Macrophages polarize to the pro-inflammatory phenotype and delay neutrophil efferocytosis to augment Aggregatibacter actinomycetemcomitance JP2 clearance.

OBJECTIVE: The study examines how neutrophils cross-talk with macrophages during JP2 Aggregatibacter actinomycetemcomitance infection and factors that are involved in inflammatory resolution and efferocytosis. BACKGROUND: Although sub-gingival bacteria constitute the primary initiating factor in the pathogenesis of molar-incisor pattern periodontitis (MIPP), the non-resolved host response has a major role in tissue destruction. While evidence links neutrophils to MIPP pathogenesis, their clearance during inflammatory resolution, governed by macrophages, is poorly understood. METHODS: Human neutrophils (differentiated from HL60 cells) and macrophages (differentiated from THP1 cells) were inoculated with JP2. The supernatants were collected and exposed to naïve neutrophils or macrophages with or without exposure to JP2. Reactive oxygen species (ROS) were measured with 2'-7'-dichlorofluorescein-diacetate and a fluorescent plate reader. Immunofluorescence labeling of CD47 and cell vitality were examined using flow cytometry. Macrophage polarization was tested by immunofluorescence staining for CD163 and CD68 and a fluorescent microscope, and TNFα and IL-10 secretion was tested using ELISA and RT-PCR. Efferocytosis was examined by pHrodo and carboxyfluorescein succinimidyl ester staining and fluorescent microscopy. In vivo, macrophages were depleted from C57Bl/6 mice and neutrophil CD47 levels were tested using the subcutaneous chamber model. RESULTS: Neutrophils exposed to macrophage supernatant show increased ROS, mainly extracellularly, that increased during JP2 infection. Macrophages showed pro-inflammatory M1 phenotype polarization during JP2 infection, and their supernatants prolonged neutrophil survival by inhibiting CD47 down-expression and reducing neutrophil necrosis and apoptosis. Also, the macrophages delay neutrophil efferocytosis during JP2 infection which, in turn, enhanced JP2 clearance. Depletion of macrophages in mice mildly prevented neutrophils CD47 reduction and reduced JP2 clearance. The JP2 infection in mice also led to macrophage M1 polarization similar to the in vitro results. CONCLUSIONS: As shown in this study, neutrophil efferocytosis potentially may be reduced during JP2 infection, promoting JP2 clearance, which may contribute to the inflammatory-mediated periodontal tissue damage.

Neutrophils modulate natural killer-mediated osteoclastogenesis during Aggregatibacteractinomycetemcomitans (JP2 clone) infection.

The study investigates the interplay of neutrophils and natural-killer cells (NK) in mediating osseoresorption during infection of molar-incisor-pattern-periodontitis (MIPP). Human neutrophils from periodontally healthy and MIPP patients were inoculated with the periopathogen Aggregatibacter-actinomycetemcomitans (JP2) and their supernatants were exposed to NK to study their function and osteoclastogenesis promotion. A mouse MIPP model was used to compare disease progression following NK versus neutrophils depletion. The exposure of primary NK to supernatants of neutrophils inoculated with JP2 led to NK cell arrest and activation with enhanced osteoprotegerin expression. Incubation of monocytes with NK led to osteoclastogenesis, whereas NK that were pre-exposed to healthy neutrophil supernatant showed reduced osteoclastogenesis. In mice, NK depletion led to the similar bone phenotype as the neutrophil's depletion highlighting their role on osseoprotection. The present study portrays a key crosstalk between neutrophils and NK cells during JP2 infection as a central mechanism that regulates bone loss.

CD18 Mediates Neutrophil Imperviousness to the Aggregatibacter actinomycetemcomitans JP2 Clone in Molar-Incisor Pattern Periodontitis.

Introduction: Molar-incisor pattern periodontitis (MIPP) in the absence of significant local risk factors or systemic disease, is a rare, early onset periodontal disease phenotype, with 0.5% to 2.5% global prevalence. The condition is characterized by impaired neutrophil function and persistent Aggregatibacter actinomycetemcomitans (JP2 clone) infection. The aim of this study was to characterize neutrophil functional responses to JP2 and to investigate the neutrophil receptors involved. Materials and Methods: Neutrophils were obtained from whole blood samples of periodontally healthy and MIPP subjects and incubated with the JP2 clone or a non-JP2 clone of A. actinomycetemcomitans. Bacterial survival was tested by blood agar culture; neutrophil death was tested with propidium iodide and flow cytometry; Reactive oxygen production (ROS) was measured with 2',7'-dichlorofluorescein diacetate and a fluorescence plate reader; the cytokinome was analysed using an array profiler, ELISA and RT-PCR. Receptors binding to JP2 were isolated using a novel immunoprecipitation assay and validated functionally using specific blocking antibodies. Results: JP2 and non-JP2 survival was comparable between all the neutrophil groups. Resistance to neutrophil necrosis following exposure to JP2 was significantly lower in the MIPP group, than in all the other groups (p<0.0001). Conversely, MIPP neutrophils showed lower levels of ROS production in response to JP2 infection compared with that of healthy neutrophils (p<0.001). Furthermore, significantly lower levels of cytokines, such as IL8, IL10 and TNFα, were observed during JP2 incubation with MIPP neutrophils than upon incubation with periodontally healthy neutrophils. Various proteins expressed on neutrophils bind to JP2. Of these, CD18 was found to mediate neutrophil necrosis. The CD18 receptor on MIPP neutrophils acts differently from that on periodontally healthy patients neutrophils, and appears to reflect differential neutrophil reactions to JP2. Conclusion: This study portrays a fundamental difference in neutrophil response to JP2 infection between periodontally healthy and MIPP patients. This was evident in the resistance to necrosis, and lower ROS and cytokine production, despite the persistent presence of viable JP2. Whilst in periodontally healthy neutrophils, JP2 binds to CD18 on cell surfaces, this is not the case in MIPP neutrophils, suggesting a potential role for CD18 in the periodontal susceptibility of MIPP patients.