Search

Periodontal diseases are common inflammatory conditions characterized by bone loss in response to simultaneous bacterial aggression and host defenses. The etiology of such diseases is still not completely understood, however. It has been shown that specific pathogens involved in the build-up of dysbiotic biofilms participate actively in the establishment of periodontitis. This multifactorial pathology also depends on environmental factors and host characteristics, especially defenses. The immune response to the pathogens seems to be critical in preventing the disease from starting but also contributes to tissue damage. It is known that small molecules known as antimicrobial peptides (AMPs) are key actors in the innate immune response. They not only target microbes, but also act as immuno-modulators. They can help to recruit or activate cells such as neutrophils, monocytes, dendritic cells, or lymphocytes. AMPs have already been described in the periodontium, and their expression seems to be connected to disease activity. Alpha and beta defensins and LL37 are the AMPs most frequently linked to periodontitis. Additionally, leukocyte infiltrates, especially B-cells, have also been linked to the severity of periodontitis. Indeed, the particular subpopulations of B-cells in these infiltrates have been linked to inflammation and bone resorption. A link between B-cells and AMP could be relevant to understanding B-cells’ action. Some AMP receptors, such as chemokines receptors, toll-like receptors, or purinergic receptors, have been shown to be expressed by B-cells. Consequently, the action of AMPs on B—cell subpopulations could participate to B-cell recruitment, their differentiation, and their implication in both periodontal defense and destruction.

Rationale: Acute lung injury (ALI) causes high mortality, but its molecular mechanisms and therapeutic options remain ill-defined. Gram-negative bacterial infections are the main cause of ALI, leading to lung neutrophil infiltration, permeability increases, deterioration of gas exchange, and lung damage. Platelets are activated during ALI, but insights into their mechanistic contribution to neutrophil accumulation in the lung are elusive. Objectives: To determine mechanisms of platelet-mediated neutrophil recruitment in ALI. Methods: Interference with platelet-neutrophil interactions using antagonists to P-selectin and glycoprotein IIb/IIIa or a small peptide antagonist disrupting platelet chemokine heteromer formation in mouse models of ALI. Measurements and Main Results: In a murine model of LPS-induced ALI, we uncover important roles for neutrophils and platelets in permeability changes and subsequent lung damage. Furthermore, platelet depletion abrogated lung neutrophil infiltration, suggesting a sequential participation of platelets and neutrophils. Whereas antagonists to P-selectin and glycoprotein IIb/IIIa had no effects on LPS-mediated ALI, antibodies to the platelet-derived chemokines CCL5 and CXCL4 strongly diminished neutrophil eflux and permeability changes. The two chemokines were found to form heteromers in human and murine ALI samples, positively correlating with leukocyte influx into the lung. Disruption of CCL5-CXCL4 heteromers in LPS-, acid-, and sepsis-induced ALI abolished lung edema, neutrophil infiltration, and tissue damage, thereby revealing a causal contribution. Conclusions: Taken together, our data identify a novel function of platelet-derived chemokine heteromers during ALI and demonstrate means for therapeutic interference.

Anti-endothelial cell (EC) antibodies (AECA) are a heterogeneous group of antibodies directed against a variety of EC membrane proteins. A pathogenic role for AECA in diseases that involve the vascular system has not been clearly demonstrated. Induction of EC apoptosis appears to be one of the mechanisms by which AECA may exert their effect. AECA from some patients trigger the translocation of anionic phospholipids, most notably phosphatidylserine, from the inner to the outer leaflet of the plasma membrane, and thereafter activation of caspase 3 and cleavage of poly (ADP-ribose) polymerase, hallmarks of apoptosis. Apoptotic cell death generates oxidatively modified moieties, which can induce autoimmune and local inflammatory responses. While a sole AECA target involved in the apoptotic process of ECs has not been identified, some evidence suggests that Heat Shock Proteins may be an outstanding antigen.

