Are Artificial Intelligence-Assisted Three-Dimensional Histological Reconstructions Reliable for the Assessment of Trabecular Microarchitecture?

Objectives: This study aimed to create a three-dimensional histological reconstruction through the AI-assisted classification of tissues and the alignment of serial sections. The secondary aim was to evaluate if the novel technique for histological reconstruction accurately replicated the trabecular microarchitecture of bone. This was performed by conducting micromorphometric measurements on the reconstruction and comparing the results obtained with those of microCT reconstructions. Methods: A bone biopsy sample was harvested upon re-entry following sinus floor augmentation. Following microCT scanning and histological processing, a modified version of the U-Net architecture was trained to categorize tissues on the sections. Detector-free local feature matching with transformers was used to create the histological reconstruction. The micromorphometric parameters were calculated using Bruker's CTAn software (version 1.18.8.0, Bruker, Kontich, Belgium) for both histological and microCT datasets. Results: Correlation coefficients calculated between the micromorphometric parameters measured on the microCT and histological reconstruction suggest a strong linear relationship between the two with p-values of 0.777, 0.717, 0.705, 0.666, and 0.687 for BV/TV, BS/TV, Tb.Pf Tb.Th, and Tb.Sp, respectively. Bland-Altman and mountain plots suggest good agreement between BV/TV measurements on the two reconstruction methods. Conclusions: This novel method for three-dimensional histological reconstruction provides researchers with a tool that enables the assessment of accurate trabecular microarchitecture and histological information simultaneously.

The Syk Inhibitor Entospletinib Abolishes Dermal-Epidermal Separation in a Fully Human Ex Vivo Model of Bullous Pemphigoid.

Bullous pemphigoid (BP) is an autoantibody-mediated blistering skin disease characterized by local inflammation and dermal-epidermal separation, with no approved targeted therapy. The Syk tyrosine kinase is critical for various functions of the immune response. Second-generation Syk inhibitors such as entospletinib are currently being tested for hematological malignancies. Our aim was to test the effect of entospletinib in a fully human model system of BP. Incubating BP serum-treated human frozen skin sections with normal human granulocytes and fresh plasma triggered dermal-epidermal separation that was dependent on complement, NADPH oxidase, and protease activity. Entospletinib dramatically reduced dermal-epidermal separation with a half-maximal inhibitory concentration of ≈16 nM. Entospletinib also reduced ROS production, granule release, and spreading of human granulocytes plated on immobilized immune complexes consisting either of a generic antigen-antibody pair or of recombinant collagen type XVII (BPAg2) and BP serum components (supposedly autoantibodies). However, entospletinib did not affect the chemotactic migration of human granulocytes or their responses to nonphysiological stimulation by phorbol esters. Entospletinib had no effect on the survival of granulocytes either. Taken together, entospletinib abrogates dermal-epidermal separation, likely through inhibition of granulocyte responsiveness to deposited immune complexes. Entospletinib or other Syk inhibitors may provide therapeutic benefits in BP.

The selective inhibition of the Syk tyrosine kinase ameliorates experimental autoimmune arthritis.

Autoimmune arthritis - such as rheumatoid arthritis - affect a significant proportion of the population, which can cause everyday joint pain, decreased mobility and reduced quality of life. Despite having more and more therapeutic options available, there are still a lot of patients who cannot reach remission or low disease activity by current therapies. This causes an urgent need for the development of new treatment options. The Syk tyrosine kinase plays an essential role in B cell receptor, Fc receptor and integrin signaling. It has been shown that the hematopoietic cell-specific deletion of Syk resulted in a complete protection against autoantibody-induced experimental arthritis. This prompted us to test the effect of entospletinib, a second generation, Syk-selective inhibitor, which has a tolerable safety profile according to hematological clinical trials, in experimental autoimmune arthritis. We found that entospletinib dose-dependently decreased the macroscopic signs of joint inflammation, while it did not affect the health status of the animals. In line with these findings, local neutrophil accumulation and cytokine levels were reduced compared to the vehicle-treated group, while macrophage accumulation and synovial fibroblast numbers were not significantly altered. Meanwhile, entospletinib dose-dependently decreased the cell responses of immune complex- or integrin ligand-activated neutrophils. Overall, we found that selective Syk inhibition by entospletinib reduced the activity of autoantibody-induced experimental arthritis, which seems to be based mainly on the effect of the inhibitor on neutrophil functions. Our data raise the possibility that entospletinib could be a good drug candidate in the treatment of human autoimmune arthritis.

