Classical monocyte ontogeny dictates their functions and fates as tissue macrophages.

Classical monocytes (CMs) are ephemeral myeloid immune cells that circulate in the blood. Emerging evidence suggests that CMs can have distinct ontogeny and originate from either granulocyte-monocyte- or monocyte-dendritic-cell progenitors (GMPs or MDPs). Here, we report surface markers that allowed segregation of murine GMP- and MDP-derived CMs, i.e., GMP-Mo and MDP-Mo, as well as their functional characterization, including fate definition following adoptive cell transfer. GMP-Mo and MDP-Mo yielded an equal increase in homeostatic CM progeny, such as blood-resident non-classical monocytes and gut macrophages; however, these cells differentially seeded various other selected tissues, including the dura mater and lung. Specifically, GMP-Mo and MDP-Mo differentiated into distinct interstitial lung macrophages, linking CM dichotomy to previously reported pulmonary macrophage heterogeneity. Collectively, we provide evidence for the existence of two functionally distinct CM subsets in the mouse that differentially contribute to peripheral tissue macrophage populations in homeostasis and following challenge.

Early-onset pulmonary and cutaneous vasculitis driven by constitutively active SRC-family kinase HCK.

BACKGROUND: Inborn errors of immunity are genetic disorders characterized by various degrees of immune dysregulation that can manifest as immune deficiency, autoimmunity, or autoinflammation. The routine use of next-generation sequencing in the clinic has facilitated the identification of an ever-increasing number of inborn errors of immunity, revealing the roles of immunologically important genes in human pathologies. However, despite this progress, treatment is still extremely challenging. OBJECTIVE: We sought to report a new monogenic autoinflammatory disorder caused by a de novo activating mutation, p.Tyr515∗, in hematopoietic cell kinase (HCK). The disease is characterized by cutaneous vasculitis and chronic pulmonary inflammation that progresses to fibrosis. METHODS: Whole-exome sequencing, Sanger sequencing, mass spectrometry, and western blotting were performed to identify and characterize the pathogenic HCK mutation. Dysregulation of mutant HCK was confirmed ex vivo in primary cells and in vitro in transduced cell lines. RESULTS: Mutant HCK lacking the C-terminal inhibitory tyrosine Tyr522 exhibited increased kinase activity and enhanced myeloid cell priming, migration and effector functions, such as production of the inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α, and production of reactive oxygen species. These aberrant functions were reflected by inflammatory leukocyte infiltration of the lungs and skin. Moreover, an overview of the clinical course of the disease, including therapies, provides evidence for the therapeutic efficacy of the Janus kinase 1/2 inhibitor ruxolitinib in inflammatory lung disease. CONCLUSIONS: We propose HCK-driven pulmonary and cutaneous vasculitis as a novel autoinflammatory disorder of inborn errors of immunity.

The receptor-type protein tyrosine phosphatase CD45 promotes onset and severity of IL-1ß-mediated autoinflammatory osteomyelitis.

A number of human autoinflammatory diseases manifest with severe inflammatory bone destruction. Mouse models of these diseases represent valuable tools that help us to understand molecular mechanisms triggering this bone autoinflammation. The Pstpip2cmo mouse strain is among the best characterized of these; it harbors a mutation resulting in the loss of adaptor protein PSTPIP2 and development of autoinflammatory osteomyelitis. In Pstpip2cmo mice, overproduction of interleukin-1ß (IL-1ß) and reactive oxygen species by neutrophil granulocytes leads to spontaneous inflammation of the bones and surrounding soft tissues. However, the upstream signaling events leading to this overproduction are poorly characterized. Here, we show that Pstpip2cmo mice deficient in major regulator of Src-family kinases (SFKs) receptor-type protein tyrosine phosphatase CD45 display delayed onset and lower severity of the disease, while the development of autoinflammation is not affected by deficiencies in Toll-like receptor signaling. Our data also show deregulation of pro-IL-1ß production by Pstpip2cmo neutrophils that are attenuated by CD45 deficiency. These data suggest a role for SFKs in autoinflammation. Together with previously published work on the involvement of protein tyrosine kinase spleen tyrosine kinase, they point to the role of receptors containing immunoreceptor tyrosine-based activation motifs, which after phosphorylation by SFKs recruit spleen tyrosine kinase for further signal propagation. We propose that this class of receptors triggers the events resulting in increased pro-IL-1ß synthesis and disease initiation and/or progression.

