Reduced Leukocyte Infiltration Research Articles

Conditional over-expression of heme-oxygenase 1 in myelomonocytic cells reduces AngII-induced hypertension and vascular inflammation in mice

Abstract Background Systemic arterial Hypertension is one of the most important risk factor responsible for morbidity and mortality world-wide. Angiotensin II (Ang II), a potent vasoconstrictor and key player in the renin-angiotensin-aldosterone system (RAAS) is responsible for vascular dysfunction, inflammation, and tissue damage. Heme oxygenase-1 (HO-1) overexpression may confer antioxidant and anti-inflammatory properties in Ang II-induced hypertension through its action on heme catabolism, generating carbon monoxide (CO), ferritin and biliverdin/bilirubin by the action of biliverdin reductase A (BLVRA). Methods and results Male 9–12-weeks-old HO-1indxLySMCre/wt and LySMCre/wt mice were infused with AngII (1mgAngII/Day/Kg) for 7 days to induce hypertension and compared to sham treatment. Blood pressure monitoring by tail-cuff method, and endothelium-dependent vasodilator studies via organ chamber system using acetylcholine (ACh) in isolated aortic segments (~4 mm), revealed that overexpression of HO-1 in myeloid cells resulted in decreased systolic blood pressure and improved endothelial function in AngII-infused HO-1indxLySMCre/wt mice compared to controls. Concurrently, increased BLVRA expression in liver tissue and elevated plasma bilirubin levels were detected by transcriptome analysis and high-performance liquid chromatography, respectively. These results were supported by a reduction in the expression of inducible cytokines and cell adhesion molecules (CAMs) in the aorta, assessed using qPCR. Bone marrow transplant experiments demonstrated that bone marrow from LysMcre mice in HO-1indxLySMCre/wt mice abrogated the protective effect of HO-1, resulting in endothelial damage and increased blood pressure, potentially due to decreased liver BLVRA expression and the accumulation of bone marrow-derived cells in the vessel wall in response to AngII. Additionally, adeno-associated viral vector (AAV) transfection targeting specifically BLVRA activity in liver lead to an increase in blood pressure values and worse endothelium relaxation, in parallel with higher oxidative stress and the blood neutrophils concentration, as assessed in whole blood by using oxidative burst method and blood count system respectively. Conclusion The overexpression of HO-1 in myeloid cells confers anti-inflammatory protection in AngII-induced arterial hypertension in mice. Myeloid cells bone marrow-derived overexpressing HO-1 regulate the differentiation and infiltration of pro-inflammatory immune cells in the vasculature. BLVRA expression and bilirubin levels downstream of HO-1 play a critical role in protecting against vascular damage by reducing leukocyte infiltration.

Bacteriophage Therapy for Staphylococcus aureus Induced Postoperative Acute Endophthalmitis In Vivo

Background: Postoperative acute endophthalmitis is a rare but severe complication following cataract surgery, with bacterial pathogens such as Staphylococcus aureus being the leading cause. Despite declining incidence rates globally, visual outcomes remain poor in many cases. Recent studies have explored the potential of bacteriophage therapy to treat bacterial infections, particularly for antimicrobial-resistant strains. This study aimed to assess the therapeutic effectiveness of bacteriophage treatment in a rabbit model of S. aureus-induced acute postoperative endophthalmitis. Methods: The study employed a randomized, controlled, posttest-only design using New Zealand white rabbits. Following extracapsular lens extraction surgery, rabbits were injected with S. aureus intracamerally to induce endophthalmitis. The treatment group received additional bacteriophage therapy. Ocular inflammation was assessed by measuring two biomarkers, myeloperoxidase (MPO) and intercellular adhesion molecule-1 (ICAM-1), using enzyme-linked immunosorbent assay (ELISA) from vitreous fluid samples. Histopathological analysis of retinal and scleral structures was performed post-enucleation. Statistical analyses were conducted using independent t-tests. Results: The treatment group showed significantly lower ICAM-1 levels (p < 0.001), indicating reduced leukocyte infiltration and inflammation compared to the control group. MPO levels were slightly elevated in the treatment group but were not statistically significant (p = 0.261). Histopathological examination revealed that retinal structures in the treatment group were preserved with reduced inflammatory cell infiltration, whereas the control group displayed extensive retinal damage, including neovascularization and retinal detachment. Conclusion: Bacteriophage therapy significantly reduced ocular inflammation and tissue damage in S. aureus-induced endophthalmitis, as evidenced by lower ICAM-1 levels and better-preserved retinal structures in the treatment group. These findings suggest that bacteriophage therapy may be an effective alternative to conventional treatments for postoperative acute endophthalmitis, particularly in cases involving antibiotic-resistant bacteria. Further studies are warranted to explore its broader clinical applicability.