Analysis of intracellular tyrosine phosphorylation in circulating neutrophils as a rapid assay for the in vivo effect of oral tyrosine kinase inhibitors.

Tyrosine kinases are crucial signaling components of diverse biological processes and are major therapeutic targets in various malignancies and immune-mediated disorders. A critical step of development of novel tyrosine kinase inhibitors is the transition from the confirmation of the in vitro effects of drug candidates to the analysis of their in vivo efficacy. To facilitate this transition, we have developed a rapid in vivo assay for the analysis of the effect of oral tyrosine kinase inhibitors on basal tyrosine phosphorylation of circulating mouse neutrophils. The assay uses a single drop of peripheral blood without sacrificing the mice. Flow cytometry using intracellular staining by fluorescently labeled anti-phosphotyrosine antibodies revealed robust basal tyrosine phosphorylation in resting circulating neutrophils. This signal was abrogated by the use of isotype control antibodies or by pre-saturation of the anti-phosphotyrosine antibodies with soluble phosphotyrosine amino acids or tyrosine-phosphorylated peptides. Basal tyrosine phosphorylation was dramatically reduced in neutrophils of triple knockout mice lacking the Src-family tyrosine kinases Hck, Fgr, and Lyn. Neutrophil tyrosine phosphorylation was also abrogated by oral administration of the Abl/Src-family inhibitor dasatinib, a clinically used anti-leukemic agent. Detailed dose-response and kinetic studies revealed half-maximal reduction of neutrophil tyrosine phosphorylation by 2.9 mg/kg dasatinib, with maximal reduction observed 2 h after inhibitor administration. Taken together, our assay allows highly efficient analysis of the in vivo effect of orally administered tyrosine kinase inhibitors, and may be used as a suitable alternative to other existing approaches.

Myeloid Src-family kinases are critical for neutrophil-mediated autoinflammation in gout and motheaten models.

Autoinflammatory diseases include a number of monogenic systemic inflammatory diseases, as well as acquired autoinflammatory diseases such as gout. Here, we show that the myeloid Src-family kinases Hck, Fgr, and Lyn are critical for experimental models of gout, as well as for genetically determined systemic inflammation in the Ptpn6me-v/me-v (motheaten viable) mouse model. The Hck-/-Fgr-/-Lyn-/- mutation abrogated various monosodium urate (MSU) crystal-induced pro-inflammatory responses of neutrophils, and protected mice from the development of gouty arthritis. The Src-family inhibitor dasatinib abrogated MSU crystal-induced responses of human neutrophils and reduced experimental gouty arthritis in mice. The Hck-/-Fgr-/-Lyn-/- mutation also abrogated spontaneous inflammation and prolonged the survival of the Ptpn6me-v/me-v mice. Spontaneous adhesion and superoxide release of Ptpn6me-v/me-v neutrophils were also abolished by the Hck-/-Fgr-/-Lyn-/- mutation. Excessive activation of tyrosine phosphorylation pathways in myeloid cells may characterize a subset of autoinflammatory diseases.

Myeloid expression of the anti-apoptotic protein Mcl1 is required in anti-myeloperoxidase vasculitis but myeloperoxidase inhibition is not protective.