Dysregulated NADPH Oxidase Promotes Bone Damage in Murine Model of Autoinflammatory Osteomyelitis.

Autoinflammatory diseases are characterized by dysregulation of the innate immune system, leading to spontaneous inflammation. Pstpip2cmo mouse strain is a well-characterized model of this class of disorders. Because of the mutation leading to the lack of adaptor protein PSTPIP2, these animals suffer from autoinflammatory chronic multifocal osteomyelitis similar to several human syndromes. Current evidence suggests that it is driven by hyperproduction of IL-1ß by neutrophil granulocytes. In this study, we show that in addition to IL-1ß, PSTPIP2 also negatively regulates pathways governing reactive oxygen species generation by neutrophil NOX2 NADPH oxidase. Pstpip2cmo neutrophils display highly elevated superoxide production in response to a range of stimuli. Inactivation of NOX2 NADPH oxidase in Pstpip2cmo mice did not affect IL-1ß levels, and the autoinflammatory process was initiated with similar kinetics. However, the bone destruction was almost completely alleviated, suggesting that dysregulated NADPH oxidase activity is a key factor promoting autoinflammatory bone damage in Pstpip2cmo mice.

Synthesis and biological evaluation of cationic TopFluor cholesterol analogues.

Cholesterol is not only a major component of the cell membrane, but also plays an important role in a wide range of biological processes and pathologies. It is therefore crucial to develop appropriate tools for visualizing intracellular cholesterol transport. Here, we describe new cationic analogues of BODIPY-Cholesterol (TopFluor-Cholesterol, TF-Chol), which combine a positive charge on the sterol side chain and a BODIPY group connected via a C-4 linker. In contrast to TF-Chol, the new analogues TF-1 and TF-3 possessing acetyl groups on the A ring (C-3 position on steroid) internalized much faster and displayed slightly different levels of intracellular localization. Their applicability for cholesterol monitoring was indicated by the fact that they strongly label compartments with accumulated cholesterol in cells carrying a mutation of the Niemann-Pick disease-associated cholesterol transporter, NPC1.

Transmembrane adaptor protein WBP1L regulates CXCR4 signalling and murine haematopoiesis.

WW domain binding protein 1-like (WBP1L), also known as outcome predictor of acute leukaemia 1 (OPAL1), is a transmembrane adaptor protein, expression of which correlates with ETV6-RUNX1 (t(12;21)(p13;q22)) translocation and favourable prognosis in childhood leukaemia. It has a broad expression pattern in haematopoietic and in non-haematopoietic cells. However, its physiological function has been unknown. Here, we show that WBP1L negatively regulates signalling through a critical chemokine receptor CXCR4 in multiple leucocyte subsets and cell lines. We also show that WBP1L interacts with NEDD4-family ubiquitin ligases and regulates CXCR4 ubiquitination and expression. Moreover, analysis of Wbp1l-deficient mice revealed alterations in B cell development and enhanced efficiency of bone marrow cell transplantation. Collectively, our data show that WBP1L is a novel regulator of CXCR4 signalling and haematopoiesis.

Coumarin Tröger's base derivatives with cyanine substitution as selective and sensitive fluorescent lysosomal probes.

The fluorescent probes based on Tröger's base motive with both coumarin and cyanine substitution 11-13 have been synthesized by multi-step synthesis in high overall yields. Intracellular localization of prepared probes have been tested using four different cell lines (HF-P4, BLM, U-2 OS and A-2058). Prepared probes have intensive green and red fluorescence. Co-localization with commercial lysosome specific marker LysoTracker Blue DND 22 has been confirmed that all prepared fluorescent probes labeled lysosomal compartment with high selectivity and probes show excellent brightness at low concentration.

Pentamethinium salts as ligands for cancer: Sulfated polysaccharide co-receptors as possible therapeutic target.

A series of pentamethinium salts with benzothiazolium and indolium side units comprising one or two positive charges were designed and synthesized to determine the relationships among the molecular structure, charge density, affinity to sulfated polysaccharides, and biological activity. Firstly, it was found that the affinity of the pentamethinium salts to sulfated polysaccharides correlated with their biological activity. Secondly, the side heteroaromates displayed a strong effect on the cytotoxicity and selectivity towards cancer cells. Finally, doubly charged pentamethinium salts possessing benzothiazolium side units exhibited remarkably high efficacy against a taxol-resistant cancer cell line.

