Supplementary Figure 2 from Chemotherapy-Induced Genotoxic Stress Promotes Sensitivity to Natural Killer Cell Cytotoxicity by Enabling Missing-Self Recognition

Supplementary Figure 3 from Chemotherapy-Induced Genotoxic Stress Promotes Sensitivity to Natural Killer Cell Cytotoxicity by Enabling Missing-Self Recognition

Although sepsis in burn patients is a major contributor to mortality, treatments are not always effective and underlying mechanisms have yet to be completely elucidated. NLRP3 inflammasome orchestrates burn‐induced, inflammatory‐driven pathophysiologic processes. Here, we determined the mechanism of NLRP3 inflammasome activation on bacterial clearance and mortality in burn sepsis. We obtained tissue and blood from 30 wild‐type and 30 Nlrp3 −/− mice. Mice were subjected to a two‐hit model of 25–30% TBSA scald burn followed by Pseudomonas aeruginosa wound infection 72 hours after injury. We also obtained tissue from 34 adult burn patients (≥18 years of age) with early (0–11 days post‐burn) and later (≥12 days post‐burn) surgical time‐points and ten healthy controls. Murine studies indicated that Nlrp3 −/− had 30% improved survival and bacterial clearance at the site of injury and is systemically relative to burn sepsis wild type. Greater macrophage and neutrophil infiltration occurred acutely after infection (12 hours) to the site of injury and adipose tissue. This was followed by increased macrophage and neutrophil infiltration to lymphoid organs and liver beyond the acute phase (24 and 72 hours). Interestingly, Nlrp3 ablation increased acute systemic inflammation (IL‐6, TNF‐α, IL‐1β). Septic burn patients had persistently increased adipose NLRP3 by‐product expression beyond the acute phase that was more pronounced in late‐onset sepsis. Our findings suggest that Nlrp3 genetic ablation enhanced acute tissue‐specific inflammatory responsiveness. Likely, this occurs by paradoxically increasing acute immune infiltration and inflammation with a non‐persistent response. Clinically, persistent NLRP3‐mediated inflammation occurs in septic versus normal burn patients and potentially detrimentally impacts patient outcomes.

Natural killer (NK) cells play a key role in innate immunity by detecting alterations in self and non-self ligands via NK cell receptors (NKRs). They serve an important role in controlling viruses and have been described as being particularly important in managing herpesvirus infections. Among the various NKRs, inhibitory NKR-P1 receptors have recently being shown to be targeted by virally-encoded proteins of cytomegalovirus (CMV), a member of the beta-herpesvirus family. In rodents, the NKR-P1B receptor interacts with Clec2d/Clr-b, an inhibitory ligand abundantly expressed on healthy cells. Clr-b is downregulated during stress and serves to alert NK cells of pathogenesis. Interestingly, both rat and mouse CMVs encode viral ligands (RCTL and m12 respectively) that directly engage the NKR-P1B receptor and serve as decoys to subvert NK recognition. In humans, the homologous receptor is NKR-P1A/CD161 and binds CLEC2D/LLT-1. Given the findings in rodents, we are investigating if human CMV (HCMV) similarly employs a mechanism to escape NK detection through NKR-P1A/CD161. Using cell reporters capable of detecting receptor-ligand interactions and various HCMV strains, we are undergoing a systematic approach address this hypothesis.

BACKGROUND:During the past decades' sepsis has become the major cause of death in severely burned patients. Despite the importance of burn sepsis, its diagnosis, let alone its prediction, is difficult if not impossible. Recently, we have demonstrated burn patients have increased NLRP3 inflammasome activation in white adipose tissue. We aimed to delineate a unique immune profile that can be used to identify septic outcomes in severely burned patients. METHODS:Adult burn patients (n = 37) admitted to our burn center between June 2013-2015 were enrolled in this study. White adipose tissue from the site of injury and plasma were collected from severely burned patients (>20% total body surface area) within 96 hours after thermal injury, indiscriminate of sex or age. RESULTS:We found that patients exhibiting aberrantly high levels of proinflammatory interleukin-1β and decreased macrophages at the site of injury are highly susceptible to development of sepsis. Septic patients also had increased anti-inflammatory (interleukin-10, interleukin-1RA) cytokines in plasma. The Septic Predictor Index was generated as a quotient for the site of injury macrophage proportion and interleukin-1β production. All patients who eventually develop sepsis had septic predictor index values >0.5. Septic patients with Septic Predictor Index values >1 all had sepsis onset within 12 days post-injury, whereas patients with Septic Predictor Index values between 0.5-1 all had later onset (>12 days). CONCLUSION:The Septic Predictor Index can determine sepsis onset accurately in thermally injured patients a priori and further enables surgeons to develop clinical studies and focused therapies specifically designed for septic cohorts.

