Knockout of the KH-Type Splicing Regulatory Protein Drives Glomerulonephritis in MRL-Faslpr Mice.

KH-type splicing regulatory protein (KSRP) is an RNA-binding protein that promotes mRNA decay and thereby negatively regulates cytokine expression at the post-transcriptional level. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by dysregulated cytokine expression causing multiple organ manifestations; MRL-Faslpr mice are an established mouse model to study lupus disease pathogenesis. To investigate the impact of KSRP on lupus disease progression, we generated KSRP-deficient MRL-Faslpr mice (MRL-Faslpr/KSRP-/- mice). In line with the predicted role of KSRP as a negative regulator of cytokine expression, lupus nephritis was augmented in MRL-Faslpr/KSRP-/- mice. Increased infiltration of immune cells, especially of IFN-γ producing T cells and macrophages, driven by enhanced expression of T cell-attracting chemokines and adhesion molecules, seems to be responsible for worsened kidney morphology. Reduced expression of the anti-inflammatory interleukin-1 receptor antagonist may be another reason for severe inflammation. The increase of FoxP3+ T cells detected in the kidney seems unable to dampen the massive kidney inflammation. Interestingly, lymphadenopathy was reduced in MRL-Faslpr/KSRP-/- mice. Altogether, KSRP appears to have a complex role in immune regulation; however, it is clearly able to ameliorate lupus nephritis.

The RNA-Binding Protein KSRP Modulates Cytokine Expression of CD4+ T Cells.

The KH-type splicing regulatory protein (KSRP) is a RNA-binding protein, which regulates the stability of many mRNAs encoding immune-relevant proteins. As KSRP regulates innate immune responses, for instance by the modulation of type I interferon mRNA stability, we were interested whether knockdown of the protein (KSRP-/-) interferes with T cell activation and polarization. Polyclonally stimulated KSRP-/- CD4+ T cells proliferated at a higher extent and higher frequency and expressed the activation marker CD25 more than wild-type T cells. In supernatants of stimulated KSRP-/- CD4+ T cells, levels of IL-5, IL-9, IL-10, and IL-13 were observed to be increased compared to those of the control group. KSRP-/- CD8+ T cells showed no altered proliferative capacity upon polyclonal stimulation, but supernatants contained lower levels of interferon-γ. Similar changes in the cytokine expression patterns were also detected in T cells derived from KSRP-/- mice undergoing arthritis induction indicative of a pathophysiological role of KSRP-dependent T cell polarization. We demonstrated the direct binding of KSRP to the 3' untranslated region of IL-13, IL-10, and IFN-γ mRNA in in vitro experiments. Moreover, since IL-4 mRNA decay was reduced in KSRP-/- CD4+ T cells, we identify KSRP as a negative regulator of IL-4 expression. These data indicate that overexpression of IL-4, which constitutes the primary inducer of Th2 polarization, may cause the Th2 bias of polyclonally stimulated KSRP-/- CD4+ T cells. This is the first report demonstrating that KSRP is involved in the regulation of T cell responses. We present strong evidence that T cells derived from KSRP-/- mice favor Th2-driven immune responses.

The KH-type splicing regulatory protein (KSRP) regulates type III interferon expression post-transcriptionally.

Type III interferons (IFNs) are the latest members of the IFN family. They play an important role in immune defense mechanisms, especially in antiviral responses at mucosal sites. Moreover, they control inflammatory reactions by modulating neutrophil and dendritic cell functions. Therefore, it is important to identify cellular mechanisms involved in the control of type III IFN expression. All IFN family members contain AU-rich elements (AREs) in the 3'-untranslated regions (3'-UTR) of their mRNAs that determine mRNA half-life and consequently the expressional level of these cytokines. mRNA stability is controlled by different proteins binding to these AREs leading to either stabilization or destabilization of the respective target mRNA. The KH-type splicing regulatory protein KSRP (also named KHSRP) is an important negative regulator of ARE-containing mRNAs. Here, we identify the interferon lambda 3 (IFNL3) mRNA as a new KSRP target by pull-down and immunoprecipitation experiments, as well as luciferase reporter gene assays. We characterize the KSRP-binding site in the IFNL3 3'-UTR and demonstrate that KSRP regulates the mRNA half-life of the IFNL3 transcript. In addition, we detect enhanced expression of IFNL3 mRNA in KSRP-/- mice, establishing a negative regulatory function of KSRP in type III IFN expression also in vivo Besides KSRP the RNA-binding protein AUF1 (AU-rich element RNA-binding protein 1) also seems to be involved in the regulation of type III IFN mRNA expression.

Divergent roles of the Drosophila melanogaster globins.

