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 has been 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, inability to fight bacterial infection, heightened levels of pro-inflammatory cytokines, overt granulocyte/monocyte progenitor cell apoptosis and failure to reconstitute peripheral myeloid populations. Anti-TNF antibody administration improved peripheral immune recovery. These data suggest that NLRP12 is essential for survival after RCI by regulating myelopoiesis and immune reconstitution

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

Burn mice, but not sham mice, exhibit dose-dependent mortality and develop a systemic infection following a <i>P. aeruginosa</i> wound inoculation.

<p>Wildtype <i>P. aeruginosa</i> (PAK) was administered subcutaneously at 24 hours after burn or sham treatment. A) Various doses of bacteria (2×10³, 2×10⁴, or 2×10⁵ CFU/100 ul) were given and survival was monitored for 120 hours post infection (hpi). B–F) Using a dose of 2×10⁴ CFU, bacterial load at the injection site and distal organs was assessed at 48 hpi in sham (open circles) and burn (closed circles) mice. (n = 4–9 per group) *, p≤0.05. **, p≤0.005. These experiments were repeated three times with similar results.</p

Administration of flagellin at burn resuscitation and prior to wound infection with wildtype <i>P. aeruginosa</i> (PAK) reduces bacterial load in the periphery and increases the percentage of IL-12 producing neutrophils within the spleen.

<p>Burn mice were given given an intraperitoneal injection of flagellin (circles/solid bars) or left untreated (squared/striped bars) twenty-two hours after burn injury. Twenty-four hours after burn injury, mice were challenged with subcutaneous wound infection with PAK. Forty-eight hours following the bacterial challenge, various organs were harvested. Bacterial load in A) liver and B) lung samples was determined by colony forming unit (CFU) assay. The percentage of splenic neutrophils producing C) IL-10 and D) IL-12 was determined by flow cytometric analysis. Data expressed as mean ± SEM. (n = 8–10) *, p≤0.05. **, p≤0.005. These experiments were repeated three times with similar results.</p

Burn mice, but not sham mice, mount a robust serum IL-10 response after <i>P. aeruginosa</i> wound inoculation.

<p>Twenty-four hours after sham (open) or burn (solid) treatment, mice were given a subcutaneous injection of wild-type <i>P.aeruginosa</i> PAK. Forty-hours following infection, serum was collected to determine circulating levels of A) IL-10 and B) IL-12 by cytometric bead array. Data expressed as mean ± SEM. (n = 10–15) *, p≤0.05.</p

Infected burn mice have a higher percentage of IL-10⁺ neutrophils and a lower percentage of IL-12⁺ neutrophils, dendritic cells, and macrophages than infected sham mice.

<p>A) Splenocytes were harvested at 48 hours post infection and underwent intracellular staining for cytokine analysis without further stimulation <i>in vitro</i>. Shown is a representative histogram from an infected burn mouse, which indicates that IL-10 is being produced by Gr1⁺ CD11b⁺ cells within the spleen. B–E) Splenocytes were collected at 48 following infection and underwent CD11b enrichment by magnetic selection. CD11b⁺ cells were cultured in the presence of LPS and brefeldin-A then were subjected to cell surface and intracellular staining. Percentage of B) IL-10⁺ neutrophils, as well as IL-12⁺ C) neutrophils, D) dendritic cells, and E) macrophages were measured for infected sham (open) and burn (solid) mice. Data expressed as mean ± SEM. (n = 6, 7) **, p≤0.005. ***, p≤0.0005. ****, p<0.0001. These experiments were repeated three times with similar results.</p

<i>Pseudomonas aeruginosa</i> exoproducts determine antibiotic efficacy against <i>Staphylococcus aureus</i>

<div><p>Chronic coinfections of <i>Staphylococcus aureus</i> and <i>Pseudomonas aeruginosa</i> frequently fail to respond to antibiotic treatment, leading to significant patient morbidity and mortality. Currently, the impact of interspecies interaction on <i>S</i>. <i>aureus</i> antibiotic susceptibility remains poorly understood. In this study, we utilize a panel of <i>P</i>. <i>aeruginosa</i> burn wound and cystic fibrosis (CF) lung isolates to demonstrate that <i>P</i>. <i>aeruginosa</i> alters <i>S</i>. <i>aureus</i> susceptibility to bactericidal antibiotics in a variable, strain-dependent manner and further identify 3 independent interactions responsible for antagonizing or potentiating antibiotic activity against <i>S</i>. <i>aureus</i>. We find that <i>P</i>. <i>aeruginosa</i> LasA endopeptidase potentiates lysis of <i>S</i>. <i>aureus</i> by vancomycin, rhamnolipids facilitate proton-motive force-independent tobramycin uptake, and 2-heptyl-4-hydroxyquinoline <i>N</i>-oxide (HQNO) induces multidrug tolerance in <i>S</i>. <i>aureus</i> through respiratory inhibition and reduction of cellular ATP. We find that the production of each of these factors varies between clinical isolates and corresponds to the capacity of each isolate to alter <i>S</i>. <i>aureus</i> antibiotic susceptibility. Furthermore, we demonstrate that vancomycin treatment of a <i>S</i>. <i>aureus</i> mouse burn infection is potentiated by the presence of a LasA-producing <i>P</i>. <i>aeruginosa</i> population. These findings demonstrate that antibiotic susceptibility is complex and dependent not only upon the genotype of the pathogen being targeted, but also on interactions with other microorganisms in the infection environment. Consideration of these interactions will improve the treatment of polymicrobial infections.</p></div

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 has been 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, inability to fight bacterial infection, heightened levels of pro-inflammatory cytokines, overt granulocyte/monocyte progenitor cell apoptosis and failure to reconstitute peripheral myeloid populations. Anti-TNF antibody administration improved peripheral immune recovery. These data suggest that NLRP12 is essential for survival after RCI by regulating myelopoiesis and immune reconstitution

LC-MS/MS quantification of HQNO production in <i>P</i>. <i>aeruginosa</i> strains.

<p>LC-MS/MS quantification of HQNO production in <i>P</i>. <i>aeruginosa</i> strains.</p