Sepsis is one of the most aggressive, life threatening situations, represented by a deregulated response to infection, impaired with immune suppression and multi-organ collapse. Nowadays, sepsis is being considered as an immunosuppressive disorder and therefore, immunostimulatory therapies are envisaged as potential treatments. Among the various experimental models, the present study focused on the LPS-induced endotoxemia model that displayed major sepsis-related immunosuppressive and inflammatory markers and survived long enough to allow application of a therapeutic treatment, which consisted of a PG-activated implant, previously described to exert a mild mitogenic effect to the host. The results showed that indeed implantation of a PG-activated scaffold to LPS-treated mice could reverse all tested markers of endotoxemia, while also significantly ameliorating animal morbidity.
Implant fabrication followed the previously described “vaccine-on-chip” technology according to which 3D laser micro-textured Si scaffolds are used to support autologous macrophage adherence, antigen seeding and natural antigen presentation in vitro and further activation of the immune response in vivo . For the construction of the implants used herein, laser micro-structured Si scaffolds were loaded with naïve macrophages and the best conditions for PG-induced proliferation and subsequent lymphocyte activation were determined. Thus, seeding with 1 µg/ml PG provided the highest proliferation and spreading of macrophages onto the Si-scaffolds as visualized by CFSE staining, while also supporting non-specific antibody production upon addition of autologous naïve T and B lymphocytes to the system. The interaction of macrophages with T cells was visualized by double fluorescence experiments, followed by confocal microscopy analysis, indicating that this system could indeed activate T cells, which in turn could stimulate B cells, as verified by the presence of IgG antibodies to the culture supernatants. The ability of this system to induce non-specific antibody production was mandatory, since previous results had demonstrated the ability of IgG to decrease sepsis markers and allow animal survival , while inoculation of IgM-enriched IgG to patients has been considered to reverse septic shock conditions .
The experimental model used herein consisted of 5-day intraperitoneal injections of 5µg LPS per day. As previously described, such manipulation allows half of the animals to survive long enough to allow the application of a treatment . Most protocols in the literature using higher doses of LPS ranging from 1 to 25 mg/kg of body weight of BALB/c mice study endotoxemia 24 hours after LPS injection, which is not convenient for a treatment application [21, 23]. Following the above experimental model, animals showed a statistically significant increase of immunosuppressive cell populations in the spleen as evaluated by the expression of CD11b/Gr1 and CD25/Foxp3 markers, which characterize MDSCs and Tregs respectively, while also increasing arginase-1 activity that is known to mediate the suppressive effect of MDSCs [6–9]. These cell populations have been shown to expand in various pathological conditions including cancer and acute infectious diseases and in particular sepsis, when migrating to the periphery . The LPS-induced endotoxemia also resulted in a decrease of effector CD4- and CD8-positive cells in the spleen, which also correlates with the septic profile . In addition, such treatment increased the levels of CRP and PCT as well as the inflammatory cytokines IL-6, IL-18 and TNF-a in the serum, which classically increase during inflammation and are also associated with sepsis [21, 24]. The treatment also increased the immunosuppressive cytokine IL-10 in the serum, facilitating thus the establishment of the immunosuppressive state to the animals.
The application of the PG-activated implant one day after the LPS treatment initiation rescued all inflammatory and suppressive markers tested to control levels, while also restoring the levels of effector CD4- and CD8-positive cells in the spleen. Except from restoring endotoxemia markers, the PG-implant has been previously shown to induce non-specific IgG production , which could also play an additional therapeutic role to the model.
The application of the PG-activated implant to control untreated mice did not alter any of the markers in the spleen, except from the expression of class II MHC protein, which argues in favor of the mild immunostimulatory activity previously described . However, such treatment of control mice increased the levels of IL-6 and TNF-a, but not IL18, IL-10, CRP or PCT, which could be proved useful to other pathologic conditions.
Interestingly, the application of non-activated implant to the LPS-treated animals, which was used as control to the system, could also rescue sepsis-associated markers in the spleen except from the effector CD8-positive cell population and arginase-1 activity, while also rescuing the levels of TNF-a, CRP and PCT, but not IL-6, IL-18 and IL-10 in the serum. Despite the similarities, when the non-activated implant was excised from the animals and submitted to SEM analysis, it displayed a quite different histology as compared to the PG-activated implant. The PG-activated implant showed thick cellular and membrane structures covering the scaffold with important collagen depositions, which as previously shown in the case of a conventional antigenic stimulus will support the development of blood vessels . Non-activated scaffolds, although becoming populated by adherent cells (morphologically defined as macrophages and fibroblasts), they failed to induce collagen depositions and protective membranes, at least at the specific time point tested.
Most importantly, the non-activated scaffolds failed to rescue the septic phenotype as evaluated by the MSS score, which, however, could be rescued almost to control levels by the PG-activated implants.
In conclusion, the results presented in this study showed that the “vaccine-on-chip” technology, using PG as the antigenic stimulus, could rescue the LPS-induced endotoxemia. In the context of personalized therapy, the “PG-vaccine-on-chip” could provide a controllable and safe management of the systematic inflammatory response characterizing sepsis. The rapid effectiveness of the proposed immunotherapy could be proved to rescue fatal morbidity to humans.