James A. Lederer

Major injury leads to predominance of the T helper-2 lymphocyte phenotype and diminished interleukin-12 production associated with decreased resistance to infection.

Objective Patients with serious traumatic injury and major burns and an animal model of burn injury were studied to determine the effect of injury on the production of cytokines typical of the T helper‐2 lymphocyte phenotype as opposed to the T helper‐1 phenotype and on the production of interleukin‐12. Summary Background Data Perturbations of natural and adoptive immunity are related to the increased susceptibility to infection manifested by seriously injured and burn patients. Earlier work has shown that impaired adoptive immunity after injury is characterized by diminished production of interleukin‐2 (IL‐2), a product of Th lymphocytes. Exposure of naive Th cells to certain antigens and cytokines causes conversion to either the Th‐1 or the Th‐2 phenotype. Th‐1 cells produce IL‐2 and interferon‐gamma (IFN‐τ) and initiate cellular immunity. Th‐2 cells secrete interleukin‐4 (IL‐4) and interleukin‐10 (IL‐10) and stimulate production of certain antibodies. Conversion to the Th‐1 phenotype is facilitated by IL‐12, and conversion to the Th‐2 phenotype is promoted by IL‐4. The authors believed that serious injury might cause conversion of Th cells to the Th‐2 as opposed to the Th‐1 phenotype rather than generalized Th suppression. Methods The authors studied circulating peripheral blood mononuclear cells (PBMC) from 16 major burn and 8 trauma patients on 32 occasions early after injury and from 13 age‐ and sex‐matched healthy individuals for cytokine production after phytohemagglutinin stimulation. Also studied was a mouse model of 20% burn injury known to mimic the immune abnormalities seen in humans with burns. Splenocytes from burn mice, 10 to 12 per group, were studied after activation by concanavalin A or by the bacterial antigen Staphylococcus aureus Cowan strain I for cytokine production and cytokine messenger RNA expression as determined by reverse transcriptase polymerase chain reaction. Burn mice were compared with sham‐burn controls and attention was focused on day 10 after burn injury, a time when IL‐2 production and resistance to infection are highly suppressed. Finally, burn and sham‐burn animals, 20 per group, were treated in vivo with IL‐12 (25 ng daily for 5 days) and observed for mortality after septic challenge (cecal ligation and puncture [CLP]) performed on day 10 after injury. Results Peripheral blood mononuclear cells from burn and trauma patients produced less IFN‐τ, the index cytokine of Th‐1 cells, than PBMCs from healthy individuals 1 to 14 days after burn injury (SE = 77.6 ± 16 pg/mL patients vs. 141.3 ± 35 pg/mL controls, p < 0.05). However, production of IL‐4, the index cytokine of Th‐2 cells, by patient PBMCs was increased (51.0 ± 13.0 pg/mL patients vs. 26.9 ± 2.5 controls, p < 0.05). Splenocytes from mice 10 days after burn injury, when compared with sham‐burn controls, showed diminished production of IL‐2 (1.04 ± 0.91 units/mL burns vs. 5.8 ± 0.55 units/mL controls, p < 0.05) and IFN‐τ (1.05 ± 0.7 units/mL burns vs. 12.0 ± 8.9 units/ mL controls, p < 0.05). However, burn splenocytes produced more IL‐4 (2492 ± 157.0 pg/mL burns vs. 672.0 ± 22.7 pg/mL controls, p < 0.01) and IL‐10 (695.2 ± 20.8 pg/mL burns vs. 567.0 ± 16.7 pg/mL controls, p < 0.05). Splenocyte production of IL‐12 was also reduced after burn (0.20 ± 0.035 units/mL) as compared with sham burn (0.46 ± 0.08 units/mL, p < 0.05). The reduction in IL‐2, IFN‐τ, and IL‐12 production by burn splenocytes was reflected by a tenfold decrease in expression of their respective cytokine mRNAs. In vivo IL‐12 treatment of burn animals decreased mortality from CLP on day 10 after injury from 85% to 15% (sham‐burn mortality after CLP, 15%, p < 0.05) and increased splenocyte IFN‐τ production to supranormal levels. Conclusions Serious injury induced diminished production of IL‐12 and a shift to the Th‐2 phenotype with increased production of IL‐4 and IL‐10, cytokines known to inhibit Th‐1 function. The ability of exogenous IL‐12 to restore Th‐1 cytokine production and resistance to infection suggests a therapeutic role for IL‐12 in the immune dysfunction seen after major injury.

