Toll-like receptor 2 pathway drives streptococcal cell wall-induced joint inflammation: critical role of myeloid differentiation factor 88

The IL-1R/Toll-like receptor (TLR) superfamily of receptors has a key role in innate immunity and inflammation. In this study, we report that streptococcal cell wall (SCW)-induced joint inflammation is predominantly dependent on TLR-2 signaling, since TLR-2-deficient mice were unable to develop either joint swelling or inhibition of cartilage matrix synthesis. Myeloid differentiation factor 88 (MyD88) is a Toll/IL-1R domain containing adaptor molecule known to have a central role in both IL-1R/IL-18R and TLR signaling. Mice deficient for MyD88 did not develop SCW-induced arthritis; both joint swelling and disturbance of cartilage chondrocyte anabolic function was completely abolished. Local levels of proinflammatory cytokines and chemokines in synovial tissue washouts were strongly reduced in MyD88-deficient mice. Histology confirmed the pivotal role of MyD88 in acute joint inflammation. TLR-2-deficient mice still allow influx of inflammatory cells into the joint cavity, although the number of cells was markedly reduced. No influx of inflammatory cells was seen in joints of MyD88-deficient mice. In addition, cartilage matrix proteoglycan loss was completely absent in MyD88 knockout mice. These findings clearly demonstrated that MyD88 is a key component in SCW-induced joint inflammation. Since agonists of the Toll-like pathway are abundantly involved in both septic and rheumatoid arthritis, targeting of MyD88 may be a novel therapy in inflammatory joint diseases.     

Dynein-mediated vesicle transport controls intracellular Salmonella replication

Salmonella typhimurium survives and replicates intracellular in a membrane-bound compartment, the Salmonella-containing vacuole (SCV). In HeLa cells, the SCV matures through interactions with the endocytic pathway, but Salmonella avoids fusion with mature lysosomes. The exact mechanism of the inhibition of phagolysosomal fusion is not understood. Rab GTPases control several proteins involved in membrane fusion and vesicular transport. The small GTPase Rab7 regulates the transport of and fusion between late endosomes and lysosomes and associates with the SCV. We show that the Rab7 GTPase cycle is not affected on the SCV. We then manipulated a pathway downstream of the small GTPase Rab7 in HeLa cells infected with Salmonella. Expression of the Rab7 effector RILP induces recruitment of the dynein/dynactin motor complex to the SCV. Subsequently, SCV fuse with lysosomes. As a result, the intracellular replication of Salmonella is inhibited. Activation of dynein-mediated vesicle transport can thus control intracellular survival of Salmonella.     

Cloning and characterization of cDNA clones encoding membrane-bound and potentially secreted major histocompatibility class I receptors from walleye (Stizostedion vitreum)

Major histocompatibility (MH) gene polymorphism has been used to type populations of humans, mice, and fish. Walleye ( Stizostedion vitreum) comprise an economically important fishery in Lake Erie, but whether those in the western basin form a single population or separate shoal- and river-breeding populations is not known. To develop MH gene markers for use in defining their population structure, we constructed a head kidney cDNA library from which five full-length class I heavy-chain clones were isolated and sequenced. Although they came in roughly three sizes, 1300, 1400, and over 2000 bp, the clones all exhibited a high degree of sequence similarity to each other and to known teleost MH class I cDNAs in the area encoding the extracellular domains, but showed dramatic differences in their transmembrane and cytoplasmic domains. One clone had an AG repeat that eliminated the hydrophobicity of the transmembrane domain, indicating that it may encode a secreted class I receptor. The other four clones encode three distinctly different cytoplasmic domains. The two clones that encode the same cytoplasmic domain resemble those of the known teleost MH class I sequences the most. Southern blotting indicated that there were four copies of the gene present in the walleye genome. Northern blotting showed that class I MH genes are expressed in most tissues and mRNAs of all three size classes can be detected. A preliminary survey of the polymorphism of these genes indicates that they will provide useful markers for differentiating fish stocks.     

