G-protein coupled receptor kinase-5 (GRK5) is a serine/threonine kinase discovered for its role in the regulation of G-protein coupled receptor signaling. Recent studies have shown that GRK5 is also an important regulator of signaling pathways stimulated by non-GPCRs. This study was undertaken to determine the physiological role of GRK5 in Toll-like receptor-4-induced inflammatory signaling pathways in vivo and in vitro. Using mice genetically deficient in GRK5 (GRK5 −/− ) we demonstrate here that GRK5 is an important positive regulator of lipopolysaccharide (LPS, a TLR4 agonist)-induced inflammatory cytokine and chemokine production in vivo. Consistent with this role, LPS-induced neutrophil infiltration in the lungs (assessed by myeloperoxidase activity) was markedly attenuated in the GRK5 −/− mice compared to the GRK5 +/+ mice. Similar to the in vivo studies, primary macrophages from GRK5 −/− mice showed attenuated cytokine production in response to LPS. Our results also identify TLR4-induced NFκB pathway in macrophages to be selectively regulated by GRK5. LPS-induced IκBα phosphorylation, NFκB p65 nuclear translocation and NFκB binding were markedly attenuated in GRK5 −/− macrophages. Together, our findings demonstrate that GRK5 is a positive regulator of TLR4-induced IκBα-NFκB pathway as well as a key modulator of lipopolysaccharide-induced inflammatory response.

BackgroundRapeseed-mustard is the second most important source of edible oil in India. Several species of Brassica are grown in different parts of country for its oilseeds.ObjectiveThe objective was to investigate allergenicity to antigenic extracts of pollen of 4 species of Brassica.MethodsBrassica campestris, Brassica juncea, Brassica nigra, and Brassica napus were selected for the detailed investigation. Pollen samples from each of the four species were collected from the polliniferous materials. The antigenic and allergenic profiles of these extracts were evaluated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Skin prick test, enzyme linked immuno sorbent assay and Western blot on atopic individuals.ResultsOut of the 159 atopic subjects tested, 21.38% were positive to at least one or other species of Brassica pollen, with highest skin positivity (13.20%) to B. campestris extract. Raised IgE with significant linear correlation with intensity of skin reactions was obtained. Protein fractions of 20, 25, 32, 37, 56, and 90 kDa were recognized by B. campestris and B. juncea whereas 56, 76, 87, and 90 kDa were recognized by B. nigra and B. napus as major IgE binding protein fractions. The patients also showed positivity to other inhalant pollen allergens tested.ConclusionIgE mediated hypersensitivity varied from 4.40% to 13.20% in Indian atopic subjects to pollen of one or the other species of Brassica. Protein fractions of 47, 56, 76, 87, and 90 kDa were identified as IgE binding by all the four species, however individual heterogeneity exists. Thus a local species may be more pertinent for immunotherapy. The major allergen needs to be further characterized.

A study on heterogeneity in water-soluble and non-water-soluble protein profiles of different species of Brassica pollen, seeds, and industrial flour for efficient allergy detection and immunotherapy has been lacking in India. The purpose of this study was to examine heterogeneity in the protein profile of antigenic extracts of different species of pollen, seeds, and seed flour of Brassica. A comparison of water-soluble and non-water-soluble protein profiles of seed extracts was studied. Water-soluble and non-water-soluble proteins were extracted, concentration was estimated by Lowry's method, and biochemical characterization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was analyzed. Pollen from four species, four species of Brassica seeds comprising 25 varieties, and industrial seed flour were included for water-soluble studies, whereas four species of seeds were included for non-water-soluble protein extraction. Significant variation in protein content was observed among four different species of pollen, seeds, and industrial seed flour, respectively. No significant variation was observed in non-water-soluble extracts of four species or among water-soluble and non-water-soluble content of seeds. Heterogeneity in the protein profile of different species of pollen was not observed. However, variation in banding pattern of water-soluble as well as non-water-soluble protein extracts among four different species of seeds was observed. Interestingly, the industrial seed flour also showed a rich protein banding pattern. Variation in protein content as well as protein profile among different species of seeds of Brassica is recorded.

Toll-like receptor-4 (TLR4) activated by lipopolysaccharide (LPS) is involved in a number of inflammatory diseases, in part via activation of NFκB pathway. G-protein coupled receptor kinase-5 (GRK5), a serine-threonine kinase originally discovered for its role in desensitization of G-protein coupled receptors has recently been shown to regulate NFκB pathway stimulated by non-GPCRs. This study was undertaken to determine the potential role of GRK5 in inflammatory signaling in primary macrophages and in vivo using GRK5 knockout (KO) mice. We demonstrate here that TLR4-induced IκBα-NFκB pathway is selectively inhibited in primary macrophage from GRK5 KO mice compared to cells from GRK5 wild type (WT) mice. Consistent with the effects on the NFκB pathway, LPS-induced production of many inflammatory cytokines/chemokines (including Interleukin-2, -3, -4, -5, -12, -17, MCP-1, IFNγ, KC, GMCSF and Eotaxin) are inhibited in GRK5 KO macrophages. Confirming this in vitro data, LPS-induced cytokines/chemokines levels in the plasma are also significantly inhibited in the GRK5 KO mice in vivo. Associated with these effects LPS-mediated liver injury is markedly decreased in the GRK5 KO mice. Taken together, our findings demonstrate that GRK5 acts as a positive regulator of TLR4 signaling in vivo and further suggest that these findings could have potential implications for drug development in inflammatory diseases.

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