Antibodies to neutrophil and monocyte myeloperoxidase and proteinase 3 are a feature of anti-neutrophil cytoplasmic antibody vasculitis, a disease with significant morbidity for which new treatments are needed. Mice with a myeloid-specific deletion of the anti-apoptotic protein Mcl1 have reduced numbers of circulating neutrophils. Here, we assessed if myeloid-specific Mcl1 was required in murine anti-myeloperoxidase vasculitis and whether inhibition of myeloperoxidase was protective. In a murine model of anti-neutrophil cytoplasmic antibody vasculitis, induced by anti-myeloperoxidase antibody, mice with a myeloid-specific deletion of Mcl1 were protected from disease. They had fewer crescents, neutrophils, and macrophages in the glomeruli, lower serum creatinine levels and reduced albuminuria compared with controls. At baseline and day six after disease induction they had fewer circulating neutrophils than controls. At day six there were also fewer circulating monocytes. Myeloperoxidase inhibition with AZD5904 had no effect on histological or biochemical parameters of disease, and there was also no reduction in albuminuria at day one, two, five or seven after disease induction. These findings persisted when disease was induced without granulocyte-colony stimulating factor, which increases disease severity. A second myeloperoxidase inhibitor, AZM198, also showed no evidence of an effect, although both AZD5904 and AZM198 inhibited human neutrophil extracellular trap formation in vitro. Thus, our results show that while myeloid-specific Mcl1 is required in this model of anti-myeloperoxidase vasculitis, myeloperoxidase inhibition is not protective.

Phospholipase Cγ2 Is Essential for Experimental Models of Epidermolysis Bullosa Acquisita.

Phospholipase Cγ2 (PLCγ2) mediates tyrosine kinase‒coupled receptor signaling in various hematopoietic lineages. Although PLCγ2 has been implicated in certain human and mouse inflammatory disorders, its contribution to autoimmune and inflammatory skin diseases is poorly understood. In this study, we tested the role of PLCγ2 in a mouse model of epidermolysis bullosa acquisita triggered by antibodies against type VII collagen (C7), a component of the dermo-epidermal junction. PLCγ2-deficient (Plcg2-/-) mice and bone marrow chimeras with a Plcg2-/- hematopoietic system were completely protected from signs of anti-C7-induced skin disease, including skin erosions, dermal‒epidermal separation, and inflammation, despite normal circulating levels and skin deposition of anti-C7 antibodies. PLCγ2 was required for the tissue infiltration of neutrophils, eosinophils, and monocytes/macrophages as well as for the accumulation of proinflammatory mediators (including IL-1ß, MIP-2, and LTB4) and reactive oxygen species. Mechanistic experiments revealed a role for PLCγ2 in the release of proinflammatory mediators and reactive oxygen species but not in the intrinsic migratory capacity of leukocytes. The phospholipase C inhibitor U73122 inhibited dermal-epidermal separation of human skin sections incubated with human neutrophils in the presence of anti-C7 antibodies. Taken together, our results suggest a critical role for PLCγ2 in the pathogenesis of the inflammatory form of epidermolysis bullosa acquisita.

Signaling through Syk or CARD9 Mediates Species-Specific Anti-Candida Protection in Bone Marrow Chimeric Mice.

The spleen tyrosine kinase (Syk) and the downstream adaptor protein CARD9 are crucial signaling molecules in antimicrobial immunity. Candida parapsilosis is an emerging fungal pathogen with a high incidence in neonates, while Candida albicans is the most common agent of candidiasis. While signaling through Syk/CARD9 promotes protective host mechanisms in response to C. albicans, its function in immunity against C. parapsilosis remains unclear. Here, we generated Syk-/- and CARD9-/- bone marrow chimeric mice to study the role of Syk/CARD9 signaling in immune responses to C. parapsilosis compared to C. albicans. We demonstrate various functions of this pathway (e.g., phagocytosis, phagosome acidification, and killing) in Candida-challenged, bone marrow-derived macrophages with differential involvement of Syk and CARD9 along with species-specific differences in cytokine production. We report that Syk-/- or CARD9-/- chimeras rapidly display high susceptibility to C. albicans, while C. parapsilosis infection exacerbates over a prolonged period in these animals. Thus, our results establish that Syk and CARD9 contribute to systemic resistance to C. parapsilosis and C. albicans differently. Additionally, we confirm prior studies but also detail new insights into the fundamental roles of both proteins in immunity against C. albicans. Our data further suggest that Syk has a more prominent influence on anti-Candida immunity than CARD9. Therefore, this study reinforces the Syk/CARD9 pathway as a potential target for anti-Candida immune therapy. IMPORTANCE While C. albicans remains the most clinically significant Candida species, C. parapsilosis is an emerging pathogen with increased affinity to neonates. Syk/CARD9 signaling is crucial in immunity to C. albicans, but its role in in vivo responses to other pathogenic Candida species is largely unexplored. We used mice with hematopoietic systems deficient in Syk or CARD9 to comparatively study the function of these proteins in anti-Candida immunity. We demonstrate that Syk/CARD9 signaling has a protective role against C. parapsilosis differently than against C. albicans. Thus, this study is the first to reveal that Syk can exert immune responses during systemic Candida infections species specifically. Additionally, Syk-dependent immunity to a nonalbicans Candida species in an in vivo murine model has not been reported previously. We highlight that the contribution of Syk and CARD9 to fungal infections are not identical and underline this pathway as a promising immune-therapeutic target to fight Candida infections.

