, the biodegradable LTB(4)/CFAgs-loaded MS were able to efficiently activate murine BMDM and thereby have the potential to be used in an effective vaccine against H. capsulatum infection.

Hyaluronidase recruits mesenchymal-like cells to the lung and ameliorates fibrosis.

Hyaluronidases (HYALs) comprise a group of enzymes that degrade hyaluronic acid (HA). In this report, we reveal that a single intranasal inoculation of HYAL induces an increase in mononuclear cells within the bronchoalveolar space demonstrating a mesenchymal-like phenotype, expressing stem cell antigen-1 (SCA-1), CD44 and CD73 but not CD34, CD45, CD3, CD4, CD8 or CD19. This influx of mesenchymal stem cell (MSC)-like cells was dependent on leukotriene production within the lung parenchyma. These findings prompted experiments demonstrating that HYAL treatment potently blocked bleomycin-induced lung injury and fibrosis while decreasing transforming growth factor (TGF)-ß production and collagen deposition. These data suggest that HYAL is a novel and promising tool to use autologous MSC-like cells in the treatment of pulmonary fibrosis.