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.     

IL-15 Superagonist Expands mCD8+ T,NK and NKT Cells after Burn Injury but Fails to Improve Outcome during Burn Wound Infection

Background

Severely burned patients are highly susceptible to opportunistic infections and sepsis, owing to the loss of the protective skin barrier and immunological dysfunction. Interleukin-15 (IL-15) belongs to the IL-2 family of common gamma chain cytokines and stimulates the proliferation and activation of T (specifically memory CD8), NK and NKT cells. It has been shown to preserve T cell function and improve survival during cecal ligation and puncture (CLP)-induced sepsis in mice. However, the therapeutic efficacy of IL-15 or IL-15 superagonist (SA) during infection after burn injury has not been evaluated. Moreover, very few, if any, studies have examined, in detail, the effect of burn injury and infection on the adaptive immune system. Thus, we examined the effect of burn and sepsis on adaptive immune cell populations and the effect of IL-15 SA treatment on the host response to infection.

Methods

Mice were subjected to a 35% total body surface area burn, followed by wound infection with Pseudomonas aeruginosa. In some experiments, IL-15 SA was administered after burn injury, but before infection. Leukocytes in spleen, liver and peritoneal cavity were characterized using flow cytometry. Bacterial clearance, organ injury and survival were also assessed.

Results

Burn wound infection led to a significant decline in total white blood cell and lymphocyte counts and induced organ injury and sepsis. Burn injury caused decline in CD4⁺ and CD8⁺ T cells in the spleen, which was worsened by infection. IL-15 treatment inhibited this decline and significantly increased cell numbers and activation, as determined by CD69 expression, of CD4⁺, CD8⁺, B, NK and NKT cells in the spleen and liver after burn injury. However, IL-15 SA treatment failed to prevent burn wound sepsis-induced loss of CD4⁺, CD8⁺, B, NK and NKT cells and failed to improve bacterial clearance and survival.

Conclusion

Cutaneous burn injury and infection cause significant adaptive immune dysfunction. IL-15 SA does not augment host resistance to burn wound sepsis in mice despite inducing proliferation and activation of lymphocyte subsets.     

Plasmacytoid Dendritic Cells Contribute to Systemic but Not Local Antiviral Responses to HSV Infections

Plasmacytoid dendritic cells (pDC) produce type I interferons (IFN-I) and proinflammatory cytokines in response to viruses; however, their contribution to antiviral immunity in vivo is unclear. In this study, we investigated the impact of pDC depletion on local and systemic antiviral responses to herpes simplex virus (HSV) infections using CLEC4C-DTR transgenic mice. We found that pDC do not appear to influence viral burden or survival after vaginal HSV-2 infection, nor do they seem to contribute to virus-specific CD8 T cell responses following subcutaneous HSV-1 infection. In contrast, pDC were important for early IFN-I production, proinflammatory cytokine production, NK cell activation and CD8 T cell responses during systemic HSV-2 and HSV-1 infections. Our data also indicate that unlike pDC, TLR3-expressing cells are important for promoting antiviral responses to HSV-1 regardless of the route of virus administration.     

The role of nasal carriage in Staphylococcus aureus burn wound colonization

Thermal injury destroys the physical skin barrier that normally prevents invasion of microorganisms. This and concomitant depression of local and systemic host cellular and humoral immune responses are important factors that contribute to colonization and infection of the burn wound. One of the most common burn wound pathogens is Staphylococcus aureus. Staphylococcus aureus is both a human commensal and a frequent cause of infections leading to mild to life-threatening diseases. Despite a variety of infection control measures, for example patient cohorting and contact precaution at burn centres, S. aureus is still frequently encountered in burn wounds. Colonization with S. aureus has been associated with delayed wound healing, increased need for surgical interventions, and prolonged length of stay at burn centres. In this minireview, we focus on S. aureus nasal carriage in relation to S. aureus burn wound colonization and subsequent infection, and its impact on strategies for infection control.     