Fluorescence-Based Real-Time Analysis of Osteoclast Development.

Osteoclasts are multinucleated cells of hematopoietic origin which are critically involved in physiological and pathological bone resorption. They develop from myeloid progenitors through characteristic gene expression changes and intercellular fusion. This process is directed by M-CSF and RANKL which are also able to trigger osteoclast development from bone marrow cells in vitro. Osteoclasts are conventionally visualized by histochemical staining followed by manual counting, which hinders kinetic studies and automated quantification. Here we describe two fluorescence-based assays for the real-time analysis of myeloid cell to osteoclast development (FRAMCO) in primary mouse bone marrow cell cultures. Both assays rely on red-to-green fluorescence conversion of the membrane-targeted tdTomato/membrane-targeted eGFP (mTmG) transgene by Cre recombinase driven by the osteoclast-specific cathepsin K promoter (Ctsk-Cre). In the first assay (FRAMCO1.1), osteoclast-specific gene expression triggers red-to-green color conversion of cells carrying both the Ctsk-Cre and mTmG transgenes. In the second assay (FRAMCO1.2), red-to-green fluorescence conversion is triggered by fusion of neighboring co-cultured bone marrow cells separately carrying either the Ctsk-Cre or the mTmG transgenes. The two assays were tested using a high-content confocal fluorescence imaging system, followed by automated quantification. The FRAMCO1.1 assay showed robust red-to-green fluorescence conversion of more than 50% of the culture (including mononuclear cells) within 3 days under osteoclastogenic conditions. The FRAMCO1.2 assay showed a less robust but still readily measurable red-to-green color conversion in multinuclear cells within 5 days of differentiation. The assays required both the Ctsk-Cre and the mTmG transgenes and gave no signals in parallel macrophage cultures. The proper functioning of the two assays was also confirmed at the DNA, mRNA and bulk protein level. The assay systems were validated using lisophosphatidylcholine, a previously reported inhibitor of preosteoclast fusion. Taken together, our assays allow high-throughput automated real-time analysis of two critical aspects of osteoclast development, facilitating the screening for novel drug candidates for the pharmacological control of osteoclast-mediated bone resorption.

Complement receptor 3 mediates both sinking phagocytosis and phagocytic cup formation via distinct mechanisms.