CYP7B1 deficiency impairs myeloid cell activation in autoimmune disease of the central nervous system.

Dysregulation of cholesterol metabolism underlies neurodegenerative disease and is increasingly implicated in neuroinflammatory diseases, such as multiple sclerosis (MS). Cytochrome P450 family 7 subfamily B member 1 (CYP7B1) is a key enzyme in alternative cholesterol metabolism. A recessive mutation in the gene CYP7B1 is known to cause a neurodegenerative disease, hereditary spastic paraplegia type 5 and oxysterol accumulation. However, the role of CYP7B1 in neuroinflammation has been little revealed. In this study, we induced experimental autoimmune encephalomyelitis (EAE), as a murine model of MS, using CYP7B1 homozygous knockout (KO) mice. We found that CYP7B1 deficiency can significantly attenuate EAE severity. CYP7B1 deficiency is sufficient to reduce leukocyte infiltration into the central nervous system, suppress proliferation of pathogenic CD4+ T cells, and decrease myeloid cell activation during EAE. Additionally, live-animal imaging targeting translocator protein expression, an outer mitochondrial membrane protein biomarker of neuroinflammation, showed that CYP7B1 deficiency results in suppressed neuroinflammation. Using human monocyte-derived microglia-like cellular disease model and primary microglia of CYP7B1 KO mice, we also found that activation of microglia of CYP7B1 deficiency was impaired. These cumulative results suggest that CYP7B1 can regulate neuroinflammation, thus providing potential new targets for therapeutic intervention.

Treatment with lipoxin A4 improves influenza A infection outcome, induces macrophage reprogramming, anti-inflammatory and pro-resolutive responses.

Influenza A is a virus from theOrthomixoviridaefamily responsible for high lethality rates and morbidity, despite clinically proven vaccination strategies and some anti-viral therapies. The eicosanoid Lipoxin A4 (LXA4) promotes the resolution of inflammation by decreasing cell recruitment and pro-inflammatory cytokines release, but also for inducing activation of apoptosis, efferocytosis, and macrophage reprogramming. Here, we evaluated whether a synthetic lipoxin mimetic, designated AT-01-KG, would improve the course of influenza A infection in a murine model. Mice were infected with influenza A/H1N1 and treated with AT-01-KG (1.7μg/kg/day, i.p.) at day 3 post-infection. AT-01-KG attenuated mortality, reducing leukocyte infiltration and lung damage at day 5 and day 7 post-infection. AT-01-KG is a Formyl Peptide Receptor 2 (designated FPR2/3 in mice) agonist, and the protective responses were not observed in fpr2/3-/-animals. In mice treated with LXA4(50μg/kg/day, i.p., days 3-6 post-infection), at day 7, macrophage reprogramming was observed, as seen by a decrease in classically activated macrophages and an increase in alternatively activated macrophages in the lungs. Furthermore, the number of apoptotic cells and cells undergoing efferocytosis was increased in the lavage of treated mice. Treatment also modulated the adaptive immune response, increasing the number of T helper 2 cells (Th2) and regulatory T (Tregs) cells in the lungs of the treated mice. Therefore, treatment with a lipoxin A4analog was beneficial in a model of influenza A infection in mice. The drug decreased inflammation and promoted resolution and beneficial immune responses, suggesting it may be useful in patients with severe influenza.

Treatment with lipoxin A 4 improves influenza A infection outcome through macrophage reprogramming, anti-inflammatory and pro-resolutive responses.