A Cyclic Pentamethinium Salt Induces Cancer Cell Cytotoxicity through Mitochondrial Disintegration and Metabolic Collapse.

Cancer cells preferentially utilize glycolysis for ATP production even in aerobic conditions (the Warburg effect) and adapt mitochondrial processes to their specific needs. Recent studies indicate that altered mitochondrial activities in cancer represent an actionable target for therapy. We previously showed that salt 1-3C, a quinoxaline unit (with cytotoxic activity) incorporated into a meso-substituted pentamethinium salt (with mitochondrial selectivity and fluorescence properties), displayed potent cytotoxic effects in vitro and in vivo, without significant toxic effects to normal tissues. Here, we investigated the cytotoxic mechanism of salt 1-3C compared to its analogue, salt 1-8C, with an extended side carbon chain. Live cell imaging demonstrated that salt 1-3C, but not 1-8C, is rapidly incorporated into mitochondria, correlating with increased cytotoxicity of salt 1-3C. The accumulation in mitochondria led to their fragmentation and loss of function, accompanied by increased autophagy/mitophagy. Salt 1-3C preferentially activated AMP-activated kinase and inhibited mammalian target of rapamycin (mTOR) signaling pathways, sensors of cellular metabolism, but did not induce apoptosis. These data indicate that salt 1-3C cytotoxicity involves mitochondrial perturbation and disintegration, and such compounds are promising candidates for targeting mitochondria as a weak spot of cancer.

The Transmembrane Adaptor Protein SCIMP Facilitates Sustained Dectin-1 Signaling in Dendritic Cells.

Transmembrane adaptor proteins are molecules specialized in recruiting cytoplasmic proteins to the proximity of the cell membrane as part of the signal transduction process. A member of this family, SLP65/SLP76, Csk-interacting membrane protein (SCIMP), recruits a complex of SLP65/SLP76 and Grb2 adaptor proteins, known to be involved in the activation of PLCγ1/2, Ras, and other pathways. SCIMP expression is restricted to antigen-presenting cells. In a previous cell line-based study, it was shown that, in B cells, SCIMP contributes to the reverse signaling in the immunological synapse, downstream of MHCII glycoproteins. There it mainly facilitates the activation of ERK MAP kinases. However, its importance for MHCII glycoprotein-dependent ERK signaling in primary B cells has not been analyzed. Moreover, its role in macrophages and dendritic cells has remained largely unknown. Here we present the results of our analysis of SCIMP-deficient mice. In these mice, we did not observe any defects in B cell signaling and B cell-dependent responses. On the other hand, we found that, in dendritic cells and macrophages, SCIMP expression is up-regulated after exposure to GM-CSF or the Dectin-1 agonist zymosan. Moreover, we found that SCIMP is strongly phosphorylated after Dectin-1 stimulation and that it participates in signal transduction downstream of this important pattern recognition receptor. Our analysis of SCIMP-deficient dendritic cells revealed that SCIMP specifically contributes to sustaining long-term MAP kinase signaling and cytokine production downstream of Dectin-1 because of an increased expression and sustained phosphorylation lasting at least 24 h after signal initiation.

Optical probes and sensors as perspective tools in epigenetics.

Modifications of DNA cytosine bases and histone posttranslational modifications play key roles in the control of gene expression and specification of cell states. Such modifications affect many important biological processes and changes to these important regulation mechanisms can initiate or significantly contribute to the development of many serious pathological states. Therefore, recognition and determination of chromatin modifications is an important goal in basic and clinical research. Two of the most promising tools for this purpose are optical probes and sensors, especially colourimetric and fluorescence devices. The use of optical probes and sensors is simple, without highly expensive instrumentation, and with excellent sensitivity and specificity for target structural motifs. Accordingly, the application of various probes and sensors in the recognition and determination of cytosine modifications and structure of histones and histone posttranslational modifications, are discussed in detail in this review.

PSTPIP2, a Protein Associated with Autoinflammatory Disease, Interacts with Inhibitory Enzymes SHIP1 and Csk.