The Role of CHI3L1 (Chitinase-3-Like-1) in the Pathogenesis of Infections in Burns in a Mouse Model

In severe burn injury the unique setting of a depleted, dysfunctional immune system along with a loss of barrier function commonly results in opportunistic infections that eventually proof fatal. Unfortunately, the dynamic sequence of bacterial contamination, colonization and eventually septic invasion with bacteria such as Pseudomonas species is still poorly understood although a limiting factor in clinical decision making. Increasing evidence supports the notion that inhibition of bacterial translocation into the wound site may be an effective alternative to prevent infection. In this context we investigated the role of the mammalian Chitinase-3-Like-1 (CHI3L1) non-enyzmatic protein predominately expressed on epithelial as well as innate immune cells as a potential bacterial-translocation-mediating factor. We show a strong trend that a modulation of chitinase expression is likely to be effective in reducing mortality rates in a mouse model of burn injury with superinfection with the opportunistic PA14 Pseudomonas strain, thus demonstrating possible clinical leverage.     

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.     

Gene expression analysis in burn wounds of rats

The events occurring early in the burn wound trigger a sequence of local and systemic responses that influence cell and tissue survival and, consequently, wound healing and recovery. Using high-density oligonucleotide arrays we identified gene expression patterns in skin samples taken from a region of injury in the burn rat model. The associated genomic events include the differential expression of genes involved in cell survival and death, cell growth regulation, cell metabolism, inflammation, and immune response. The functional gene cluster detected and their time appearance matched the time sequence known to occur in burn wound healing.     

Synergistic action of fms-like tyrosine kinase 3 ligand and CD40 ligand in the induction of dendritic cells and generation of antitumor immunity in vivo.

Daily treatment of mice with fms-like tyrosine kinase 3 ligand (Flt3L) leads to a significant increase in the number of dendritic cells and induces antitumor immunity. Here, we show that Flt3L and CD40 ligand (CD40L) synergize in the generation of immune responses against two poorly immunogenic tumors, leading to complete tumor rejection in a high proportion of mice. Rechallenge of the Flt3L + CD40L-treated mice with the immunizing tumor resulted in complete inhibition of tumor growth, indicating that these animals had developed long-lasting antitumor immunity. In addition, we demonstrate that endogenous CD40L plays a critical role in antitumor immunity, since blockade of CD40-CD40L interactions in vivo prevents the generation of antitumor immunity in therapeutic and vaccination protocols. Dendritic cells generated in mice treated with Flt3L alone or in combination with CD40L were equally potent in stimulating allogeneic T cells and expressed similar levels of MHC class II, CD80, and CD86. However, mice treated with Flt3L + CD40L had significantly more dendritic cells than mice treated with either of the cytokines alone, suggesting that CD40L promotes the proliferation and/or survival of dendritic cells generated by Flt3L treatment. Dendritic cells generated in this manner are likely to be involved in the priming of antitumor immune responses.     

Lupus-Prone Mice Fail to Raise Antigen-Specific T Cell Responses to Intracellular Infection

Systemic lupus erythematosus (SLE) is characterized by multiple cellular abnormalities culminating in the production of autoantibodies and immune complexes, resulting in tissue inflammation and organ damage. Besides active disease, the main cause of morbidity and mortality in SLE patients is infections, including those from opportunistic pathogens. To understand the failure of the immune system to fend off infections in systemic autoimmunity, we infected the lupus-prone murine strains B6.lpr and BXSB with the intracellular parasite Toxoplasma gondii and survival was monitored. Furthermore, mice were sacrificed days post infection and parasite burden and cellular immune responses such as cytokine production and cell activation were assessed. Mice from both strains succumbed to infection acutely and we observed greater susceptibility to infection in older mice. Increased parasite burden and a defective antigen-specific IFN-gamma response were observed in the lupus-prone mice. Furthermore, T cell:dendritic cell co-cultures established the presence of an intrinsic T cell defect responsible for the decreased antigen-specific response. An antigen-specific defect in IFN- gamma production prevents lupus-prone mice from clearing infection effectively. This study reveals the first cellular insight into the origin of increased susceptibility to infections in SLE disease and may guide therapeutic approaches.     