The effects of injury on the adaptive immune response

For more than thirty years it has been apparent that serious injury in humans and experimental animals is associated with a decrease in immune functions dependent upon T cells, the principal cells involved in initiating adaptive immune responses. This review focuses on more recent evidence that T helper cell function is altered after serious injury with loss of T helper 1 function and cytokine production and with preservation of T helper 2 function and an increased production of T helper 2 cytokines. Emphasis is placed on the importance of interactions between the innate and adaptive immune systems in the perturbed immune responses seen following injury. Immunomodulatory strategies are mentioned that have had success in animal models in ameliorating the diminished resistance to infection commonly seen after major traumatic or thermal injury. Finally, it is emphasized that immunomodulatory treatments that are successful in preventing infection may be contraindicated once infection is manifest.

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-1β, IL-6, and TNF-α 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.

The immunologic response to injury.

Research on the immune consequences of shock and trauma by multiple laboratories over more than 20 years has resulted in the following paradigm, which is currently accepted by most investigators in this field: serious traumatic or thermal injury is quickly followed, after initial resuscitation, by the systemic inflammatory response syndrome (SIRS) which, in a sizeable minority of patients, will lead inexorably to multiple organ dysfunction syndrome (early MODS), with an attendant high mortality. The majority of seriously injured patients survive the initial SIRS response without developing early MODS, and after a period of relative clinical stability, manifest a compensatory antiinflammatory response syndrome (CARS) with suppressed immunity and diminished resistance to infection. Resultant infection and its attendant inflammation in turn can lead to multiple organ dysfunction (late MODS) and death (Fig. 1). This paradigm has several implications of potential importance in interpreting the sometimes conflicting results of research in this area:

Impaired Regulatory T-Cell Response and Enhanced Atherosclerosis in the Absence of Inducible Costimulatory Molecule

Background— T-cell–mediated immunity contributes to the pathogenesis of atherosclerosis, but little is known about how these responses are regulated. We explored the influence of the inducible costimulatory molecule (ICOS) on atherosclerosis and associated immune responses. Methods and Results— Bone morrow chimeras were generated by transplanting ICOS-deficient or wild-type bone marrow into irradiated atherosclerosis-prone, LDR receptor–deficient mice, and the chimeric mice were fed a high-cholesterol diet for 8 weeks. Compared with controls, mice transplanted with ICOS-deficient marrow had a 43% increase in the atherosclerotic burden, and importantly, their lesions had a 3-fold increase in CD4+ T cells, as well as increased macrophage, smooth muscle cell, and collagen content. CD4+ T cells from ICOS-deficient chimeras proliferated more and secreted more interferon-γ and tumor necrosis factor-α than T cells from control mice, which suggests a lack of regulation. FoxP3+ regulatory T cells (Treg) were found to constitutively express high ICOS levels, which suggests a role for ICOS in Treg function. ICOS-deficient mice had decreased numbers of FoxP3+ Treg and impaired in vitro Treg suppressive function compared with control mice. Conclusions— ICOS has a key role in regulation of atherosclerosis, through its effect on regulatory T-cell responses.

CD4+CD25+ Regulatory T Cells Control Innate Immune Reactivity after Injury

Major injury initiates a systemic inflammatory response that can be detrimental to the host. We have recently reported that burn injury primes innate immune cells for a progressive increase in TLR4 and TLR2 agonist-induced proinflammatory cytokine production and that this inflammatory phenotype is exaggerated in adaptive immune system-deficient (Rag1−/−) mice. The present study uses a series of adoptive transfer experiments to determine which adaptive immune cell type(s) has the capacity to control innate inflammatory responses after injury. We first compared the relative changes in TLR4- and TLR2-induced TNF-α, IL-1β, and IL-6 production by spleen cell populations prepared from wild-type (WT), Rag1−/−, CD4−/−, or CD8−/− mice 7 days after sham or burn injury. Our findings indicated that splenocytes prepared from burn-injured CD8−/− mice displayed TLR-induced cytokine production levels similar to those in WT mice. In contrast, spleen cells from burn-injured CD4−/− mice produced cytokines at significantly higher levels, equivalent to those in Rag1−/− mice. Moreover, reconstitution of Rag1−/− or CD4−/− mice with WT CD4+ T cells reduced postinjury cytokine production to WT levels. Additional separation of CD4+ T cells into CD4+CD25+ and CD4+CD25− subpopulations before their adoptive transfer into Rag1−/− mice showed that CD4+CD25+ T cells were capable of reducing TLR-stimulated cytokine production levels to WT levels, whereas CD4+CD25− T cells had no regulatory effect. These findings suggest a previously unsuspected role for CD4+CD25+ T regulatory cells in controlling host inflammatory responses after injury.

