Lena Svensson

Exosome uptake depends on ERK1/2-heat shock protein 27 signalling and lipid raft-mediated endocytosis negatively regulated by caveolin-1

Background: Exosome vesicles can transfer molecular information previously shown to stimulate tumor development; however, the mechanism of exosome uptake is unknown. Results: Mammalian cells internalize exosomes through lipid raft-mediated endocytosis negatively regulated by caveolin-1. Conclusion: Our findings provide novel insights into cellular uptake of exosomes. Significance: Our data provide potential strategies for how the exosome uptake pathway may be targeted. The role of exosomes in cancer can be inferred from the observation that they transfer tumor cell derived genetic material and signaling proteins, resulting in e.g. increased tumor angiogenesis and metastasis. However, the membrane transport mechanisms and the signaling events involved in the uptake of these virus-like particles remain ill-defined. We now report that internalization of exosomes derived from glioblastoma (GBM) cells involves nonclassical, lipid raft-dependent endocytosis. Importantly, we show that the lipid raft-associated protein caveolin-1 (CAV1), in analogy with its previously described role in virus uptake, negatively regulates the uptake of exosomes. We find that exosomes induce the phosphorylation of several downstream targets known to associate with lipid rafts as signaling and sorting platforms, such as extracellular signal-regulated kinase-1/2 (ERK1/2) and heat shock protein 27 (HSP27). Interestingly, exosome uptake appears dependent on unperturbed ERK1/2-HSP27 signaling, and ERK1/2 phosphorylation is under negative influence by CAV1 during internalization of exosomes. These findings significantly advance our general understanding of exosome-mediated uptake and offer potential strategies for how this pathway may be targeted through modulation of CAV1 expression and ERK1/2 signaling.

Integrins in immunity

A successful immune response depends on the capacity of immune cells to travel from one location in the body to another–these cells are rapid migrators, travelling at speeds of μm/minute. Their ability to penetrate into tissues and to make contacts with other cells depends chiefly on the β2 integrin known as LFA-1. For this reason, we describe the control of its activity in some detail. For the non-immunologist, the fine details of an immune response often seem difficult to fathom. However, the behaviour of immune cells, known as leukocytes (Box 1), is subject to the same biological rules as many other cell types, and this holds true particularly for the functioning of the integrins on these cells. In this Commentary, we highlight, from a cell-biology point of view, the integrin-mediated immune-cell migration and cell-cell interactions that occur during the course of an immune response.

Why is PTPN22 a good candidate susceptibility gene for autoimmune disease

The PTPN22 locus is one of the strongest risk factors outside of the major histocompatability complex that associates with autoimmune diseases. PTPN22 encodes lymphoid protein tyrosine phosphatase (Lyp) which is expressed exclusively in immune cells. A single base change in the coding region of this gene resulting in an arginine to tryptophan amino acid substitution within a polyproline binding motif associates with type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosis, Hashimotos thyroiditis, Graves disease, Addisons disease, Myasthenia Gravis, vitiligo, systemic sclerosis juvenile idiopathic arthritis and psoriatic arthritis. Here, we review the current understanding of the PTPN22 locus from a genetic, geographical, biochemical and functional perspective.

Lack of the phosphatase PTPN22 increases adhesion of murine regulatory T cells to improve their immunosuppressive function

Loss of the phosphatase PTPN22 enhances the functions of both effector and regulatory T cells. Tipping Both Sides of the Inflammatory Scales Autoimmune disorders arise because of an imbalance in the immune response. For example, effector T cells can exhibit enhanced activation, causing damaging, proinflammatory responses, whereas regulatory T cells (Tregs), which inhibit effector T cell responses, may become less effective at immunosuppression. Brownlie et al. investigated a role in the immune response for PTPN22, a protein tyrosine phosphatase implicated in autoimmunity in humans and mice. Although PTPN22-deficient mice had more potent effector T cells, they did not develop autoimmunity because they also had Tregs with greater immunosuppressive function than those of wild-type mice. Together, these data suggest that manipulating PTPN22 function in human Tregs may help in the treatment of autoimmune diseases. The cytoplasmic phosphatase PTPN22 (protein tyrosine phosphatase nonreceptor type 22) plays a key role in regulating lymphocyte homeostasis, which ensures that the total number of lymphocytes in the periphery remains relatively constant. Mutations in PTPN22 confer an increased risk of developing autoimmune diseases; however, the precise function of PTPN22 and how mutations contribute to autoimmunity remain controversial. Loss-of-function mutations in PTPN22 are associated with increased numbers of effector T cells and autoreactive B cells in humans and mice; however, the complete absence of PTPN22 in mice does not result in spontaneous autoimmunity. We found that PTPN22 was a key regulator of regulatory T cell (Treg) function that fine-tuned the signaling of the T cell receptor and integrins. PTPN22−/− Tregs were more effective at immunosuppression than were wild-type Tregs, and they suppressed the activity of PTPN22−/− effector T cells, preventing autoimmunity. Compared to wild-type Tregs, PTPN22−/− Tregs produced increased amounts of the immunosuppressive cytokine interleukin-10 and had enhanced adhesive properties mediated by the integrin lymphocyte function–associated antigen-1, processes that are critical for Treg function. This previously undiscovered role of PTPN22 in regulating integrin signaling and Treg function suggests that PTPN22 may be a useful therapeutic target for manipulating Treg function in human disease.

