Erle D. Murphey

Enhancement of Dendritic Cell Production by Fms-Like Tyrosine Kinase-3 Ligand Increases the Resistance of Mice to a Burn Wound Infection

Fms-like tyrosine kinase-3 ligand (Flt3L) is a hemopoietic cytokine that stimulates the production of dendritic cells. This study evaluated the ability of Flt3L-enhanced dendritic cell production to increase the resistance of mice to a burn wound infection with Pseudomonas aeruginosa, a common source of infections in burn patients that have impaired immunity and are susceptible to opportunistic microorganisms. Treatment of mice with Flt3L for 5 days caused a significant increase in dendritic cell numbers in the spleen and significantly increased survival upon a subsequent burn wound infection. Improved survival in Flt3L-treated mice was associated with limited bacterial growth and spread within the burn wounds and a decrease in systemic dissemination of P. aeruginosa. Resistance to burn wound infection could also be conferred to recipient mice by the adoptive transfer of dendritic cells that had been isolated from spleens of Flt3L-treated mice. Adoptive transfer of the same number of splenic dendritic cells from nontreated mice did not confer resistance to burn wound infection. These data indicate that Flt3L can increase the resistance of mice to a P. aeruginosa burn wound infection through both stimulation of dendritic cell production and enhancement of dendritic cell function.

Diminished bacterial clearance is associated with decreased IL-12 and interferon-gamma production but a sustained proinflammatory response in a murine model of postseptic immunosuppression.

After a major illness or injury, immune status in critically ill patients may fluctuate between a marked proinflammatory response and an immunosuppressed state. Postinflammatory immunosuppression can result in increased susceptibility to infection. Alterations of cytokine production, such as suppression of IFNγ and elevation of the anti-inflammatory cytokine IL-10, are believed to contribute to postinflammatory immunosuppression. We examined antimicrobial immunity in mice that had previously been subjected to a sublethal cecal ligation and puncture (CLP) as a model of major injury. Mice were challenged with Pseudomonas aeruginosa (5 × 107 CFU i.v.) on day 5 after CLP or sham surgery. Bacterial clearance in mice after CLP was impaired and associated with decreased production of IFNγ and increased production of IL-10 in the early response to the Pseudomonas challenge. Pseudomonas-induced production of the IFNγ-inducing factor IL-12 was also decreased in post-CLP mice. However, splenocytes from post-CLP mice remained responsive to exogenous stimulation with the IFNγ-inducing cytokines IL-12, IL-15, and IL-18 as well as T-cell receptor activation. Furthermore, production of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 were as high, or higher, in the post-CLP group compared with sham mice after P. aeruginosa challenge. Blockade of IL-10 did not reverse IL-12 and IFNγ suppression in splenocytes from post-CLP mice. These studies show that suppressed bacterial clearance in post-CLP mice is associated with decreased production of IFNγ and IL-12 and with increased production of IL-10 and proinflammatory cytokines.

Mice depleted of CD8 + T and NK cells are resistant to injury caused by cecal ligation and puncture

We previously showed that beta 2 microglobulin knockout mice depleted of NK cells by treatment with anti-asialoGM1 (β2MKO/αAsGM1 mice) are resistant to sepsis caused by cecal ligation and puncture (CLP). β2MKO mice possess multiple immunological defects including depletion of CD8+ T cells. This study was designed to determine the contribution of CD8+ T and NK cell deficiency to the resistance of β2MKO/αAsGM1 mice to CLP-induced injury. β2MKO/αAsGM1 mice and CD8 knockout mice treated with anti-asialoGM1 (CD8KO/αAsGM1 mice) survived significantly longer than wild-type mice following CLP. Improved long-term survival was also observed in wild-type mice rendered CD8+ T/NK cell-deficient by treatment with both anti-CD8α and anti-asialoGM1. Blood gas analysis and body temperature measurements showed that CD8+ T and NK cell-deficient mice have significantly reduced metabolic acidosis and less hypothermia compared to control mice at 18 h after CLP. CD8+ T/NK cell-deficient mice also showed an attenuated proinflammatory response as indicated by decreased expression of mRNAs for IL-1, IL-6 and MIP-2 in spleen and heart. IL-6, KC and MIP-2 levels in blood and peritoneal fluid were also significantly decreased CD8+ T/NK cell-deficient mice compared to controls. CD8+ T/NK cell-deficient mice exhibited decreased bacterial concentrations in blood, but not in peritoneal fluid or lung, compared to wild-type controls. These data show that mice depleted of CD8+ T and NK cells exhibit survival benefit, improved physiologic function and an attenuated proinflammatory response following CLP that is comparable to β2M/αAsGM1 mice.