A long-standing hypothesis is that complement receptors (CRs), especially CR3, mediate sinking phagocytosis, but evidence is lacking. Alternatively, CRs have been reported to induce membrane ruffles or phagocytic cups, akin to those induced by Fcγ receptors (FcγRs), but the details of these events are unclear. Here we used real-time 3D imaging and KO mouse models to clarify how particles (human red blood cells) are internalized by resident peritoneal F4/80+ cells (macrophages) via CRs and/or FcγRs. We first show that FcγRs mediate highly efficient, rapid (2-3 min) phagocytic cup formation, which is completely abolished by deletion or mutation of the FcR γ chain or conditional deletion of the signal transducer Syk. FcγR-mediated phagocytic cups robustly arise from any point of cell-particle contact, including filopodia. In the absence of CR3, FcγR-mediated phagocytic cups exhibit delayed closure and become aberrantly elongated. Independent of FcγRs, CR3 mediates sporadic ingestion of complement-opsonized particles by rapid phagocytic cup-like structures, typically emanating from membrane ruffles and largely prevented by deletion of the immunoreceptor tyrosine-based activation motif (ITAM) adaptors FcR γ chain and DAP12 or Syk. Deletion of ITAM adaptors or Syk clearly revealed that there is a slow (10-25 min) sinking mode of phagocytosis via a restricted orifice. In summary, we show that (1) CR3 indeed mediates a slow sinking mode of phagocytosis, which is accentuated by deletion of ITAM adaptors or Syk, (2) CR3 induces phagocytic cup-like structures, driven by ITAM adaptors and Syk, and (3) CR3 is involved in forming and closing FcγR-mediated phagocytic cups.

A New Zebrafish Model for Pseudoxanthoma Elasticum.

Calcification of various tissues is a significant health issue associated with aging, cancer and autoimmune diseases. There are both environmental and genetic factors behind this phenomenon and understanding them is essential for the development of efficient therapeutic approaches. Pseudoxanthoma elasticum (PXE) is a rare genetic disease, a prototype for calcification disorders, resulting from the dysfunction of ABCC6, a transport protein found in the membranes of cells. It is identified by excess calcification in a variety of tissues (e.g., eyes, skin, arteries) and currently it has no cure, known treatments target the symptoms only. Preclinical studies of PXE have been successful in mice, proving the usefulness of animal models for the study of the disease. Here, we present a new zebrafish (Danio rerio) model for PXE. By resolving some ambiguous assemblies in the zebrafish genome, we show that there are two functional and one non-functional paralogs for ABCC6 in zebrafish (abcc6a, abcc6b.1, and abcc6b.2, respectively). We created single and double mutants for the functional paralogs and characterized their calcification defects with a combination of techniques. Zebrafish deficient in abcc6a show defects in their vertebral calcification and also display ectopic calcification foci in their soft tissues. Our results also suggest that the impairment of abcc6b.1 does not affect this biological process.

Neutrophil Phospholipase Cγ2 Drives Autoantibody-Induced Arthritis Through the Generation of the Inflammatory Microenvironment.

OBJECTIVE: Gain-of-function mutations and genome-wide association studies have linked phospholipase Cγ2 (PLCγ2) to various inflammatory diseases, including arthritis in humans and mice. PLCγ2-deficient (Plcg2-/- ) mice are also protected against experimental arthritis. This study was undertaken to test how PLCγ2 triggers autoantibody-induced arthritis in mice. METHODS: PLCγ2 was deleted from various mouse cellular lineages. Deletion efficacy and specificity were tested by immunoblotting and intracellular flow cytometry. Autoantibody-induced arthritis was triggered by K/BxN serum transfer. The role of neutrophil PLCγ2 was further investigated by analysis of the inflammatory exudate, competitive in vivo migration assays, and in vitro functional studies. RESULTS: PLCγ2 deficiency in the entire hematopoietic compartment completely blocked autoantibody-induced arthritis. Arthritis development was abrogated by deletion of PLCγ2 from myeloid cells or neutrophils but not from mast cells or platelets. Neutrophil infiltration was reduced in neutrophil-specific PLCγ2-deficient (Plcg2Δ PMN ) mice. However, this was not due to an intrinsic migration defect since Plcg2Δ PMN neutrophils accumulated normally when wild-type cells were also present in mixed bone marrow chimeras. Instead, the Plcg2Δ PMN mutation blocked the accumulation of interleukin-1ß, macrophage inflammatory protein 2 (MIP-2), and leukotriene B4 (LTB4 ) in synovial tissues and reduced the secondary infiltration of macrophages. These findings were supported by in vitro studies showing normal chemotactic migration but defective immune complex-induced respiratory burst and MIP-2 or LTB4 release in PLCγ2-deficient neutrophils. CONCLUSION: Neutrophil PLCγ2 is critical for arthritis development, supposedly through the generation of the inflammatory microenvironment. PLCγ2-expressing neutrophils exert complex indirect effects on other inflammatory cells. PLCγ2-targeted therapies may provide particular benefit in inflammatory diseases with a major neutrophil component.

