Mast cell-derived factor XIIIA contributes to sexual dimorphic defense against group B streptococcal infections.

Invasive bacterial infections remain a major cause of human morbidity. Group B streptococcus (GBS) are Gram-positive bacteria that cause invasive infections in humans. Here, we show that factor XIIIA-deficient (FXIIIA-deficient) female mice exhibited significantly increased susceptibility to GBS infections. Additionally, female WT mice had increased levels of FXIIIA and were more resistant to GBS infection compared with isogenic male mice. We observed that administration of exogenous FXIIIA to male mice increased host resistance to GBS infection. Conversely, administration of a FXIIIA transglutaminase inhibitor to female mice decreased host resistance to GBS infection. Interestingly, male gonadectomized mice exhibited decreased sensitivity to GBS infection, suggesting a role for gonadal androgens in host susceptibility. FXIIIA promoted GBS entrapment within fibrin clots by crosslinking fibronectin with ScpB, a fibronectin-binding GBS surface protein. Thus, ScpB-deficient GBS exhibited decreased entrapment within fibrin clots in vitro and increased dissemination during systemic infections. Finally, using mice in which FXIIIA expression was depleted in mast cells, we observed that mast cell-derived FXIIIA contributes to host defense against GBS infection. Our studies provide insights into the effects of sexual dimorphism and mast cells on FXIIIA expression and its interactions with GBS adhesins that mediate bacterial dissemination and pathogenesis.

Mast cell surfaceome characterization reveals CD98 heavy chain is critical for optimal cell function.

BACKGROUND: Mast cells are involved in many distinct pathologic conditions, suggesting that they recognize and respond to various stimuli and thus require a rich repertoire of cell surface proteins. However, mast cell surface proteomes have not been comprehensively characterized. OBJECTIVE: We aimed to further characterize the mast cell surface proteome to obtain a better understanding of how mast cells function in health and disease. METHODS: We enriched for glycosylated surface proteins expressed in mouse bone marrow-derived cultured mast cells (BMCMCs) and identified them using mass spectrometry analysis. The presence of novel surface proteins in mast cells was validated by real-time quantitative PCR and flow cytometry analysis in BMCMCs and peritoneal mast cells (PMCs). We developed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to disrupt genes of interest in BMCMCs. RESULTS: The glycoprotein enrichment approach resulted in the identification of 1270 proteins in BMCMCs, 378 of which were localized to the plasma membrane. The most common protein classes among plasma membrane proteins were small GTPases, receptors, and transporters. One such cell surface protein was CD98 heavy chain (CD98hc), encoded by the Slc3a2 gene. Slc3a2 gene disruption resulted in a significant reduction in CD98hc expression, adhesion, and proliferation. CONCLUSIONS: Glycoprotein enrichment coupled with mass spectrometry can be used to identify novel surface molecules in mast cells. Moreover, CD98hc plays an important role in mast cell function.

E3 Ubiquitin Ligase RNF125 Activates Interleukin-36 Receptor Signaling and Contributes to Its Turnover.

Signaling by the interleukin-36 receptor (IL-36R) is linked to inflammatory diseases such as psoriasis. However, the regulation of IL-36R signaling is poorly understood. Activation of IL-36R signaling in cultured cells results in an increased polyubiquitination of the receptor subunit, IL-1Rrp2. Treatment with deubiquitinases shows that the receptor subunit of IL-36R, IL-1Rrp2, is primarily polyubiquitinated at the K63 position, which is associated with endocytic trafficking and signal transduction. A minor amount of ubiquitination is at the K48 position that is associated with protein degradation. A focused siRNA screen identified RNF125, an E3 ubiquitin ligase, to ubiquitinate IL-1Rrp2 upon activation of IL-36R signaling while not affecting the activated IL-1 receptor. Knockdown of RNF125 decreases signal transduction by the IL-36R. Overexpression of RNF125 in HEK293T cells activates IL-36R signaling and increases the ubiquitination of IL-1Rrp2 and its subsequent turnover. RNF125 can coimmunoprecipitate with the IL-36R, and it traffics with IL-1Rrp2 from the cell surface to lysosomes. Mutations of Lys568 and Lys569 in the C-terminal tail of IL-1Rrp2 decrease ubiquitination by RNF125 and increase the steady-state levels of IL-1Rrp2. These results demonstrate that RNF125 has multiple regulatory roles in the signaling, trafficking, and turnover of the IL-36R.

Structural and Functional Attributes of the Interleukin-36 Receptor.

