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Penetrating traumatic brain injury (PTBI) is a significant cause of death and disability in the United States. Inflammasomes are one of the key regulators of the interleukin (IL)-1β mediated inflammatory responses after traumatic brain injury (TBI). However, the contribution of inflammasome signaling after PTBI has not been determined. In this study, adult male Sprague-Dawley rats were subjected to sham procedures or penetrating ballistic-like brain injury (PBBI) and sacrificed at various time points. Tissues were assessed by immunoblot analysis for expression of IL-1β, IL-18 and components of the inflammasome: apoptosis-associated speck-like protein containing a caspase-activation and recruitment domain (ASC), caspase-1, X-linked inhibitor of apoptosis protein (XIAP), NOD-like receptor protein 3 (NLRP3), and gasdermin-D (GSDMD). Specific cell types expressing inflammasome proteins were also evaluated immunohistochemically and assessed quantitatively. After PBBI, expression of IL-1β, IL-18, caspase-1, ASC, XIAP, and NLRP3 peaked around 48h. Brain protein lysates from PTBI animals showed pyroptosome formation evidenced by ASC laddering, and also contained increased expression of GSDMD at 48h after injury. ASC-positive immunoreactive neurons within the perilesional cortex were observed at 24 hrs. At 48hrs, ASC expression was concentrated in morphologically activated cortical microglia. This expression of ASC in activated microglia persisted until 12 weeks following PBBI. This is the first report of inflammasome activation after PBBI. Our results demonstrate cell-specific patterns of inflammasome activation and pyroptosis predominantly in microglia suggesting a sustained pro-inflammatory state following PBBI, thus offering a therapeutic target for this type of brain injury.

Potential applications of neural stem cells (NSCs) for transplantation requires understanding myosin heavy chain (MHC) expression and the ability of T cells and natural killer (NK) cells to recognize this progenitor population. Cells from the cortices of day-13 embryonic (E13) B6 (H-2(b)) mice were explanted and cultured to expand NSCs. Analysis of P2-P17-cultured cells using anti-MHC class I/II monoclonal antibodies (mAbs) showed marginal expression of both products. Although recombinant murine interferon-gamma (rmIFN gamma) exposure did not alter the multipotential capacity of these stem cells, titration of mrIFN gamma NSC cultures demonstrated that MHC molecules could be strongly upregulated after addition of 3 ng/ml rmIFN gamma for 60 hours. To assess the susceptibility of NSCs with low or absent versus high levels of MHC expression to lysis by cytotoxic T lymphocyte (CTL) and NK populations, untreated and rmIFN gamma-treated NSC target cells were examined. Untreated NSCs were not recognized by BALB/c (H-2(d)) allospecific anti-H-2(b) CTL, consistent with the mAb findings; however, upregulation of MHC products on both early and later passaged NSCs resulted in their efficient lysis by CTL. NK cells were prepared from syngeneic B6 or allogeneic BALB/c mice. Although NK cells effectively killed control YAC-1 target cells, these effectors did not kill MHC-deficient (or expressing) NSC targets. Thus, similar to hematopoietic, embryonic, and mesenchymal stem cell populations, unmanipulated NSCs are not readily killed by T and NK cells. These findings suggest that following transplant into syngeneic or allogeneic recipients, NSCs may exhibit diminished susceptibility to clearance by host T- and NK-cell populations.