Here, we evaluated whether a synthetic lipoxin mimetic, designated AT-01-KG, would improve the course of influenza A infection in a murine model. Mice were infected with influenza A/H1N1 and treated with AT-01-KG (1.7 mg/kg/day, i.p.) at day 3 post-infection. Mortality rate was assessed up to day 21 and inflammatory parameters were assessed at days 5 and 7. AT-01-KG attenuated mortality, reducing leukocyte infiltration and lung damage at day 5 and day 7 post-infection. AT-01-KG is a Formyl Peptide Receptor 2 (designated FPR2/3 in mice) agonist, and the protective responses were not observed in FPR2/3 -/- animals. In mice treated with LXA4 (50mg/kg/day, i.p., days 3-6 post-infection), at day 7, macrophage reprogramming was observed, as seen by a decrease in classically activated macrophages and an increase in alternatively activated macrophages in the lungs. Furthermore, the number of apoptotic cells and cells undergoing efferocytosis was increased in the lavage of treated mice. Treatment also modulated the adaptive immune response, increasing the number of anti-inflammatory T cells (Th2) and regulatory T (Tregs) cells in the lungs of the treated mice. Therefore, treatment with a lipoxin A4 analog was beneficial in a model of influenza A infection in mice. The drug decreased inflammation and promoted resolution and beneficial immune responses, suggesting it may be useful in patients with severe influenza.

(Homo-)harringtonine prevents endothelial inflammation through IRF-1 dependent downregulation of VCAM1 mRNA expression and inhibition of cell adhesion molecule protein biosynthesis

The plant alkaloid homoharringtonine (HHT) is a Food and Drug Administration (FDA)-approved drug for the treatment of hematologic malignancies. In addition to its well-established antitumor activity, accumulating evidence attributes anti-inflammatory effects to HHT, which have mainly been studied in leukocytes to date. However, a potential influence of HHT on inflammatory activation processes in endothelial cells, which are a key feature of inflammation and a prerequisite for the leukocyte-endothelial cell interaction and leukocyte extravasation, remains poorly understood. In this study, the anti-inflammatory potential of HHT and its derivative harringtonine (HT) on the TNF-induced leukocyte-endothelial cell interaction was assessed, and the underlying mechanistic basis of these effects was elucidated. HHT affected inflammation in vivo in a murine peritonitis model by reducing leukocyte infiltration and proinflammatory cytokine expression as well as ameliorating abdominal pain behavior. In vitro, HT and HHT impaired the leukocyte-endothelial cell interaction by decreasing the expression of the endothelial cell adhesion molecules intracellular adhesion molecule −1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). This effect was mediated by a bipartite mechanism. While HHT did not affect the prominent TNF-induced pro-inflammatory NF-ĸB signaling cascade, the compound downregulated the VCAM1 mRNA expression in an IRF-1-dependent manner and diminished active ICAM1 mRNA translation as determined by polysome profiling. This study highlights HHT as an anti-inflammatory compound that efficiently hampers the leukocyte-endothelial cell interaction by targeting endothelial activation processes.

Role of ADAR2-mediated innate immune responses in vascular inflammation and atherosclerosis

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG) Background Adenosine-to-inosine (A-to-I) RNA editing is an epitranscriptional regulator of RNA metabolism, catalyzed by the adenosine deaminases acting on RNA-1/-2 (ADAR1/2). Whether ADAR2 play a role in atherosclerotic processes remains elusive. Aim We examined the role of ADAR2 in vascular endothelial cell (EC) immunomodulatory responses and its biological relevance in murine and human atherosclerosis. Methods The role of ADAR2 in atherosclerosis was determined in a double ADAR2 and ApoE knockout mice fed with Western diet for 20 weeks. Three mouse genetic models (global and inducible vascular endothelial cell (iEC)-restricted ADAR2 and IL6ST knockout) were used to determine the role of ADAR2 in IL-6-related innate immunity. Expression levels of ADAR2 were measured in peripheral blood mononuclear cells derived from 533 individuals at risk for atherosclerotic vascular disease. Structural vascular measurements were used, including intima-media thickness (IMT), carotid wall maximum thickness (maxWT), and the number of atheromatous plaques by carotid and femoral artery ultrasonography as surrogate markers of subclinical arterial disease. Study participants were followed for major adverse cardiovascular events (MACE). Results Genetic ablation of ADAR2 resulted in a reduction of atherosclerotic plaque burden and necrotic core in the aorta and brachiocephalic trunk as well as reduced leukocyte infiltration in atherosclerotic plaques without affecting cholesterol levels in mice. Blood analysis revealed decreased pro-inflammatory cytokine levels including IL-6 after treatment of ADAR2/APOE KO mice compared to littermate control mice. Intravital imaging of IL-6-inflamed vascular endothelium showed that rolling and adhesion of leukocyte subsets to vascular wall were impaired by 2-fold in ADAR2-/- or iEC-ADAR2-/- KO mice. In vivo and ex vivo histological examination revealed that endothelial ADAR2 controls endothelial vascular adhesion molecule expression induced by gp130 signaling. ADAR2-deficient transcriptome revealed a selective upregulation of a conserved group of miRNAs targeting the IL6ST mRNA. Multilayered rescue studies including miR-inhibitors restored IL6ST levels after ADAR2 deficiency. In humans, increased ADAR2 expression was independently associated with the presence of CAD, higher C-reactive protein and mean carotid IMT after adjustment for traditional risk factors. Increased ADAR2 at baseline was prospectively associated with accelerated progression of subclinical atherosclerosis reflected by the increased number of carotid plaques at follow-up (OR=3.74, P<0.05). Finally, high ADAR2 levels were independently associated with higher MACE incidence across a median follow-up period of 54 months. Conclusion Posttranscriptional control of vascular inflammation by ADAR2 is integral for the development and progression of atherosclerosis in mice and humans. Our findings suggest that ADAR2 may serve as a promising therapeutic target for atherosclerosis.Role of ADAR2 in atherosclerosis

