Fas Signalling Promotes Intercellular Communication in T Cells

Cell-to-cell communication is a fundamental process for development and maintenance of multicellular organisms. Diverse mechanisms for the exchange of molecular information between cells have been documented, such as the exchange of membrane fragments (trogocytosis), formation of tunneling nanotubes (TNTs) and release of microvesicles (MVs). In this study we assign to Fas signalling a pivotal role for intercellular communication in CD4+ T cells. Binding of membrane-bound FasL to Fas expressing target cells triggers a well-characterized pro-apoptotic signalling cascade. However, our results, pairing up flow cytometric studies with confocal microscopy data, highlight a new social dimension for Fas/FasL interactions between CD4+ T cells. Indeed, FasL enhances the formation of cell conjugates (8 fold of increase) in an early time-frame of stimulation (30 min), and this phenomenon appears to be a crucial step to prime intercellular communication. Our findings show that this communication mainly proceeds along a cytosolic material exchange (ratio of exchange >10, calculated as ratio of stimulated cells signal divided by that recorded in control cells) via TNTs and MVs release. In particular, inhibition of TNTs genesis by pharmacological agents (Latruculin A and Nocodazole) markedly reduced this exchange (inhibition percentage: >40% and >50% respectively), suggesting a key role for TNTs in CD4+ T cells communication. Although MVs are present in supernatants from PHA-activated T cells, Fas treatment also leads to a significant increase in the amount of released MVs. In fact, the co-culture performed between MVs and untreated cells highlights a higher presence of MVs in the medium (1.4 fold of increase) and a significant MVs uptake (6 fold of increase) by untreated T lymphocytes. We conclude that Fas signalling induces intercellular communication in CD4+ T cells by different mechanisms that seem to start concomitantly with the main pathway (programmed cell death) promoted by FasL

Discovery and Characterization of an Endogenous CXCR4 Antagonist

CXCL12-CXCR4 signaling controls multiple physiological processes and its dysregulation is associated with cancers and inflammatory diseases. To discover as-yet-unknown endogenous ligands of CXCR4, we screened a blood-derived peptide library for inhibitors of CXCR4-tropic HIV-1 strains. This approach identified a 16 amino acid fragment of serum albumin as an effective and highly specific CXCR4 antagonist. The endogenous peptide, termed EPI-X4, is evolutionarily conserved and generated from the highly abundant albumin precursor by pH-regulated proteases. EPI-X4 forms an unusual lasso-like structure and antagonizes CXCL12-induced tumor cell migration, mobilizes stem cells, and suppresses inflammatory responses in mice. Furthermore, the peptide is abundant in the urine of patients with inflammatory kidney diseases and may serve as a biomarker. Our results identify EPIX4 as a key regulator of CXCR4 signaling and introduce proteolysis of an abundant precursor protein as an alternative concept for chemokine receptor regulation

Blocking TLR2 Activity Attenuates Pulmonary Metastases of Tumor

Background: Metastasis is the most pivotal cause of mortality in cancer patients. Immune tolerance plays a crucial role in tumor progression and metastasis. Methods and Findings: In this study, we investigated the potential roles and mechanisms of TLR2 signaling on tumor metastasis in a mouse model of intravenously injected B16 melanoma cells. Multiple subtypes of TLRs were expressed on B16 cells and several human cancer cell lines; TLR2 mediated the invasive activity of these cells. High metastatic B16 cells released more heat shock protein 60 than poor metastatic B16-F1 cells. Importantly, heat shock protein 60 released by tumor cells caused a persistent activation of TLR2 and was critical in the constitutive activation of transcription factor Stat3, leading to the release of immunosuppressive cytokines and chemokines. Moreover, targeting TLR2 markedly reduced pulmonary metastases and increased the survival of B16-bearing mice by reversing B16 cells induced immunosuppressive microenvironment and restoring tumor-killing cells such as CD8 + T cells and M1 macrophages. Combining an anti-TLR2 antibody and a cytotoxic agent, gemcitabine, provided a further improvement in the survival of tumor-bearing mice. Conclusions and Significance: Our results demonstrate that TLR2 is an attractive target against metastasis and that targeting immunosuppressive microenvironment using anti-TLR2 antibody is a novel therapeutic strategy for combating

Chitohexaose Activates Macrophages by Alternate Pathway through TLR4 and Blocks Endotoxemia

