Crystal Neely

Functional Modularity of the Arginine Catabolic Mobile Element Contributes to the Success of USA300 Methicillin-Resistant Staphylococcus aureus

The USA300 community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) lineage causes the majority of skin and soft tissue infections (SSTIs) and is highly associated with the carriage of the arginine catabolic mobile element (ACME). However, the contribution of ACME to USA300s success in SSTIs is not completely understood. We show that the constitutive ACME-encoded arginine-deiminase system (Arc) allows USA300 to thrive in acidic environments that mimic human skin. Consequently, the ACME-Arc system drives excessive production of host polyamines, compounds uniquely toxic to S. aureus. To mitigate this, ACME also encodes SpeG, a polyamine-resistance enzyme that is essential for combating excess host polyamines in a murine SSTI model. Inhibiting host polyamine production not only restored ΔspeG persistence within infected wounds but also severely altered the host healing process, implying that polyamines play an integral role in coordinating the wound-healing response. Together, these data underscore the functional modularity of ACME and its contribution to the success of USA300 CA-MRSA.

Flagellin Treatment Prevents Increased Susceptibility to Systemic Bacterial Infection after Injury by Inhibiting Anti-Inflammatory IL-10+ IL-12- Neutrophil Polarization

Severe trauma renders patients susceptible to infection. In sepsis, defective bacterial clearance has been linked to specific deviations in the innate immune response. We hypothesized that innate immune modulations observed during sepsis also contribute to increased bacterial susceptibility after severe trauma. A well-established murine model of burn injury, used to replicate infection following trauma, showed that wound inoculation with P. aeruginosa quickly spreads systemically. The systemic IL-10/IL-12 axis was skewed after burn injury with infection as indicated by a significant elevation in serum IL-10 and polarization of neutrophils into an anti-inflammatory (“N2”; IL-10+ IL-12−) phenotype. Infection with an attenuated P. aeruginosa strain (ΔCyaB) was cleared better than the wildtype strain and was associated with an increased pro-inflammatory neutrophil (“N1”; IL-10−IL-12+) response in burn mice. This suggests that neutrophil polarization influences bacterial clearance after burn injury. Administration of a TLR5 agonist, flagellin, after burn injury restored the neutrophil response towards a N1 phenotype resulting in an increased clearance of wildtype P. aeruginosa after wound inoculation. This study details specific alterations in innate cell populations after burn injury that contribute to increased susceptibility to bacterial infection. In addition, for the first time, it identifies neutrophil polarization as a therapeutic target for the reversal of bacterial susceptibility after injury.

Th17 (IFNγ- IL17+) CD4+ T cells generated after burn injury may be a novel cellular mechanism for postburn immunosuppression

BACKGROUND The mechanism responsible for initiating and controlling the immunosuppressive response after burn injury remains unknown. Interleukin-17 (IL-17) secreting Th17 (interferon [IFN]γ IL17) cells are a novel subset of CD4 T cells associated with a weak, proinflammatory response that antagonizes the proinflammatory Th1 (IFNγ IL17) response. Given that transforming growth factor-β and IL6 mediate Th17 cell development, we hypothesized that burn injury may generate Th17 cells that could mediate postburn immunosuppression. METHODS After a 20% total body surface area burn in female C57BL/6 mice, wound-draining lymph nodes were harvested 3 days, 7 days, or 14 days after injury. CD4 T cells were enriched by magnetic selection, and flow cytometry was used to identify intracellular IL17 and IFNγ in CD3CD4 T cells. Additional purified CD3CD4 T cells were cultured with Th17 polarizing IL6 and transforming growth factor-β for 4 days, and flow cytometry was again used to identify intracellular IL17 and IFNγ in CD4 T cells. RESULTS The number and percentage of preformed Th17 cells was significantly greater in burn mice compared with sham at all time points. In addition, the ratio of Th17 cells to Th1 cells was always significantly higher in burn mice compared with sham. These differences were eliminated in Th17 polarizing conditions in vitro. CD4 T cells never generated both IL17 and IFNγ. CONCLUSION These results demonstrate for the first time that Th17 cells (IFNγ IL17) are spontaneously generated after burn injury. Given that Th17 cells (IFNγ IL17) are antagonistic to Th1 (IFNγ IL17) cells, these results suggest a novel mechanism for initiating and controlling postburn immunosuppression that deserves further investigation.

Radiation combined with thermal injury induces immature myeloid cells.

ABSTRACT The continued development of nuclear weapons and the potential for thermonuclear injury necessitates the further understanding of the immune consequences after radiation combined with injury (RCI). We hypothesized that sublethal ionization radiation exposure combined with a full-thickness thermal injury would result in the production of immature myeloid cells. Mice underwent either a full-thickness contact burn of 20% total body surface area or sham procedure followed by a single whole-body dose of 5-Gy radiation. Serum, spleen, and peripheral lymph nodes were harvested at 3 and 14 days after injury. Flow cytometry was performed to identify and characterize adaptive and innate cell compartments. Elevated proinflammatory and anti-inflammatory serum cytokines and profound leukopenia were observed after RCI. A population of cells with dual expression of the cell surface markers Gr-1 and CD11b were identified in all experimental groups, but were significantly elevated after burn alone and RCI at 14 days after injury. In contrast to the T-cell–suppressive nature of myeloid-derived suppressor cells found after trauma and sepsis, myeloid cells after RCI augmented T-cell proliferation and were associated with a weak but significant increase in interferon &ggr; and a decrease in interleukin 10. This is consistent with previous work in burn injury indicating that a myeloid-derived suppressor cell–like population increases innate immunity. Radiation combined injury results in the increase in distinct populations of Gr-1+CD11b+ cells within the secondary lymphoid organs, and we propose these immature inflammatory myeloid cells provide innate immunity to the severely injured and immunocompromised host.

Innate immune cell recovery is positively regulated by NLRP12 during emergency hematopoiesis

With enhanced concerns of terrorist attacks, dual exposure to radiation and thermal combined injury (RCI) has become a real threat with devastating immunosuppression. NLRP12, a member of the NOD-like receptor family, is expressed in myeloid and bone marrow cells and was implicated as a checkpoint regulator of inflammatory cytokines, as well as an inflammasome activator. We show that NLRP12 has a profound impact on hematopoietic recovery during RCI by serving as a checkpoint of TNF signaling and preventing hematopoietic apoptosis. Using a mouse model of RCI, increased NLRP12 expression was detected in target tissues. Nlrp12−/− mice exhibited significantly greater mortality, an inability to fight bacterial infection, heightened levels of proinflammatory cytokines, overt granulocyte/monocyte progenitor cell apoptosis, and failure to reconstitute peripheral myeloid populations. Anti-TNF Ab administration improved peripheral immune recovery. These data suggest that NLRP12 is essential for survival after RCI by regulating myelopoiesis and immune reconstitution.