Fms-Like Tyrosine Kinase 3 Ligand Controls Formation of Regulatory T Cells in Autoimmune Arthritis

Fms-like tyrosine kinase 3 ligand (Flt3L) is known as the primary differentiation and survival factor for dendritic cells (DCs). Furthermore, Flt3L is involved in the homeostatic feedback loop between DCs and regulatory T cell (Treg). We have previously shown that Flt3L accumulates in the synovial fluid in rheumatoid arthritis (RA) and that local exposure to Flt3L aggravates arthritis in mice, suggesting a possible involvement in RA pathogenesis. In the present study we investigated the role of Flt3L on DC populations, Tregs as well as inflammatory responses in experimental antigen-induced arthritis. Arthritis was induced in mBSA-immunized mice by local knee injection of mBSA and Flt3L was provided by daily intraperitoneal injections. Flow cytometry analysis of spleen and lymph nodes revealed an increased formation of DCs and subsequently Tregs in mice treated with Flt3L. Flt3L-treatment was also associated with a reduced production of mBSA specific antibodies and reduced levels of the pro-inflammatory cytokines IL-6 and TNF-α. Morphological evaluation of mBSA injected joints revealed reduced joint destruction in Flt3L treated mice. The role of DCs in mBSA arthritis was further challenged in an adoptive transfer experiment. Transfer of DCs in combination with T-cells from mBSA immunized mice, predisposed naïve recipients for arthritis and production of mBSA specific antibodies. We provide experimental evidence that Flt3L has potent immunoregulatory properties. Flt3L facilitates formation of Treg cells and by this mechanism reduces severity of antigen-induced arthritis in mice. We suggest that high systemic levels of Flt3L have potential to modulate autoreactivity and autoimmunity.     

Persistent virus infection inhibits type I interferon production by plasmacytoid dendritic cells to facilitate opportunistic infections

Emerging studies indicate an association between virus-induced impairment in type I interferon (IFN-I) production and enhanced susceptibility to opportunistic infections, which represent a major health problem. Here, we provide in vivo evidence that lymphocytic choriomeningitis virus (LCMV) infection of its natural murine host dramatically diminishes the unique capacity of plasmacytoid dendritic cells (pDCs) to secrete high levels of systemic IFN-I. While both acute and persistent LCMV infections suppress pDC IFN-I response, only the persistent virus induces a long-lasting diversion of this innate immune pathway. The consequent reduction in IFN-I production serves to impair natural killer cell responses in LCMV-infected mice challenged subsequently with murine cytomegalovirus (MCMV) as an opportunistic pathogen. This innate defect also compromises the host's ability to counteract early MCMV spread. These findings provide a mechanistic explanation for the occurrence of opportunistic infections following viral insults and have important implications for treating such medical complications.     

Injury primes the innate immune system for enhanced Toll-like receptor reactivity

Severe injury causes a dramatic host response that disrupts immune homeostasis and predisposes the injured host to opportunistic infections. Because Toll-like receptors (TLRs) recognize conserved microbial Ags and endogenous danger signals that may be triggered by injury, we wanted to determine how injury influences TLR responses. Using an in vivo injury model, we demonstrate that injury significantly increased TLR2- and TLR4-induced IL-1beta, IL-6, and TNF-alpha production by spleen cells. This influence of injury on TLR reactivity was observed as early as 1 day after injury and persisted for at least 7 days. The outcome of similar studies performed using TLR4-mutant C57BL/10ScN/Cr mice revealed that TLR2 responses remained primed, thus suggesting that injury-induced priming can occur independently of endogenous TLR4 signaling. Increased TLR4 reactivity was also observed in vivo, because LPS-challenged injured mice demonstrated significantly higher cytokine expression levels in the lung, liver, spleen, and plasma. Macrophages and dendritic cells were the major source of these cytokines as judged by intracellular cytokine staining. Moreover, ex vivo studies using enriched macrophage and dendritic cell populations confirmed that T cells did not contribute to the enhanced TLR2 and TLR4 responses. The results of flow cytometry studies using TLR2- and TLR4-MD-2-specific Abs indicated that injury did not markedly alter cell surface TLR2 or TLR4-MD-2 expression. Taken together, these findings establish that injury primes the innate immune system for enhanced TLR2- and TLR4-mediated responses and provides evidence to suggest that augmented TLR reactivity might contribute to the development of heightened systemic inflammation following severe injury.     

Why is HIV a pathogen?

