Toll-like receptors: New targets for multiple myeloma treatment?

Despite recent biomedical improvements in treating multiple myeloma, this disease still remains incurable. Toll-like receptors (TLRs) are key immune receptors that recognize conserved molecular patterns expressed by pathogens and damaged cells. Activation of TLRs can induce several effects including inflammatory responses, modulation of cell cycle, apoptosis, or regulation of cell metabolism. In multiple myeloma there is a dysregulated signalling of TLRs due to an abnormal presence of certain pathogens and release of molecules from damaged cells. Thus, TLRs could be critical players for tumour microenvironment and multiple myeloma progression. This haematological malignancy is characterized by a high percentage of recurrences, where many patients can develop residual drug-resistant malignant cells. Strategic targeting of TLRs might result in novel therapeutic combinations that improve the response to current treatments, reducing relapses. This review examines the potential of TLRs as targets for the treatment of multiple myeloma, making a particular emphasis on their therapeutic applications.

Astrocyte senescence may drive alterations in GFAPα, CDKN2A p14ARF, and TAU3 transcript expression and contribute to cognitive decline.

The accumulation of senescent cells in tissues is causally linked to the development of several age-related diseases; the removal of senescent glial cells in animal models prevents Tau accumulation and cognitive decline. Senescent cells can arise through several distinct mechanisms; one such mechanism is dysregulation of alternative splicing. In this study, we characterised the senescent cell phenotype in primary human astrocytes in terms of SA-ß-Gal staining and SASP secretion, and then assessed splicing factor expression and candidate gene splicing patterns. Finally, we assessed associations between expression of dysregulated isoforms and premature cognitive decline in 197 samples from the InCHIANTI study of ageing, where expression was present in both blood and brain. We demonstrate here that senescent astrocytes secrete a modified SASP characterised by increased IL8, MMP3, MMP10, and TIMP2 but decreased IL10 levels. We identified significant changes in splicing factor expression for 10/20 splicing factors tested in senescent astrocytes compared with early passage cells, as well as dysregulation of isoform levels for 8/13 brain or senescence genes tested. Finally, associations were identified between peripheral blood GFAPα, TAU3, and CDKN2A (P14ARF) isoform levels and mild or severe cognitive decline over a 3-7-year period. Our data are suggestive that some of the features of cognitive decline may arise from dysregulated splicing of important genes in senescent brain support cells, and that defects in alternative splicing or splicing regulator expression deserve exploration as points of therapeutic intervention in the future.

TLR2 and TLR4 interact with sulfide system in the modulation of mouse colonic motility.

BACKGROUND: H2 S is a neuromodulator that may inhibit intestinal motility. H2 S production in colon is yielded by cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) enzymes and sulfate-reducing bacteria (SRB). Toll-like receptors (TLRs) recognize intestinal microbiota. The aim of this work was to evaluate the influence of TLR2 and TLR4 on the endogenous and SRB-mediated synthesis of H2 S and its consequences on the colonic motility of mouse. METHODS: Muscle contractility studies were performed in colon from WT, Tlr2-/- , and Tlr4-/- mice. The mRNA levels of TLR2, TLR4, CBS, CSE, and SRB were measured by real-time PCR. Free sulfide levels in colon and feces were determined by colorimetric assays. RESULTS: NaHS and GYY4137, donors of H2 S, reduced the contractility of colon. Aminooxyacetic acid (AOAA), inhibitor of CBS, and D-L propargylglycine (PAG), inhibitor of CSE, increased the contractility of colon. In vivo treatment with NaHS or GYY4137 inhibited the spontaneous contractions and upregulated TLR2 expression. The in vivo activation of TLR4 with lipopolysaccharide increased the contractile response to PAG, mRNA levels of CSE, and the free sulfide levels of H2 S in colon. In Tlr2-/- and Tlr4-/-  mice, the contractions induced by AOAA and PAG and mRNA levels of CBS and CSE were lower with respect to WT mice. Deficiency of TLR2 or TLR4 provokes alterations in free sulfide levels and SRB of colon. CONCLUSIONS AND INFERENCES: Our study demonstrates interaction between TLR2 and TLR4 and the sulfide system in the regulation of colonic motility and contributes to the pathophysiology knowledge of intestinal motility disorders.

FOXO1 and ETV6 genes may represent novel regulators of splicing factor expression in cellular senescence.

