Neutrophil IL-26 fuels autoinflammation.

Pustular psoriasis is an inflammatory skin disease with features of neutrophil-mediated sterile autoinflammation. In this issue of JEM, Baldo et al. (https://doi.org/10.1084/jem.20231464) show that this autoinflammation is driven by a vicious cycle through neutrophil-derived IL-26.

Neutrophils and NADPH Oxidases Are Major Contributors to Mild but Not Severe Ischemic Acute Kidney Injury in Mice.

Upregulation of free radical-generating NADPH oxidases (NOX), xanthine oxidoreductase (XOR), and neutrophil infiltration-induced, NOX2-mediated respiratory burst contribute to renal ischemia-reperfusion injury (IRI), but their roles may depend on the severity of IRI. We investigated the role of NOX, XOR, and neutrophils in developing IRI of various severities. C57BL/6 and Mcl-1ΔMyelo neutrophil-deficient mice were used. Oxidases were silenced by RNA interference (RNAi) or pharmacologically inhibited. Kidney function, morphology, immunohistochemistry and mRNA expression were assessed. After reperfusion, the expression of NOX enzymes and XOR increased until 6 h and from 15 h, respectively, while neutrophil infiltration was prominent from 3 h. NOX4 and XOR silencing or pharmacological XOR inhibition did not protect the kidney from IRI. Attenuation of NOX enzyme-induced oxidative stress by apocynin and neutrophil deficiency improved kidney function and ameliorated morphological damage after mild but not moderate/severe IRI. The IR-induced postischemic renal functional impairment (BUN, Lcn-2), tubular necrosis score, inflammation (TNF-α, F4/80), and decreases in the antioxidant enzyme (GPx3) mRNA expression were attenuated by both apocynin and neutrophil deficiency. Inhibition of NOX enzyme-induced oxidative stress or the lack of infiltration by NOX2-expressing neutrophils can attenuate reperfusion injury after mild but not moderate/severe renal IR.

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.

Lacking ARHGAP25 mitigates the symptoms of autoantibody-induced arthritis in mice.

Objective: Despite intensive research on rheumatoid arthritis, the pathomechanism of the disease is still not fully understood and the treatment has not been completely resolved. Previously we demonstrated that the GTPase-activating protein, ARHGAP25 has a crucial role in the regulation of basic phagocyte functions. Here we investigate the role of ARHGAP25 in the complex inflammatory process of autoantibody-induced arthritis. Methods: Wild-type and ARHGAP25 deficient (KO) mice on a C57BL/6 background, as well as bone marrow chimeric mice, were treated i.p. with the K/BxN arthritogenic or control serum, and the severity of inflammation and pain-related behavior was measured. Histology was prepared, leukocyte infiltration, cytokine production, myeloperoxidase activity, and superoxide production were determined, and comprehensive western blot analysis was conducted. Results: In the absence of ARHGAP25, the severity of inflammation, joint destruction, and mechanical hyperalgesia significantly decreased, similarly to phagocyte infiltration, IL-1ß, and MIP-2 levels in the tibiotarsal joint, whereas superoxide production or myeloperoxidase activity was unchanged. We observed a significantly mitigated phenotype in KO bone marrow chimeras as well. In addition, fibroblast-like synoviocytes showed comparable expression of ARHGAP25 to neutrophils. Significantly reduced ERK1/2, MAPK, and I-κB protein signals were detected in the arthritic KO mouse ankles. Conclusion: Our findings suggest that ARHGAP25 has a key role in the pathomechanism of autoantibody-induced arthritis in which it regulates inflammation via the I-κB/NF-κB/IL-1ß axis with the involvement of both immune cells and fibroblast-like synoviocytes.

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.

Neutrophil-Specific Syk Expression Is Crucial for Skin Disease in Experimental Epidermolysis Bullosa Acquisita.

Autoantibodies against the dermal-epidermal junction component type VII collagen (C7) trigger skin disease in the inflammatory form of epidermolysis bullosa acquisita. We have previously identified the Syk tyrosine kinase as a crucial participant in anti-C7 antibody-induced experimental epidermolysis bullosa acquisita. However, it is still unclear which cellular lineage needs to express Syk during the disease process. In this study, we show that the loss of Syk, specifically from neutrophils, results in complete protection from the anti-C7 antibody-initiated skin disease both macroscopically and microscopically. Mice with a neutrophil-specific Syk deletion had decreased neutrophil accumulation and abrogated CXCL2 and IL-1ß levels in the skin upon anti-C7 treatment, whereas isolated Syk-deficient neutrophils had decreased superoxide release, cell spreading, and cytokine release on C7-anti-C7 immune complex surfaces. Entospletinib and lanraplenib, two second-generation Syk-specific inhibitors, effectively abrogated immune complex-induced responses of human neutrophils and decreased the anti-C7 antibody-initiated, neutrophil-mediated ex vivo dermal-epidermal separation in human skin samples. Taken together, these results point to a crucial role for Syk in neutrophils in the development and progression of epidermolysis bullosa acquisita and suggest Syk inhibition as a potential therapeutic strategy.