Astrocytic RIPK3 exerts protective anti-inflammatory activity during viral encephalitis via induction of serpin protease inhibitors.

Flaviviruses pose a significant threat to public health due to their ability to infect the central nervous system (CNS) and cause severe neurologic disease. Astrocytes play a crucial role in the pathogenesis of flavivirus encephalitis through their maintenance of blood-brain barrier (BBB) integrity and their modulation of immune cell recruitment and activation within the CNS. We have previously shown that receptor interacting protein kinase-3 (RIPK3) is a central coordinator of neuroinflammation during CNS viral infection, a function that occurs independently of its canonical function in inducing necroptotic cell death. To date, however, roles for necroptosis-independent RIPK3 signaling in astrocytes are poorly understood. Here, we use mouse genetic tools to induce astrocyte-specific deletion, overexpression, and chemogenetic activation of RIPK3 to demonstrate an unexpected anti-inflammatory function for astrocytic RIPK3. RIPK3 activation in astrocytes was required for host survival in multiple models of flavivirus encephalitis, where it restricted neuropathogenesis by limiting immune cell recruitment to the CNS. Transcriptomic analysis revealed that, despite inducing a traditional pro-inflammatory transcriptional program, astrocytic RIPK3 paradoxically promoted neuroprotection through the upregulation of serpins, endogenous protease inhibitors with broad immunomodulatory activity. Notably, intracerebroventricular administration of SerpinA3N in infected mice preserved BBB integrity, reduced leukocyte infiltration, and improved survival outcomes in mice lacking astrocytic RIPK3. These findings highlight a previously unappreciated role for astrocytic RIPK3 in suppressing pathologic neuroinflammation and suggests new therapeutic targets for the treatment of flavivirus encephalitis.

C57BL/6J-Trex1em3Lutzy/J KO mouse as a type 1 IFN dependent model for SLE preclinical drug discovery

Abstract Systemic Lupus Erythematosus is a chronic autoimmune disease characterized by a broad spectrum of clinical manifestations. An important role of the type I interferon (IFN) pathway has been identified in SLE patients. No one mouse model completely recapitulates the complexity of the human disease. Nonetheless, mouse models of SLE have been instrumental in our understanding of the genetic and cellular requirements for SLE induction and represent a valuable resource for testing SLE therapies and drug candidates in vivo. Here we present preclinical reference data in a Trex1KO mouse model generated at the Jackson Laboratory using CRISPR technology. Trex1KO mice have a median survival of approximately 165 – 185 days. Body weight is significantly reduced in both male and female KO versus WT. Unlike NZBWF1/J and MRL/MpJ mouse models that develop lupus nephritis, the Trex1 KO develops dilated cardiomyopathy due to leukocyte infiltration and cardiomyocyte degeneration. Here we treated KO mice with anti-IFNα antibodies through 32 weeks of age. We observed a significant treatment effect on survival, reduction in leukocyte infiltration and degeneration of the heart, and percentage of T peripheral helper T cells in spleen. A mouse model recapitulating a role for type I IFN in disease pathogenesis has been lacking in the common models of SLE, and these data indicate that the Trex1KO model is relevant for the screening of targeted therapies of type I IFN and related pathways.