Signal transduction by the IL-36 receptor (IL-36R) is linked to several human diseases. However, the structure and function of the IL-36R is not well understood. A molecular model of the IL-36R complex was generated and a cell-based reporter assay was established to assess the signal transduction of recombinant subunits of the IL-36R. Mutational analyses and functional assays have identified residues of the receptor subunit IL-1Rrp2 needed for cytokine recognition, stable protein expression, disulfide bond formation and glycosylation that are critical for signal transduction. We also observed that, overexpression of ectodomain (ECD) of Il-1Rrp2 or IL-1RAcP exhibited dominant-negative effect on IL-36R signaling. The presence of IL-36 cytokine significantly increased the interaction of IL-1Rrp2 ECD with the co-receptor IL-1RAcP. Finally, we found that single nucleotide polymorphism A471T in the Toll-interleukin 1 receptor domain (TIR) of the IL-1Rrp2 that is present in ∼2% of the human population, down-regulated IL-36R signaling by a decrease of interaction with IL-1RAcP.

Signal Transduction and Intracellular Trafficking by the Interleukin 36 Receptor.

Improper signaling of the IL-36 receptor (IL-36R), a member of the IL-1 receptor family, has been associated with various inflammation-associated diseases. However, the requirements for IL-36R signal transduction remain poorly characterized. This work seeks to define the requirements for IL-36R signaling and intracellular trafficking. In the absence of cognate agonists, IL-36R was endocytosed and recycled to the plasma membrane. In the presence of IL-36, IL-36R increased accumulation in LAMP1+ lysosomes. Endocytosis predominantly used a clathrin-mediated pathway, and the accumulation of the IL-36R in lysosomes did not result in increased receptor turnover. The ubiquitin-binding Tollip protein contributed to IL-36R signaling and increased the accumulation of both subunits of the IL-36R.

Protective effect of conjugated linolenic acid isomers present in vegetable oils against arsenite-induced renal toxicity in rat model.

OBJECTIVE: The aim of the present study was to investigate the protective effect of conjugated linolenic acid (CLnA), present in vegetable oils against arsenite-induced renal oxidative stress. METHODS: Albino rats were divided into six groups. Group 1 was control and group 2 was treated with sodium arsenite (Sa; 10 mg/kg BW). Rats in groups 3 and 4 were treated with mixture of α-eleostearic acid and punicic acid (1:1) (0.5% and 1.0%, respectively), whereas rats in the groups 5 and 6 were treated with 0.5% of α-eleostearic acid and 0.5% of punicic acid, respectively, along with Sa by oral gavage once daily. RESULTS: Results revealed that activity of antioxidant enzymes and total reduced glutathione content, total protein content, and phospholipid content in kidney were decreased significantly in arsenite-treated group compared with control. Activity of nitric oxide synthase, peroxidation of lipid, protein oxidation, total cholesterol content, total lipid content of kidney, and plasma creatinine level were increased significantly (P < 0.05) in arsenite-treated rats compared with control. Fatty-acid composition of renal lipids showed significant decrease in monounsaturated fatty acid, polyunsaturated fatty acid (PUFA) content, and increase in saturated fatty acid content due to oxidative stress. PUFA such as γ-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid decreased significantly with significant (P < 0.05) increase in arachidonic acid content after Sa treatment. Administration of blended product of both the isomers caused better restoration of renal fatty acids and other altered parameters. CONCLUSION: CLnA isomers caused amelioration of renal oxidative stress and the isomers showed synergistic activity.

Synergistic effect of conjugated linolenic acid isomers against induced oxidative stress, inflammation and erythrocyte membrane disintegrity in rat model.

BACKGROUND: α-Eleostearic acid and punicic acid, two typical conjugated linolenic acid (CLnA) isomers present in bitter gourd and snake gourd oil respectively, exhibit contrasting cis-trans configuration which made them biologically important. METHODS: Rats were divided into six groups. Group 1 was control and group 2 was treated control. Rats in the groups 3 and 4 were treated with mixture of α-eleostearic acid and punicic acid (1:1) (0.5% and 1.0% respectively) while rats in the groups 5 and 6 were treated with 0.5% of α-eleostearic acid and 0.5% of punicic acid respectively along with sodium arsenite by oral gavage once per day. RESULTS: Results showed that increase in nitric oxide synthase (NOS) activity, inflammatory markers expression, platelet aggregation, lipid peroxidation, protein oxidation, DNA damage and altered expression of liver X receptor-α (LXR-α) after arsenite treatment were restored with the supplementation of oils containing CLnA isomers. Altered activities of different antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and ferric reducing ability of plasma (FRAP) also restored after oil supplementation. Altered morphology and fluidity of erythrocyte membrane studied by atomic force and scanning electron microscopy, after stress induction were significantly improved due to amelioration in cholesterol/phospholipid ratio and fatty acid profile of membrane. Oils treatment also improved morphology of liver and fatty acid composition of hepatic lipid. CONCLUSIONS: Overall two isomers showed synergistic antioxidant and anti-inflammatory effect against induced perturbations and membrane disintegrity. GENERAL SIGNIFICANCE: Synergistic antioxidant and anti-inflammatory role of these CLnA isomers were established by this study.

Antioxidant and anti-inflammatory effect of conjugated linolenic acid isomers against streptozotocin-induced diabetes.

