Activation of endothelial roundabout receptor 4 reduces the severity of virus-induced keratitis

Antiangiogenic molecules exert a feedback control to restrain pathological angiogenesis, which includes physical binding or inhibition of angiogenic signaling in blood vessel endothelial cells. The latter is the case in which Slit2 ligand-dependent activation of the blood vessel endothelial cell receptor roundabout 4 (Robo4) occurs. In this study, we demonstrate that Robo4 receptors are upregulated following HSV infection of the eye on the majority of the new blood vessel endothelial cells that occur in the corneal stroma. However, expression levels of the ligand for Robo4 receptors, Slit2, was not significantly increased during the disease process, and the knockdown of Slit2 gene expression using lentiviral short hairpin RNAs had no effect on the extent of pathological angiogenesis. In contrast, providing additional Slit2 protein by subconjunctival administration resulted in significantly reduced angiogenesis. The Slit2 binding to Robo4 was shown to block the downstream vascular endothelial growth factor signaling molecules Arf 6 and Rac 1 and reduce the antiapoptotic molecule Bcl-xL in blood vessel endothelial cells. Our results indicate that augmenting the host Robo4/Slit2 system could provide a useful therapeutic approach to control pathological angiogenesis associated with HSV induced stromal keratitis.     

Galectin-1 reduces the severity of herpes simplex virus-induced ocular immunopathological lesions

Stromal keratitis is a chronic immunopathological lesion of the eye caused by HSV-1 infection that can result in blindness. Because the inflammatory lesions are primarily orchestrated by Th1 cells, and to a lesser extent by Th17 cells, inhibiting their activity represents a useful form of therapy. In this study we evaluated the therapeutic potential of galectin-1 (gal-1), an endogenous lectin that in some autoimmune diseases was shown to suppress the functions of Th1 and Th17 cells. Treatment was begun at different times after ocular infection with HSV and the outcome was assessed clinically as well as for effects on various immune parameters. Treatment with recombinant gal-1 significantly diminished stromal keratitis lesion severity and the extent of corneal neovascularization. Treated mice had reduced numbers of IFN-γ- and IL-17-producing CD4(+) T cells, as well as neutrophil infiltration in the cornea. Furthermore, disease severity was greater in gal-1 knockout mice compared with their wild-type counterparts. The many effects of gal-1 treatment include reduction in the production of proinflammatory cytokines and chemokines, increased production of IL-10, and inhibitory effects on molecules involved in neovascularization. To our knowledge, our findings are the first to show that gal-1 treatment represents a useful approach to control lesion severity in a virally induced immunopathological disease.     

IL-11 receptor alpha in the pathogenesis of IL-13-induced inflammation and remodeling

IL-13 is a major stimulator of inflammation and tissue remodeling at sites of Th2 inflammation. In Th2-dominant inflammatory disorders such as asthma, IL-11 is simultaneously induced. However, the relationship(s) between IL-11 and IL-13 in these responses has not been defined, and the role(s) of IL-11 in the genesis of the tissue effects of IL-13 has not been evaluated. We hypothesized that IL-11, signaling via the IL-11Ralpha-gp130 receptor complex, plays a key role in IL-13-induced tissue responses. To test this hypothesis we compared the expression of IL-11, IL-11Ralpha, and gp130 in lungs from wild-type mice and transgenic mice in which IL-13 was overexpressed in a lung-specific fashion. We simultaneously characterized the effects of a null mutation of IL-11Ralpha on the tissue effects of transgenic IL-13. These studies demonstrate that IL-13 is a potent stimulator of IL-11 and IL-11Ralpha. They also demonstrate that IL-13 is a potent stimulator of inflammation, fibrosis, hyaluronic acid accumulation, myofibroblast accumulation, alveolar remodeling, mucus metaplasia, and respiratory failure and death in mice with wild-type IL-11Ralpha loci and that these alterations are ameliorated in the absence of IL-11Ralpha. Lastly, they provide insight into the mechanisms of these processes by demonstrating that IL-13 stimulates CC chemokines, matrix metalloproteinases, mucin genes, and gob-5 and stimulates and activates TGF-beta1 via IL-11Ralpha-dependent pathways. When viewed in combination, these studies demonstrate that IL-11Ralpha plays a key role in the pathogenesis of IL-13-induced inflammation and remodeling.     