Intrapersonal and Interpersonal Concomitants of Facial Blushing during Everyday Social Encounters

Facial blushing may usually be undesirable but may have an ameliorative function for some individuals under some circumstances. Researchers have studied the blush in laboratory settings, but not in daily life. In the present research, conducted with young adults, we employed for the first time an event-contingent recording method for assessing facial blushing during every-day social encounters. Blushing was associated with feeling embarrassed, ashamed, and exposed. These findings, though based on correlational analyses, are consistent with the idea that blushing is often unpleasant and can be maladaptive, and may contribute to the common belief that blushing is an undesirable response. Frequent blushers generally reported lower levels of dominant behavior, higher levels of submissive behavior, and perceived their social interaction partners as more powerful and less affiliative. This was independent of whether they blushed or not, suggesting that altered social behaviors and perceptions are associated with blushing-associated traits rather than with the blushing state. The experience of the blush varied as a function of the frequency with which a person blushed. Blushing was associated with higher levels of shame in frequent blushers than in infrequent blushers. In infrequent blushers, blushing was associated with higher levels of pleasant affect, suggesting that for infrequent blushers the blush may occur in positive social encounters.     

The Arabidopsis thaliana SERK1 Kinase Domain Spontaneously Refolds to an Active State In Vitro

Auto-phosphorylating kinase activity of plant leucine-rich-repeat receptor-like kinases (LRR-RLK''s) needs to be under tight negative control to avoid unscheduled activation. One way to achieve this would be to keep these kinase domains as intrinsically disordered protein (IDP) during synthesis and transport to its final location. Subsequent folding, which may depend on chaperone activity or presence of interaction partners, is then required for full activation of the kinase domain. Bacterially produced SERK1 kinase domain was previously shown to be an active Ser/Thr kinase. SERK1 is predicted to contain a disordered region in kinase domains X and XI. Here, we show that loss of structure of the SERK1 kinase domain during unfolding is intimately linked to loss of activity. Phosphorylation of the SERK1 kinase domain neither changes its structure nor its stability. Unfolded SERK1 kinase has no autophosphorylation activity and upon removal of denaturant about one half of the protein population spontaneously refolds to an active protein in vitro. Thus, neither chaperones nor interaction partners are required during folding of this protein to its catalytically active state.     

The In-Vivo Use of Superparamagnetic Iron Oxide Nanoparticles to Detect Inflammation Elicits a Cytokine Response but Does Not Aggravate Experimental Arthritis

BackgroundSuperparamagnetic Iron Oxide Nanoparticles (SPION) are used in diagnostic imaging of a variety of different diseases. For such in-vivo application, an additional coating with a polymer, for example polyvinyl alcohol (PVA), is needed to stabilize the SPION and prevent aggregation. As the particles are foreign to the body, reaction against the SPION could occur. In this study we investigated the effects that SPION may have on experimental arthritis after intra-articular (i.a.) or intravenous (i.v.) injection.MethodsPVA-coated SPION were injected either i.a. (6 or 24 μg iron) or i.v. (100 μg or 1 mg iron) into naïve Toll-like receptor-4 deficient (TLR4-/-) or wild-type C57Bl/6 mice, or C57Bl/6 mice with antigen-induced arthritis. As control, some mice were injected with PVA or PBS. MR imaging was performed at 1 and 7 days after injection. Mice were sacrificed 2 hours and 1, 2, 7, 10 and 14 days after injection of the SPION, and RNA from synovium and liver was isolated for pro-inflammatory gene expression analysis. Serum cytokine measurements and whole knee joint histology were also performed.ResultsInjection of a high dose of SPION or PVA into naïve knee joints resulted in an immediate upregulation of pro-inflammatory gene expression in the synovium. A similar gene expression profile was observed after SPION or PVA injection into knee joints of TLR4-/- mice, indicating that this effect is not due to LPS contamination. Histological analysis of the knee joints also revealed synovial inflammation after SPION injection. Two hours after i.v. injection of SPION or PVA into naïve mice, an upregulation of pro-inflammatory gene expression was detected in the liver. Administration of SPION or PVA into arthritic mice via i.a. injection did not result in an upregulation in gene expression and also no additional effects were observed on histology. MR imaging and histology showed long-term retention of SPION in the inflamed joint. However, 14 days after the injections no long-term effects were evident for gene expression, histology or serum cytokine concentrations.ConclusionsInjection of SPION, either locally or systemically, gives an acute inflammatory response. In the long term, up to 14 days after the injection, while the SPION reside in the joint, no further activating effects of SPION were observed. Hence, we conclude that SPION do not aggravate arthritis and can therefore be used safely to detect joint inflammation by MR imaging.     

