Sonic Hedgehog acts as a macrophage chemoattractant during regeneration of the gastric epithelium

Sonic Hedgehog (Shh), secreted from gastric parietal cells, contributes to the regeneration of the epithelium. The recruitment of macrophages plays a central role in the regenerative process. The mechanism that regulates macrophage recruitment in response to gastric injury is largely unknown. Here we tested the hypothesis that Shh stimulates macrophage chemotaxis to the injured epithelium and contributes to gastric regeneration. A mouse model expressing a myeloid cell-specific deletion of Smoothened (LysMcre/+;Smof/f) was generated using transgenic mice bearing loxP sites flanking the Smo gene (Smo loxP) and mice expressing a Cre recombinase transgene from the Lysozyme M locus (LysMCre). Acetic acid injury was induced in the stomachs of both control and LysMcre/+;Smof/f (SmoKO) mice and gastric epithelial regeneration and macrophage recruitment analyzed over a period of 7 days post-injury. Bone marrow-derived macrophages (BM-Mø) were collected from control and SmoKO mice. Human-derived gastric organoid/macrophage co-cultures were established, and macrophage chemotaxis measured. Compared to control mice, SmoKO animals exhibited inhibition of ulcer repair and normal epithelial regeneration, which correlated with decreased macrophage infiltration at the site of injury. Bone marrow chimera experiments using SmoKO donor cells showed that control chimera mice transplanted with SmoKO bone marrow donor cells exhibited a loss of ulcer repair, and transplantation of control bone marrow donor cells to SmoKO mice rescued epithelial cell regeneration. Histamine-stimulated Shh secretion in human organoid/macrophage co-cultures resulted in macrophage migration toward the gastric epithelium, a response that was blocked with Smo inhibitor Vismodegib. Shh-induced macrophage migration was mediated by AKT signaling. In conclusion, Shh signaling acts as a macrophage chemoattractant via a Smo-dependent mechanism during gastric epithelial regeneration in response to injury.

Identification and validation of candidate genes dysregulated in alveolar macrophages of acute respiratory distress syndrome

Background Acute respiratory distress syndrome (ARDS) is a common cause of death in ICU patients and its underlying mechanism remains unclear, which leads to its high mortality rate. This study aimed to identify candidate genes potentially implicating in the pathogenesis of ARDS and provide novel therapeutic targets. Methods Using bioinformatics tools, we searched for differentially expressed genes (DEGs) in an ARDS microarray dataset downloaded from the Gene Expression Omnibus (GEO) database. Afterwards, functional enrichment analysis of GO, KEGG, GSEA and WGCNA were carried out to investigate the potential involvement of these DEGs. Moreover, the Protein–protein interaction (PPI) network was constructed and molecular complexes and hub genes were identified, followed by prognosis analysis of the hub genes. Further, we performed qRT-PCR, Western Blot and flow cytometry analysis to detect candidate genes of CCR2 and FPR3 in macrophage model of LPS-induced ARDS and primary alveolar macrophages(AMs). Macrophage chemotaxis was evaluated using Transwell assay. Results DEGs mainly involved in myeloid leukocyte activation, cell chemotaxis, adenylate cyclase-modulating G protein-coupled receptor signaling pathway and cytokine-cytokine receptor interaction. Basing on the constructed PPI network, we identified five molecular complexes and 10 hub genes potentially participating in the pathogenesis of ARDS. It was observed that candidate genes of CCR2 and FPR3 were significantly over-expressed in primary alveolar macrophages from ARDS patients and macrophgae model of LPS-induced ARDS. Moreover, in vitro transwell assay demonstrated that CCR2 and FPR3 down-regulation, respectively, inhibited LPS-triggered macrophage chemotaxis toward CCL2. Finally, a positive correlation between FPR3 and CCR2 expression was confirmed using pearson correlation analysis and Western Blot assay. Conclusions Our study identified CCR2 and FPR3 as the candidate genes which can promote macrophage chemotaxis through a possible interaction between FPR3 and CCL2/CCR2 axis and provided novel insights into ARDS pathogenesis.

