[Autoimmunity and oral cavity, where are we in 2023?] / Auto-immunité et cavité orale, où en est-on en 2023 ?

Autoimmune diseases (AIDs) remain an enigma to the current understanding of immune system functioning. Identifying their etiologies remains a major challenge, despite growing knowledge. The oral cavity has a very special place in regard to AIDs. The oral mucosa, the most exposed body's natural barrier to pathogens, plays a role in both education of the immune system and the organism's daily protection. On the one hand, systemic disturbance of the immune system can impact the oral sphere with early signs which are useful diagnostic tools. On the other hand, the current research efforts on interactions between microbiota and the immune system allow an update of the old hypothesis involving an initial infection to trigger autoimmunity. Dysbiosis of our microbiota, especially in the oral sphere, could lead to a breakdown in tolerance mechanisms. Immune tolerance has to maintain the integrity of the organism but also cohabitation with commensal microbiota. The relationship between periodontitis, a chronic infectious disease, and rheumatoid arthritis, one of the most common systemic autoimmune disorders, illustrates the possible relationship between chronic infections and the etiopathogenesis of autoimmunity. Indeed, its association with oral pathogens involved in periodontal damage raises questions about a possible infectious etiology of rheumatoid arthritis (RA) which would place the management of periodontitis not only as mandatory RA's support therapy but also as a prophylactic gesture to prevent autoimmunity.

Title: Auto-immunité et cavité orale, où en est-on en 2023 ? Abstract: Les maladies auto-immunes (MAI) restent une énigme dans notre compréhension du système immunitaire. L'identification de leurs étiologies demeure un défi majeur en dépit d'une augmentation exponentielle de nos connaissances sur le fonctionnement du système immunitaire. La cavité orale a une place particulière vis-à-vis des MAI. La muqueuse buccale présente les barrières naturelles de l'organisme parmi les plus exposées à des agents pathogènes. À ce titre, elles jouent un rôle dans l'éducation du système immunitaire, puis dans la protection quotidienne de l'organisme. Les perturbations du système immunitaire se manifestent fréquemment par des conséquences au niveau de la sphère buccale, le plus souvent précocement, permettant d'initier une démarche diagnostique. L'effort de recherche actuel sur les interactions entre microbiotes et système immunitaire permet de moderniser l'hypothèse historique liant une origine infectieuse à l'apparition de l'auto-immunité, en y apportant quelques nuances.

Could AMPs and B-cells be the missing link in understanding periodontitis?

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.

Structure-Based Design of Peptidic Inhibitors of the Interaction between CC Chemokine Ligand 5 (CCL5) and Human Neutrophil Peptides 1 (HNP1).

Protein-protein interactions (PPIs) are receiving increasing interest, much sparked by the realization that they represent druggable targets. Recently, we successfully developed a peptidic inhibitor, RRYGTSKYQ ("SKY" peptide), that shows high potential in vitro and in vivo to interrupt a PPI between the platelet-borne chemokine CCL5 and the neutrophil-derived granule protein HNP1. This PPI plays a vital role in monocyte adhesion, representing a key mechanism in acute and chronic inflammatory diseases. Here, we present extensive and detailed computational methods applied to develop the SKY peptide. We combined experimentally determined binding affinities (KD) of several orthologs of CCL5 with HNP1 with in silico studies to identify the most likely heterodimeric CCL5-HNP1 complex which was subsequently used as a starting structure to rationally design peptidic inhibitors. Our method represents a fast and simple approach that can be widely applied to determine other protein-protein complexes and moreover to design inhibitors or stabilizers of protein-protein interaction.

Recruitment of classical monocytes can be inhibited by disturbing heteromers of neutrophil HNP1 and platelet CCL5.

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, α-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.

Neutrophil-derived cathelicidin promotes adhesion of classical monocytes.

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 ß1- and ß2-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.

Disruption of platelet-derived chemokine heteromers prevents neutrophil extravasation in acute lung injury.