Role of 5-lipoxygenase in IL-13-induced pulmonary inflammation and remodeling

Exaggerated levels of IL-13 and leukotriene (LT) pathway activation frequently coexist at sites of Th2 inflammation and in tissue fibrotic responses. However, the relationship(s) between the IL-13 and LTs in these responses have not been defined. We hypothesized that the 5-lipoxygenase (5-LO) pathway of LT metabolism plays an important role in the pathogenesis of IL-13-induced chronic inflammation and remodeling. To test this hypothesis, we evaluated the effects of IL-13 on components of the 5-LO metabolic and activation pathways. We also compared the effects of transgenic IL-13 in C57BL/6 mice with wild-type and null 5-LO genetic loci. These studies demonstrate that IL-13 increases the levels of mRNA encoding cytosolic phospholipase A(2), LTA(4) hydrolase, and 5-LO-activating protein without altering the expression of 5-LO, LTC(4) synthase, LTB(4) receptors 1 and 2, and cysteinyl-LT receptors 1 and 2. They also demonstrate that this activation is associated with the enhanced accumulation of LTB(4) but not of cysteinyl-LTs. Furthermore, they demonstrate that this stimulation plays a critical role in the pathogenesis of IL-13-induced inflammation, tissue fibrosis, and respiratory failure-induced death while inhibiting alveolar remodeling. Lastly, mechanistic insights are provided by demonstrating that IL-13-induced 5-LO activation is required for optimal stimulation and activation of TGF-beta(1) and the inhibition of matrix metalloproteinase-12. When viewed in combination, these studies demonstrate that 5-LO plays an important role in IL-13-induced inflammation and remodeling.     

Role of CCR5 in the pathogenesis of IL-13-induced inflammation and remodeling

IL-13 is a major effector at sites of Th2 inflammation and tissue remodeling. In these locations, it frequently coexists with the CCR5 chemokine receptor and its ligands MIP-1alpha/CCL3 and MIP-1beta/CCL4. We hypothesized that CCR5 induction and activation play important roles in the pathogenesis of IL-13-induced tissue responses. To test this hypothesis, we evaluated the effects of IL-13 on the expression of CCR5 in the murine lung. We also compared the effects of lung-targeted transgenic IL-13 in mice treated with anti-CCR5 or an Ab control and mice with wild-type or null CCR5 loci. These studies demonstrate that IL-13 is a potent stimulator of epithelial cell CCR5 expression. They also demonstrate that CCR5 neutralization or a deficiency of CCR5 significantly decreases IL-13-induced inflammation, alveolar remodeling, structural and inflammatory cell apoptosis, and respiratory failure and death. Lastly, these studies provide mechanistic insights by demonstrating that CCR5 is required for optimal IL-13 stimulation of select chemokines (MIP-1alpha/CCL3, MIP-1beta/CCL4, MCP-1/CCL-2), matrix metalloproteinase-9 and cell death regulators (Fas, TNF, TNFR1, TNFR2, Bid), optimal IL-13 inhibition of alpha1-antitrypsin, and IL-13-induction of and activation of caspases-3, -8, and-9. Collectively, these studies demonstrate that CCR5 plays a critical role in the pathogenesis of IL-13-induced inflammation and tissue remodeling.     

IL-10 reduces grain dust-induced airway inflammation and airway hyperreactivity.

To determine whether interleukin-10 (IL-10) could alter the development of grain dust-induced airway disease, we pretreated mice with either saline or IL-10 intravenously, exposed the mice to an inhalation challenge with corn dust extract (CDE), and measured inflammation and the development of airway hyperreactivity. Pretreatment with IL-10, in comparison to saline, reduced the concentration and percentage of polymorphonuclear cells in the lavage fluid 30 min after the inhalation challenge with CDE (P < 0. 05). In comparison to saline-treated mice, IL-10 did not significantly alter the degree of airway hyperreactivity 30 min after the exposure to CDE. IL-10-treated mice lavaged 18 h after challenge with CDE also exhibited a lower percentage of polymorphonuclear cells in the lavage fluid (P < 0.05) and had significantly less airway hyperreactivity than did mice pretreated with the saline placebo (P < 0.05). These findings indicate that exogenous IL-10 is effective in reducing airway inflammation and airway hyperreactivity due to the inhalation of CDE.     