OBJECTIVE:Comparing the inflammatory and immunological trajectories in burned adults versus burned elderly patients to gain novel insights and better understanding why elderly have poor outcomes.SUMMARY BACKGROUND DATA:Despite receiving the same treatment and clinical consideration as all other burn patients, elderly patients continue to have substantially poorer outcomes compared with adults. In light of an aging population, gaining a better understanding of their susceptibility to complications and creating new treatment strategies is imperative.METHODS:We included 130 burn patients (94 adults: <65 years old and 36 elderly: ≥65 years old) and 10 healthy controls in this study. Immune activity and expression was assessed using bioplex at various time points. Clinical outcomes such as infection, sepsis, and mortality were prospectively collected.RESULTS:Elderly burn patients had significantly lower burn size but significantly higher Baux scores. Morbidity and mortality was significantly increased in the elderly cohort. Immune biomarkers indicated that elderly are immune compromised and unable to respond with the expected inflammatory response during the early phase after injury. This trajectory changes to a hyperinflammatory pattern during the later phase after burn. These findings are even more pronounced when comparing sepsis versus nonsepsis patients as well as survivors versus nonsurvivors in the elderly.CONCLUSIONS:Elderly burned patients mount a delayed immune and dampened inflammatory response early after burn injury that changes to an augmented response at later time points. Late-onset sepsis and nonsurvivors had an immune exhaustion phenotype, which may represent one of the main mediators responsible for the striking mortality in elderly.

The hypermetabolic stress response after burn contributes to multi-organ failure, sepsis, morbidity, and mortality. The cytokine interleukin 6 (IL-6) has been hypothesized to mediate not only white adipose tissue (WAT) browning in burns, but also other hypermetabolic conditions. In addition to its inflammatory effects, IL-6 also acts as a metabolic mediator that affects metabolic tissues. Therefore, we sought to uncover the origin of circulating IL-6 post burn injury that regulates WAT browning. WAT and sera samples were collected from both adult burn patients admitted to the Ross Tilley Burn Centre at Sunnybrook Hospital and mice subjected to a burn injury. Collected tissues were analyzed for browning markers and metabolic state via histology, gene expression, and resting energy expenditure. Increased WAT browning was observed in burn patients as well as mice subjected to burn injury. Circulating IL-6 levels were significantly elevated post burn injury in mice (<0.05) and in burn patients (<0.05), the latter of which was positively correlated with elevated REE. Genetic loss of whole body IL-6 in mice prevented burn-induced WAT browning. Transplanting IL-6 knockout (KO) mice with bone marrow (BM) from wild-type (WT) mice, recovered the browning phenotype in these mice, as evaluated by increased uncoupling protein 1 (UCP1) expression (<0.05). Conversely, transplanting irradiated WT mice with BM from IL-6 KO mice impaired burn induced browning with no significant expression of UCP1. Together, our findings implicate BM derived IL-6 as the source controlling browning of WAT post burn injury. Thus, targeting IL-6 is a promising target for hypermetabolism in burns.

NKR-P1B is a homodimeric type II transmembrane C-type lectinlike receptor that inhibits natural killer (NK) cell function upon interaction with its cognate C-type lectin-related ligand, Clr-b. The NKR-P1B:Clr-b interaction represents a major histocompatibility complex class I (MHC-I)-independent missing-self recognition system that monitors cellular Clr-b levels. We have generated NKR-P1B(B6)-deficient (Nkrp1b(-/-)) mice to study the role of NKR-P1B in NK cell development and function in vivo. NK cell inhibition by Clr-b is abolished in Nkrp1b(-/-) mice, confirming the inhibitory nature of NKR-P1B(B6). Inhibitory receptors also promote NK cell tolerance and responsiveness to stimulation; hence, NK cells expressing NKR-P1B(B6) and Ly49C/I display augmented responsiveness to activating signals vs NK cells expressing either or none of the receptors. In addition, Nkrp1b(-/-) mice are defective in rejecting cells lacking Clr-b, supporting a role for NKR-P1B(B6) in MHC-I-independent missing-self recognition of Clr-b in vivo. In contrast, MHC-I-dependent missing-self recognition is preserved in Nkrp1b(-/-) mice. Interestingly, spontaneous myc-induced B lymphoma cells may selectively use NKR-P1B:Clr-b interactions to escape immune surveillance by wild-type, but not Nkrp1b(-/-), NK cells. These data provide direct genetic evidence of a role for NKR-P1B in NK cell tolerance and MHC-I-independent missing-self recognition.