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.

Time restricted feeding modifies leukocyte responsiveness and improves inflammation outcome.

Time restricted eating, the dietary approach limiting food intake to a maximal 10-hour period of daytime is considered beneficial in metabolic dysfunctions, such as obesity and diabetes. Rhythm of food intake and parallel changes in serum nutrient levels are also important entrainment signals for the circadian clock, particularly in tissues involved in metabolic regulation. As both the metabolic state and the circadian clock have large impact on immune functions, we investigated in mice whether time restricted feeding (TRF) affects systemic inflammatory potential. TRF slackened the symptoms in K/BxN serum-transfer arthritis, an experimental model of human autoimmune joint inflammation. Compared to ad libitum conditions TRF reduced the expression of inflammatory mediators in visceral adipose tissue, an integrator and coordinator of metabolic and inflammatory processes. Furthermore, TRF strengthened the oscillation of peripheral leukocyte counts and alongside decreased the pool of both marginated and tissue leukocytes. Our data suggest that the altered leukocyte distribution in TRF mice is related to the attenuated expression of adhesion molecules on the surface of neutrophils and monocytes. We propose that TRF modifies both rhythm and inflammatory potential of leukocytes which contribute to the milder reactivity of the immune system and therefore time-restricted eating could serve as an effective complementary tool in the therapy of autoinflammatory processes.

Investigation of the Role of the TRPA1 Ion Channel in Conveying the Effect of Dimethyl Trisulfide on Vascular and Histological Changes in Serum-Transfer Arthritis.

Rheumatoid arthritis (RA) is one of the most prevalent autoimmune diseases. Its therapy is often challenging, even in the era of biologicals. Previously, we observed the anti-inflammatory effects of garlic-derived organic polysulfide dimethyl trisulfide (DMTS). Some of these effects were mediated by activation of the TRPA1 ion channel. TRPA1 was mostly expressed in a subset of nociceptor neurons. We decided to investigate the action of DMTS in K/BxN serum-transfer arthritis, which is a relevant model of RA. TRPA1 gene knockout (KO) and wild-type (WT) mice were used. The interaction of DMTS and TRPA1 was examined using a patch clamp in CHO cells. Arthritis was characterized by mechanical hyperalgesia, paw swelling, movement range of the ankle joint, hanging performance, plasma extravasation rate, myeloperoxidase activity, and histological changes in the tibiotarsal joint. DMTS activated TRPA1 channels dose-dependently. DMTS treatment reduced paw swelling and plasma extravasation in both TRPA1 WT and KO animals. DMTS-treated TRPA1 KO animals developed milder collagen deposition in the inflamed joints than WT ones. TRPA1 WT mice did not exhibit significant cartilage damage compared to ones administered a vehicle. We concluded that DMTS and related substances might evolve into novel complementary therapeutic aids for RA patients.

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.

Siglec-H-Deficient Mice Show Enhanced Type I IFN Responses, but Do Not Develop Autoimmunity After Influenza or LCMV Infections.

Siglec-H is a DAP12-associated receptor on plasmacytoid dendritic cells (pDCs) and microglia. Siglec-H inhibits TLR9-induced IFN-α production by pDCs. Previously, it was found that Siglec-H-deficient mice develop a lupus-like severe autoimmune disease after persistent murine cytomegalovirus (mCMV) infection. This was due to enhanced type I interferon responses, including IFN-α. Here we examined, whether other virus infections can also induce autoimmunity in Siglec-H-deficient mice. To this end we infected Siglec-H-deficient mice with influenza virus or with Lymphocytic Choriomeningitis virus (LCMV) clone 13. With both types of viruses we did not observe induction of autoimmune disease in Siglec-H-deficient mice. This can be explained by the fact that both types of viruses are ssRNA viruses that engage TLR7, rather than TLR9. Also, Influenza causes an acute infection that is rapidly cleared and the chronicity of LCMV clone 13 may not be sufficient and may rather suppress pDC functions. Siglec-H inhibited exclusively TLR-9 driven type I interferon responses, but did not affect type II or type III interferon production by pDCs. Siglec-H-deficient pDCs showed impaired Hck expression, which is a Src-family kinase expressed in myeloid cells, and downmodulation of the chemokine receptor CCR9, that has important functions for pDCs. Accordingly, Siglec-H-deficient pDCs showed impaired migration towards the CCR9 ligand CCL25. Furthermore, autoimmune-related genes such as Klk1 and DNase1l3 are downregulated in Siglec-H-deficient pDCs as well. From these findings we conclude that Siglec-H controls TLR-9-dependent, but not TLR-7 dependent inflammatory responses after virus infections and regulates chemokine responsiveness of pDCs.

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.