In Vivo Functions of Mouse Neutrophils Derived from HoxB8-Transduced Conditionally Immortalized Myeloid Progenitors.

Although neutrophils play important roles in immunity and inflammation, their analysis is strongly hindered by their short-lived and terminally differentiated nature. Prior studies reported conditional immortalization of myeloid progenitors using retroviral expression of an estrogen-dependent fusion protein of the HoxB8 transcription factor. This approach allowed the long-term culture of mouse myeloid progenitors (HoxB8 progenitors) in estrogen-containing media, followed by differentiation toward neutrophils upon estrogen withdrawal. Although several reports confirmed the in vitro functional responsiveness of the resulting differentiated cells (HoxB8 neutrophils), little is known about their capacity to perform in vivo neutrophil functions. We have addressed this issue by an in vivo transplantation approach. In vitro-generated HoxB8 neutrophils showed a neutrophil-like phenotype and were able to perform conventional neutrophil functions, like respiratory burst, chemotaxis, and phagocytosis. The i.v. injection of HoxB8 progenitors into lethally irradiated recipients resulted in the appearance of circulating donor-derived HoxB8 neutrophils. In vivo-differentiated HoxB8 neutrophils were able to migrate to the inflamed peritoneum and to phagocytose heat-killed Candida particles. The reverse passive Arthus reaction could be induced in HoxB8 chimeras but not in irradiated, nontransplanted control animals. Repeated injection of HoxB8 progenitors also allowed us to maintain stable circulating HoxB8 neutrophil counts for several days. Injection of arthritogenic K/B×N serum triggered robust arthritis in HoxB8 chimeras, but not in irradiated, nontransplanted control mice. Taken together, our results indicate that HoxB8 progenitor-derived neutrophils are capable of performing various in vivo neutrophil functions, providing a framework for using the HoxB8 system for the in vivo analysis of neutrophil function.

Osteoclast Signal Transduction During Bone Metastasis Formation.

Osteoclasts are myeloid lineage-derived bone-resorbing cells of hematopoietic origin. They differentiate from myeloid precursors through a complex regulation process where the differentiation of preosteoclasts is followed by intercellular fusion to generate large multinucleated cells. Under physiological conditions, osteoclastogenesis is primarily directed by interactions between CSF-1R and macrophage colony-stimulating factor (M-CSF, CSF-1), receptor activator of nuclear factor NF-κB (RANK) and RANK ligand (RANKL), as well as adhesion receptors (e.g., integrins) and their ligands. Osteoclasts play a central role in physiological and pathological bone resorption and are also required for excessive bone loss during osteoporosis, inflammatory bone and joint diseases (such as rheumatoid arthritis) and cancer cell-induced osteolysis. Due to the major role of osteoclasts in these diseases the better understanding of their intracellular signaling pathways can lead to the identification of potential novel therapeutic targets. Non-receptor tyrosine kinases and lipid kinases play major roles in osteoclasts and small-molecule kinase inhibitors are emerging new therapeutics in diseases with pathological bone loss. During the last few years, we and others have shown that certain lipid (such as phosphoinositide 3-kinases PI3Kß and PI3Kδ) and tyrosine (Src-family and Syk) kinases play a critical role in osteoclast differentiation and function in humans and mice. Some of these signaling pathways shows similarity to immunoreceptor-like receptor signaling and involves important other enzymes (e.g., PLCγ2) and adapter proteins (such as the ITAM-bearing adapters DAP12 and the Fc-receptor γ-chain). Here, we review recently identified osteoclast signaling pathways and their role in osteoclast differentiation and function as well as pathological bone loss associated with osteolytic tumors of the bone. A better understanding of osteoclast signaling may facilitate the design of novel and more efficient therapies for pathological bone resorption and osteolytic skeletal metastasis formation.

VISTA deficiency protects from immune complex-mediated glomerulonephritis by inhibiting neutrophil activation.