Cellular plasticity is a key facet of cellular homeostasis requiring correct temporal and spatial patterns of alternative splicing. Splicing factors, which orchestrate this process, demonstrate age-related dysregulation of expression; they are emerging as potential influences on aging and longevity. The upstream drivers of these alterations are still unclear but may involve aberrant cellular signaling. We compared the phosphorylation status of proteins in multiple signaling pathways in early and late passage human primary fibroblasts. We then assessed the impact of chemical inhibition or targeted knockdown of direct downstream targets of the ERK and AKT pathways on splicing factor expression, cellular senescence, and proliferation kinetics in senescent primary human fibroblasts. Components of the ERK and AKT signaling pathways demonstrated altered activation during cellular aging. Inhibition of AKT and ERK pathways led to up-regulation of splicing factor expression, reduction in senescent cell load, and partial reversal of multiple cellular senescence phenotypes in a dose-dependent manner. Furthermore, targeted knockdown of the genes encoding the downstream targets FOXO1 or ETV6 was sufficient to mimic these observations. Our results suggest that age-associated dysregulation of splicing factor expression and cellular senescence may derive in part from altered activity of ERK and AKT signaling and may act in part through the ETV6 and FOXO1 transcription factors. Targeting the activity of downstream effectors of ERK and AKT may therefore represent promising targets for future therapeutic intervention.-Latorre, E., Ostler, E. L., Faragher, R. G. A., Harries, L. W. FOXO1 and ETV6 genes may represent novel regulators of splicing factor expression in cellular senescence.

Mitochondria-targeted hydrogen sulfide attenuates endothelial senescence by selective induction of splicing factors HNRNPD and SRSF2.

Cellular senescence is a key driver of ageing, influenced by age-related changes to the regulation of alternative splicing. Hydrogen sulfide (H2S) has similarly been described to influence senescence, but the pathways by which it accomplishes this are unclear.We assessed the effects of the slow release H2S donor Na-GYY4137 (100 µg/ml), and three novel mitochondria-targeted H2S donors AP39, AP123 and RT01 (10 ng/ml) on splicing factor expression, cell proliferation, apoptosis, DNA replication, DNA damage, telomere length and senescence-related secretory complex (SASP) expression in senescent primary human endothelial cells.All H2S donors produced up to a 50% drop in senescent cell load assessed at the biochemical and molecular level. Some changes were noted in the composition of senescence-related secretory complex (SASP); IL8 levels increased by 24% but proliferation was not re-established in the culture as a whole. Telomere length, apoptotic index and the extent of DNA damage were unaffected. Differential effects on splicing factor expression were observed depending on the intracellular targeting of the H2S donors. Na-GYY4137 produced a general 1.9 - 3.2-fold upregulation of splicing factor expression, whereas the mitochondria-targeted donors produced a specific 2.5 and 3.1-fold upregulation of SRSF2 and HNRNPD splicing factors only. Knockdown of SRSF2 or HNRNPD genes in treated cells rendered the cells non-responsive to H2S, and increased levels of senescence by up to 25% in untreated cells.Our data suggest that SRSF2 and HNRNPD may be implicated in endothelial cell senescence, and can be targeted by exogenous H2S. These molecules may have potential as moderators of splicing factor expression and senescence phenotypes.

The VEGFA156b isoform is dysregulated in senescent endothelial cells and may be associated with prevalent and incident coronary heart disease.

Coronary heart disease (CHD) is a leading cause of morbidity in people over 65 years of age; >40% of all deaths are due to this condition. The association between increasing age and CHD is well documented; the accumulation of senescent cells in cardiac and vascular tissues may represent one factor underpinning this observation. We aimed to identify senescence-related expression changes in primary human senescent cardiomyocytes and endothelial cells and to relate transcript expression in peripheral blood leucocytes to prevalent and incident CHD in the InCHIANTI study of aging. We quantified splicing factor expression and splicing patterns of candidate transcripts in proliferative and senescent later passage endothelial cells and cardiomyocytes using qRTPCR. Senescence-associated isoforms also expressed in peripheral blood leucocytes were then examined for associations with CHD status in 134 pairs of age, sex and BMI-matched CHD cases and controls. Splicing factor expression was dysregulated in senescent cardiomyocytes, as previously reported for endothelial cells, as was the expression of alternatively expressed cardiac and vascular candidate genes in both cell types. We found nominal associations between the expression of VEGFA156b and FNI-EIIIIA isoforms in peripheral blood mRNA and CHD status. Dysregulated splicing factor expression is a key feature of senescent cardiomyocytes and endothelial cells. Altered splicing of key cardiac or endothelial genes may contribute to the risk of CHD in the human population.

Splicing regulatory factors, ageing and age-related disease.

Alternative splicing is a co-transcriptional process, which allows for the production of multiple transcripts from a single gene and is emerging as an important control point for gene expression. Alternatively expressed isoforms often have antagonistic function and differential temporal or spatial expression patterns, yielding enormous plasticity and adaptability to cells and increasing their ability to respond to environmental challenge. The regulation of alternative splicing is critical for numerous cellular functions in both pathological and physiological conditions, and deregulated alternative splicing is a key feature of common chronic diseases. Isoform choice is controlled by a battery of splicing regulatory proteins, which include the serine arginine rich (SRSF) proteins and the heterogeneous ribonucleoprotein (hnRNP) classes of genes. These important splicing regulators have been implicated in age-related disease, and in the ageing process itself. This review will outline the important contribution of splicing regulator proteins to ageing and age-related disease.

IL-10 counteracts proinflammatory mediator evoked oxidative stress in Caco-2 cells.

