Structural Basis of β2 Integrin Inside—Out Activation

β2 integrins are expressed on all leukocytes. Precise regulation of the β2 integrin is critical for leukocyte adhesion and trafficking. In neutrophils, β2 integrins participate in slow rolling. When activated by inside–out signaling, fully activated β2 integrins mediate rapid leukocyte arrest and adhesion. The two activation pathways, starting with selectin ligand engagement and chemokine receptor ligation, respectively, converge on phosphoinositide 3-kinase, talin-1, kindlin-3 and Rap1. Here, we focus on recent structural insights into autoinhibited talin-1 and autoinhibited trimeric kindlin-3. When activated, both talin-1 and kindlin-3 can bind the β2 cytoplasmic tail at separate but adjacent sites. We discuss possible pathways for talin-1 and kindlin-3 activation, recruitment to the plasma membrane, and their role in integrin activation. We propose new models of the final steps of integrin activation involving the complex of talin-1, kindlin-3, integrin and the plasma membrane

Hsp70 and nf-kb mediated control of innate inflammatory responses in a canine macrophage cell line

The pathogenesis of many inflammatory diseases is associated with the uncontrolled activation of nuclear factor kappa B (NF-κB) in macrophages. Previous studies have shown that in various cell types, heat shock protein 70 (Hsp70) plays a crucial role in controlling NF-κB activity. So far, little is known about the role of Hsp70 in canine inflammatory processes. In this study we investigated the potential anti-inflammatory effects of Hsp70 in canine macrophages as well as the mechanisms underlying these effects. To this end, a canine macrophage cell line was stressed with arsenite, a chemical stressor, which upregulated Hsp70 expression as detected by flow cytometry and qPCR. A gene-edited version of this macrophage cell line lacking inducible Hsp70 was generated using CRISPR-Cas9 technology. To determine the effects of Hsp70 on macrophage inflammatory properties, arsenite-stressed wild-type and Hsp70 knockout macrophages were exposed to lipopolysaccharide (LPS), and the expression of the inflammatory cytokines IL-6, IL-1β and tumor necrosis factor-α (TNF-α) and levels of phosphorylated NF-κB were determined by qPCR and Western Blotting, respectively. Our results show that non-toxic concentrations of arsenite induced Hsp70 expression in canine macrophages; Hsp70 upregulation significantly inhibited the LPS-induced expression of the pro-inflammatory mediators TNF-α and IL-6, as well as NF-κB activation in canine macrophages. Furthermore, the gene editing of inducible Hsp70 by CRISPR-Cas9-mediated gene editing neutralized this inhibitory effect of cell stress on NF-κB activation and pro-inflammatory cytokine expression. Collectively, our study reveals that Hsp70 may regulate inflammatory responses through NF-κB activation and cytokine expression in canine macrophages.http://www.mdpi.com/journal/ijmspm2021Veterinary Tropical Disease