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.

Autoantibodies to endothelial cell surface ATP synthase, the endogenous receptor for hsp60, might play a pathogenic role in vasculatides.

BACKGROUND: Heat shock protein (hsp) 60 that provides "danger signal" binds to the surface of resting endothelial cells (EC) but its receptor has not yet been characterized. In mitochondria, hsp60 specifically associates with adenosine triphosphate (ATP) synthase. We therefore examined the possible interaction between hsp60 and ATP synthase on EC surface. METHODOLOGY/PRINCIPAL FINDINGS: Using Far Western blot approach, co-immunoprecipitation studies and surface plasmon resonance analyses, we demonstrated that hsp60 binds to the ß-subunit of ATP synthase. As a cell surface-expressed molecule, ATP synthase is potentially targeted by anti-EC-antibodies (AECAs) found in the sera of patients suffering vasculitides. Based on enzyme-linked immunosorbent assay and Western blotting techniques with F1-ATP synthase as substrate, we established the presence of anti-ATP synthase antibodies at higher frequency in patients with primary vasculitides (group I) compared with secondary vasculitides (group II). Anti-ATP synthase reactivity from group I patients was restricted to the ß-subunit of ATP synthase, whereas those from group II was directed to the α-, ß- and γ-subunits. Cell surface ATP synthase regulates intracellular pH (pHi). In low extracellular pH medium, we detected abnormal decreased of EC pHi in the presence of anti-ATP synthase antibodies, irrespective of their fine reactivities. Interestingly, soluble hsp60 abrogated the anti-ATP synthase-induced pHi down-regulation. CONCLUSIONS/SIGNIFICANCE: Our results indicate that ATP synthase is targeted by AECAs on the surface of EC that induce intracellular acidification. Such pathogenic effect in vasculitides can be modulated by hsp60 binding on ATP synthase which preserves ATP synthase activity.

TLR2 is one of the endothelial receptors for beta 2-glycoprotein I.

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.

Are autoantibodies triggering endothelial cell apoptosis really pathogenic?

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.

Mitochondrial heat shock protein (HSP) 70 synergizes with HSP60 in transducing endothelial cell apoptosis induced by anti-HSP60 autoantibody.

Heat shock protein (HSP) 60, up-regulated by endothelial cells (ECs) to resist stress, is the target of a subgroup of apoptosis-inducing anti-EC autoantibodies (Abs) in human vasculitides. Given that HSP60 is not a transmembrane protein, the mechanism by which these auto-Abs induces apoptosis is unclear. EC membrane proteins were analyzed using bidimensional electrophoresis and Far Western blot, and the HSP60 receptor was identified by mass spectrometry. Heat stress-dependent synthesis of HSP60 and receptor was examined by semiquantitative RT-PCR, and expression was examined by flow cytometry and indirect immunofluorescence. Interaction was demonstrated by coimmunoprecipitations. Lipid rafts were purified to evaluate specific localization, and the apoptotic response was investigated by blocking monoclonal Ab. Mitochondrial HSP70 (mtHSP70) was identified as an HSP60 receptor. Stress was required for ECs to up-regulate mRNA and express mtHSP70 on their surface. HSP60 and mtHSP70 colocalized and interacted within lipid rafts. They were associated with chemokine CC motif receptor 5 (CCR5), also induced at the mRNA and protein levels in stressed ECs. CCR5 was involved in the anti-HSP60-triggered apoptosis of ECs. These results provide new insights into the mechanism by which anti-EC auto-Abs from vasculitides induce apoptosis of ECs.

HSP60 and anti-HSP60 antibodies in vasculitis: they are two of a kind.

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.

Modulation of endothelial cell damages by anti-Hsp60 autoantibodies in systemic autoimmune diseases.

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.

Induction of endothelial cell apoptosis by the binding of anti-endothelial cell antibodies to Hsp60 in vasculitis-associated systemic autoimmune diseases.

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.