The present study was undertaken to evaluate the effect of α-eleostearic acid and punicic acid, two isomers of conjugated linolenic acid (CLnA) present in bitter gourd (Momordica charantia) and snake gourd oil (Trichosanthes anguina), respectively, against oxidative stress, inflammatory challenge and aberration in erythrocyte morphology due to streptozotocin (STZ)-induced diabetes. Male albino rats were divided into four groups consisting of eight animals in each group. The first group served as control and diabetes was induced in rats in groups 2-4 by a single intraperitoneal injection of STZ. Moreover, rats in groups 3 and 4 were treated with 0·5 % of α-eleostearic acid and 0·5 % of punicic acid of the total lipid given, respectively, by oral administration once per d. After administration, CLnA isomers had significantly reduced oxidative stress, lipid peroxidation and restored antioxidant and pro-inflammatory enzymes such as superoxide dismutase, catalase, and glutathione peroxidase, reduced glutathione, NO synthase level in pancreas, blood and erythrocyte lysate. The ferric reducing antioxidant power (FRAP) assay of plasma showed that CLnA treatment caused improvement in the FRAP value which was altered after STZ treatment due to an increased level of free radicals. Expression of inflammatory cytokines such as TNF-α and IL-6 in blood and expression of hepatic NF-κB (p65) increased significantly after STZ treatment due to increased inflammation which was restored with the administration of CLnA isomers. From the obtained results, it could be concluded that α-eleostearic acid and punicic acid showed potent antioxidant and anti-inflammatory activity with varying effectivity.

Comparative study of hypocholesterolemic and hypolipidemic effects of conjugated linolenic acid isomers against induced biochemical perturbations and aberration in erythrocyte membrane fluidity.

AIM OF STUDY: The purpose of the study was to evaluate hypolipidemic and hypocholesterolemic activities of conjugated linolenic acid (CLnA) isomers, present in bitter gourd and snake gourd seed, in terms of amelioration of plasma lipid profile, lipoprotein oxidation and erythrocyte membrane fluidity after oral administration. METHODS: Male albino rats were divided into six groups. Group 1 was control, and others were induced with oxidative stress by oral gavage of sodium arsenite (Sa). Group 2 was kept as treated control, and groups 3-6 were further treated with different oral doses of seed oils to maintaining definite concentration of CLnA isomers (0.5 and 1.0% of total lipid for each CLnA isomer). RESULTS: CLnA isomers normalized cholesterol, LDL-cholesterol, HDL-cholesterol and triglyceride contents in plasma and body weight of experimental rats and decreased cholesterol synthesis by reducing hepatic HMG-CoA reductase activity. Administration of Sa caused alteration in erythrocyte membrane fluidity due to increase in cholesterol and decrease in phospholipid content. Tissue cholesterol and lipid contents were also increased by Sa administration. These altered parameters were reversed by experimental oil administration. Protective effect of CLnA isomers on erythrocyte morphology was observed by atomic force microscopy (AFM). Fatty acid composition of erythrocyte membrane showed decrease in polyunsaturated fatty acid (PUFA) and increase in arachidonic acid content after Sa administration, which was normalized with the treatment of these oils. Supplementation of CLnA isomers restored erythrocyte membrane (EM) lipid peroxidation and lipoprotein oxidation. CONCLUSION: CLnA isomers, present in vegetable oils, showed potent hypolipidemic and hypocholesterolemic activities against biochemical perturbations.

Antioxidant effect of vegetable oils containing conjugated linolenic acid isomers against induced tissue lipid peroxidation and inflammation in rat model.

The purpose of the present study was to examine the antioxidant activity of two typical oils obtained from two vegetables, bitter gourd seed and snake gourd seed, containing two different isomers of conjugated linolenic acid (CLnA) against oxidative stress induced by sodium arsenite in relation to tissue lipid peroxidation and inflammation. Male albino rats were taken as subject and divided into six groups: Group 1 was control and Group 2 was treated with sodium arsenite (Sa; 10mg/Kg BW); Groups 3-6 were orally treated with different doses of seed oils maintaining definite concentration of CLnA isomers (0.5% and 1.0% of total lipid for each CLnA isomer) along with sodium arsenite. There was significant increase in lipid peroxidation, pro-oxidant enzyme activity and decrease in antioxidant enzyme activity in brain due to Sa administration. Decrease in total protein content was also observed in plasma, liver and brain of Sa treated group. Significant decrease in phospholipid content and increase in total lipid content and cholesterol content were observed in arsenite treated group. There was significant increase in relative organ weight of liver due to Sa administration. Fatty acid profile of liver and brain lipid shows significant (P<0.05) reduction in most of the polyunsaturated fatty acids and increase in arachidonic acid (20:4n-6) (75.23%) due to inflammation after arsenite treatment. Administration of experimental oils made almost complete restoration of those altered parameters. Overall, these two oils were effective in protecting tissue lipid profiles which were altered due to oxidative stress.