Mice Depleted Of αβ But Not γδ T Cells Are Resistant To Mortality Caused By Cecal Ligation And Puncture

The present study was undertaken to determine whether the mice depleted of &agr;&bgr; or &ggr;&dgr; T cells show resistance to acute polymicrobial sepsis caused by cecal ligation and puncture (CLP). T-cell receptor &bgr; knockout (&bgr;TCRKO) and T-cell receptor &dgr; knockout (&dgr;TCRKO) mice were used. An additional group of mice was treated with an antibody against the &agr;&bgr; T-cell receptor to induce &agr;&bgr; T-cell depletion; a subset of &agr;&bgr; T cell-deficient mice was also treated with anti-asialoGM1 to deplete natural killer (NK) cells. The mice underwent CLP and were monitored for survival, temperature, acid-base balance, bacterial counts, and cytokine production. The &bgr;TCRKO mice and the wild-type mice treated with anti-&bgr; T-cell receptor (anti-TCR&bgr;) antibody showed improved survival after CLP compared with wild-type mice. The treatment of &agr;&bgr; T cell-deficient mice with anti-asialoGM1further improved survival after CLP, especially when the mice were treated with imipenem. The improved survival observed in &agr;&bgr; T cell-deficient mice was associated with less hypothermia, improved acid-base balance, and decreased production of the proinflammatory cytokines interleukin (IL) 6 and macrophage inflammatory protein (MIP) 2. Compared with wild-type controls, the overall survival was not improved in &dgr;TCRKO mice. The concentrations of IL-6 and MIP-2 in plasma and cytokine mRNA expression in tissues were not significantly different between wild-type and &dgr;TCRKO mice. These studies indicate that mice depleted of &agr;&bgr; but not of &ggr;&dgr; T cells are resistant to mortality in an acutely lethal model of CLP. The depletion of NK cells caused further survival benefit in &agr;&bgr; T cell-deficient mice. These findings suggest that &agr;&bgr; T and NK cells mediate or facilitate CLP-induced inflammatory injury.

Endotoxin Priming Improves Clearance of Pseudomonas aeruginosa in Wild-Type and Interleukin-10 Knockout Mice

ABSTRACT Endotoxin (lipopolysaccharide [LPS]) tolerance is an altered state of immunity caused by prior exposure to LPS, in which production of many cytokines, including gamma interferon (IFN-γ) and interleukin-12 (IL-12), are reduced but secretion of the anti-inflammatory cytokine IL-10 is increased in response to a subsequent LPS challenge. This pattern of cytokine production is also characteristic of postinflammatory immunosuppression. Therefore, we hypothesized that LPS-primed mice would exhibit an impaired ability to respond to systemic infection with the opportunistic pathogen Pseudomonas aeruginosa. We further hypothesized that depletion of IL-10 would reverse the endotoxin-tolerant state. To test this hypothesis, systemic clearance of Pseudomonas aeruginosa was measured for LPS-primed wild-type and IL-10-deficient mice. LPS-primed wild-type mice exhibited significant suppression of LPS-induced IFN-γ and IL-12 but increased IL-10 production in blood and spleen compared to levels exhibited by saline-primed wild-type mice. The suppressed production of IFN-γ and IL-12 caused by LPS priming was ablated in the spleens, but not blood, of IL-10 knockout mice. LPS-primed wild-type mice cleared Pseudomonas aeruginosa from lungs and blood more effectively than saline-primed mice. LPS-primed IL-10-deficient mice were particularly efficient in clearing Pseudomonas aeruginosa after systemic challenge. These studies show that induction of LPS tolerance enhanced systemic clearance of Pseudomonas aeruginosa and that this effect was augmented by neutralization of IL-10.

Improved bacterial clearance and decreased mortality can be induced by LPS tolerance and is not dependent upon IFN-gamma.