Acute vaping exacerbates microbial pneumonia due to calcium (Ca2+) dysregulation

As electronic cigarette (E-cig) use, also known as “vaping”, has rapidly increased in popularity, data regarding potential pathologic effects are recently emerging. Recent associations between vaping and lung pathology have led to an increased need to scrutinize E-cigs for adverse health impacts. Our previous work (and others) has associated vaping with Ca2+-dependent cytotoxicity in cultured human airway epithelial cells. Herein, we develop a vaped e-liquid pulmonary exposure mouse model to evaluate vaping effects in vivo. Using this model, we demonstrate lung pathology through the use of preclinical measures, that is, the lung wet: dry ratio and lung histology/H&E staining. Further, we demonstrate that acute vaping increases macrophage chemotaxis, which was ascertained using flow cytometry-based techniques, and inflammatory cytokine production, via Luminex analysis, through a Ca2+-dependent mechanism. This increase in macrophage activation appears to exacerbate pulmonary pathology resulting from microbial infection. Importantly, modulating Ca2+ signaling may present a therapeutic direction for treatment against vaping-associated pulmonary inflammation.

miR-223 Inhibits the Polarization and Recruitment of Macrophages via NLRP3/IL-1β Pathway to Meliorate Neuropathic Pain

Background. miRNA is an essential factor in neuropathic pain. However, the underlying mechanism of miRNA in neuropathic pain remains unclear. Objective. To explore the potential role of miR-223 in neuropathic pain in a mice model of chronic sciatic nerve injury. Methods. Mice were divided into the sham group, CCI group, CCI + Lenti-vector group, and CCI + Lenti-miR-223 group. Flow cytometry was used to detect the neuronal apoptosis and the proportion of M1/M2 macrophages in each group. Western blot was used to detect the protein expression levels of ASC, caspase-1, IL-1β, and IL-18 in each group. Luciferase activity assay detects the binding of miR-223 and NLRP3. Macrophage chemotaxis experiments verified the anti-inflammatory effect of miR-223 in vitro. Results. The overexpression of miR-233 significantly reduced the neuropathic pain caused by CCI and reduced the apoptosis and inflammatory factor expression. miR-223 inhibits the expression of NLRP3 by directly binding to the 3′-untranslated region. Overexpression of miR-223 reduces the protein levels of NLRP3, ASC, caspase-1, IL-1β, and IL-18 in the spinal cord of CCI mice, increases the proportion of M2-type macrophages, and reduces the proportion of M1-type macrophages. Conclusion. miR-223 may facilitate the development of neuropathic pain in CCI mice by inhibiting NLRP3-mediated neuroinflammation.

Neuropilin-1 Mediates SARS-CoV-2 Infection in Bone Marrow-derived Macrophages

SARS-CoV-2 infection in human can cause medical complications across various tissues and organs. Despite of the advances to understanding the pathogenesis of SARS-CoV-2, its tissue tropism and interactions with host cells have not been fully understood. Existing clinical data have suggested possible SARS-CoV-2 infection in human skeleton system. In the present study, we found that authentic SARS-CoV-2 could efficiently infect human and mouse bone marrow-derived macrophages (BMMs) and alter the expression of macrophage chemotaxis and osteoclast-related genes. Importantly, in a mouse SARS-CoV-2 infection model that was enabled by the intranasal adenoviral (AdV) delivery of human angiotensin converting enzyme 2 (hACE2), SARS-CoV-2 was found to be present in femoral BMMs as determined by in situ immunofluorescence analysis. Using single-cell RNA sequencing (scRNA-Seq), we characterized SARS-CoV-2 infection in BMMs. Importantly, SARS-CoV-2 entry on BMMs appeared to be dependent on the expression of neuropilin-1 (NRP1) rather than the widely recognized receptor ACE2. It was also noted that unlike brain macrophages which displayed aging-dependent NRP1 expression, BMMs from neonatal and aged mice had constant NRP1 expression, making BMMs constantly vulnerable target cells for SARS-CoV-2. Furthermore, it was found that the abolished SARS-CoV-2 entry in BMM-derived osteoclasts was associated with the loss of NRP1 expression during BMM-to-osteoclast differentiation. Collectively, our study has suggested that NRP1 can mediate SARS-CoV-2 infection in BMMs, which precautions the potential impact of SARS-CoV-2 infection on human skeleton system.