Healing effect of the polysaccharide extract from Cissus sicyoides leaves in a rat model of excisional cutaneous wounds

The healing process is a physiological event involving cellular and molecular mechanisms to remove and replace damaged tissues, allowing new extracellular matrix synthesis. Based on previous studies showing antinociceptive, anti-inflammatory and antioxidative effects of the polysaccharide-rich extract of Cissus sicyoides leaves (PE-Cs) in classical inflammation models, this study evaluated the PE-Cs effect in the rat model of excisional cutaneous wound. The topical treatment with PE-Cs (0.01–1%) for 14 days reduced (43–50%, 5 – 7th day) the wound area and increased the nociceptive threshold (38.85–47.65%, 6th hour - 7th day). PE-Cs reduced leukocyte infiltration (42% - 2nd day and 84% - 5th day) and increased fibroblasts/myofibroblasts (59% - 5th day and 24% - 7th day), collagen deposition (30% - 5th day and 23% - 7th day) and blood vessels (60% - 5th day and 50% - 7th day). PE-Cs reduced the oxidative stress marker MDA (33% - 2nd day), and increased GSH levels (35–27%, 2 – 7th day). In conclusion, PE-Cs acts in the inflammatory and proliferative phases of the rat healing process via acceleration of wound closure, reduction of inflammatory signs, oxidative stress markers and stimulation of fibroplasia, collagenesis and increased blood vessel count.

Human-induced pluripotent stem cell-derived neural stem cell exosomes improve blood-brain barrier function after intracerebral hemorrhage by activating astrocytes via PI3K/AKT/MCP-1 axis.

JOURNAL/nrgr/04.03/01300535-202502000-00029/figure1/v/2024-05-28T214302Z/r/image-tiff Cerebral edema caused by blood-brain barrier injury after intracerebral hemorrhage is an important factor leading to poor prognosis. Human-induced pluripotent stem cell-derived neural stem cell exosomes (hiPSC-NSC-Exos) have shown potential for brain injury repair in central nervous system diseases. In this study, we explored the impact of hiPSC-NSC-Exos on blood-brain barrier preservation and the underlying mechanism. Our results indicated that intranasal delivery of hiPSC-NSC-Exos mitigated neurological deficits, enhanced blood-brain barrier integrity, and reduced leukocyte infiltration in a mouse model of intracerebral hemorrhage. Additionally, hiPSC-NSC-Exos decreased immune cell infiltration, activated astrocytes, and decreased the secretion of inflammatory cytokines like monocyte chemoattractant protein-1, macrophage inflammatory protein-1α, and tumor necrosis factor-α post-intracerebral hemorrhage, thereby improving the inflammatory microenvironment. RNA sequencing indicated that hiPSC-NSC-Exo activated the PI3K/AKT signaling pathway in astrocytes and decreased monocyte chemoattractant protein-1 secretion, thereby improving blood-brain barrier integrity. Treatment with the PI3K/AKT inhibitor LY294002 or the monocyte chemoattractant protein-1 neutralizing agent C1142 abolished these effects. In summary, our findings suggest that hiPSC-NSC-Exos maintains blood-brain barrier integrity, in part by downregulating monocyte chemoattractant protein-1 secretion through activation of the PI3K/AKT signaling pathway in astrocytes.

Bone marrow-derived mesenchymal stem cells mitigate chronic colitis and enteric neuropathy via anti-inflammatory and anti-oxidative mechanisms

Current treatments for inflammatory bowel disease (IBD) are often inadequate due to limited efficacy and toxicity, leading to surgical resection in refractory cases. IBD’s broad and complex pathogenesis involving the immune system, enteric nervous system, microbiome, and oxidative stress requires more effective therapeutic strategies. In this study, we investigated the therapeutic potential of bone marrow-derived mesenchymal stem cell (BM-MSC) treatments in spontaneous chronic colitis using the Winnie mouse model which closely replicates the presentation and inflammatory profile of ulcerative colitis. The 14-day BM-MSC treatment regimen reduced the severity of colitis, leading to the attenuation of diarrheal symptoms and recovery in body mass. Morphological and histological abnormalities in the colon were also alleviated. Transcriptomic analysis demonstrated that BM-MSC treatment led to alterations in gene expression profiles primarily downregulating genes related to inflammation, including pro-inflammatory cytokines, chemokines and other biomarkers of inflammation. Further evaluation of immune cell populations using immunohistochemistry revealed a reduction in leukocyte infiltration upon BM-MSC treatment. Notably, enteric neuronal gene signatures were the most impacted by BM-MSC treatment, which correlated with the restoration of neuronal density in the myenteric ganglia. Moreover, BM-MSCs exhibited neuroprotective effects against oxidative stress-induced neuronal loss through antioxidant mechanisms, including the reduction of mitochondrial-derived superoxide and attenuation of oxidative stress-induced HMGB1 translocation, potentially relying on MSC-derived SOD1. These findings suggest that BM-MSCs hold promise as a therapeutic intervention to mitigate chronic colitis by exerting anti-inflammatory effects and protecting the enteric nervous system from oxidative stress-induced damage.

