Self-adhesive hydrogel meshes reduce tissue incorporation and mechanical behavior versus microgrips self-fixation: a preclinical study

Purpose Atraumatic mesh fixation for abdominal hernia repair has been developed to avoid the disadvantages of classical fixation with sutures, which is considered a cause of chronic pain and discomfort. This study was designed to analyze, in the short and medium term, the biological and mechanical behavior of two self-fixing meshes compared to that of a polypropylene (PP) mesh fixed with a cyanoacrylate (CA) tissue adhesive. Methods Partial abdominal wall defects (6 × 4 cm) were created in New Zealand rabbits (n = 36) and repaired using a self-adhesive hydrogel mesh (Adhesix™), a self-gripping mesh (ProGrip™) or a PP mesh fixed with CA (Surgipro™ CA). After 14 and 90 days, the host tissue incorporation, macrophage response and biomechanical strength were examined. Results At 14 and 90 days, the ProGrip and Surgipro CA meshes showed good host tissue incorporation; however, the Adhesix implants presented poor integration, seroma formation and a higher degree of shrinkage. The Adhesix hydrogel was completely reabsorbed at 14 days, whereas ProGrip microhooks were observed at all study times. The macrophage response was higher in the ProGrip and Surgipro CA groups at 14 and 90 days, respectively, and decreased over time. At 90 days, the ProGrip implants showed the highest tensile strength values and the Adhesix implants showed the highest failure stretch. Conclusion Meshes with mechanical microgrip self-fixation (ProGrip) show better biological and mechanical behavior than those with adhesive hydrogel (Adhesix) in a preclinical model of abdominal hernia repair in rabbits.

Differently Charged P (VDF-TrFE) Membranes Influence Osteogenesis Through Differential Immunomodulatory Function of Macrophages

A biomaterial-mediated immune response is a critical factor to determine the cell fate as well as the tissue-regenerative outcome. Although piezoelectric-membranes have attracted considerable interest in the field of guided bone regeneration thanks to their biomimetic electroactivity, the influence of their different surface-charge polarities on the immune-osteogenic microenvironment remains obscure. The present study aimed at investigating the interaction between piezoelectric poly (vinylidene fluoridetrifluoroethylene) [P (VDF-TrFE)] membranes with different surface polarities (negative or positive) and macrophage response, as well as their subsequent influence on osteogenesis from an immunomodulating perspective. Specifically, the morphology, wettability, crystal phase, piezoelectric performance, and surface potential of the synthetic P (VDF-TrFE) samples were systematically characterized. In addition, RAW 264.7 macrophages were seeded onto differently charged P (VDF-TrFE) surfaces, and the culture supernatants were used to supplement cultures of rat bone marrow mesenchymal stem cells (rBMSCs) on the corresponding P (VDF-TrFE) surfaces. Our results revealed that oppositely charged surfaces had different abilities in modulating the macrophage-immune-osteogenic microenvironment. Negatively charged P (VDF-TrFE), characterized by the highest macrophage elongation effect, induced a switch in the phenotype of macrophages from M0 (inactivated) to M2 (anti-inflammatory), thus promoting the osteogenic differentiation of rBMSCs by releasing anti-inflammatory cytokine IL-10. Interestingly, positively charged P (VDF-TrFE) possessed pro-inflammatory properties to induce an M1 (pro-inflammatory) macrophage-dominated reaction, without compromising the subsequent osteogenesis as expected. In conclusion, these findings highlighted the distinct modulatory effect of piezoelectric-P (VDF-TrFE) membranes on the macrophage phenotype, inflammatory reaction, and consequent immune-osteogenic microenvironment depending on their surface-charge polarity. This study provides significant insight into the design of effective immunoregulatory materials for the guided bone regeneration application.

Cell-based screen identifies human type I interferon-stimulated regulators of Toxoplasma gondii Infection

Macrophages activated with interferons (IFNs) respond with transcriptional changes that enhance clearance of intracellular pathogens such as Toxoplasma, a ubiquitous apicomplexan parasite that infects more than a billion people worldwide. Although IFNs generally inhibit Toxoplasma, the parasite can also induce components of the host IFN signalling pathway to enhance survival in host cells. Compared to the type II IFN gamma (IFNγ), the role of type I IFNs in macrophage response to Toxoplasma is relatively not well characterized. Here, using fluorescent Toxoplasma and a CRISPR/Cas9 knockout library that only targets interferon-stimulated genes (ISGs), we adapted a loss-of-function flow cytometry-based approach to systematically identify type I ISGs that control Toxoplasma growth in THP-1 cells, a human macrophage cell line. The system enabled the rapid screening of more than 1900 ISGs for type I (IFNα)-induced inhibitors and enhancers of Toxoplasma growth in THP-1 cells. We identified 26 genes that are associated with Toxoplasma growth arrest out of which we confirmed MAX, SNX5, F2RL2, and SSB, as potent IFNα-induced inhibitors of Toxoplasma in THP1 cells. These findings provide a genetic and experimental roadmap to elucidate type I IFN-induced cell-autonomous responses to Toxoplasma.

