The posttraumatic response of CD4+ regulatory T cells is modulated by direct cell-cell contact via CD40L- and P-selectin-dependent pathways.

CD4+ FoxP3+ regulatory T cells (CD4+ Tregs) are important for the posttraumatic anti-inflammatory host response. As described previously, platelets are able to modulate CD4+ Treg activity in a reciprocally activating interaction following injury. The underlying mechanisms of the posttraumatic interaction between platelets and CD4+ Tregs remain unclear. We investigated the potential influence of CD40L and P-selectin, molecules known to be involved in direct cell contact of these cell types. In a murine burn injury model, the potential interaction pathways were addressed using CD40L- and P-selectin-deficient mice. Draining lymph nodes were harvested following trauma (1 h) and following a sham procedure. Early rapid activation of CD4+ Tregs was assessed by phospho-flow cytometry (signaling molecules (p)PKC-δ and (p)ZAP-70). Platelet function was analyzed performing rotational thromboelastometry (ROTEM). We hypothesized that disruption of the direct cell-cell contact via CD40L and P-selectin would affect posttraumatic activation of CD4+ Tregs and influence the hemostatic function of platelets. Indeed, while injury induced early activation of CD4+ Tregs in wild-type mice (ZAP-70: p = 0.13, pZAP-70: p < 0.05, PKC-δ: p < 0.05, pPKC-δ: p < 0.05), disruption of CD40L-dependent interaction (ZAP-70: p = 0.57, pZAP-70: p = 0.68, PKC-δ: p = 0.68, pPKC-δ: p = 0.9) or P-selectin-dependent interaction (ZAP-70: p = 0.78, pZAP-70: p = 0.58, PKC-δ: p = 0.81, pPKC-δ: p = 0.73) resulted in reduced posttraumatic activation. Furthermore, hemostatic function was impaired towards hypocoagulability in either deficiency. Our results suggest that the posttraumatic activation of CD4+ Tregs and hemostatic function of platelets are affected by direct cell-cell-signaling via CD40L and P-selectin.

Platelets differentially modulate CD4+ Treg activation via GPIIa/IIIb-, fibrinogen-, and PAR4-dependent pathways.

CD4+FoxP3+ regulatory T cells (CD4+ Tregs) are known to dampen inflammation following severe trauma. Platelets were shown to augment their posttraumatic activation in burn injury, but the exact mechanisms remain unclear. We hypothesized that platelet activation mechanisms via GPIIb/IIIa, fibrinogen, and PAR4 have an immunological effect and modulate CD4+ Treg activation early after trauma. Therefore, C57Bl/6 N mice were injected with tirofiban (GPIIb/IIIa inhibition), ancrod (fibrinogen splitting enzyme), or tcY-NH2 (selective PAR4 antagonist peptide) before inducing a third-degree burn injury of 25% of the total body surface area. Changes in coagulation, and local and systemic CD4+ Treg activity were assessed via rotational thromboelastometry (ROTEM®) and phospho-flow cytometry 1 h post intervention. The inhibition of GPIIb/IIIa and fibrinogen locally led to a higher basic activity of CD4+ Tregs compared to non-inhibited animals. In contrast, PAR4 disruption on platelets locally led to an increased posttraumatic activation of CD4+ Tregs. Fibrinogen led to complete elimination of coagulation, whereas GPIIb/IIIa or PAR4 inhibition did not. GPIIb/IIIa receptor and fibrinogen inhibition increase CD4+ Tregs activity independently of trauma. Both are crucial for thrombus formation. We suggest platelets trapped in thrombi are unable to interact with CD4+ Tregs but augment their activity when circulating freely. In contrast, PAR4 seems to reduce CD4+ Treg activation following trauma. In summary, GPIIb/IIIa-, PAR4-, and fibrinogen-dependent pathways in platelets modulate CD4+ Treg baseline activity, independently from their hemostatic functionality. PAR4-dependent pathways modulate the posttraumatic interplay of platelets and CD4+ Tregs.

Traumatic Brain Injury Induces a Differential Immune Response in Polytrauma Patients; Prospective Analysis of CD69 Expression on T Cells and Platelet Expansion.

BACKGROUND: Accidents and injuries are the leading causes of mortality in young people. CD4+ regulatory T cells (CD4+ Tregs), Th17 cells and platelets could be identified as key players in post-traumatic immunological dysfunction, which is a common cause of late mortality in trauma patients. The mechanisms of activation of these cell types and their interaction remain mostly unclear. Since CD69 is not only a leukocyte marker but has also immunoregulatory functions, we postulate a role for CD69 after trauma. The present study investigates the expression of CD69 on CD4+ Tregs and Th17 cells, as well as the posttraumatic expansion of platelets and hemostatic function. Subgroup analysis was performed to assess the differences between polytrauma patients with and without severe traumatic brain injury (TBI). METHODS: In this non-interventional prospective clinical trial, we analyzed sequential blood samples over a period of 10 days from 30 patients after multiple traumas with an ISS ≥ 16. Platelet function was assessed by rotational thromboelastometry (ROTEM analysis). CD4+ Tregs and Th17 cells were stained with surface markers and analyzed by flow cytometry. RESULTS: We were able to demonstrate a significantly increased expression of CD69 on CD4+ Tregs after trauma. Subgroup analysis revealed that the absence of severe TBI is associated with a significantly higher expression of CD69 on CD4+ Tregs and on Th17 cells. Platelets expanded and showed signs of dysfunction, while an overall tendency of posttraumatic hypercoagulation was detected. CONCLUSIONS: Our results support the concept of injury-specific immune responses and add to a further understanding of the complex pathophysiology of post-traumatic immune dysfunction.

Oxygen-distribution within 3-D collagen I hydrogels for bone tissue engineering.

Tissue engineering (TE) approaches typically envisage the structural and functional reconstitution of previously damaged tissue in situ. An adequate three-dimensional environment is therefore of fundamental importance for the designated cells associated to the scaffold material. The sufficient supply with nutrients and oxygen in vitro and in vivo mark thereby critical challenges of TE. In this study, we intended to analyse the level of locally dissolved oxygen within 3-D cell-loaded collagen I gels in vitro. For the analysis of the oxygen levels in situ, we employed an optical fibre-based micro sensor setup, as well as a camera supported non-invasive optical sensor foil based technique. These complementary analytical tools enable the identification, localization, and temporal follow-up investigation of specified regions of interest within TE constructs. Human adipose-derived mesenchymal stem cells (hAdMSCs) cultured in collagen I gels under normoxic conditions were analysed periodically and kinetically up to 70 days - thereby revealing dynamic changes of the level of dissolved oxygen inside the gel constructs. Dependent on the applied cell concentration, the in vitro oxygen concentration (cO2) within the gels reached physiological ranges (7-9%) after 21 days, or 35 days of culture. The minimal cO2 was measured after 35 days in vitro, featuring an oxygen level of 4.8 ±â€¯1.3%. Upon prolonged culture, a plateau-like status of the cO2 around 8-9% established, indicating a change in the physiological activity of the cells under investigation. The expression patterns of BCL2, CASP3 and MCM5 revealed significant differences among the proliferative and apoptotic stages of the cell-loaded samples at the investigated time points of 7 and 70 days in culture. In summary, these data show the temporary dynamic nature of the oxygen distribution in cell-loaded gel constructs. The applied technique is an ideal tool for the evaluation of multiple parameters affecting the oxygen distribution in vitro. We conclude that it takes 5 weeks for establishing an equilibrium of cO2. Levels reached in a 3-D gel construct are comparable with physiological oxygenation ranges in bone-associated tissues.