Phosphatase and tensin homolog (PTEN) in antigen-presenting cells controls Th17-mediated autoimmune arthritis

IntroductionAutoreactive T cells are a central element in many systemic autoimmune diseases. The generation of these pathogenic T cells is instructed by antigen-presenting cells (APCs). However, signaling pathways in APCs that drive autoimmune diseases, such as rheumatoid arthritis, are not understood.MethodsWe measured phenotypic maturation, cytokine production and induction of T cell proliferation of APCs derived from wt mice and mice with a myeloid-specific deletion of PTEN (myeloid PTEN^-/-) in vitro and in vivo. We induced collagen-induced arthritis (CIA) and K/BxN serum transfer arthritis in wt and myeloid-specific PTEN^-/- mice. We measured the cellular composition of lymph nodes by flow cytometry and cytokines in serum and after ex vivo stimulation of T cells.ResultsWe show that myeloid-specific PTEN^-/- mice are almost protected from CIA. Myeloid-specific deletion of PTEN leads to a significant reduction of cytokine expression pivotal for the induction of systemic autoimmunity such as interleukin (IL)-23 and IL-6, leading to a significant reduction of a Th17 type of immune response characterized by reduced production of IL-17 and IL-22. In contrast, myeloid-specific PTEN deficiency did not affect K/BxN serum transfer arthritis, which is independent of the adaptive immune system and solely depends on innate effector functions.ConclusionsThese data demonstrate that the presence of PTEN in myeloid cells is required for the development of CIA. Deletion of PTEN in myeloid cells inhibits the development of autoimmune arthritis by preventing the generation of a pathogenic Th17 type of immune response.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0742-y) contains supplementary material, which is available to authorized users.     

Autophagy Controls BCG-Induced Trained Immunity and the Response to Intravesical BCG Therapy for Bladder Cancer

The anti-tuberculosis-vaccine Bacillus Calmette-Guérin (BCG) is the most widely used vaccine in the world. In addition to its effects against tuberculosis, BCG vaccination also induces non-specific beneficial effects against certain forms of malignancy and against infections with unrelated pathogens. It has been recently proposed that the non-specific effects of BCG are mediated through epigenetic reprogramming of monocytes, a process called trained immunity. In the present study we demonstrate that autophagy contributes to trained immunity induced by BCG. Pharmacologic inhibition of autophagy blocked trained immunity induced in vitro by stimuli such as β–glucans or BCG. Single nucleotide polymorphisms (SNPs) in the autophagy genes ATG2B (rs3759601) and ATG5 (rs2245214) influenced both the in vitro and in vivo training effect of BCG upon restimulation with unrelated bacterial or fungal stimuli. Furthermore, pharmacologic or genetic inhibition of autophagy blocked epigenetic reprogramming of monocytes at the level of H3K4 trimethylation. Finally, we demonstrate that rs3759601 in ATG2B correlates with progression and recurrence of bladder cancer after BCG intravesical instillation therapy. These findings identify a key role of autophagy for the nonspecific protective effects of BCG.