The pathogenesis of HIV begins with a profound depletion of CD4+ T cells in the gut followed by a long period of clinically silent but dynamic virus replication and diversification with high host cell turnover before the onset of AIDS. The AIDS-defining opportunistic infections and tumors mark the end-point of a long balancing act between virus and host that occurs when CD4+ T cell numbers fall below a level that can sustain immunity. Comparative studies of lentivirus infections in other species show that AIDS is not an inevitable outcome of infection because simian immunodeficiency virus in natural hosts seldom causes disease. What distinguishes pathogenic from 'passenger' infection is a systemic activation of immune responses followed by destruction of the integrity of lymphoid follicles. Macrophage and dendritic cell infection also contribute to pathogenesis. Maedi-Visna virus infection in sheep, which targets these cells but not T lymphocytes, also leads to progressive disease and death that resembles the wasting and brain diseases of HIV without the T cell immunodeficiency. Thus, lessons from pathogenic and nonpathogenic lentivirus infections provide insight into the complex syndrome called AIDS.     

Burn injury suppresses human dermal dendritic cell and Langerhans cell function

Human skin contains epidermal Langerhans cells (LCs) and dermal dendritic cells (DCs) that are key players in induction of adaptive immunity upon infection. After major burn injury, suppressed adaptive immunity has been observed in patients. Here we demonstrate that burn injury affects adaptive immunity by altering both epidermal LC and dermal DC functions. We developed a human ex vivo burn injury model to study the function of DCs in thermally injured skin. No differences were observed in the capacity of both LCs and dermal DCs to migrate out of burned skin compared to unburned skin. Similarly, expression levels of co-stimulatory molecules were unaltered. Notably, we observed a strong reduction of T cell activation induced by antigen presenting cell (APC) subsets that migrated from burned skin through soluble burn factors. Further analyses demonstrated that both epidermal LCs and dermal DCs have a decreased T cell stimulatory capacity after burn injury. Restoring the T cell stimulatory capacity of DC subsets might improve tissue regeneration in patients with burn wounds.     

Antifungal Pharmacokinetics and Dosing Considerations in Burn Patients

Patients with burn injuries are at high risk of developing invasive fungal infections leading to increased morbidity and mortality. Burn patients undergo major physiologic changes, which produce significant alterations in the pharmacokinetics and pharmacodynamics of antimicrobial agents. These changes result from the breakdown of the body’s natural barriers to infection and the systemic responses that subsequently ensue after burn injury, including systemic inflammatory responses, third spacing, and development of a hypermetabolic state. Severe burn injuries often lead to larger volumes of distribution and increased drug clearance. Limited data are available to guide the clinician in optimizing the dosing regimen of antifungals in patients with burn injuries. We present a review of antifungal pharmacokinetics and describe how these properties can be used to design rational therapeutic regimens tailored to the pharmacodynamic alterations characteristic of burn patients.     

Nasal administration of Lactococcus lactis improves local and systemic immune responses against Streptococcus pneumoniae

Lactococcus lactis NZ9000 is a non-pathogenic non-invasive bacterium extensively used for the delivery of antigens and cytokines at the mucosal level. However, there are no reports concerning the per se immunomodulatory capacity of this strain. The aim of the present study was to investigate the intrinsic immunostimulating properties of the nasal administration of L. lactis NZ9000 in a pneumococcal infection model. Mice were preventively treated with L. lactis (2, 5 or 7 days with 10(8) cells/day per mouse) and then challenged with Streptococcus pneumoniae. The local and the systemic immune responses were evaluated. Our results showed that nasal administration of L. lactis for 5 days (LLN5d) increased the clearance rate of S. pneumoniae from lung and prevented the dissemination of pneumococci into blood. This effect coincided with an upregulation of the innate and specific immune responses in both local and systemic compartments. LLN5d increased phagocyte activation in lung, blood and bone marrow, determined by NBT and peroxidase tests. Anti-pneumococcal immunoglobulin (Ig)A in bronchoalveolar lavages (BAL) and IgG in BAL and serum were increased in the LLN5d group. Lung tissue injury was reduced by LLN5d treatment as revealed by histopathological examination and albumin concentration and lactate dehydrogenase activity in BAL. The adjuvant effect of L. lactis in our infection model would be an important advantage for its use as a delivery vehicle of pneumococcal proteins and nasal immunization with recombinant L. lactis emerges as an effective route of vaccination for both systemic and mucosal immunity against pneumococcal infection.     