Burn is accompanied by long-lasting immuno-metabolic alterations referred to as hypermetabolism that are characterized by a considerable increase in resting energy expenditure and substantial whole-body catabolism. In burned patients, the length and magnitude of the hypermetabolic state is the highest of all patients and associated with profoundly increased morbidity and mortality. Unfortunately, the mechanisms involved in hypermetabolism are essentially unknown. We hypothesized that the adipose tissue plays a central role for the induction and persistence of hypermetabolism post-burn injury. Here, we show that burn induces a switch in the phenotype of the subcutaneous fat from white to beige, with associated characteristics such as increased mitochondrial mass and UCP1 expression. Our results further demonstrate the significant role of catecholamines and interleukin-6 in this process. We conclude that subcutaneous fat remodeling and browning represent an underlying mechanism that explains the elevated energy expenditure in burn-induced hypermetabolism.

MHC-I–specific receptors play a vital role in NK cell–mediated “missing-self” recognition, which contributes to NK cell activation. In contrast, MHC-independent NK recognition mechanisms are less well characterized. In this study, we investigated the role of NKR-P1B:Clr-b (Klrb1:Clec2d) interactions in determining the outcome of murine hematopoietic cell transplantation in vivo. Using a competitive transplant assay, we show that Clr-b−/− bone marrow (BM) cells were selectively rejected by wild-type B6 recipients, to a similar extent as H-2Db−/− MHC-I–deficient BM cells. Selective rejection of Clr-b−/− BM cells was mitigated by NK depletion of recipient mice. Competitive rejection of Clr-b−/− BM cells also occurred in allogeneic transplant recipients, where it was reversed by selective depletion of NKR-P1Bhi NK cells, leaving the remaining NKR-P1Blo NK subset and MHC-I–dependent missing-self recognition intact. Moreover, competitive rejection of Clr-b−/− hematopoietic cells was abrogated in Nkrp1b-deficient recipients, which lack the receptor for Clr-b. Of interest, similar to MHC-I–deficient NK cells, Clr-b−/− NK cells were hyporesponsive to both NK1.1 (NKR-P1C)–stimulated and IL-12/18 cytokine–primed IFN-γ production. These findings support a unique and nonredundant role for NKR-P1B:Clr-b interactions in missing-self recognition of normal hematopoietic cells and suggest that optimal BM transplant success relies on MHC-independent tolerance mechanisms. These findings provide a model for human NKR-P1A:LLT1 (KLRB1:CLEC2D) interactions in human hematopoietic cell transplants.

Viruses are known to induce pathological cellular states that render infected cells susceptible or resistant to immune recognition. Here, we characterize an MHC-I-independent natural killer (NK) cell recognition mechanism that involves modulation of inhibitory NKR-P1B:Clr-b receptor-ligand interactions in response to mouse cytomegalovirus (MCMV) infection. We demonstrate that mouse Clr-b expression on healthy cells is rapidly lost at the cell surface and transcript levels in a time- and dose-dependent manner upon MCMV infection. In addition, cross-species infections using rat cytomegalovirus (RCMV) infection of mouse fibroblasts and MCMV infection of rat fibroblasts suggest that this response is conserved during host-pathogen interactions. Active viral infection appears to be necessary for Clr-b loss, as cellular stimulation using UV-inactivated whole virus or agonists of many innate pattern recognition receptors failed to elicit efficient Clr-b downregulation. Notably, Clr-b loss could be partially blocked by titrated cycloheximide treatment, suggesting that early viral or nascent host proteins are required for Clr-b downregulation. Interestingly, reporter cell assays suggest that MCMV may encode a novel Clr-b-independent immunoevasin that functionally engages the NKR-P1B receptor. Together, these data suggest that Clr-b modulation is a conserved innate host cell response to virus infection that is subverted by multiple CMV immune evasion strategies.