Apilimod inhibits the production of IL-12 and IL-23 and reduces dendritic cell infiltration in psoriasis

Psoriasis is characterized by hyperplasia of the epidermis and infiltration of leukocytes into both the dermis and epidermis. IL-23, a key cytokine that induces T(H)17 cells, has been found to play a critical role in the pathogenesis of psoriasis. Apilimod is a small-molecule compound that selectively suppresses synthesis of IL-12 and IL-23. An open-label clinical study of oral administration of apilimod was conducted in patients with psoriasis. Substantial improvements in histology and clinical measurements were observed in patients receiving 70 mg QD. The expression of IL-23p19 and IL-12/IL-23p40 in skin lesions was significantly reduced in this dose group, with a simultaneous increase in IL-10 observed. A decrease in the levels of T(H)1 and T(H)17 cytokines/chemokines in skin lesions followed these p19 and p40 changes. In parallel, a reduction in skin-infiltrating CD11c(+) dendritic cells and CD3(+) T cells was seen, with a greater decrease in the CD11c(+) population. This was accompanied by increases in T and B cells, and decreases in neutrophils and eosinophils in the periphery. This study demonstrates the immunomodulatory activity of apilimod and provides clinical evidence supporting the inhibition of IL-12/IL-23 synthesis for the treatment of T(H)1- and T(H)17-mediated inflammatory diseases.     

Endogenous hydrogen sulfide reduces airway inflammation and remodeling in a rat model of asthma

Endogenous hydrogen sulfide (H₂S) is hypothesized to have an important role in systemic inflammation. We investigated if endogenous H₂S may be a crucial mediator in airway inflammation and airway remodeling in a rat model of asthma and if endogenous H₂S may exert its anti-inflammatory effect by inhibiting inducible nitric oxide synthase (iNOS)/NO pathway. Cystathionine-γ-lyase (CSE; a H₂S-synthesizing enzyme) was mainly expressed in airway and vascular smooth muscle cells in rat lung tissue. Levels of endogenous H₂S was decreased in pulmonary tissue in ovalbumin (OVA)-treated rats. Exogenous administration of NaHS alleviated airway inflammation and airway remodeling: peak expiratory flow (PEF) increased, goblet cell hyperplasia and collagen deposition score decreased, with decreased total cells recovered from bronchoalveolar fluid (BALF) and influx of eosinophils and neutrophils. The H₂S levels of serum and lung tissue were positively correlated with PEF and negatively correlated with the level of eosinophils and neutrophils in BALF, score of lung pathology. NaHS treatment significantly attenuated pulmonary iNOS activation in OVA-treated rats. These results suggest that the CSE/H₂S pathway plays an anti-inflammatory and anti-remodeling part in asthma pathogenesis and could be a novel target in prevention and treatment of asthma.     

Studies on the interaction of Sendai virus with liposomal membranes. Sendai virus-induced agglutination of liposomes containing glycophorin

Liposomes constituted with the major sialoglycoprotein of human erythrocytes, glycophorin, were used as models for studies on cell-virus interactions. Liposomes composed of egg yolk phosphatidylcholine, cholesterol and glycophorin were found to interact with the paramyxovirus HVJ to form aggregates. The aggregation process was temperature dependent: it was maximal at 0 degrees C and decreased with increase of the incubation temperature. The activity of viral neuraminidase is also temperature dependent, and it increases with increase of the incubation temperature; release of N-acetylneuraminic acid was negligible at 0 degrees C. Shift-up of the incubation temperature immediately cancelled HVJ-induced agglutination of liposomes. Viruses attached to liposomes seemed to be released into the supernatant when the 'virus-liposome' complex formed at 0 degrees C was incubated at 37 degrees C, possibly as a result of breakdown of the 'binding' site by neuraminidase. The characteristics of the interaction of HVJ with liposomes containing glycophorin appeared to be phenomenologically similar to those of HVJ-cell interaction.     

IL-12 p40 homodimer-dependent macrophage chemotaxis and respiratory viral inflammation are mediated through IL-12 receptor beta 1

Leukocyte recruitment to the airway lumen is a central feature of inflammatory conditions such as asthma and respiratory viral infection. Characterization of mediators that regulate leukocyte recruitment in these conditions revealed increased IL-12 p40 homodimer (p80) levels were associated with enhanced airway macrophage accumulation. To examine this association, we used in vivo and in vitro assays to demonstrate p80, but not IL-12 or p40, provided a macrophage chemoattractant signal. Macrophages from genetically deficient mice indicated p80-dependent chemotaxis was independent of IL-12 and required IL-12Rbeta1 (Rbeta1) expression. Furthermore, analysis of murine cell lines and primary culture macrophages revealed Rbeta1 expression, with an intact cytoplasmic tail, was necessary and sufficient to mediate p80-dependent chemotaxis. To examine the role for Rbeta1 in mediating macrophage accumulation in vivo, we contrasted Sendai virus-driven airway inflammation in wild-type and Rbeta1-deficient mice. Despite similar viral burden and production of the macrophage chemoattractant p80, the Rbeta1-deficient mice displayed a selective decrease in airway macrophage accumulation and resistance to viral-dependent mortality. Thus, Rbeta1 mediates p80-dependent macrophage chemotaxis and inhibition of the p80-Rbeta1 interaction may provide a novel anti-inflammatory strategy to manipulate the inflammation associated with asthma and respiratory viral infection.     