Endotoxin (LPS) tolerance is induced by exposure to sublethal doses of LPS, resulting in a suppressed proinflammatory response and an improved survival rate after challenge with a normally lethal dose of LPS. We studied the effects of tolerance induced by either Escherichia coli-derived LPS or Pseudomonas aeruginosa-derived LPS on the innate immune response to a subsequent P. aeruginosa bacterial challenge and determined if the induction of tolerance was dependent on interferon &ggr; (IFN-&ggr;) activity. LPS tolerance was induced in wild-type (WT) and IFN-&ggr; knockout mice by i.p. injection of 1 &mgr;g of LPS on 2 consecutive days. Mice were challenged with an i.p. injection of live P. aeruginosa (1 × 108 colony-forming units) 2 days after the second LPS dose. LPS tolerance in WT mice was associated with diminished serum IFN-&ggr; and IL-12 and increased serum IL-10 responses to the Pseudomonas challenge. Both clearance of the bacterial challenge and survival were improved in WT animals pretreated with either E. coli LPS or P. aeruginosa LPS compared with saline-pretreated control mice. Similarly, IFN-&ggr; knockout mice exposed to LPS before the Pseudomonas challenge also had improved bacterial clearance of the challenge and an improved survival rate. In separate experiments, priming with IFN-&ggr; at a dose that approximated the serum concentration induced by LPS priming did not alter cytokine production or bacterial clearance after a Pseudomonas challenge. Finally, administration of IFN-&ggr; at the time of Pseudomonas challenge amplified cytokine production in LPS-tolerant animals but did not affect bacterial clearance. These results suggest that IFN-&ggr; is not necessary for the induction of LPS tolerance. Furthermore, IFN-&ggr; seems to play a role in propagating the inflammatory cytokine response to Pseudomonas challenge, but it did not seem to have any role in bacterial clearance.

Bacterial clearance and mortality are not improved by a combination of IL-10 neutralization and IFN-gamma administration in a murine model of post-CLP immunosuppression.

ABSTRACT Immunocompromise after a major injury is presumed to be a predisposing factor for sepsis. Mice subjected to sublethal cecal ligation and puncture (CLP) and challenged 5 days later with Pseudomonas aeruginosa had more bacterial growth in lung tissue, lower serum interferon &ggr; (IFN-&ggr;) and interleukin (IL) 12,and higher serum IL-10 when compared with sham CLP mice challenged with Pseudomonas. To test the functional significance of these alterations in cytokine production in the immune response to bacteria, we administered IFN-&ggr; and anti-IL-10 to post-CLP mice before the Pseudomonas challenge. Administration of IFN-&ggr; and anti-IL-10 did not improve bacterial clearance or mortality in post-CLP mice. In further studies, we administered IFN-&ggr; to IL-10 knockout mice before a challenge with P. aeruginosa. Our results showed no significant differences in bacterial clearance or mortality in IL-10 knockout mice with or without IFN-&ggr; treatment compared with wild-type controls. Finally, because most mortality occurred within 2 to 3 days of the Pseudomonas challenge in the aforementioned studies and was likely associated with a marked proinflammatory response, we investigated the effect of IFN-&ggr; and anti-IL-10 on clearance of Pseudomonas in C3H/HeJ mice, which do not mount an exaggerated proinflammatory response to endotoxin or Gram-negative bacteria. Neither clearance of the Pseudomonas bacteria nor mortality was improved in C3H/HeJ mice receiving anti-IL-10 and IFN-&ggr;. These results suggest that the suppressed IFN-&ggr; and IL-12 responses, in combination with an exaggerated IL-10 response to P. aeruginosa challenge after injury, do not correlate with bacterial clearance or survival.

Gamma interferon does not enhance clearance of Pseudomonas aeruginosa but does amplify a proinflammatory response in a murine model of postseptic immunosuppression.

ABSTRACT Patients that have suffered a major injury may sustain a period of immunocompromise and altered Th1/Th2 cytokine balance that can predispose them to opportunistic infections. Pseudomonas aeruginosa is frequently a causative organism for nosocomial infections in critically ill patients and is associated with high mortality. We previously mimicked this clinical scenario by challenging mice with P. aeruginosa 5 days after a cecal ligation and puncture (CLP) procedure. Mice that were subjected to CLP had reduced ability to clear bacteria, significantly lower gamma interferon (IFN-γ) concentrations in plasma, and significantly elevated levels of interleukin 10 (IL-10) in plasma in response to the Pseudomonas challenge compared to uninjured control mice. We investigated the significance of the alteration in IFN-γ by administering recombinant IFN-γ to post-CLP mice at the time of Pseudomonas challenge and by challenging IFN-γ knockout (IFN-γ KO) mice with Pseudomonas. Administration of IFN-γ to post-CLP mice attenuated IL-10 secretion and enhanced IL-12 secretion but did not improve bacterial clearance or survival after Pseudomonas challenge. Furthermore, IFN-γ KO mice had significantly higher plasma IL-10 concentrations but did not exhibit impaired bacterial clearance or increased mortality following Pseudomonas challenge. These data indicate that systemic administration of IFN-γ effectively reverses alterations in immune function that are commonly associated with immunosuppression in critically injured mice but does not improve bacterial clearance or survival following Pseudomonas challenge. Further, endogenous IFN-γ does not appear to contribute significantly to early clearance of Pseudomonas bacteremia, nor does it affect the mortality rate after a lethal Pseudomonas challenge.