Objective: The aim of this study was to compare the hypermetabolic, and inflammatory trajectories in burned adults to gain insight into the pathophysiological alterations and outcomes after injury. Summary of Background Data: Burn injury leads to a complex response that is associated with hypermetabolism, morbidity, and mortality. The underlying pathophysiology and the correlations between humoral changes and organ function have not been well delineated in adult burn patients. Methods: Burned adult patients (n = 1288) admitted to our center from 2006 to 2016 were enrolled in this prospective study. Demographics, clinical data, metabolic and inflammatory markers, hypermetabolism, organ function, and clinical outcomes were obtained throughout acute hospitalization. We then stratified patients according to burn size (<20%, 20% to 40%, and >40% total body surface area [TBSA]) and compared biomedical profiles and clinical outcomes for these patients. Results: Burn patients were hypermetabolic with elevated resting energy expenditure (REE) associated with increased browning of white adipose tissue from weeks 2 to 4. Hyperglycemia and hyperinsulinemia peaked 7 to 14 days after injury. Oral glucose tolerance and insulin resistance (QUICKI, HOMA2) tests further confirmed these findings with similar areas under the curve for moderate (20% to 40% TBSA) and severe burn (>40% TBSA). Lipid metabolism in sera revealed elevated pro-inflammatory stearic and linoleic acid, with complementary increases in anti-inflammatory free fatty acids. Similar increases were observed for inflammatory cytokines, chemokines, and metabolic hormones. White adipose tissue from the site of injury had increased ER stress, mitochondrial damage, and inflammasome activity, which was exacerbated with increasing burn severity. Conclusions: In this large prospective trial, we delineated the complexity of the pathophysiologic responses postburn in adults and concluded that these profound responses are time and burn size dependent. Patients with medium-size (20% to 40% TBSA) burn demonstrated a very robust response that is similar to large burns.

Sepsis can be defined as a systemic inflammatory response syndrome occurring in the presence of an infectious source. Over the past 25 years, numerous guidelines have been established to clarify definitions and improve the overall management of clinical sepsis. In light of these multiple paradigm shifts, this review attempts to summarize the innate immunologic alterations that manifest during sepsis, establish and compare mouse models of sepsis with the clinical course, and discuss the authors’ views on additional elements that should be considered in modeling and predicting clinical sepsis from the standpoint of a basic research setting.

Objectives Severe thermal injury is associated with extreme and prolonged inflammatory and hypermetabolic responses, resulting in significant catabolism that delays recovery or even leads to multiple organ failure and death. Burned patients exhibit many symptoms of stress-induced diabetes, including hyperglycemia, hyperinsulinemia, and hyperlipidemia. Recently, the nucleotide-binding domain, leucine-rich family (NLR), pyrin-containing 3 (NLRP3) inflammasome has received much attention as the sensor of endogenous danger signals and mediator of sterile inflammation in type II diabetes. Therefore, we investigated whether the NLRP3 inflammasome is activated in the adipose tissue of burned patients, as we hypothesize that, similar to the scenario observed in chronic diabetes, the cytokines produced by the inflammasome mediate insulin resistance and metabolic dysfunction.Design Prospective cohort study.Setting Ross Tilley Burn Centre & Sunnybrook Research Institute.Patients We enrolled 76 patients with burn sizes ranging from 1% to 70% total body surface area. All severely burned patients exhibited burn-induced insulin resistance and hyperglycemia.Interventions None.Measurements and Main Results: We examined the adipose tissue of control and burned patients and found, via flow cytometry and gene expression studies, increased infiltration of leukocytesespecially macrophagesand evidence of inflammasome priming and activation. Furthermore, we observed increased levels of interleukin-1 in the plasma of burned patients when compared to controls.Conclusions: In summary, our study is the first to show activation of the inflammasome in burned humans, and our results provide impetus for further investigation of the role of the inflammasome in burn-induced hypermetabolism and, potentially, developing novel therapies targeting this protein complex for the treatment of stress-induced diabetes.