Characterization of polarization states of canine monocyte derived macrophages

DATA AVAILABILITY STATEMENT : All relevant data are within the manuscript and its Supporting Information files.SUPPORTING INFPRMATION : TABLE S1. Gene counts of M0 monocytes and M1, M2 macrophages and CD14+ and CD14D cells. TABLE S2. Differentially expressed genes of M0 monocytes, M1, M2 macrophages and CD14+ and CD14D cells. TABLE S3. Top 50 DEG pairwise comparisons M0, M1, M2 and CD14+ cells. FIGURE S1. Identification of non-viable cells by back gating. FIGURE S2. Selection of Immunity-related DEG’s. VIDE0 S1. A. Bead uptake M0 monocytes visualized by confocal microscopy. B. Bead uptake M1 MDMs visualized by confocal microscopy. C. Bead uptake M2 MDMs visualized by confocal microscopy.Macrophages can reversibly polarize into multiple functional subsets depending on their micro-environment. Identification and understanding the functionality of these subsets is relevant for the study of immune-related diseases. However, knowledge about canine macrophage polarization is still in its infancy. In this study, we polarized canine monocytes using GM-CSF/IFN- γ and LPS towards M1 macrophages or M-CSF and IL-4 towards M2 macrophages and compared them to undifferentiated monocytes (M0). Polarized M1 and M2 macrophages were thoroughly characterized for morphology, surface marker features, gene profiles and functional properties. Our results showed that canine M1-polarized macrophages obtained a characteristic large, roundish, or amoeboid shape, while M2-polarized macrophages were smaller and adopted an elongated spindle-like morphology. Phenotypically, all macrophage subsets expressed the pan-macrophage markers CD14 and CD11b. M1-polarized macrophages expressed increased levels of CD40, CD80 CD86 and MHC II, while a significant increase in the expression levels of CD206, CD209, and CD163 was observed in M2-polarized macrophages. RNAseq of the three macrophage subsets showed distinct gene expression profiles, which are closely associated with immune responsiveness, cell differentiation and phagocytosis. However, the complexity of the gene expression patterns makes it difficult to assign clear new polarization markers. Functionally, undifferentiated -monocytes, and M1- and M2- like subsets of canine macrophages can all phagocytose latex beads. M2-polarized macrophages exhibited the strongest phagocytic capacity compared to undifferentiated monocytes- and M1-polarized cells. Taken together, this study showed that canine M1 and M2-like macrophages have distinct features largely in parallel to those of well-studied species, such as human, mouse and pig. These findings enable future use of monocyte derived polarized macrophages particularly in studies of immune related diseases in dogs.The China Scholarship Council (CSC).https://journals.plos.org/plosone/am2024Veterinary Tropical DiseasesSDG-03:Good heatlh and well-bein

Characterization of polarization states of canine monocyte derived macrophages

Macrophages can reversibly polarize into multiple functional subsets depending on their micro-environment. Identification and understanding the functionality of these subsets is relevant for the study of immune‑related diseases. However, knowledge about canine macrophage polarization is still in its infancy. In this study, we polarized canine monocytes using GM-CSF/IFN- γ and LPS towards M1 macrophages or M-CSF and IL-4 towards M2 macrophages and compared them to undifferentiated monocytes (M0). Polarized M1 and M2 macrophages were thoroughly characterized for morphology, surface marker features, gene profiles and functional properties. Our results showed that canine M1-polarized macrophages obtained a characteristic large, roundish, or amoeboid shape, while M2-polarized macrophages were smaller and adopted an elongated spindle-like morphology. Phenotypically, all macrophage subsets expressed the pan-macrophage markers CD14 and CD11b. M1-polarized macrophages expressed increased levels of CD40, CD80 CD86 and MHC II, while a significant increase in the expression levels of CD206, CD209, and CD163 was observed in M2-polarized macrophages. RNAseq of the three macrophage subsets showed distinct gene expression profiles, which are closely associated with immune responsiveness, cell differentiation and phagocytosis. However, the complexity of the gene expression patterns makes it difficult to assign clear new polarization markers. Functionally, undifferentiated -monocytes, and M1- and M2- like subsets of canine macrophages can all phagocytose latex beads. M2-polarized macrophages exhibited the strongest phagocytic capacity compared to undifferentiated monocytes- and M1-polarized cells. Taken together, this study showed that canine M1 and M2-like macrophages have distinct features largely in parallel to those of well-studied species, such as human, mouse and pig. These findings enable future use of monocyte derived polarized macrophages particularly in studies of immune related diseases in dogs

Regulation of inflammation in canine species: a role for macrophages and Hsp70

Summary This thesis can be roughly divided into two parts. In the first three chapters, we discussed and studied the role of Hsp70 in retinal pigment epithelial (RPE) cells, and the innate and adaptive immune response. We reviewed the relationship of T cell-mediated diseases and HSPs, and pointed out that the induction of Hsp70 may contribute to therapeutic tolerance (chapter 2). Then, we explored a new Hsp70 co-inducer, leucinostatin, and its role in canine RPE cells (chapter 3). Further, we studied the anti-inflammatory effects of Hsp70 in canine macrophages (chapter 4). In the last two chapters, we focus on various differently activated canine macrophage subsets originating from both primary monocytes and a monocyte-like cell line (030D cell) (chapter 5 and 6). We successfully polarized canine monocyte-derived macrophages (MDMs) into M1 and M2 cells and thoroughly characterized their features (chapter 5). Meanwhile, we demonstrated that 030D cells can be differentiated into M1 and M2 macrophages and that each subset shares the characteristics of the corresponding canine monocyte-derived macrophage subset