Sepsis is a consequence of systemic bacterial infections leading to hyper activation of immune cells by bacterial products resulting in enhanced release of mediators of inflammation. Endotoxin (LPS) is a major component of the outer membrane of Gram negative bacteria and a critical factor in pathogenesis of sepsis. Development of antagonists that inhibit the storm of inflammatory molecules by blocking Toll like receptors (TLR) has been the main stay of research efforts. We report here that a filarial glycoprotein binds to murine macrophages and human monocytes through TLR4 and activates them through alternate pathway and in the process inhibits LPS mediated classical activation which leads to inflammation associated with endotoxemia. The active component of the nematode glycoprotein mediating alternate activation of macrophages was found to be a carbohydrate residue, Chitohexaose. Murine macrophages and human monocytes up regulated Arginase-1 and released high levels of IL-10 when incubated with chitohexaose. Macrophages of C3H/HeJ mice (non-responsive to LPS) failed to get activated by chitohexaose suggesting that a functional TLR4 is critical for alternate activation of macrophages also. Chitohexaose inhibited LPS induced production of inflammatory molecules TNF-α, IL-1β and IL-6 by macropahges in vitro and in vivo in mice. Intraperitoneal injection of chitohexaose completely protected mice against endotoxemia when challenged with a lethal dose of LPS. Furthermore, Chitohexaose was found to reverse LPS induced endotoxemia in mice even 6/24/48 hrs after its onset. Monocytes of subjects with active filarial infection displayed characteristic alternate activation markers and were refractory to LPS mediated inflammatory activation suggesting an interesting possibility of subjects with filarial infections being less prone to develop of endotoxemia. These observations that innate activation of alternate pathway of macrophages by chtx through TLR4 has offered novel opportunities to cell biologists to study two mutually exclusive activation pathways of macrophages being mediated through a single receptor

Plasmodium falciparum Adhesion on Human Brain Microvascular Endothelial Cells Involves Transmigration-Like Cup Formation and Induces Opening of Intercellular Junctions

Cerebral malaria, a major cause of death during malaria infection, is characterised by the sequestration of infected red blood cells (IRBC) in brain microvessels. Most of the molecules implicated in the adhesion of IRBC on endothelial cells (EC) are already described; however, the structure of the IRBC/EC junction and the impact of this adhesion on the EC are poorly understood. We analysed this interaction using human brain microvascular EC monolayers co-cultured with IRBC. Our study demonstrates the transfer of material from the IRBC to the brain EC plasma membrane in a trogocytosis-like process, followed by a TNF-enhanced IRBC engulfing process. Upon IRBC/EC binding, parasite antigens are transferred to early endosomes in the EC, in a cytoskeleton-dependent process. This is associated with the opening of the intercellular junctions. The transfer of IRBC antigens can thus transform EC into a target for the immune response and contribute to the profound EC alterations, including peri-vascular oedema, associated with cerebral malaria

T cells at the site of autoimmune inflammation show increased potential for trogocytosis

CD4+ T cells acquire membrane fragments from antigen-presenting-cells via a process termed trogocytosis. Identifying which CD4+ T cells undergo trogocytosis in co-culture with Ag-loaded APC can enrich for antigen-reactive T cells without knowledge of their fine specificity or cytokine-production profiles. We sought to assess the suitability of this method to identify disease relevant effector and regulatory T cells during autoimmune inflammation. Trogocytosis efficiently identified MBP-reactive T cells in vitro and ex-vivo following immunization. However, Foxp3+ regulatory T cells constitutively displayed a higher rate of trogocytosis than their Foxp3- counterparts which limits the potential of trogocytosis to identify antigen-reactive Treg cells. During inflammation a locally elevated rate of trogocytosis (seen in both effector and regulatory T cells isolated from the inflamed CNS) precludes the use of trogocytosis as a measure of antigenic reactivity among cells taken from inflammatory sites. Our results indicate trogocytosis detection can enrich for Ag-reactive conventional T cells in the periphery but is limited in its ability to identify Ag-reactive Treg or T effector cells at sites of inflammation. Increased trogocytosis potential at inflammatory sites also draws into the question the biological significance of this phenomenon during inflammation, in Treg mediated suppression and for the maintenance of tolerance in health and disease

TLR4/MD-2 monoclonal antibody therapy affords protection in experimental models of septic shock.

Overactivation of the immune system upon acute bacterial infection leads to septic shock. Specific bacterial products potently stimulate immune cells via toll-like receptors (TLRs). Gram-negative bacteria induce a predominantly TLR4-driven signal through LPS release. To neutralize LPS signaling in experimental models of sepsis, we generated mAbs toward the TLR4/myeloid differentiation protein-2 (MD-2) complex. The binding properties of an array of selected rat mAbs differed in respect to their specificity for TLR4/MD-2 complex. The specificity of one such mAb, 5E3, to murine TLR4 was confirmed by its recognition of an epitope within the second quarter of the ectodomain. 5E3 inhibited LPS-dependent cell activation in vitro and prevented proinflammatory cytokine production in vivo following LPS challenge in a dose-dependent manner. Furthermore, 5E3 protected mice from lethal shock-like syndrome when applied using both preventative and therapeutic protocols. Most notably, in the colon ascendens stent peritonitis model of polymicrobial abdominal sepsis, administration of a single dose of 5E3 (50 mug) protected mice against mortality. These results demonstrate that neutralizing TLR4/MD-2 is highly efficacious in protecting against bacterial infection-induced toxemia and offers TLR4/MD-2 mAb treatment as a potential therapy for numerous clinical indications