SLIT2-ROBO signaling in tumor-associated microglia/macrophages drives glioblastoma immunosuppression and vascular dysmorphia

SLIT2 is a secreted polypeptide that guides migration of cells expressing ROBO1&2 receptors. Herein, we investigated SLIT2/ROBO signaling effects in gliomas. In patients with glioblastoma (GBM), SLIT2 expression increased with malignant progression and correlated with poor survival and immunosuppression. Knockdown of SLIT2 in mouse glioma cells and patient derived GBM xenografts reduced tumor growth and synergized with immunotherapy to prolong survival. Tumor cell SLIT2 knockdown inhibited macrophage invasion and promoted a cytotoxic gene expression profile, which improved tumor vessel function and enhanced efficacy of chemotherapy and immunotherapy. Mechanistically, SLIT2 promoted microglia/macrophage chemotaxis and tumor-supportive polarization via ROBO1&2-mediated PI3Kγ activation. Macrophage Robo1&2 deletion and systemic SLIT2 trap delivery mimicked SLIT2 knockdown effects on tumor growth and the tumor microenvironment (TME), revealing SLIT2 signaling through macrophage ROBOs as a novel regulator of the GBM microenvironment and a potential immunotherapeutic target for brain tumors.

Macrophage depletion impairs neonatal tendon regeneration

Tendons are dense connective tissues that transmit muscle forces to the skeleton. After adult injury, healing potential is generally poor and dominated by scar formation. Although the immune response is a key feature of healing, the specific immune cells and signals that drive tendon healing have not been fully defined. In particular, the immune regulators underlying tendon regeneration are almost completely undetermined due to a paucity of tendon regeneration models. Using a mouse model of neonatal tendon regeneration, we screened for immune-related markers and identified upregulation of several genes associated with inflammation, macrophage chemotaxis, and TGFβ signaling after injury. Depletion of macrophages using AP20187 treatment of MaFIA mice resulted in impaired functional healing, reduced cell proliferation, reduced ScxGFP+ neo-tendon formation, and altered tendon gene expression. Collectively, these results show that inflammation is a key component of neonatal tendon regeneration and demonstrate a requirement for macrophages in effective functional healing.

How Have Leukocyte In Vitro Chemotaxis Assays Shaped Our Ideas about Macrophage Migration?

Macrophage chemotaxis is crucial during both onset and resolution of inflammation and unique among all leukocytes. Macrophages are able to switch between amoeboid and mesenchymal migration to optimise their migration through 3D environments. This subtle migration phenotype has been underappreciated in the literature, with macrophages often being grouped and discussed together with other leukocytes, possibly due to the limitations of current chemotaxis assays. Transwell assays were originally designed in the 1960s but despite their long-known limitations, they are still one of the most popular methods of studying macrophage migration. This review aims to critically evaluate transwell assays, and other popular chemotaxis assays, comparing their advantages and limitations in macrophage migration studies.

Prokineticin receptors (version 2020.4) in the IUPHAR/BPS Guide to Pharmacology Database

Prokineticin receptors, PKR1 and PKR2 (provisional nomenclature as recommended by NC-IUPHAR [23]) respond to the cysteine-rich 81-86 amino-acid peptides prokineticin-1 (also known as endocrine gland-derived vascular endothelial growth factor, mambakine) and prokineticin-2 (protein Bv8 homologue). An orthologue of PROK1 from black mamba (Dendroaspis polylepis) venom, mamba intestinal toxin 1 (MIT1, [65]) is a potent, non-selective agonist at prokineticin receptors [41], while Bv8, an orthologue of PROK2 from amphibians (Bombina sp., [44]), is equipotent at recombinant PKR1 and PKR2 [48], and has high potency in macrophage chemotaxis assays, which are lost in PKR1-null mice.