Mutations in the adaptor protein PSTPIP2 are the cause of the autoinflammatory disease chronic multifocal osteomyelitis in mice. This disease closely resembles the human disorder chronic recurrent multifocal osteomyelitis, characterized by sterile inflammation of the bones and often associated with inflammation in other organs, such as the skin. The most critical process in the disease's development is the enhanced production of IL-1ß. This excessive IL-1ß is likely produced by neutrophils. In addition, the increased activity of macrophages, osteoclasts, and megakaryocytes has also been described. However, the molecular mechanism of how PSTPIP2 deficiency results in this phenotype is poorly understood. Part of the PSTPIP2 inhibitory function is mediated by protein tyrosine phosphatases from the proline-, glutamic acid-, serine- and threonine-rich (PEST) family, which are known to interact with the central part of this protein, but other regions of PSTPIP2 not required for PEST-family phosphatase binding were also shown to be indispensable for PSTPIP2 function. In this article, we show that PSTPIP2 binds the inhibitory enzymes Csk and SHIP1. The interaction with SHIP1 is of particular importance because it binds to the critical tyrosine residues at the C terminus of PSTPIP2, which is known to be crucial for its PEST-phosphatase-independent inhibitory effects in different cellular systems. We demonstrate that in neutrophils this region is important for the PSTPIP2-mediated suppression of IL-1ß processing and that SHIP1 inhibition results in the enhancement of this processing. We also describe deregulated neutrophil response to multiple activators, including silica, Ab aggregates, and LPS, which is suggestive of a rather generalized hypersensitivity of these cells to various external stimulants.

Synthesis and biological activity evaluation of hydrazone derivatives based on a Tröger's base skeleton.

We report the design and synthesis of novel anticancer agents based on bis-hydrazones separated by a rigid Tröger's base skeleton. This novel approach combines a biologically active moiety (hydrazone) with this scaffold (Tröger's base) to construct DNA intercalators. Evaluation of the anticancer activity of these agents using seven cancer cell lines and two healthy cell lines found that several derivatives had potent anticancer activity and excellent selectivity indexes toward cancer cells. The antimicrobial activities were tested on a set of thirteen bacterial stains, but the prepared compounds were not active. Complexation studies using biologically important metal ions demonstrated that these compounds are able to bind Cu(2+), Fe(3+), Co(2+), Ni(2+) and Zn(2+). DNA intercalation studies showed that the compounds themselves do not interact with DNA, but their metallocomplexes do interact, most likely via intercalation into DNA.

Caffeine-hydrazones as anticancer agents with pronounced selectivity toward T-lymphoblastic leukaemia cells.

We report design and synthesis of set of novel anticancer agents based on caffeine-hydrazones bearing 2-hydroxyaryl- or 2-N-heteroaryl moiety. Anticancer activity evaluation using seven cancer cell lines and two non-malignant cell lines demonstrated that several derivatives display significant anticancer activity and great selectivity index toward T-lymphoblastic leukaemia cells. In general, hydrazones bearing 2-N-heteroaryl moiety are more active and selective than those with 2-hydroxyaryl moiety. Tested compounds exhibit dose-dependent inhibition of both RNA and DNA synthesis, with some exceptions. Antimicrobial activities were tested on set of twelve bacterial and yeast strains, however prepared compounds were not active, suggesting for a molecular target specific for eukaryotic cells.

Draft genome of Kazachstania heterogenica var. weizmannii, a commensal fungus of the mouse digestive tract.

Kazachstania heterogenica is a member of the K. telluris complex, where all members to date are reported to be pathogenic fungi. We have isolated a strain, K. heterogenica var. weizmannii, from the gut of mice that seems to be a commensal strain and sequenced its genome.

Competitive fungal commensalism mitigates candidiasis pathology.

The mycobiota are a critical part of the gut microbiome, but host-fungal interactions and specific functional contributions of commensal fungi to host fitness remain incompletely understood. Here, we report the identification of a new fungal commensal, Kazachstania heterogenica var. weizmannii, isolated from murine intestines. K. weizmannii exposure prevented Candida albicans colonization and significantly reduced the commensal C. albicans burden in colonized animals. Following immunosuppression of C. albicans colonized mice, competitive fungal commensalism thereby mitigated fatal candidiasis. Metagenome analysis revealed K. heterogenica or K. weizmannii presence among human commensals. Our results reveal competitive fungal commensalism within the intestinal microbiota, independent of bacteria and immune responses, that could bear potential therapeutic value for the management of C. albicans-mediated diseases.

Rational design of chemical ligands for selective mitochondrial targeting.