Clinical and pathological manifestations present as heterogeneous in vasculitides. Thus, inflammation can affect arteries, arterioles, capillaries, venules, and veins toward major body regions. One common feature of vascular diseases appears to be the presence of anti-HSP60 autoantibodies arising either consecutively to infection and molecular mimicry reaction with bacterial HSP60, or following recognition of endogenous HSP60 translocated or bound onto the surface of stressed endothelial cells. Because their levels are very low in some diseases but strikingly upregulated in others, and because their frequencies vary from one vasculitis to another, anti-HSP60 autoantibodies might play a role in the pathological mechanisms that likely differ among the vascular diseases. Identification of the variety of HSP60 epitope specificities along with each vasculitis would help to understand such discrepancies.

In acute and chronic inflammation, neutrophils and platelets, both of which promote monocyte recruitment, are often activated simultaneously. We investigated how secretory products of neutrophils and platelets synergize to enhance the recruitment of monocytes. We found that neutrophil-borne human neutrophil peptide 1 (HNP1, a-defensin) and platelet-derived CCL5 form heteromers. These heteromers stimulate monocyte adhesion through CCR5 ligation. We further determined structural features of HNP1-CCL5 heteromers and designed a stable peptide that could disturb proinflammatory HNP1-CCL5 interactions. This peptide attenuated monocyte and macrophage recruitment in a mouse model of myocardial infarction. These results establish the in vivo relevance of heteromers formed between proteins released from neutrophils and platelets and show the potential of targeting heteromer formation to resolve acute or chronic inflammation.

Heat shock proteins (HSPs) are a wide family of molecular chaperones. The first members of HSPs were observed in cellular stress reactions, especially in thermic changes ascribing their generic names. HSPs can be induced not only following temperature shift but also after other stress pressures such as shear force, cellular injury, and heavy metal intoxication, which all upregulate their expression. In fact, any changes of cellular microenvironment leading to a disturbance of protein integrity will trigger an increased expression of HSPs. Many of them, such as HSP70 and HSP60 subfamilies, are expressed in basal condition as actors of synthesis, protection, and transport of proteins. In all these processes, HSPs will conduct, restore, or maintain the proper folding of proteins to preserve or help to acquire their functions. These purposes make most of them ubiquitously expressed in a similar way to housekeeping proteins. Indeed, knockout animals for HSP60 or GRP75 develop trouble during embryogenesis and are barely viable. Most HSP classifications sort them according to their molecular weights, including subfamilies of HSP90, HSP70, HSP60, HSP40, HSP27, and HSP10. Unfortunately, this classification is not physiologically relevant, especially regarding their implication in autoimmune processes. All HSPs do not derive from the same phylogenic origins. Eukaryotic cells derived from a fusion of eukaryote and mitochondria ancestors. During evolution, both pools of genes mixed with each other to be located either in mitochondria or in the nucleus. Nevertheless, the protein structures of HSPs overall preserve the characteristics of their source. Moreover, HSPs gather proteins from both eukaryotic and bacterial origin, which will later determine their structure and their potential immunogenicity. This feature enlightens the capacity of many HSPs to stimulate pathogen recognition receptors (PRR), which are key elements of innate immunity to detect pathogens during infection.

Rationale: The leukocyte response in acute inflammation is characterized by an initial recruitment of neutrophils preceding a second wave of monocytes. Neutrophil-derived granule proteins were suggested to hold an important role in this cellular switch. The exact mechanisms by which neutrophils mediate these processes are only partially understood. Objective: To investigate the role of neutrophils and their granule contents in the adhesion of monocyte subpopulations in acute inflammation. Methods and Results: Here, we show that neutrophil-derived cathelicidins (human: LL37, mouse: CRAMP) induce adhesion of classical monocytes but not of nonclassical monocytes in the mouse cremaster muscle and in in vitro flow chamber assays. CRAMP is released from emigrated neutrophils and then transported across the endothelium, where it is presented to rolling leukocytes. Endothelial-bound cathelicidin activates formyl-peptide receptor 2 on classical monocytes, resulting in monocytic β 1 - and β 2 -integrin conformational change toward an extended, active conformation that allows for adhesion to their respective ligands, vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. Conclusions: These data elucidate a novel mechanism of neutrophil-mediated monocyte recruitment, which could be targeted in conditions where recruitment of classical monocytes plays an unfavorable role.