Oxidative stress is thought to play a key role in the development of intestinal damage in intestinal inflammatory diseases. Several molecules are involved in the intestinal inflammation, either as pro- or anti-inflammatory factors; however, their effects on intestinal oxidative stress seem to be controversial. This work analyzes the contribution of pro- and anti-inflammatory molecules to the balance of oxidative damage in intestinal epithelial cells, as well as their effects on cellular antioxidant enzyme activity. With this purpose, the lipid and protein oxidation, together with the activity of catalase, superoxide dismutase, and glutathione peroxidase, were determined in the Caco-2 cells treated with serotonin, adenosine, melatonin, and TNFα, as proinflammatory factors, and IL-10, as an anti-inflammatory cytokine. The results have shown that all the proinflammatory factors assayed increased oxidative damage. In addition, these factors also inhibited the activity of antioxidant enzymes in the cells, except melatonin. In contrast, IL-10 did not alter these parameters but was able to reduce the prooxidant effects yielded by serotonin, adenosine, melatonin, or TNFα, in part by restoring the antioxidant enzymes activities. In summary, proinflammatory factors may induce oxidative damage in intestinal epithelial cells, whereas IL-10 seems to be able to restore the altered redox equilibrium in Caco-2 cells.

TLR2, TLR3, and TLR4 activation specifically alters the oxidative status of intestinal epithelial cells.

Intestinal inflammatory diseases are the result of multiple processes, including mucosal oxidative stress and perturbed homeostasis between commensal bacteria and mucosal immunity. Toll-like receptors (TLRs) recognize molecular-associated microorganisms' patterns and trigger innate immunity responses contributing to intestinal homeostasis and inflammatory responses. However, TLRs effects on redox balance in intestinal mucosa remain unknown. Therefore, the present study analyzes the effect of TLR2, TLR3, and TLR4 on both oxidative damage of lipids and proteins, and the activity of antioxidant enzymes in enterocyte-like Caco-2 cells. The results show that the activation of these TLRs increased lipid and protein oxidation levels; however, the effect on the antioxidant enzymes activity is different depending on the TLR activated. These results suggest that the activation of TLR2, TLR3, and TLR4 might affect intestinal inflammation by not only their inherent innate immunity responses, but also their pro-oxidative effects on intestinal epithelial cells.

IL-10 modulates serotonin transporter activity and molecular expression in intestinal epithelial cells.

Serotonin is a neuromodulator mainly synthesized by intestinal enterochromaffin cells that regulate overall intestinal physiology. The serotonin transporter (SERT) determines the final serotonin availability and has been described as altered in inflammatory bowel diseases. IL-10 is an anti-inflammatory cytokine that is involved in intestinal inflammatory processes and also contributes to intestinal mucosa homeostasis. The regulation of SERT by pro-inflammatory factors is well known; however, the effect of IL-10 on the intestinal serotoninergic system mediated by SERT remains unknown. Therefore, the aim of the present study is to determine whether IL-10 affects SERT activity and expression in enterocyte-like Caco-2 cells. Treatment with IL-10 was assessed and SERT activity was determined by 5-HT uptake. SERT mRNA and protein expression was analyzed using quantitative RT-PCR and western blotting. The results showed that IL-10 induced a dual effect on SERT after 6h of treatment. On one hand, IL-10, at a low concentration, inhibited SERT activity, and this effect might be explained by a non-competitive inhibition of SERT. On the other hand, IL-10, at a high concentration, increased SERT activity and molecular expression in the membrane of the cells. This effect was mediated by the IL-10 receptor and triggered by the PI3K intracellular pathway. Our results demonstrate that IL-10 modulates SERT activity and expression, depending on its extracellular conditions. This study may contribute to understand serotoninergic responses in intestinal pathophysiology.

Toll-like receptor 3 activation affects serotonin transporter activity and expression in human enterocyte-like Caco-2 cells.

Serotonin, a neurotransmitter/autocrineagent mainly synthesized by intestinal enterochromaffin cells, regulates the whole intestinal physiology. Toll-like receptor 3 (TLR3) also contributes to the intestinal physiology by modulating intestinal innate immunity responses. Both serotonin and TLR3 are involved in intestinal inflammatory processes; however, the role of TLR3 in the regulation of intestinal 5-HT availability remains unexplored. The present study analyzes the effect of TLR3 activation on serotonin transporter (SERT) activity in Caco-2 cells. Treatment with poly(I:C), dsRNA synthetic analogue and TLR3 ligand, was assayed and SERT activity determined by 5-HT uptake and transepithelial flux. SERT expression was analyzed by qRT-PCR and western blotting. Poly(I:C) short-term treatment inhibited SERT activity in the apical and basal membrane of epithelial cells and diminished SERT protein content in the membrane. SERT total protein and mRNA levels were not affected by poly(I:C), suggesting a post-translational alteration of SERT. The poly(I:C) effect on SERT activity did not appear to be mediated by PKC, cAMP, PKR or JNK signaling pathways; however, the p38 MAPK pathway seemed to be involved. Our results demonstrate that TLR3 inhibits SERT activity, which may increase 5-HT extracellular levels and contribute to the inflammatory response; however, 5-HT treatment did not affect TLR3 expression.