Danger signal extracellular calcium initiates differentiation of monocytes into SPP1/osteopontin-producing macrophages

The danger signal extracellular calcium is pathophysiologically increased in the synovial fluid of patients with rheumatoid arthritis (RA). Calcium activates the NLRP3-inflammasome via the calcium-sensing receptor in monocytes/macrophages primed by lipopolysaccharide, and this effect is mediated by the uptake of calciprotein particles (CPPs) formed out of calcium, phosphate, and fetuin-A. Aim of the study was to unravel the influence of calcium on monocytes when the priming signal is not present. Monocytes were isolated from the blood of healthy controls and RA patients. Macrophages were characterized using scRNA-seq, DNA microarray, and proteomics. Imaging flow cytometry was utilized to study intracellular events. Here we show that extracellular calcium and CPPs lead to the differentiation of monocytes into calcium-macrophages when the priming signal is absent. Additional growth factors are not needed, and differentiation is triggered by calcium-dependent CPP-uptake, lysosomal alkalization due to CPP overload, and TFEB- and STAT3-dependent increased transcription of the lysosomal gene network. Calcium-macrophages have a needle-like shape, are characterized by excessive, constitutive SPP1/osteopontin production and a strong pro-inflammatory cytokine response. Calcium-macrophages differentiated out of RA monocytes show a stronger manifestation of this phenotype, suggesting the differentiation process might lead to the pro-inflammatory macrophage response seen in the RA synovial membrane.

Lipidome profiling with Raman microspectroscopy identifies macrophage response to surface topographies of implant materials

Biomaterial characteristics such as surface topographies have been shown to modulate macrophage phenotypes. The standard methodologies to measure macrophage response to biomaterials are marker-based and invasive. Raman microspectroscopy (RM) is a marker-independent, noninvasive technology that allows the analysis of living cells without the need for staining or processing. In the present study, we analyzed human monocyte-derived macrophages (MDMs) using RM, revealing that macrophage activation by lipopolysaccharides (LPS), interferons (IFN), or cytokines can be identified by lipid composition, which significantly differs in M0 (resting), M1 (IFN-γ/LPS), M2a (IL-4/IL-13), and M2c (IL-10) MDMs. To identify the impact of a biomaterial on MDM phenotype and polarization, we cultured macrophages on titanium disks with varying surface topographies and analyzed the adherent MDMs with RM. We detected surface topography–induced changes in MDM biochemistry and lipid composition that were not shown by less sensitive standard methods such as cytokine expression or surface antigen analysis. Our data suggest that RM may enable a more precise classification of macrophage activation and biomaterial–macrophage interaction.

NOTCH4 Exhibits Anti-Inflammatory Activity in Activated Macrophages by Interfering With Interferon-γ and TLR4 Signaling

NOTCH4 is a member of the NOTCH family of receptors whose expression is intensively induced in macrophages after their activation by Toll-like receptors (TLR) and/or interferon-γ (IFN-γ). In this work, we show that this receptor acts as a negative regulator of macrophage activation by diminishing the expression of proinflammatory cytokines, such as IL-6 and IL-12, and costimulatory proteins, such as CD80 and CD86. We have observed that NOTCH4 inhibits IFN-γ signaling by interfering with STAT1-dependent transcription. Our results show that NOTCH4 reprograms the macrophage response to IFN-γ by favoring STAT3 versus STAT1 phosphorylation without affecting their expression levels. This lower activation of STAT1 results in diminished transcriptional activity and expression of STAT1-dependent genes, including IRF1, SOCS1 and CXCL10. In macrophages, NOTCH4 inhibits the canonical NOTCH signaling pathway induced by LPS; however, it can reverse the inhibition exerted by IFN-γ on NOTCH signaling, favoring the expression of NOTCH-target genes, such as Hes1. Indeed, HES1 seems to mediate, at least in part, the enhancement of STAT3 activation by NOTCH4. NOTCH4 also affects TLR signaling by interfering with NF-κB transcriptional activity. This effect could be mediated by the diminished activation of STAT1. These results provide new insights into the mechanisms by which NOTCH, TLR and IFN-γ signal pathways are integrated to modulate macrophage-specific effector functions and reveal NOTCH4 acting as a new regulatory element in the control of macrophage activation that could be used as a target for the treatment of pathologies caused by an excess of inflammation.