Aprepitant Restores Corneal Sensitivity and Reduces Pain inDED.

This study aims to assess the efficacy of two aprepitant formulations (X1 and X2), in a preclinical model of dry eye disease (DED) induced by benzalkonium chloride (BAK). Two aprepitant formulations were tested on 7 to 8-week-old male mice for their efficacy. In vivo corneal fluorescein staining assessed epithelial damage as the primary end point on days 0, 3, 5, 7, 9, 12, and 14 using slit-lamp microscopy. The DED model was induced with 0.2% BAK twice daily for the first week and once daily for the next week. Mice were randomly assigned to 5 treatment groups: Aprepitant X1 (n = 10) and X2 (n = 10) formulation, 2mg/mL dexamethasone (n = 10), control vehicle X (n = 10), 0.2% hyaluronic acid (n = 10), or no treatment (n = 10). Eye wiping, phenol red, and Cochet Bonnet tests assessed ocular pain, tear fluid secretion, and nerve function. After 7days, the mice were euthanized to quantify leukocyte infiltration and corneal nerve density. Topical aprepitant X1 reduced BAK-induced corneal damage and pain compared to gel vehicle X (P = 0.007) and dexamethasone (P = 0.021). Aprepitant X1 and X2 improved corneal sensitivity versus gel vehicle X and dexamethasone (P < 0.001). Aprepitant X1 reduced leukocyte infiltration (P < 0.05) and enhanced corneal nerve density (P < 0.001). Tear fluid secretion remained statistically unchanged in both the X1 and X2 groups. Aprepitant formulation X1 reduced pain, improved corneal sensitivity and nerve density, ameliorated epitheliopathy, and reduced leukocyte infiltration in male mouse corneas. Aprepitant emerges as a safe, promising therapeutic prospect for the amelioration of DED's associated symptoms.

Emodin inhibits HDAC6 mediated NLRP3 signaling and relieves chronic inflammatory pain in mice.

Chronic pain reduces the quality of life and ability to function of individuals suffering from it, making it a common public health problem. Neuroinflammation which is mediated by the Nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation in the spinal cord participates and modulates chronic pain. A chronic inflammatory pain mouse model was created in the current study by intraplantar injection of complete Freund's adjuvant (CFA) into C57BL/6J left foot of mice. Following CFA injection, the mice had enhanced pain sensitivities, decreased motor function, increased spinal inflammation and activated spinal astrocytes. Emodin (10 mg/kg) was administered intraperitoneally into the mice for 3 days. As a result, there were fewer spontaneous flinches, higher mechanical threshold values and greater latency to fall. Additionally, in the spinal cord, emodin administration reduced leukocyte infiltration level, downregulated protein level of IL-1β, lowered histone deacetylase (HDAC)6 and NLRP3 inflammasome activity and suppressed astrocytic activation. Emodin also binds to HDAC6 via four electrovalent bonds. In summary, emodin treatment blocked the HDAC6/NLRP3 inflammasome signaling, suppresses spinal inflammation and alleviates chronic inflammatory pain.

Beneficial Effect of Fingolimod in a Lafora Disease Mouse Model by Preventing Reactive Astrogliosis-Derived Neuroinflammation and Brain Infiltration of T-lymphocytes.