Regulation of inflammation in canine species: a role for macrophages and Hsp70

Summary This thesis can be roughly divided into two parts. In the first three chapters, we discussed and studied the role of Hsp70 in retinal pigment epithelial (RPE) cells, and the innate and adaptive immune response. We reviewed the relationship of T cell-mediated diseases and HSPs, and pointed out that the induction of Hsp70 may contribute to therapeutic tolerance (chapter 2). Then, we explored a new Hsp70 co-inducer, leucinostatin, and its role in canine RPE cells (chapter 3). Further, we studied the anti-inflammatory effects of Hsp70 in canine macrophages (chapter 4). In the last two chapters, we focus on various differently activated canine macrophage subsets originating from both primary monocytes and a monocyte-like cell line (030D cell) (chapter 5 and 6). We successfully polarized canine monocyte-derived macrophages (MDMs) into M1 and M2 cells and thoroughly characterized their features (chapter 5). Meanwhile, we demonstrated that 030D cells can be differentiated into M1 and M2 macrophages and that each subset shares the characteristics of the corresponding canine monocyte-derived macrophage subset

Hsp70 and NF-kB Mediated Control of Innate Inflammatory Responses in a Canine Macrophage Cell Line

The pathogenesis of many inflammatory diseases is associated with the uncontrolled activation of nuclear factor kappa B (NF-κB) in macrophages. Previous studies have shown that in various cell types, heat shock protein 70 (Hsp70) plays a crucial role in controlling NF-κB activity. So far, little is known about the role of Hsp70 in canine inflammatory processes. In this study we investigated the potential anti-inflammatory effects of Hsp70 in canine macrophages as well as the mechanisms underlying these effects. To this end, a canine macrophage cell line was stressed with arsenite, a chemical stressor, which upregulated Hsp70 expression as detected by flow cytometry and qPCR. A gene-edited version of this macrophage cell line lacking inducible Hsp70 was generated using CRISPR-Cas9 technology. To determine the effects of Hsp70 on macrophage inflammatory properties, arsenite-stressed wild-type and Hsp70 knockout macrophages were exposed to lipopolysaccharide (LPS), and the expression of the inflammatory cytokines IL-6, IL-1β and tumor necrosis factor-α (TNF-α) and levels of phosphorylated NF-κB were determined by qPCR and Western Blotting, respectively. Our results show that non-toxic concentrations of arsenite induced Hsp70 expression in canine macrophages; Hsp70 upregulation significantly inhibited the LPS-induced expression of the pro-inflammatory mediators TNF-α and IL-6, as well as NF-κB activation in canine macrophages. Furthermore, the gene editing of inducible Hsp70 by CRISPR-Cas9-mediated gene editing neutralized this inhibitory effect of cell stress on NF-κB activation and pro-inflammatory cytokine expression. Collectively, our study reveals that Hsp70 may regulate inflammatory responses through NF-κB activation and cytokine expression in canine macrophages

T Cell-Mediated Chronic Inflammatory Diseases Are Candidates for Therapeutic Tolerance Induction with Heat Shock Proteins

Failing immunological tolerance for critical self-antigens is the problem underlying most chronic inflammatory diseases of humans. Despite the success of novel immunosuppressive biological drugs, the so-called biologics, in the treatment of diseases such rheumatoid arthritis (RA) and type 1 diabetes, none of these approaches does lead to a permanent state of medicine free disease remission. Therefore, there is a need for therapies that restore physiological mechanisms of self-tolerance. Heat shock proteins (HSPs) have shown disease suppressive activities in many models of experimental autoimmune diseases through the induction of regulatory T cells (Tregs). Also in first clinical trials with HSP-based peptides in RA and diabetes, the induction of Tregs was noted. Due to their exceptionally high degree of evolutionary conservation, HSP protein sequences (peptides) are shared between the microbiota-associated bacterial species and the self-HSP in the tissues. Therefore, Treg mechanisms, such as those induced and maintained by gut mucosal tolerance for the microbiota, can play a role by targeting the more conserved HSP peptide sequences in the inflamed tissues. In addition, the stress upregulated presence of HSP in these tissues may well assist the targeting of the HSP induced Treg specifically to the sites of inflammation

