Integrin-mediated adhesive properties of neutrophils are reduced by hyperbaric oxygen therapy in patients with chronic non-healing wound

Abstract

In recent years, several studies suggested that the ability of hyperbaric oxygen therapy (HBOT) to promote healing in patients with diabetic ulcers and chronic wounds is due to the reduction of inflammatory cytokines and to a significant decrease in neutrophils recruitment to the damaged area. α4 and β2 integrins are receptors mediating the neutrophil adhesion to the endothelium and the comprehension of the effects of hyperbaric oxygenation on their expression and functions in neutrophils could be of great importance for the design of novel therapeutic protocols focused on anti-inflammatory agents. In this study, the α4 and β2 integrins' expression and functions have been evaluated in human primary neutrophils obtained from patients with chronic non-healing wounds and undergoing a prolonged HBOT (150 kPa per 90 minutes). The effect of a peptidomimetic α4β1 integrin antagonist has been also analyzed under these conditions. A statistically significant decrease (68%) in β2 integrin expression on neutrophils was observed during the treatment with HBO and maintained one month after the last treatment, while α4 integrin levels remained unchanged. However, cell adhesion function of both neutrophilic integrins α4β1 and β2 was significantly reduced 70 and 67%, respectively), but α4β1 integrin was still sensitive to antagonist inhibition in the presence of fibronectin, suggesting that a combined therapy between HBOT and integrin antagonists could have greater antinflammatory efficacy.

Fig 1. HBOT does not modify α₄ integrin levels (panels a and b) but reduces β₂ expression (panels c and d) in neutrophils deriving from patients receiving standard wound care alone (control group) and patients undergoing HBOT for 15 sessions (HBOT group).

The decrement of β₂ integrins was maintained up to one month after ending HBOT. The effects of HBOT on integrin expression were evaluated both by qPCR (mRNA levels; panel a, c) and by flow cytometry (measuring integrin expressed on cell surface; panel b, d). Results from qPCR are expressed as mean ± standard deviation of individual samples, carried out in triplicate at each time point (control group n = 15; HBOT group n = 15). Data from flow cytometry analysis are expressed as mean fluorescence intensity (MFI) ± standard deviation of individual samples carried out in triplicate at each time point (control group n = 15; HBOT group n = 15). MFI values for respective isotype control monoclonal antibody were set to 0. *** p < 0.001 versus T₀, both control and HBOT group.

Fig 2. HBOT reduces significantly integrin-mediated neutrophil adhesion to fibrinogen (Fg) or fibronectin (FN).

Integrin-mediated adhesion was decreased in neutrophils obtained from patients belonging to HBOT group; no changes were observed in control group patients. The effects of HBOT on neutrophil adhesion mediated by β₂ (panel a) or α₄β₁ (panel b) integrins were evaluated by adhesion assay to Fg or FN respectively, as described in method section. Adhesion mediated by β₂ or α₄β₁ integrin is significantly prevented in neutrophils treated with a monoclonal antibody anti-β₂ or anti-α₄, respectively. HBOT exposure significantly reduced anti-β₁ HUTS-21 mAb binding to neutrophils, namely changing α₄β₁ integrin conformation (panel c). Mean fluorescence intensity (MFI) due to the anti-β1 integrin mAb PE conjugated HUTS-21 binding in the presence of fibronectin (10 μg/mL) was measured. Non-specific binding of an isotype control PE conjugated mAb added to neutrophils produced an MFI of 12 ± 3 that was subtracted from all samples. Data are expressed as mean ± standard deviation of individual samples, carried out in triplicate at each time point (control group: n = 15; HBOT group n = 15). ** p < 0.01; *** p < 0.001; **** p < 0.0001 versus T₀, both control and HBOT group.

Fig 3. Variation of ulcer sizes at different time points during and after HBO therapy in patients with chronic non-healing wounds.

Ulcer sizes were determined for control group patients during the first evaluation (T₀) and after fifteen days of conventional wound therapy (T₁₅); for patients in HBOT group before (T₀), after 15 HBO sessions (T₁₅) and one month after the last HBO treatment (T_1M). Data, expressed as variation of the percentage in ulcer sizes and related to the basal value (T₀), represent the mean ± standard deviation of individual samples (control group n = 15; HBOT group n = 15). **p<0.01, **** p < 0.0001 versus T₀, both control and HBOT group.

Fig 4. Wound healing progression during and after HBO therapy in patients with chronic non-healing wounds, shown qualitatively.

Images of the same ulcer were acquired before (T₀), after 15 HBO sessions (T₁₅) and one month after the last HBO treatment (T_1M) for patients of HBOT group; during the first evaluation (T₀) and after fifteen days of standard wound therapy (T₁₅) for control group patients. Three representative sets of wound images for each group are shown.

Fig 5. Effects of HBOT on TNF-α and IL-1β evaluated as mRNA levels in human primary neutrophils and circulating protein levels in plasma.

HBO treatment significantly reduced TNF-α (panel a) and IL-1β (panel b) mRNA levels in neutrophils isolated from blood samples of patients with chronic non-healing wounds undergoing HBOT or from control group. Moreover, HBOT induced a significant reduction in circulating inflammatory cytokines TNF-α (panel c) and IL-1β (panel d) starting from the twelfth HBO session up to one month after the last HBO treatment in HBOT group patients. No changes in pro-inflammatory cytokine levels, both at neutrophil mRNA and circulating protein levels, were observed in control patients. Data are expressed as mean ± standard deviation of individual samples, carried out in triplicate (control group n = 15; HBOT group n = 15). * p < 0.05; ** p < 0.01; *** p < 0.001 versus T₀, both control and HBOT group.

Fig 6. Effects of integrin antagonist RG66 on integrin-mediated neutrophil adhesion.

Integrin antagonist RG66 does not influence neutrophil adhesion mediated by β₂ integrins (panel a); on the contrary, RG66 reduces α₄β₁ integrin-mediated neutrophil adhesion to fibronectin (FN) in a concentration-dependent manner (IC₅₀ 0.17 ± 0.03 μM) (panel b). The effects of RG66 (10⁻⁴–10⁻¹⁰ M) on neutrophil adhesion mediated by β₂ (panel a) or α₄ (panel b) integrins were evaluated by adhesion assay to Fg or FN respectively, as described in method section. Neutrophils were isolated from blood samples of patients (with chronic non-healing wounds) undergoing HBOT. A representative concentration-response curve at T₈ is reported. Data are expressed as mean ± standard deviation, carried out in triplicate (HBOT group, n = 15).