Glucan phosphate treatment attenuates burn-induced inflammation and improves resistance to Pseudomonas aeruginosa burn wound infection.

These studies evaluated the effects treatment with glucan phosphate, a soluble polysaccharide immunomodulator, on the inflammatory response induced by burn injury and on resistance to Pseudomonas aeruginosa burn wound infection. Mice were exposed to 35% total body surface area burns and were resuscitated with lactated Ringers (LR) solution alone or LR supplemented with glucan phosphate (40 mg/kg). Glucan phosphate treatment attenuated burn-induced expression of interleukin (IL)-1β, IL-6, and IL-10 mRNAs in spleen, lung, and heart. Plasma concentrations of IL-1β, IL-6, macrophage inflammatory protein (MIP)-2, and IL-10 were also decreased in burned mice treated with glucan phosphate compared with vehicle-treated controls. Early postburn mortality was not significantly different between control (20%) and glucan phosphate-treated (10%) mice, but there was a small improvement in acid-base balance in the glucan phosphate-treated group. Mice received a second injection of glucan phosphate or LR on day 4 postburn and were infected by topical application of P. aeruginosa to the burn wound on day 5. Glucan phosphate treatment significantly improved survival in mice exposed to P. aeruginosa burn wound infection. The improved survival correlated with lower bacterial burden in the burn wound, attenuated production of proinflammatory cytokines, and enhanced production of Th1 cytokines. These studies show that glucan phosphate treatment attenuates burn-induced inflammation and increases resistance to P. aeruginosa burn wound infection in an experimental model of burn injury.

Pretreatment with the Gram-positive Bacterial Cell Wall Molecule Peptidoglycan Improves Bacterial Clearance and Decreases Inflammation and Mortality in Mice Challenged with Staphylococcus aureus

Objective:To determine whether tolerance and enhancement of innate immune function can be induced by the Gram-positive cell wall component peptidoglycan. Design:Controlled, in vivo laboratory study. Subjects:Male mice, 8–12 wks (C57BL6/J; C3H/HeJ; B6.129-Tlr2tm1kir/J). Interventions:Mice were given intraperitoneal injections of 1 mg peptidoglycan on two consecutive days. Mice were then challenged with an intravenous injection of live Staphylococcus aureus (1 × 108 colony-forming units) 2 days after the second pretreatment. Measurements and Main Results:Mice pretreated with peptidoglycan had diminished plasma concentrations of tumor necrosis factor-&agr; and interferon-&ggr; in response to the bacterial challenge when compared with untreated controls. Plasma interleukin-10 after bacterial challenge was higher in peptidoglycan-pretreated mice than in controls. Clearance of bacteria after the staphylococcal challenge was improved in mice pretreated with peptidoglycan, and mortality in response to a subsequent Staphylococcus challenge was significantly attenuated. Peptidoglycan pretreatment of mice lacking intact toll-like receptor-4 signaling (C3H/HeJ) or toll-like receptor-2 signaling (toll-like receptor-2 knockouts) had similar effects on plasma cytokine balance, bacterial clearance, and mortality. Conclusions:Exposure to peptidoglycan significantly attenuated inflammation and enhanced bacterial clearance after a subsequent challenge with S. aureus. These results show that exposure to Gram-positive bacterial cell wall components can induce tolerance and enhance innate immune function and neither toll-like receptor-2 nor toll-like receptor-4 are necessary for this phenomenon. Further, although the altered cytokine balance is similar to that seen in septic patients, induced tolerance differs importantly from the clinical scenario of sepsis in that bacterial clearance and survival are improved compared with normal control animals.