IL-12 contributes to allergen-induced airway inflammation in experimental asthma

Lack of sufficient IL-12 production has been suggested to be one of the basic underlying mechanisms in atopy, but a potential role of IL-12 in established allergic airway disease remains unclear. We took advantage of a mouse model of experimental asthma to study the role of IL-12 during the development of bronchial inflammation. Administration of anti-IL-12p35 or anti-IL-12p40 mAb to previously OVA-sensitized BALB/c mice concomitantly with exposure to nebulized OVA, abolished both the development of bronchial hyperresponsiveness to metacholine as well as the eosinophilia in bronchoalveolar lavage fluid and peripheral blood. Anti-IL-12 treatment reduced CD4(+) T cell numbers and IL-4, IL-5, and IL-13 levels in the bronchoalveolar lavage fluid and the mRNA expression of IL-10, eotaxin, RANTES, MCP-1, and VCAM-1 in the lung. Anti-IL-12p35 treatment failed to show these effects in IFN-gamma knockout mice pointing to the essential role of IFN-gamma in IL-12-induced effects. Neutralization of IL-12 during the sensitization process aggravated the subsequent development of allergic airway inflammation. These data together with recent information on the role of dendritic cells in both the sensitization and effector phase of allergic respiratory diseases demonstrate a dual role of IL-12. Whereas IL-12 counteracts Th2 sensitization, it contributes to full-blown allergic airway disease upon airway allergen exposure in the postsensitization phase, with enhanced recruitment of CD4(+) T cells and eosinophils and with up-regulation of Th2 cytokines, chemokines, and VCAM-1. IFN-gamma-producing cells or cells dependent on IFN-gamma activity, play a major role in this unexpected proinflammatory effect of IL-12 in allergic airway disease.     

IL-13-induced chemokine responses in the lung: role of CCR2 in the pathogenesis of IL-13-induced inflammation and remodeling

IL-13 stimulates inflammatory and remodeling responses and contributes to the pathogenesis of human airways disorders. To further understand the cellular and molecular events that mediate these responses, we characterized the effects of IL-13 on monocyte chemotactic proteins (MCPs) and compared the tissue effects of transgenic IL-13 in mice with wild-type (+/+) and null (-/-) CCR2 loci. Transgenic IL-13 was a potent stimulator of MCP-1, -2, -3, and -5. This stimulation was not specific for MCPs because macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, MIP-3alpha, thymus- and activation-regulated chemokine, thymus-expressed chemokine, eotaxin, eotaxin 2, macrophage-derived chemokines, and C10 were also induced. The ability of IL-13 to increase lung size, alveolar size, and lung compliance, to stimulate pulmonary inflammation, hyaluronic acid accumulation, and tissue fibrosis, and to cause respiratory failure and death were markedly decreased, whereas mucus metaplasia was not altered in CCR2(-/-) mice. CCR2 deficiency did not decrease the basal or IL-13-stimulated expression of target matrix metalloproteinases or cathepsins but did increase the levels of mRNA encoding alpha1-antitrypsin, tissue inhibitor of metalloproteinase-1, -2, and -4, and secretory leukocyte proteinase inhibitor. In addition, the levels of bioactive and total TGF-beta(1) were decreased in lavage fluids from IL-13 transgenic mice with -/- CCR2 loci. These studies demonstrate that IL-13 is a potent stimulator of MCPs and other CC chemokines and document the importance of MCP-CCR2 signaling in the pathogenesis of the IL-13-induced pulmonary phenotype.     

Chilling of somatic cells unnecessary for Sendai virus-induced heterokaryon formation.

Incubation of cell suspensions or monolayers at 4 degrees C for 20 min after addition of beta-propiolactone-inactivated Sendai virus did not enhance heterokaryon formation as compared to parallel cell cultures incubated at 37 degrees C immediately after the addition of Sendai virus. These findings show that the routine chilling of somatic cells in fusion experiments can be omitted.     