The summer finale of Shock is always exciting, and this August 2014 sepsis-centric edition is certainly no exception, as it features a collection of articles describing various novel approaches in predicting clinical sepsis. In addition, a number of interesting reports pertaining to other clinical diagnostics, intervention procedures, and cardiac surgical maneuver are also incorporated in this issue. Avolio et al. (1) discuss the shortcomings of using blood cultures (BCs) as the conventional diagnosis of sepsis, specifically attributing to its inability to accurately reflect patient status in the clinic because of a lengthy turnaround time in BC assays. Furthermore, they also reiterate that although ample biomarkers have been acknowledged as positive signs for sepsis, they are also widely nonspecific. Avolio et al. offer an alternative to this problem by directly detecting the DNA of the infecting microbe in patient blood through LightCycler SeptiFast, a multipathogen probe-based real-time polymerase chain reaction (PCR) quantification method. The PCR results correlate with the BC outcomes and demonstrate that this PCR method has a specificity and predictive value of 96% and 94%, respectively. This new diagnostic method is an exciting approach for the future of clinical sepsis prediction enabling early, rapid, and accurate diagnosis, which hopefully can change the outcomes of infections and sepsis. The article by Nadler et al. (2) describes the limited sensitivity of the current triage and monitoring tools to detect life-threatening scenarios, particularly blood loss, in the clinic. To circumvent this problem, Nadler et al. implement the noninvasive Compensatory Reserve Index (CRI) as a novel method to assess the dangers associated with blood loss. Comparisons between area under the curve of CRI to changes in other vital signs (e.g., heart rate, diastolic blood pressure, oxygen saturation) showcase the 2 advantages CRI has to offer: first, it is noninvasive; second, it possesses a far superior sensitivity in contrast to the assessment of vital signs in detecting even minor blood loss. Thus, the use of CRI in combination with the conventional vital sign evaluation may be beneficial in improving the detection of life-threatening scenarios in the clinic. Ginde et al. (3) bring sepsis back into this Shock issue’s spotlight by showing age-related differences in serum inflammatory biomarkers during the first 72 h of hospitalization due to sepsis. In this study, six biomarkers (chemokine (C-C motif) ligand 23 [CCL-23], C-reactive protein, interleukin 1 receptor antagonist [IL-1ra], neutrophil gelatinase–associated lipocalin, peptidoglycan recognition protein, and tumor necrosis factor receptor 1a [TNFR-1a]) are measured in the serum of a cohort of patients with confirmed infection. It was concluded from the results that older age is associated with higher sepsis severity, and that senescence is associated with greater inflammation at baseline, but this inflammatory state resolved within 72 h. Perhaps the initial inflammatory elevation reported in the older population predisposes them to a disadvantage in combating microbial infections. Further investigation is required, as the six candidate soluble factors surveyed in this study contribute only to a part of this story. The focus shuffles from the bench back to the clinic, as Liu et al. (4) present their findings on the integration of vital signs and heart rate variability and complexity with computer decision support systems to achieve a more accurate assessment for identifying the need for lifesaving interventions during life-or-death scenarios. In terms of providing a holistic assessment of the patients’ status, it appears the traditional correlation of vital signs and heart rate variability and complexity by using a multivariate logistic regression is far inferior when compared to data that are integrated by a machine learning algorithm. Thus, the improved patient assessment through machine learning–based triage will assist physicians to promptly implement lifesaving interventions to those in need. This subsequent study by Gores et al. (5) explores the pathophysiology induced by high-dose IL-2 (HDIL-2) therapy, which is characterized by systemic inflammation and pulmonary edema. Interestingly, these observations are highly reminiscent to the symptoms reported in patients who have sepsis, specifically referring to the systemic inflammation and sepsis-induced lung injury. Because IL-2 stimulates the production of angiopoietin 2, Gores et al. postulate that patients enrolled in HDIL-2 therapy will exhibit symptoms similar to septic patients, and this pathology is attributed to the induced production of angiopoietin 2 by the HDIL-2 administered. Indeed, a progressive increase in plasma angiopoietin 2 is detected during the course of the HDIL-2 treatment. In contrast, a decrease in forced expiratory volume in 1 s is documented in these same patients who received HDIL-2 therapy. According to the authors, albeit premature, a plausible model for this inverse correlative observation is that the HDIL-2 therapy induces the secretion of angiopoietin 2, and in turn this molecule results in lung damage, consequently leading to a decreased forced expiratory volume in 1 s. Stay tuned for the sequel of this work, as Gores et al. have already alluded to the prospective plans in dissecting the mechanism of this pathophysiologic phenomenon. Whether a coronary artery bypass graft surgery (on-pump CABG) generates a postoperative systemic inflammatory response and ultimately leading to severe organ failure has been a matter of fervent debate between surgeons. However, there has been emerging evidence suggesting