Two mutations in the KINDLIN3 gene of a new leukocyte adhesion deficiency-III patient reveal distinct effects on leukocyte function in vitro

In the disorder leukocyte adhesion deficiency III (LAD-III), integrins on platelets and leukocytes are expressed but fail to function and this leads to severe bleeding and infections at an early age. Mutation in the KINDLIN3 (FERMT3) gene is the cause of LAD-III in patients from the Middle East, Malta, and Turkey. We describe 2 novel homozygous mutations in the KINDLIN3 gene of a new African-American patient that destabilize KINDLIN3 mRNA leading to loss of kindlin-3 protein. Transfection of wild-type (WT) KINDLIN3 cDNA restored integrin-related adhesion and migration in the LAD-III patients T and B lymphocytes. We analyzed the individual mutations separately in vitro to learn more about the function of the kindlin-3 protein. The first G>A mutation gives rise to a Gly308Arg change at the end of FERM (protein 4.1, ezrin, radixin, moesin) subdomain 2, and the second mutation is a base deletion causing early termination within the pleckstrin homology (PH) domain. This second mutation prevented membrane association of kindlin-3 and did not restore either adhesion or migration, whereas the FERM subdomain 2 mutation affected only migration. Thus, these LAD-III patient mutations have highlighted functionally important regions of kindlin-3 that alter leukocyte integrin-dependent function in 2 distinct ways.

Wound infiltration with ropivacaine and fentanyl: Effects on postoperative pain and PONV after breast surgery

STUDY OBJECTIVE To determine whether postoperative wound infiltration with local anesthetics combined with fentanyl improves analgesia following breast surgery; and to investigate awakening and postoperative nausea/vomiting. DESIGN Prospectively randomized clinical study. SETTING University hospital. PATIENTS 45 ASA physical status I and II patients scheduled for breast surgery. INTERVENTIONS Patients were prospectively randomized and assigned to one of three treatments during general anesthesia: postsurgical wound infiltration with ropivacaine 0.375%; wound infiltration with ropivacaine 0.375% combined with fentanyl 0.5 microg/kg; and intravenous (i.v.) fentanyl 0.5 microg/kg before skin incision and no wound infiltration. Time to first verbal response, pain at rest, postoperative nausea and vomiting, and ketobemidone and dixyrazine utilization were compared. MEASUREMENTS AND MAIN RESULTS Time to first verbal response was significantly shorter in the i.v. fentanyl group compared to both infiltration groups (8.1 +/- 4.5 min vs. 15.3 +/- 4.3, and 12.0 +/- 5.0 min; p < 0.05, respectively). Postoperative pain at rest, and nausea and vomiting occurred with similar frequencies in the groups. Ketobemidone utilization in both infiltration groups, (2.4 +/- 1.8 mg and 3.1 +/- 1.8 mg, respectively) was not different compared to the i.v. fentanyl group (2.9 +/- 2.0 mg; NS). There were no differences in postoperative antiemetic requirements during the first, second and third two-hour periods postoperatively. The dixyrazine consumption was similar in the three groups, (0.9 +/- 1.5 mg, 0.8 +/- 1.3 mg, and 1.4 +/- 1.8 mg, respectively; NS). CONCLUSION Postsurgical ropivacaine wound infiltration, with or without adding fentanyl, demonstrates no differences in postoperative pain relief and nausea/vomiting compared to a balanced general anesthetic including i.v. fentanyl.

The Gαq/11 Proteins Contribute to T Lymphocyte Migration by Promoting Turnover of Integrin LFA-1 through Recycling

The role of Gαi proteins coupled to chemokine receptors in directed migration of immune cells is well understood. In this study we show that the separate class of Gαq/11 proteins is required for the underlying ability of T cells to migrate both randomly and in a directed chemokine-dependent manner. Interfering with Gαq or Gα11 using dominant negative cDNA constructs or siRNA for Gαq causes accumulation of LFA-1 adhesions and stalled migration. Gαq/11 has an impact on LFA-1 expression at plasma membrane level and also on its internalization. Additionally Gαq co-localizes with LFA-1- and EEA1-expressing intracellular vesicles and partially with Rap1- but not Rab11-expressing vesicles. However the influence of Gαq is not confined to the vesicles that express it, as its reduction alters intracellular trafficking of other vesicles involved in recycling. In summary vesicle-associated Gαq/11 is required for the turnover of LFA-1 adhesion that is necessary for migration. These G proteins participate directly in the initial phase of recycling and this has an impact on later stages of the endo-exocytic pathway.