In contrast to long-held assumptions, the gene repertoire of most insects includes hemoglobins. Analyses of the genome of the fruitfly Drosophila melanogaster identified three distinct hemoglobin genes (glob1, glob2, and glob3). While glob1 is predominantly associated with the tracheal system and fat body, glob2 and glob3 are almost exclusively expressed in the testis. The physiological role of globins in Drosophila is uncertain. Here, we studied the functions of the three globins in a cell culture system. Drosophila Schneider 2 (S2) cells were stably transfected with each of the three globins and the empty vector as control. Under hypoxia (1% atmospheric O2), only glob1 overexpression enhanced the activity of mitochondrial oxidases and the ATP content. However, the positive effect of glob1 expression disappeared after 24h hypoxia, suggesting metabolic adaptations of the S2 cells. glob2 and glob3 had no positive effect on hypoxia-survival. After application of oxidative stress by H2O2, glob2 dramatically enhanced the viability of S2 cells. Evaluation of the intracellular localization of the globins using specific antibodies and green fluorescent protein-fusion constructs suggested that glob1 and glob2 most likely reside in the cytoplasm, while glob3 is associated with structures that may represent parts of the intracellular transport machinery. In silico analyses of public RNA-Seq data from different developmental stages provided that glob1 is co-expressed with genes of the aerobic energy metabolism, while glob2 and glob3 expression can be related to spermatogenesis and reproduction. Together, the results indicate divergent functions of the Drosophila globins: glob1 may play a role in the O2-dependent metabolism while glob2 may protect spermatogenesis from reactive oxygen species.

Inactivation of the KSRP gene modifies collagen antibody induced arthritis.

The KH type splicing regulatory protein (KSRP) is a nucleic acid binding protein, which negatively regulates the stability and/or translatability of many mRNA species encoding immune-relevant proteins. As KSRP is expressed in immune cells including T and B cells, neutrophils, macrophages and dendritic cells, we wanted to analyze its importance for the development of autoimmune diseases. We chose collagen antibody-induced arthritis (CAIA) as an appropriate autoimmune disease mouse model in which neutrophils and macrophages constitute the main effector cell populations. We compared arthritis induction in wild type (WT) and KSRP-/- mice and paws were taken for histological sections and qPCR analysis. Furthermore, we determined the frequencies of spleen immune cells by flow cytometry. Cytokine levels in spleen cell supernatants were determined by cytometric bead array analyses (CBA). After CAIA induction we unexpectedly observed in WT animals much stronger swelling of the paws than in KSRP-/- mice. In accordance, histological staining of paw sections of KSRP-/- animals revealed much lower frequencies of infiltrating immune cells in the joints compared to WT animals. Furthermore, CAIA-treatment resulted in reduced expression of several inflammatory factors (like CXCL-1, iNOS, TNF-α and S100A8) as well as immune cell marker genes (e.g. LFA-1, CD68, Ly6G) in the joints of KSRP-/- mice. Spleen cells of KSRP-/- mice showed lower frequencies of myeloid cells. On cytokine level IFN-γ production was increased in spleen cells of KSRP-/- mice compared to WT samples. These data surprisingly suggest that the absence of KSRP protects against the induction of inflammatory arthritis.

Anti-Inflammatory and Anti-Thrombotic Effects of the Fungal Metabolite Galiellalactone in Apolipoprotein E-Deficient Mice.

Patients suffering from chronic inflammatory diseases have an increased mortality risk resulting from cardiovascular disorders due to enhanced atherosclerotic and thrombotic events. Until now, it is not completely understood in which way an abnormal expression of pro-inflammatory mediators contributes to this elevated cardiovascular risk, but there is a need for new drugs that on the one hand suppress the expression of pro-inflammatory mediators and on the other hand inhibit arterial platelet adhesion. Thus, we analyzed the anti-inflammatory and anti-thrombotic capacity of the fungal metabolite Galiellalactone in atherosclerosis-prone apolipoprotein E-deficient mice. Treatment of the mice with Galiellalactone lowered the inflammatory expression profile and improved blood clotting times, as well as platelet adhesion to the injured common carotid artery. The results indicate that administration of Galiellalactone is able to reduce the extent of inflammation and arterial platelet adhesion in this mouse model.

Resveratrol post-transcriptionally regulates pro-inflammatory gene expression via regulation of KSRP RNA binding activity.

Resveratrol shows beneficial effects in inflammation-based diseases like cancer, cardiovascular and chronic inflammatory diseases. Therefore, the molecular mechanisms of the anti-inflammatory resveratrol effects deserve more attention. In human epithelial DLD-1 and monocytic Mono Mac 6 cells resveratrol decreased the expression of iNOS, IL-8 and TNF-α by reducing mRNA stability without inhibition of the promoter activity. Shown by pharmacological and siRNA-mediated inhibition, the observed effects are SIRT1-independent. Target-fishing and drug responsive target stability experiments showed selective binding of resveratrol to the RNA-binding protein KSRP, a central post-transcriptional regulator of pro-inflammatory gene expression. Knockdown of KSRP expression prevented resveratrol-induced mRNA destabilization in human and murine cells. Resveratrol did not change KSRP expression, but immunoprecipitation experiments indicated that resveratrol reduces the p38 MAPK-related inhibitory KSRP threonine phosphorylation, without blocking p38 MAPK activation or activity. Mutation of the p38 MAPK target site in KSRP blocked the resveratrol effect on pro-inflammatory gene expression. In addition, resveratrol incubation enhanced KSRP-exosome interaction, which is important for mRNA degradation. Finally, resveratrol incubation enhanced its intra-cellular binding to the IL-8, iNOS and TNF-α mRNA. Therefore, modulation of KSRP mRNA binding activity and, thereby, enhancement of mRNA degradation seems to be the common denominator of many anti-inflammatory effects of resveratrol.