The rational design of molecules with selective intracellular targeting is a great challenge for contemporary chemistry and life sciences. Here, we demonstrate a rational approach to development of compartment-specific fluorescent dyes from the γ-aryl substituted pentamethine family. These novel dyes exhibit an extraordinary affinity and selectivity for cardiolipin in inner mitochondrial membrane and possess excellent photostability, fluorescent properties, and low phototoxicity. Selective imaging of live and fixed mitochondria was achieved in various cell lines using nanomolar concentrations of these dyes. Their high localization specificity and low toxicity enables study of morphological changes, structural complexity, and dynamics of mitochondria playing a pivotal role in many pathological diseases. These far-red emitting dyes could also serve in a variety of biomedical applications.

Combination of two chromophores: synthesis and PDT application of porphyrin-pentamethinium conjugate.

A general method for the synthesis of a novel porphyrin with pentamethine periphery substitution is described. The combination of two chromophoric systems, a porphyrin macrocycle and a polymethine moiety was achieved by transformation of tetrapyridyl porphyrin. The synthetic strategy included conversion of the tetrapyridyl porphyrin to its corresponding 2,4-dinitrophenylpyridinuim salt, which was subsequently converted to tetrakis(meso-pentamethinium salt) on the porphyrin core. This novel porphyrin exhibited PDT properties as manifested by the induction of apoptosis in the myeloid cell line HL-60 and the effective reduction of amelanotic melanoma in nude mice.

Sterolight as imaging tool to study sterol uptake, trafficking and efflux in living cells.

Information about cholesterol subcellular localization and transport pathways inside cells is essential for understanding and treatment of cholesterol-related diseases. However, there is a lack of reliable tools to monitor it. This work follows the fate of Sterolight, a BODIPY-labelled sterol, within the cell and demonstrates it as a suitable probe for visualization of sterol/lipid trafficking. Sterolight enters cells through an energy-independent process and knockdown experiments suggest caveolin-1 as its potential cellular carrier. Intracellular transport of Sterolight is a rapid process, and transfer from ER and mitochondria to lysosomes and later to lipid droplets requires the participation of active microtubules, as it can be inhibited by the microtubule disruptor nocodazole. Excess of the probe is actively exported from cells, in addition to being stored in lipid droplets, to re-establish the sterol balance. Efflux occurs through a mechanism requiring energy and may be selectively poisoned with verapamil or blocked in cells with mutated cholesterol transporter NPC1. Sterolight is efficiently transferred within and between different cell populations, making it suitable for monitoring numerous aspects of sterol biology, including the live tracking and visualization of intracellular and intercellular transport.

Molecular interactions of adaptor protein PSTPIP2 control neutrophil-mediated responses leading to autoinflammation.

Introduction: Autoinflammatory diseases are characterized by dysregulation of innate immune system leading to spontaneous sterile inflammation. One of the well-established animal models of this group of disorders is the mouse strain Pstpip2cmo . In this strain, the loss of adaptor protein PSTPIP2 leads to the autoinflammatory disease chronic multifocal osteomyelitis. It is manifested by sterile inflammation of the bones and surrounding soft tissues of the hind limbs and tail. The disease development is propelled by elevated production of IL-1ß and reactive oxygen species by neutrophil granulocytes. However, the molecular mechanisms linking PSTPIP2 and these pathways have not been established. Candidate proteins potentially involved in these mechanisms include PSTPIP2 binding partners, PEST family phosphatases (PEST-PTPs) and phosphoinositide phosphatase SHIP1. Methods: To address the role of these proteins in PSTPIP2-mediated control of inflammation, we have generated mouse strains in which PEST-PTP or SHIP1 binding sites in PSTPIP2 have been disrupted. In these mouse strains, we followed disease symptoms and various inflammation markers. Results: Our data show that mutation of the PEST-PTP binding site causes symptomatic disease, whereas mice lacking the SHIP1 interaction site remain asymptomatic. Importantly, both binding partners of PSTPIP2 contribute equally to the control of IL-1ß production, while PEST-PTPs have a dominant role in the regulation of reactive oxygen species. In addition, the interaction of PEST-PTPs with PSTPIP2 regulates the production of the chemokine CXCL2 by neutrophils. Its secretion likely creates a positive feedback loop that drives neutrophil recruitment to the affected tissues. Conclusions: We demonstrate that PSTPIP2-bound PEST-PTPs and SHIP1 together control the IL-1ß pathway. In addition, PEST-PTPs have unique roles in the control of reactive oxygen species and chemokine production, which in the absence of PEST-PTP binding to PSTPIP2 shift the balance towards symptomatic disease.