Lafora disease (LD; OMIM#254780) is a rare, devastating, and fatal form of progressive myoclonus epilepsy that affects young adolescents and has no treatment yet. One of the hallmarks of the disease is the accumulation of aberrant poorly branched forms of glycogen (polyglucosans, PGs) in the brain and peripheral tissues. The current hypothesis is that this accumulation is causative of the pathophysiology of the disease. Another hallmark of LD is the presence of neuroinflammation. We have recently reported the presence of reactive glia-derived neuroinflammation in LD mouse models and defined the main inflammatory pathways that operate in these mice, mainly TNF and IL-6 signaling pathways. In addition, we described the presence of infiltration of peripheral immune cells in the brain parenchyma, which could cooperate and aggravate the neuroinflammatory landscape of LD. In this work, we have checked the beneficial effect of two compounds with the capacity to ameliorate neuroinflammation and reduce leukocyte infiltration into the brain, namely fingolimod and dimethyl fumarate. Our results indicate a beneficial effect of fingolimod in reducing reactive astrogliosis-derived neuroinflammation and T-lymphocyte infiltration, which correlated with the improved behavioral performance of the treated Epm2b-/- mice. On the contrary, dimethyl fumarate, although it was able to reduce reactive astrogliosis, was less effective in preventing neuroinflammation and T-lymphocyte infiltration and in modifying behavioral tests.

The Neuropeptide α-Melanocyte–Stimulating Hormone Prevents Persistent Corneal Edema following Injury

Corneal endothelial cells (CEnCs) regulate corneal hydration and maintain tissue transparency through their barrier and pump function. However, these cells exhibit limited regenerative capacity following injury. Currently, corneal transplantation is the only established therapy for restoring endothelial function, and there are no pharmacologic interventions available for restoring endothelial function. This study investigated the efficacy of the neuropeptide α-melanocyte-stimulating hormone (α-MSH) in promoting endothelial regeneration during the critical window between ocular injury and the onset of endothelial decompensation using an established murine model of injury using transcorneal freezing. Local administration of α-MSH following injury prevented corneal edema and opacity, reduced leukocyte infiltration, and limited CEnC apoptosis while promoting their proliferation. These results suggest that α-MSH has a proregenerative and cytoprotective function on CEnCs and shows promise as a therapy for the prevention and management of corneal endothelial dysfunction.

Analgesic and Anti-Inflammatory Effects of 1% Topical Cannabidiol Gel in Animal Models.

Introduction: Cannabidiol (CBD), a phytocannabinoid isolated from cannabis plants, is an interesting candidate for studying its anti-inflammatory effects, especially in the pre-clinical and animal models. Its anti-inflammatory effects, such as reduction of edema and arthritis, have been demonstrated in animal models. However, topical CBD administration requires further evaluation of CBD dosage and efficacy in animal models and clinical settings. Methods: This in vivo study investigated the anti-inflammatory effects of topical CBD administration in an animal model. Scientific experiments, including the formalin test, writhing test, carrageenan-induced edema, histopathological examination, and detection of various proinflammatory mediators, were performed. Results: The anti-inflammatory effects in vivo after inflammation induction, represented by decreased times of paw licking, degree of paw edema, and decreased writhing response, showed that 1% of tropical CBD use had significantly comparable or better anti-inflammatory effects when compared with tropical diclofenac, an anti-inflammatory agent. Moreover, the anti-inflammatory effects were significant compared with the placebo. In addition, the histopathological examination showed that topical CBD drastically reduced leukocyte infiltration and the degree of inflammation. This study also showed that the levels of various proinflammatory mediators in the plasma of mice treated with topical CBD did not differ from those treated with diclofenac. Conclusions: The topical administration of 1% CBD gel is a potentially effective candidate for an anti-inflammatory agent. Candidate for an anti-inflammatory agent.

Ifit2 restricts murine coronavirus spread to the spinal cord white matter and its associated myelin pathology.