V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA) is a negative checkpoint regulator of T cells. We assessed VISTA deficient mice in the murine nephrotoxic nephritis models of acute and chronic immune-complex mediated glomerulonephritis. We show that VISTA deficiency protects from crescentic glomerulonephritis, with no effect on the nephritogenic adaptive immune response. The early neutrophil influx was unaffected but proteinuria was reduced suggesting a reduction in neutrophil activation. In vivo, there was reduced neutrophil degranulation in VISTA deficienct mice and, in vitro, VISTA-deficient neutrophils had an impaired response to immune complexes but not to fMLP or PMA. Mice with a genetic deficiency of neutrophils due to myeloid-specific deletion of myeloid cell leukemia 1 (Mcl-1) were also protected from crescentic glomerulonephritis, indicating an essential role for neutrophils. Therefore, VISTA deficiency inhibits neutrophil activation by immune complexes and neutrophil-dependent crescentic glomerulonephritis. This suggests that VISTA is a therapeutic target for inflammatory disease. However, this would need to be balanced against a potential enhancing effect on autoimmunity.

Neutrophils as emerging therapeutic targets.

Neutrophils are the most abundant circulating leukocytes, being the first line of defence against bacterial and fungal infections. However, neutrophils also contribute to tissue damage during various autoimmune and inflammatory diseases, and play important roles in cancer progression. The intimate but complex involvement of neutrophils in various diseases makes them exciting targets for therapeutic intervention but also necessitates differentiation of beneficial responses from potentially detrimental side effects. A variety of approaches to therapeutically target neutrophils have emerged, including strategies to enhance, inhibit or restore neutrophil function, with several agents entering clinical trials. However, challenges and controversies in the field remain.

Src family kinase-mediated vesicle trafficking is critical for neutrophil basement membrane penetration.

Leukocyte recruitment into inflamed tissue is highly dependent on the activation and binding of integrins to their respective ligands, followed by the induction of various signaling events within the cell referred to as outside-in signaling. Src family kinases (SFK) are the central players in the outside-in signaling process, assigning them a critical role for proper immune cell function. Our study investigated the role of SFK on neutrophil recruitment in vivo using Hck-/- Fgr-/- Lyn-/- mice, which lack SFK expressed in neutrophils. We show that loss of SFK strongly reduces neutrophil adhesion and post-arrest modifications in a shear force dependent manner. Additionally, we found that in the absence of SFK, neutrophils display impaired Rab27a-dependent surface mobilization of neutrophil elastase, VLA3 and VLA6 containing vesicles. This results in a defect in neutrophil vascular basement membrane penetration and thus strongly impaired extravasation. Taken together, we demonstrate that SFK play a role in neutrophil post-arrest modifications and extravasation during acute inflammation. These findings may support the current efforts to use SFK-inhibitors in inflammatory diseases with unwanted neutrophil recruitment.

Hematopoietic or Osteoclast-Specific Deletion of Syk Leads to Increased Bone Mass in Experimental Mice.

Syk is a non-receptor tyrosine kinase critically involved in signaling by various immunoreceptors including B-cell-receptors and activating Fc-receptors. We have previously shown that Syk also mediates immunoreceptor-like signals required for the in vitro development and function of osteoclasts. However, the perinatal lethality of Syk-/- mice precluded the analysis of the role of Syk in in vivo bone metabolism. To overcome that problem, we generated mice with osteoclast-specific (SykΔOC ) or hematopoietic (SykΔHaemo ) Syk deficiency by conditional deletion of Syk using Cre recombinase expressed under the control of the Ctsk or Vav1 promoter, respectively. Micro-CT analysis revealed increased bone trabecular density in both SykΔOC and SykΔHaemo mice, although hematopoietic Syk deficiency caused a more severe phenotype than osteoclast-specific Syk deficiency. Osteoclast-specific Syk deficiency reduced, whereas hematopoietic Syk deficiency completely blocked in vitro development of osteoclasts. Both interventions inhibited the resorptive activity of osteoclasts and osteoclast-specific gene expression. Kinetic analysis of Syk protein levels, Cre expression and the genomic deletion of the Sykflox allele revealed complete and early deletion of Syk from SykΔHaemo osteoclasts whereas Syk was incompletely deleted at a later stage of osteoclast development from SykΔOC cultures. Those results provide an explanation for the in vivo and in vitro difference between the SykΔOC and SykΔHaemo mutant strains and suggest late activation of, and incomplete target gene deletion upon, osteoclast-specific Cre expression driven by the Ctsk promoter. Taken together, our results indicate that Syk plays an indispensable role in osteoclast-mediated in vivo bone resorption and suggest that Syk-specific inhibitors may provide therapeutic benefit in inflammatory and other diseases characterized by excessive osteoclast-mediated bone resorption.