International audience; During the antiphospholipid syndrome, beta2-gpI interacts with phospholipids on endothelial cell (EC) surface to allow the binding of autoantibodies. However, induced-pathogenic intracellular signals suggest that beta2-gpI associates also with a receptor that is still not clearly identified. TLR2 and TLR4 have long been suspected, yet interactions between TLRs and beta2-gpI have never been unequivocally proven. The aim of the study was to identify the TLR directly involved in the binding of beta2-gpI on EC surface. beta2-gpI was not synthesized and secreted by ECs in vitro, but rather taken up from FCS. This uptake occurred through association with TLR2 and TLR4 which partitioned together in the lipid rafts of ECs. After coimmunoprecipitation, mass-spectrometry identification of peptides demonstrated that TLR2, but not TLR4, was implicated in the beta2-gpI retention. These results were further confirmed by plasmon resonance-based studies. Finally, siRNA were used to obtain TLR2-deficient ECs that lost their ability to bind biotinylated beta2-gpI and to trigger downstream phosphorylation of kinases and activation of NFkappaB. TLR4 may upregulate TLR2 expression, thereby contributing to beta2-gpI uptake. However, our data demonstrate that direct binding of beta2-gpI on EC surface occurs through direct interaction with TLR2. Furthermore, signaling for anti-beta2-gpI may be envisioned as a multiprotein complex concentrated in lipid rafts on the EC membrane.

Heat-shock protein (Hsp) family is made up of heterogeneous proteins of which Hsp60 members are the most studied. It is now generally admitted that Hsp60 is not only a mitochondrial component but can be localized on the membrane cell surface. Considered as a signal danger following infections, Hsp60 can induce the production of anti-Hsp60 antibodies as defense mechanisms against pathogens. However, endogenous Hsp60 is also a target of autoantibodies in autoimmune disorders, atherosclerosis and vascular diseases, in which anti-endothelial cell antibodies (AECA) are generated. Hsp60 is one of the endothelial cell autoantigens able to trigger cytotoxic and apoptotic responses when recognized by the related autoantibodies. Depending on the Hsp60 epitope specificity, it appears that AECA with Hsp60 reactivity may differ in their functional effects. These observations suggest that new therapeutic approach to avoid endothelial cell damages due to anti-Hsp60 autoantibodies would be successful provided that specific Hsp60 epitopes would have been precisely characterized.

Objective Anti–endothelial cell antibodies (AECAs), which recognize a number of endothelial antigens, are seen in patients with systemic autoimmune diseases, more often in the presence of vasculitis than in its absence. Some AECAs induce apoptosis of endothelial cells (ECs), but their target antigens remain unknown. The aim of this study was to determine whether Hsp60 is a target antigen and whether AECAs induce apoptosis in ECs. Methods Two-dimensional electrophoresis and conventional Western blotting techniques were used to characterize AECA targets. Hsp60 reactivity was determined by enzyme-linked immunosorbent assay. Results Hsp60 was shown to be targeted by a proportion of AECAs. The level of reactivity was higher in patients with systemic autoimmune disease and vasculitis than in those without vasculitis and in patients with systemic lupus erythematosus than in patients with other systemic autoimmune diseases. Hsp60 was expressed on the plasma membrane of heat-stressed ECs, and this followed Hsp60 messenger RNA transcription, confinement of the protein to the cytoplasm, and translocation of the protein to the surface. Shedding of Hsp60 from ECs was induced by stress and resulted in the binding of soluble Hsp60 to the surface of ECs, particularly stressed ECs. Apoptosis of ECs was triggered by anti-Hsp60–containing AECA-positive sera and was inhibited by preincubation of the ECs with recombinant Hsp60. Conclusion Our data support the notion that Hsp60 is an important target for AECAs and that such an interaction contributes to pathogenic effects, especially in vasculitis-associated systemic autoimmune disease.