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.

Experimental lupus is aggravated in mouse strains with impaired induction of neutrophil extracellular traps.

Many effector mechanisms of neutrophils have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). Neutrophil extracellular traps (NETs) have been assigned a particularly detrimental role. Here we investigated the functional impact of neutrophils and NETs on a mouse model of lupus triggered by intraperitoneal injection of the cell death-inducing alkane pristane. Pristane-induced lupus (PIL) was aggravated in 2 mouse strains with impaired induction of NET formation, i.e., NOX2-deficient (Ncf1-mutated) and peptidyl arginine deiminase 4-deficient (PAD4-deficient) mice, as seen from elevated levels of antinuclear autoantibodies (ANAs) and exacerbated glomerulonephritis. We observed a dramatically reduced ability to form pristane-induced NETs in vivo in both Ncf1-mutated and PAD4-deficient mice, accompanied by higher levels of inflammatory mediators in the peritoneum. Similarly, neutropenic Mcl-1ΔMyelo mice exhibited higher levels of ANAs, which indicates a regulatory function in lupus of NETs and neutrophils. Blood neutrophils from Ncf1-mutated and human individuals with SLE exhibited exuberant spontaneous NET formation. Treatment with specific chemical NOX2 activators induced NET formation and ameliorated PIL. Our findings suggest that aberrant NET is one of the factors promoting experimental lupus-like autoimmunity by uncontrolled release of inflammatory mediators.

Analgesic and Anti-Inflammatory Effects of the Novel Semicarbazide-Sensitive Amine-Oxidase Inhibitor SzV-1287 in Chronic Arthritis Models of the Mouse.

Semicarbazide-sensitive amine oxidase (SSAO) catalyses oxidative deamination of primary amines. Since there is no data about its function in pain and arthritis mechanisms, we investigated the effects of our novel SSAO inhibitor SzV-1287 in chronic mouse models of joint inflammation. Effects of SzV-1287 (20 mg/kg i.p./day) were investigated in the K/BxN serum-transfer and complete Freund's adjuvant (CFA)-evoked active immunization models compared to the reference SSAO inhibitor LJP-1207. Mechanonociception was assessed by aesthesiometry, oedema by plethysmometry, clinical severity by scoring, joint function by grid test, myeloperoxidase activity by luminescence, vascular leakage by fluorescence in vivo imaging, histopathological changes by semiquantitative evaluation, and cytokines by Luminex assay. SzV-1287 significantly inhibited hyperalgesia and oedema in both models. Plasma leakage and keratinocyte chemoattractant production in the tibiotarsal joint, but not myeloperoxidase activity was significantly reduced by SzV-1287 in K/BxN-arthritis. SzV-1287 did not influence vascular and cellular mechanisms in CFA-arthritis, but significantly decreased histopathological alterations. There was no difference in the anti-hyperalgesic and anti-inflammatory actions of SzV-1287 and LJP-1207, but only SzV-1287 decreased CFA-induced tissue damage. Unlike SzV-1287, LJP-1207 induced cartilage destruction, which was confirmed in vitro. SzV-1287 exerts potent analgesic and anti-inflammatory actions in chronic arthritis models of distinct mechanisms, without inducing cartilage damage.

Capsaicin-sensitive sensory nerves exert complex regulatory functions in the serum-transfer mouse model of autoimmune arthritis.

OBJECTIVE: The K/BxN serum-transfer arthritis is a widely-used translational mouse model of rheumatoid arthritis, in which the immunological components have thoroughly been investigated. In contrast, little is known about the role of sensory neural factors and the complexity of neuro-immune interactions. Therefore, we analyzed the involvement of capsaicin-sensitive peptidergic sensory nerves in autoantibody-induced arthritis with integrative methodology. METHODS: Arthritogenic K/BxN or control serum was injected to non-pretreated mice or resiniferatoxin (RTX)-pretreated animals where capsaicin-sensitive nerves were inactivated. Edema, touch sensitivity, noxious heat threshold, joint function, body weight and clinical arthritis severity scores were determined repeatedly throughout two weeks. Micro-CT and in vivo optical imaging to determine matrix-metalloproteinase (MMP) and neutrophil-derived myeloperoxidase (MPO) activities, semiquantitative histopathological scoring and radioimmunoassay to measure somatostatin in the joint homogenates were also performed. RESULTS: In RTX-pretreated mice, the autoantibody-induced joint swelling, arthritis severity score, MMP and MPO activities, as well as histopathological alterations were significantly greater compared to non-pretreated animals. Self-control quantification of the bone mass revealed decreased values in intact female mice, but significantly greater arthritis-induced pathological bone formation after RTX-pretreatment. In contrast, mechanical hyperalgesia from day 10 was smaller after inactivating capsaicin-sensitive afferents. Although thermal hyperalgesia did not develop, noxious heat threshold was significantly higher following RTX pretreatment. Somatostatin-like immunoreactivity elevated in the tibiotarsal joints in non-pretreated, which was significantly less in RTX-pretreated mice. CONCLUSIONS: Although capsaicin-sensitive sensory nerves mediate mechanical hyperalgesia in the later phase of autoantibody-induced chronic arthritis, they play important anti-inflammatory roles at least partially through somatostatin release.

The ecotoxicological evaluation of Cylindrospermopsis raciborskii from Lake Balaton (Hungary) employing a battery of bioassays and chemical screening.

Ecotoxicity of four Cylindrospermopsis raciborskii strains (ACT 9502, ACT 9503, ACT 9504, ACT 9505) isolated from Lake Balaton (Hungary) was evaluated in four aquatic bioassays including the Thamnocephalus platyurus acute lethality test; Daphnia magna acute immobilization assay; D. magna feeding inhibition assay and Danio rerio embryo developmental toxicity assay, assisted by chemical screening for known toxins by HPLC-MS. For reference, we analyzed in parallel the toxin content and toxic effects of two previously characterized toxin-producing strains: the Australian cylindrospermopsin producer AQS C. raciborskii and the anatoxins producer Oscillatoria sp. PCC 6506. Bioassays were used to evaluate the overall toxicity of the hydrophilic bioactive metabolites pool synthesized by the selected cyanobacteria. Chemical screening has proven that the ACT C. raciborskii extracts investigated did not contained cylindrospermopsins and anatoxins. The relative toxicity of the ACT C. raciborskii aqueous extracts observed in each bioassay was comparable to the effects recorded for the anatoxins producer PCC 6506 strain while toxicity values (EC50/LC50) calculated for the AQS extract were in general one order of magnitude lower. Concerning sublethal effects of ACT C. raciborskii extracts to the D. rerio embryogenesis, the general morphological abnormality observed was a significant retardation of development. Overall, our results suggest that C. raciborskii populating Lake Balaton produce metabolites with significant bioactive potencies. Therefore, continued investigation of these unknown compounds is required.