Interferon-induced protein with tetratricopeptide repeats 2, Ifit2, is critical in restricting neurotropic murine-β-coronavirus, RSA59 infection. RSA59 intracranial injection of Ifit2-deficient (-/-) compared to wild-type (WT) mice results in impaired acute microglial activation, reduced CX3CR1 expression, limited migration of peripheral lymphocytes into the brain, and impaired virus control followed by severe morbidity and mortality. While the protective role of Ifit2 is established for acute viral encephalitis, less is known about its influence during the chronic demyelinating phase of RSA59 infection. To understand this, RSA59 infected Ifit2-/- and Ifit2+/+ (WT) were observed for neuropathological outcomes at day 5 (acute phase) and 30 post-infection (chronic phase). Our study demonstrates that Ifit2 deficiency causes extensive RSA59 spread throughout the spinal cord gray and white matter, associated with impaired CD4+ T and CD8+ T cell infiltration. Further, the cervical lymph nodes of RSA59 infected Ifit2-/- mice showed reduced activation of CD4+ T cells and impaired IFNγ expression during acute encephalomyelitis. Interestingly, BBB integrity was better preserved in Ifit2-/- mice, as evidenced by tight junction protein Claudin-5 and adapter protein ZO-1 expression surrounding the meninges and blood vessels and decreased Texas red dye uptake, which may be responsible for reduced leukocyte infiltration. In contrast to sparse myelin loss in WT mice, the chronic disease phase in Ifit2-/- mice was associated with severe demyelination and persistent viral load, even at low inoculation doses. Overall, our study highlights that Ifit2 provides antiviral functions by promoting acute neuroinflammation and thereby aiding virus control and limiting severe chronic demyelination. IMPORTANCE Interferons execute their function by inducing specific genes collectively termed as interferon-stimulated genes (ISGs), among which interferon-induced protein with tetratricopeptide repeats 2, Ifit2, is known for restricting neurotropic viral replication and spread. However, little is known about its role in viral spread to the spinal cord and its associated myelin pathology. Toward this, our study using a neurotropic murine β-coronavirus and Ifit2-deficient mice demonstrates that Ifit2 deficiency causes extensive viral spread throughout the gray and white matter of the spinal cord accompanied by impaired microglial activation and T cell infiltration. Furthermore, infected Ifit2-deficient mice showed impaired activation of T cells in the cervical lymph node and relatively intact blood-brain barrier integrity. Overall, Ifit2 plays a crucial role in mounting host immunity against neurotropic murine coronavirus in the acute phase while preventing mice from developing viral-induced severe chronic neuroinflammatory demyelination, the characteristic feature of human neurological disease multiple sclerosis (MS).

The Sigma-1 Receptor Is a Novel Target for Improving Cold Preservation in Rodent Kidney Transplants.

Kidney transplantation is the preferred treatment for patients with end-stage kidney disease. Maintaining organ viability between donation and transplantation, as well as minimizing ischemic injury, are critically important for long-term graft function and survival. Moreover, the increasing shortage of transplantable organs is a considerable problem; thus, optimizing the condition of grafts is a pivotal task. Here, rodent models of kidney transplantation and cold storage were used to demonstrate that supplementation of a preservation solution with Sigma-1 receptor (S1R) agonist fluvoxamine (FLU) reduces cold and warm ischemic injury. Post-transplant kidney function was improved, histological injury was mitigated, and mRNA expression of two tubular injury markers-kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin-was robustly reduced. In addition, renal inflammation was diminished, as shown by reduced leukocyte infiltration and pro-inflammatory cytokine expression. In the cold ischemia model, FLU ameliorated structural injury profoundly after 2 h as well as 24 h. The reduced number of TUNEL-positive and Caspase 3-positive cells suggests the anti-apoptotic effect of FLU. None of these beneficial effects of FLU were observed in S1R-/- mice. Of note, organ damage in FLU-treated kidneys after 24 h of cold storage was similar to just 2 h without FLU. These results indicate that S1R agonists can prolong storage time and have great potential in improving organ preservation and in alleviating the problem of organ shortages.

Optimization and Characterization of Novel ALCAM-Targeting Antibody Fragments for Transepithelial Delivery.

Activated leukocyte cell adhesion molecule (ALCAM) is a cell adhesion molecule that supports T cell activation, leukocyte migration, and (lymph)angiogenesis and has been shown to contribute to the pathology of various immune-mediated disorders, including asthma and corneal graft rejection. In contrast to monoclonal antibodies (mAbs) targeting ALCAM's T cell expressed binding partner CD6, no ALCAM-targeting mAbs have thus far entered clinical development. This is likely linked with the broad expression of ALCAM on many different cell types, which increases the risk of eliciting unwanted treatment-induced side effects upon systemic mAb application. Targeting ALCAM in surface-exposed tissues, such as the lungs or the cornea, by a topical application could circumvent this issue. Here, we report the development of various stability- and affinity-improved anti-ALCAM mAb fragments with cross-species reactivity towards mouse, rat, monkey, and human ALCAM. Fragments generated in either mono- or bivalent formats potently blocked ALCAM-CD6 interactions in a competition ELISA, but only bivalent fragments efficiently inhibited ALCAM-ALCAM interactions in a leukocyte transmigration assay. The different fragments displayed a clear size-dependence in their ability to penetrate the human corneal epithelium. Furthermore, intranasal delivery of anti-ALCAM fragments reduced leukocyte infiltration in a mouse model of asthma, confirming ALCAM as a target for topical application in the lungs.