Myeloid-Specific Deletion of Mcl-1 Yields Severely Neutropenic Mice That Survive and Breed in Homozygous Form.

Mouse strains with specific deficiency of given hematopoietic lineages provide invaluable tools for understanding blood cell function in health and disease. Whereas neutrophils are dominant leukocytes in humans and mice, there are no widely useful genetic models of neutrophil deficiency in mice. In this study, we show that myeloid-specific deletion of the Mcl-1 antiapoptotic protein in Lyz2 Cre/Cre Mcl1 flox/flox (Mcl1 ΔMyelo) mice leads to dramatic reduction of circulating and tissue neutrophil counts without affecting circulating lymphocyte, monocyte, or eosinophil numbers. Surprisingly, Mcl1 ΔMyelo mice appeared normally, and their survival was mostly normal both under specific pathogen-free and conventional housing conditions. Mcl1 ΔMyelo mice were also able to breed in homozygous form, making them highly useful for in vivo experimental studies. The functional relevance of neutropenia was confirmed by the complete protection of Mcl1 ΔMyelo mice from arthritis development in the K/B×N serum-transfer model and from skin inflammation in an autoantibody-induced mouse model of epidermolysis bullosa acquisita. Mcl1 ΔMyelo mice were also highly susceptible to systemic Staphylococcus aureus or Candida albicans infection, due to defective clearance of the invading pathogens. Although neutrophil-specific deletion of Mcl-1 in MRP8-CreMcl1 flox/flox (Mcl1 ΔPMN) mice also led to severe neutropenia, those mice showed an overt wasting phenotype and strongly reduced survival and breeding, limiting their use as an experimental model of neutrophil deficiency. Taken together, our results with the Mcl1 ΔMyelo mice indicate that severe neutropenia does not abrogate the viability and fertility of mice, and they provide a useful genetic mouse model for the analysis of the role of neutrophils in health and disease.

The Yin and Yang of Tyrosine Kinase Inhibition During Experimental Polymicrobial Sepsis.

Neutrophils are the first cells of our immune system to arrive at the site of inflammation. They release cytokines, e.g., chemokines, to attract further immune cells, but also actively start to phagocytose and kill pathogens. In the case of sepsis, this tightly regulated host defense mechanism can become uncontrolled and hyperactive resulting in severe organ damage. Currently, no effective therapy is available to fight sepsis; therefore, novel treatment targets that could prevent excessive inflammatory responses are warranted. Src Family tyrosine Kinases (SFK), a group of tyrosine kinases, have been shown to play a major role in regulating immune cell recruitment and host defense. Leukocytes with SFK depletion display severe spreading and migration defects along with reduced cytokine production. Thus, we investigated the effects of dasatinib, a tyrosine kinase inhibitor, with a strong inhibitory capacity on SFKs during sterile inflammation and polymicrobial sepsis in mice. We found that dasatinib-treated mice displayed diminished leukocyte adhesion and extravasation in tumor necrosis factor-α-stimulated cremaster muscle venules in vivo. In polymicrobial sepsis, sepsis severity, organ damage, and clinical outcome improved in a dose-dependent fashion pointing toward an optimal therapeutic window for dasatinib dosage during polymicrobial sepsis. Dasatinib treatment may, therefore, provide a balanced immune response by preventing an overshooting inflammatory reaction on the one side and bacterial overgrowth on the other side.