Iduronic Acid in chondroitin/dermatan sulfate affects directional migration of aortic smooth muscle cells

Aortic smooth muscle cells produce chondroitin/dermatan sulfate (CS/DS) proteoglycans that regulate extracellular matrix organization and cell behavior in normal and pathological conditions. A unique feature of CS/DS proteoglycans is the presence of iduronic acid (IdoA), catalyzed by two DS epimerases. Functional ablation of DS-epi1, the main epimerase in these cells, resulted in a major reduction of IdoA both on cell surface and in secreted CS/DS proteoglycans. Downregulation of IdoA led to delayed ability to re-populate wounded areas due to loss of directional persistence of migration. DS-epi1−/− aortic smooth muscle cells, however, had not lost the general property of migration showing even increased speed of movement compared to wild type cells. Where the cell membrane adheres to the substratum, stress fibers were denser whereas focal adhesion sites were fewer. Total cellular expression of focal adhesion kinase (FAK) and phospho-FAK (pFAK) was decreased in mutant cells compared to control cells. As many pathological conditions are dependent on migration, modulation of IdoA content may point to therapeutic strategies for diseases such as cancer and atherosclerosis.

Superresolution imaging of the cytoplasmic phosphatase PTPN22 links integrin-mediated T cell adhesion with autoimmunity

The tyrosine phosphatase PTPN22 redistributes from clusters to the leading edge in migrating T cells to inhibit integrin-mediated adhesion. Release the phosphatase! T cells need to move through the circulation, attach to endothelial cells, transmigrate into tissues, and stably interact with target cells. The phosphatase PTPN22 targets phosphorylated tyrosines in Src and Syk family kinases, many of which are phosphorylated and activated in migrating T cells in response to the binding of the integrin LFA-1 to its ligand ICAM-1. Burn et al. used superresolution microscopy to show that PTPN22 formed clusters in nonmigrating T cells, which were dispersed in T cells that migrated on surfaces coated with ICAM-1. Freed from these complexes, PTPN22 interacted with its targets near the front of the migrating T cell, which inhibited LFA-1 signaling. In contrast, clusters containing the PTPN22 R620W mutant, a variant that is associated with autoimmune diseases, failed to disaggregate in migrating T cells, and thus, LFA-1 clustering and signaling were not inhibited. Together, these data suggest how a mutation associated with autoimmunity dysregulates T cell adhesion and migration. Integrins are heterodimeric transmembrane proteins that play a fundamental role in the migration of leukocytes to sites of infection or injury. We found that protein tyrosine phosphatase nonreceptor type 22 (PTPN22) inhibits signaling by the integrin lymphocyte function-associated antigen–1 (LFA-1) in effector T cells. PTPN22 colocalized with its substrates at the leading edge of cells migrating on surfaces coated with the LFA-1 ligand intercellular adhesion molecule–1 (ICAM-1). Knockout or knockdown of PTPN22 or expression of the autoimmune disease–associated PTPN22-R620W variant resulted in the enhanced phosphorylation of signaling molecules downstream of integrins. Superresolution imaging revealed that PTPN22-R620 (wild-type PTPN22) was present as large clusters in unstimulated T cells and that these disaggregated upon stimulation of LFA-1, enabling increased association of PTPN22 with its binding partners at the leading edge. The failure of PTPN22-R620W molecules to be retained at the leading edge led to increased LFA-1 clustering and integrin-mediated cell adhesion. Our data define a previously uncharacterized mechanism for fine-tuning integrin signaling in T cells, as well as a paradigm of autoimmunity in humans in which disease susceptibility is underpinned by inherited phosphatase mutations that perturb integrin function.

CD8αβ + γδ T cells : A novel T cell subset with a potential role in inflammatory bowel disease

γδ T cells have been attributed a wide variety of functions, which in some cases may appear as contradictory. To better understand the enigmatic biology of γδ T cells it is crucial to define the constituting subpopulations. γδ T cells have previously been categorized into two subpopulations: CD8αα+ and CD8− cells. In this study we have defined and characterized a novel subset of human γδ T-cells expressing CD8αβ. These CD8αβ+ γδ T cells differed from the previously described γδ T cell subsets in several aspects, including the degree of enrichment within the gut mucosa, the activation status in blood, the type of TCRδ variant used in blood, and small but significant differences in their response to IL-2 stimulation. Furthermore, the novel subset expressed cytotoxic mediators and CD69, and produced IFN-γ and TNF-α. In patients with active inflammatory bowel disease the mucosal frequencies of CD8αβ+ γδ T cells were significantly lower as compared with healthy controls, correlated negatively with the degree of disease activity, and increased to normal levels as a result of anti–TNF-α therapy. In conclusion, our results demonstrate that CD8αβ+ γδ T cells constitute a novel lymphocyte subset, which is strongly enriched within the gut and may play an important role in gut homeostasis and mucosal healing in inflammatory bowel disease.