Quantitative proteome analysis of human plasma following in vivo lypopolysaccharide administration using 16O/18O labeling and the accurate mass and time tag approach

Identification of novel diagnostic or therapeutic biomarkers from human blood plasma would benefit significantly from quantitative measurements of the proteome constituents over a range of physiological conditions. Herein we describe an initial demonstration of proteome-wide quantitative analysis of human plasma. The approach utilizes postdigestion trypsin-catalyzed 16O/18O peptide labeling, two-dimensional LC-FTICR mass spectrometry, and the accurate mass and time (AMT) tag strategy to identify and quantify peptides/proteins from complex samples. A peptide accurate mass and LC elution time AMT tag data base was initially generated using MS/MS following extensive multidimensional LC separations to provide the basis for subsequent peptide identifications. The AMT tag data base contains >8,000 putative identified peptides, providing 938 confident plasma protein identifications. The quantitative approach was applied without depletion of high abundance proteins for comparative analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Accurate quantification of changes in protein abundance was demonstrated by both 1:1 labeling of control plasma and the comparison between the plasma samples following LPS administration. A total of 429 distinct plasma proteins were quantified from the comparative analyses, and the protein abundances for 25 proteins, including several known inflammatory response mediators, were observed to change significantly following LPS administration.

Injury, sepsis, and the regulation of Toll-like receptor responses

Although we tend to think that the immune system has evolved to protect the host from invading pathogens and to discriminate between self and nonself, there must also be an element of the immune system that has evolved to control the response to tissue injury. Moreover, these potential immune‐regulatory pathways controlling the injury response have likely coevolved in concert with self and nonself discriminatory immune‐regulatory networks with a similar level of complexity. From a clinical perspective, severe injury upsets normal immune function and can predispose the injured patient to developing life‐threatening infectious complications. This remains a significant health care problem that has driven decades of basic and clinical research aimed at defining the functional effects of injury on the immune system. This review and update on our ongoing research efforts addressing the immunological response to injury will highlight some of the most recent advances in our understanding of the impact that severe injury has on the innate and adaptive immune system focusing on phenotypic changes in innate immune cell responses to Toll‐like receptor stimulation.

High Dynamic Range Characterization of the Trauma Patient Plasma Proteome

Although human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation. Herein we describe a strategy that combines immunoaffinity subtraction and subsequent chemical fractionation based on cysteinyl peptide and N-glycopeptide captures with two-dimensional LC-MS/MS to increase the dynamic range of analysis for plasma. Application of this “divide-and-conquer” strategy to trauma patient plasma significantly improved the overall dynamic range of detection and resulted in confident identification of 22,267 unique peptides from four different peptide populations (cysteinyl peptides, non-cysteinyl peptides, N-glycopeptides, and non-glycopeptides) that covered 3654 different proteins with 1494 proteins identified by multiple peptides. Numerous low abundance proteins were identified, exemplified by 78 “classic” cytokines and cytokine receptors and by 136 human cell differentiation molecules. Additionally a total of 2910 different N-glycopeptides that correspond to 662 N-glycoproteins and 1553 N-glycosylation sites were identified. A panel of the proteins identified in this study is known to be involved in inflammation and immune responses. This study established an extensive reference protein database for trauma patients that provides a foundation for future high throughput quantitative plasma proteomic studies designed to elucidate the mechanisms that underlie systemic inflammatory responses.

Targeting Regulatory T Cells in Cancer

Infiltration of tumors by regulatory T cells confers growth and metastatic advantages by inhibiting antitumor immunity and by production of receptor activator of NF-κB (RANK) ligand, which may directly stimulate metastatic propagation of RANK-expressing cancer cells. Modulation of regulatory T cells can enhance the efficacy of cancer immunotherapy. Strategies include depletion, interference with function, inhibition of tumoral migration, and exploitation of T-cell plasticity. Problems with these strategies include a lack of specificity, resulting in depletion of antitumor effector T cells or global interruption of regulatory T cells, which may predispose to autoimmune diseases. Emerging technologies, such as RNA interference and tetramer-based targeting, may have the potential to improve selectivity and efficacy.