Innate versus adaptive immunity in Candida albicans infection

Candida albicans is a common opportunistic pathogen, causing both superficial and systemic infection. Clinical observations indicate that mucocutaneous infections are commonly associated with defective cell-mediated immune responses, whereas systemic infection is more frequently seen in patients with deficiencies in neutrophil number or function. Analysis of mechanisms of host resistance against gastrointestinal and oral infection in mouse models has demonstrated an absolute dependence on CD4(+) T cells, although clearance also involves phagocytic cells. Both IL-12 and TNF-alpha appear to be important mediators, but mouse strain-dependent variations in susceptibility to infection may be related to T-cell enhancement of production of phagocytic cells by the bone marrow. In murine systemic infection, the role of innate and adaptive responses is less well defined. Studies in immunodeficient and T-cell-depleted mice suggest that clearance of the yeast may be predominantly a function of the innate response, whereas the adaptive response may either limit tissue damage or have the potential to cause immunopathology, depending on the host genetic context in which the infection takes place.     

FMS-like tyrosine kinase 3 ligand aggravates the lung inflammatory response to Streptococcus pneumoniae infection in mice: role of dendritic cells

Pretreatment of mice with the hemopoietic growth factor, FMS-like tyrosine kinase 3 ligand (Flt3L), has been shown to increase monocyte-derived myeloid dendritic cells (DC) in lung parenchymal tissue, with possible implications for protective immunity to lung bacterial infections. However, whether Flt3L treatment improves lung innate immunity of mice to challenge with Streptococcus pneumoniae has not been investigated previously. Mice pretreated with Flt3L exhibited a peripheral monocytosis and a strongly expanded lung myeloid DC pool, but responded with a similar proinflammatory cytokine release (TNF-alpha, IL-6, keratinocyte derived cytokine, MIP-2, CCL2) and neutrophilic alveolitis upon infection with S. pneumoniae as did control mice with a normal lung DC pool. Unexpectedly, however, Flt3L-pretreated mice, but not control mice, infected with S. pneumoniae developed vasculitis and increased lung permeability by days 2-3 postinfection, and florid pneumonia accompanied by sustained increased bacterial loads by days 3-4 postinfection. This was associated with an overall increased mortality of approximately 35% by day 4 after pneumococcal challenge. Application of anti-CCR2 Ab MC21 to block inflammatory monocyte-dependent lung mononuclear phagocyte mobilization significantly reduced the lung leakage, but not vasculitis in Flt3L-pretreated mice infected with S. pneumoniae, without affecting the intra-alveolar cytokine liberation or the concomitantly developing neutrophilic alveolitis. Together, the data demonstrate that previous Flt3L-induced lung DC accumulation is not protective in lung innate immunity to challenge with S. pneumoniae, and support the concept that CCR2-dependent mononuclear phagocyte as opposed to neutrophil recruitment contributes to increased lung leakage in Flt3L-pretreated mice challenged with S. pneumoniae.     

The balance between plasmacytoid DC versus conventional DC determines pulmonary immunity to virus infections

Background

Respiratory syncytial virus (RSV) infects nearly all infants by age 2 and is a leading cause of bronchiolitis. RSV may employ several mechanisms to induce immune dysregulation, including dendritic cell (DC) modulation during the immune response to RSV.

Methods and Findings

Expansion of cDC and pDC by Flt3L treatment promoted an anti-viral response with reduced pathophysiology characterized by decreased airway hyperreactivity, reduced Th2 cytokines, increased Th1 cytokines, and a reduction in airway inflammation and mucus overexpression. These protective aspects of DC expansion could be completely reversed by depleting pDCs during the RSV infection. Expansion of DCs by Flt3L treatment enhanced in CD8+ T cell responses, which was reversed by depletion of pDC.

Conclusions

These results indicate that a balance between cDC and pDC in the lung and its lymph nodes is crucial for the outcome of a pulmonary infection. Increased pDC numbers induced by Flt3L treatment have a protective impact on the nature of the overall immune environment.