Serous Glands in the Skin of the Tungara Frog, Engystomops pustulosus (Cope, 1864) (Anura, Leptodactylidae): Degenerated Secretory Units are Selectively Removed by Macrophages

The cutaneous apparatus of Engystomops pustulosus (Cope, 1864) (the Tungara frog) includes serous glands that show impressive patterns of degeneration in their syncytial secretory units, and thus represent suitable organ models to investigate the role of macrophages in renewal processes of multicellular structures. The present case report exploits this chance and highlights that: (a) degenerating glands pertain to the Ia line of the polymorphic serous gland assortment in Tungara skin; (b) resident macrophages migrate from spongy dermis and remove syncytium debris; (c) secretory syncytium collapse results from impairment of the equilibrium between serous product manufacturing/storage and merocrine release into the dermal environment; (d) Intercalated tract (or gland neck) and myoepithelium (included its ortho-sympathetic nerve supply), are neither involved in degeneration nor affected by macrophage response. According to present evidence and current literature, it is concluded that the scavenger activity of macrophages prepares secretory unit renewal, performed by stem cells from the neck. In addition, gland functional rehabilitation may rely on effectiveness of the preexisting neuromuscular apparatus to achieve secretory bulk release onto the cutaneous surface.

Biofilm of Klebsiella Pneumoniae Minimize Phagocytosis and Cytokine Expression by Macrophage Cell Line

Infectious bacteria in biofilm mode is involved in many persistent infections. Owing to its importance in clinical settings, many in vitro and in vivo studies are being conducted to study the structural and functional properties of biofilms, their drug resistant mechanism and survival mechanism of planktonic and biofilm cells. In this regard, there is not sufficient information on the interaction between Klebsiella biofilm and macrophages. In this study, we have made an attempt to unravel the interaction between Klebsiella biofilm and macrophages in terms of phagocytic response and cytokine expression. In vitro phagocytosis assays was performed for heat inactivated and live biofilms of K. pneumoniae, together with the expression analysis of TLR2, iNOS, inflammatory cytokines such as IL-β1, IFN-γ, IL-6, IL-12, IL-4, TNF-α and anti-inflammatory cytokines, IL-10. A phagocytic rate of an average of 15% was observed against both heat inactivated and live biofilms, when LPS+IFN-γ activated macrophages were used. This was significantly higher than non-activated macrophages when tested against heat inactivated and live biofilms (average 8%). Heat-inactivated and live biofilms induced similar phagocytic response and up-regulation of pro-inflammatory genes in macrophages, indirectly conveying that macrophage response is to some extent dependent on the biofilm matrix.

Head and neck tumor cells treated with hypofractionated irradiation die via apoptosis and are better taken up by M1-like macrophages

Purpose The incidence of head and neck squamous cell carcinomas (HNSCC) is increasing worldwide, especially when triggered by the human papilloma virus (HPV). Radiotherapy has immune-modulatory properties, but the role of macrophages present in HNSCC and having contact with irradiated tumor cells remains unclear. The influence of irradiated (2 × 5Gy) HNSCC cells on the (re-)polarization and phagocytosis of human macrophages, either non-polarized or with a more M1 or M2 phenotype, was therefore investigated. Methods Human monocytes were differentiated with the hematopoietic growth factors M‑CSF (m) or GM-CSF (g) and additionally pre-polarized with either interleukin (IL)-4 and IL-10 or interferon (IFN)-γ and lipopolysaccharides (LPS), respectively. Subsequently, they were added to previously irradiated (2 × 5Gy) and mock-treated HPV-positive (UD-SCC-2) and HPV-negative (Cal33) HNSCC cells including their supernatants. Results The HNSCC cells treated with hypofractionated irradiation died via apoptosis and were strongly phagocytosed by M0m and M2 macrophages. M0g and M1 macrophages phagocytosed the tumor cells to a lesser extent. Irradiated HNSCC cells were better phagocytosed by M1 macrophages compared to mock-treated controls. The polarization status of the macrophages was not significantly changed, except for the expression of CD206 on M2 macrophages, which was reduced after phagocytosis of irradiated HPV-negative cells. Further, a significant increase in the uptake of irradiated HPV-positive cells by M0g macrophages when compared to HPV-negative cells was observed. Conclusion HNSCC cells treated with hypofractionated irradiation foster phagocytosis by anti-tumorigenic M1 macrophages. The data provide the first evidence on the impact of the HPV status of HNSCC cells on the modulation of the macrophage response to irradiated tumor cells.