Hsp70 and NF-kB Mediated Control of Innate Inflammatory Responses in a Canine Macrophage Cell Line

The pathogenesis of many inflammatory diseases is associated with the uncontrolled activation of nuclear factor kappa B (NF-κB) in macrophages. Previous studies have shown that in various cell types, heat shock protein 70 (Hsp70) plays a crucial role in controlling NF-κB activity. So far, little is known about the role of Hsp70 in canine inflammatory processes. In this study we investigated the potential anti-inflammatory effects of Hsp70 in canine macrophages as well as the mechanisms underlying these effects. To this end, a canine macrophage cell line was stressed with arsenite, a chemical stressor, which upregulated Hsp70 expression as detected by flow cytometry and qPCR. A gene-edited version of this macrophage cell line lacking inducible Hsp70 was generated using CRISPR-Cas9 technology. To determine the effects of Hsp70 on macrophage inflammatory properties, arsenite-stressed wild-type and Hsp70 knockout macrophages were exposed to lipopolysaccharide (LPS), and the expression of the inflammatory cytokines IL-6, IL-1β and tumor necrosis factor-α (TNF-α) and levels of phosphorylated NF-κB were determined by qPCR and Western Blotting, respectively. Our results show that non-toxic concentrations of arsenite induced Hsp70 expression in canine macrophages; Hsp70 upregulation significantly inhibited the LPS-induced expression of the pro-inflammatory mediators TNF-α and IL-6, as well as NF-κB activation in canine macrophages. Furthermore, the gene editing of inducible Hsp70 by CRISPR-Cas9-mediated gene editing neutralized this inhibitory effect of cell stress on NF-κB activation and pro-inflammatory cytokine expression. Collectively, our study reveals that Hsp70 may regulate inflammatory responses through NF-κB activation and cytokine expression in canine macrophages

Leucinostatin acts as a co-inducer for heat shock protein 70 in cultured canine retinal pigment epithelial cells

Dysregulation of retinal pigment epithelium (RPE) cells is the main cause of a variety of ocular diseases. Potentially heat shock proteins, by preventing molecular and cellular damage and modulating inflammatory disease, may exert a protective role in eye disease. In particular, the inducible form of heat shock protein 70 (Hsp70) is widely upregulated in inflamed tissues, and in vivo upregulation of Hsp70 expression by HSP co-inducing compounds has been shown to be a potential therapeutic strategy for inflammatory diseases. In order to gain further understanding of the potential protective effects of Hsp70 in RPE cells, we developed a method for isolation and culture of canine RPE cells. Identity of RPE cells was confirmed by detection of its specific marker, RPE65, in qPCR, flow cytometry, and immunocytochemistry analysis. The ability of RPE cells to express Hsp70 upon experimental induction of cell stress, by arsenite, was analyzed by flow cytometry. Finally, in search of a potential Hsp70 coinducer, we investigated whether the compound leucinostatin could enhance Hsp70 expression in stressed RPE cells. Canine RPE cells were isolated and cultured successfully. Purity of cells that strongly expressed RPE65 was over 90%. Arsenite-induced stress led to a time- and dose-dependent increase in Hsp70 expression in canine RPE cells in vitro. In addition, leucinostatin, which enhanced heat shock factor-1-induced transcription from the heat shock promoter in DNAJB1-luc-O23 reporter cell line, also enhanced Hsp70 expression in arsenite-stressed RPE cells, in a dose-dependent fashion. These findings demonstrate that leucinostatin can boost Hsp70 expression in canine RPE cells, most likely by activating heat shock factor-1, suggesting that leucinostatin might be applied as a new co-inducer for Hsp70 expression.Qingkang Lyu was supported by a fellowship of the China Scholarship Council (CSC).http://link.springer.com/journal/12192am2020Veterinary Tropical Disease