Manipulation of the seagrass-associated microbiome reduces disease severity

Host-associated microbes influence host health and function and can be a first line of defence against infections. While research increasingly shows that terrestrial plant microbiomes contribute to bacterial, fungal, and oomycete disease resistance, no comparable experimental work has investigated marine plant microbiomes or more diverse disease agents. We test the hypothesis that the eelgrass (Zostera marina) leaf microbiome increases resistance to seagrass wasting disease. From field eelgrass with paired diseased and asymptomatic tissue, 16S rRNA gene amplicon sequencing revealed that bacterial composition and richness varied markedly between diseased and asymptomatic tissue in one of the two years. This suggests that the influence of disease on eelgrass microbial communities may vary with environmental conditions. We next experimentally reduced the eelgrass microbiome with antibiotics and bleach, then inoculated plants with Labyrinthula zosterae, the causative agent of wasting disease. We detected significantly higher disease severity in eelgrass with a native microbiome than an experimentally reduced microbiome. Our results over multiple experiments do not support a protective role of the eelgrass microbiome against L. zosterae. Further studies of these marine host–microbe–pathogen relationships may continue to show new relationships between plant microbiomes and diseases.     

Adrenomedullin reduces the severity of cerulein-induced acute pancreatitis

We investigated the effect of Adrenomedullin (AM) on cerulein-induced acute pancreatitis in rats. AM treatment (100 ng/kg per rat, subcutaneous) after one hour of cerulein injection reduced the plasma amylase levels, pancreatic weight, pancreatic malondialdehyde (MDA) levels, and the severity of the lesions microscopically. These data suggest that AM has a protective effect on cerulein-induced acute pancreatitis. These could be due to anti-inflammatory properties of AM, inhibition of proinflammatory cytokine secretion, reducing the endothelial permeability increased by reactive oxygen species, endotoxins or cytokines.     

Serum levels of TNF-alpha, IFN-gamma, IL-6, IL-8, IL-12, IL-17, and IL-18 in patients with active psoriasis and correlation with disease severity

Recent progress in the understanding of psoriasis has shown that the regulation of local and systemic cytokines plays an important role in its pathogenesis. The most often used psoriasis score is the psoriasis area and severity index (PASI). A simple laboratory test from a blood sample would be an attractive, patient-independent, and observer-independent marker of disease severity. To this end, we evaluated the association of serum levels of some proinflammatory cytokines in vivo and their correlation with severity of psoriasis. The serum levels of cytokines levels were determined with the use of the ELISA method. All mean values except IL-17 levels of patients were significantly higher than those of controls. There was a significant correlation between serum levels of IFN-gamma, IL-12, IL-17, and IL-18, and severity of the disease. Psoriasis can be described as a T-cell-mediated disease, with a complex role for a variety of cytokines, which has led to the development of new immunomodulatory therapies. In this study, serum TNF-alpha, IFN-gamma, IL-6, IL-8, IL-12, and IL-18 levels were significantly higher in active psoriatic patients than in controls. Furthermore, high levels of IFN-gamma, IL-12, and IL-18 correlated with the clinical severity and activity of psoriasis, and those measurements of serum levels of these cytokines may be objective parameters for the disease severity.     

IL-12 induction of mRNA encoding substance P in murine macrophages from the spleen and sites of inflammation

Substance P (SP), a neuropeptide, interacts with the neurokinin 1 receptor (NK-1R) on immune cells to help control IFN-gamma production. In murine schistosomiasis mansoni, schistosome worms produce ova that incite focal Th2-type granulomatous inflammation within the liver and intestines. Normal gut is characterized by a controlled state of inflammation. IL-10 knockout mice develop chronic Th1-type colitis spontaneously. Both schistosome granulomas and gut mucosa display an SP immune regulatory circuit. However, the origin and regulation of SP production at these sites of inflammation are poorly understood. Macrophages are a potential source of SP. We therefore studied macrophages (F4/80(+)) from these models of inflammation. SP mRNA (preprotachykinin A (PPT A)) was detected within the schistosome granuloma, spleen, and lamina propria macrophages. Compared with those from wild-type mice, granuloma macrophages from STAT6(-/-) mice had 10-fold higher PPT A mRNA expression, whereas in STAT4(-/-) animals, PPT A mRNA expression was nearly abolished. IL-12 signals via STAT4 to induce Th1-type inflammation. It was demonstrated that IL-12, but not IL-18, induces SP mRNA expression in resting splenic macrophages from Schistosoma-infected mice and in wild-type lamina propria mononuclear cells. Thus, macrophages are a source for SP at these sites of chronic inflammation, and IL-12 and STAT4 are regulators of macrophage SP mRNA expression.