that endothelium activation, in response to inflammatory responses, plays a crucial role in the pathophysiology of organ dysfunction after CABG. Thus, to avoid inflammation, the off-pump CABG was reintroduced into the clinic in the 1990s. Jongman et al. (6) have dedicated this work to investigate if the off-pump approach indeed dampens the inflammatory phenotype and prevents endothelium activation. Interestingly, lower levels of numerous cytokines (TNF-α, IL-10, and myeloperoxidase) are observed in the plasma of patients who received the off-pump surgery relative to the on-pump approach. However, there appear to be no differences in the expression of soluble adhesion molecules. Collectively, these data suggest that off-pump CABG indeed reduces inflammation, but appears to have no differential influence on the activation state of the endothelium in comparison to the conventional on-pump approach. In Short Communications, Biller et al. (7) describe that the use of serum C-reactive protein and procalcitonin concentration as a predictor for survival in infected patients has been a disappointment, largely because of the false-positive or negative values these assays generate. Realizing the conundrum, Biller et al. propose cholesterol as the next promising candidate that should be incorporated into the list of biomarkers used to predict survival outcomes of hospitalized patient. As lipoproteins, in this case cholesterol, bind to the bioactive lipid A portion of lipopolysaccharide (LPS), it reduces the bioavailability of LPS for pattern recognition receptor expressed by macrophages and other innate leukocytes, thus preventing the activation of inflammation. Biller et al. predict that a decrease in proinflammatory signals as a result of cholesterol-induced inhibition may provide a survival advantage to infected patients, because most of these patients succumb to cytokine storms due to enormous surges of inflammation. Indeed, Biller et al. demonstrate that the cohort of patients who did not survive the infection had a lower cholesterol level of 69 mg/dL, whereas the survivors had 96 mg/dL. This remarkable finding has once again shed light onto the field of biomarkers for predicting survival in infected patients and should be further studied and explored. Leading off a series of upcoming basic science articles, Rani et al. (8) suggest that γδ T cells promote a wound-healing state at the site of thermal injury by regulating the trafficking of proinflammatory leukocytes. In their article, they showed that γTCR−/− mice (γδ T cells–deficient) have a significant increase in myeloid cells at the site of injury relative to its wild-type counterpart, and several proinflammatory cytokines (TNF-α, macrophage inflammatory protein 1α, macrophage inflammatory protein 1β) are also detected at higher levels in the thermally injured skin of the knockout animals in comparison to the controls. Rani et al. concluded that γδ T cells appear to have a function of promoting a wound healing state at the site of injury by attenuating inflammation, thus making it a very appealing candidate for future investigations in the field of regenerative medicine. Sumi et al. (9) show that ATP is a danger signal that activates neutrophils in mouse models of sepsis. By implementing the cecal ligation and puncture (CLP) technique in mice, these animals gradually accumulate gut-derived bacteria in their peritoneal cavity and eventually develop sepsis. The innate immune system of these rodents, particularly the neutrophil, is activated during the course of the disease modeling by “danger signals” that are released into the plasma by the invading microbes and the infected cells. Sumi et al. show that ATP is elevated in mice that received CLP and is a danger signal that potently activates polymorphonuclear neutrophils through the purinergic signaling pathway. Importantly, polymorphonuclear neutrophil activation is dampened by suramin, an ATP receptor antagonist that blocks the interaction between ATP and the ATP receptor. This work by Sumi et al. provides yet another promising candidate that can be selected to modulate the immune response of septic patients during the acute phases of their pathology. Elaborating on the theme of septic studies by using animal models, Rodríguez-González et al. (10) perform CLP in rats and foster these animals in chambers supplied with differential oxygen concentration to test if hyperoxia worsens the outcomes of these induced-septic animals. Indeed, Rodríguez-González et al. report that CLP animals receiving high oxygen dosage have elevated plasma levels of IL-6, IL-10, TNF-α, and reactive oxygen species relative to hypoxic-fostered rats. This fascinating study suggests that oxygen therapy should be administered with caution for septic patients in the clinic, as it can potentially inflict undesirable effects. Adding something “sweet” to the field, Kassim et al. (11) propose caffeic acid phenethyl ester (CAPE), an active component of honeybee product, may mitigate the physiological consequences of excessive free radicals produced during sepsis. Peroxynitrite is a free radical generated by macrophages in septic hosts, and this molecule is a potent inducer of cell death and promotes an inflammatory microenvironment. Because the pharmacological function of CAPE is to scavenge peroxynitrite, Kassim et al. hypothesized that by removing peroxynitrite via the use of CAPE during an infectious scenario, it will ameliorate cell death and inflammation, thus improving the outcomes of septic patients. In vitro data where RAW264.7 cells treated with LPS/interferon γ and CAPE showed elevated cell viability and lowered nitric oxide levels relative to its counterpart that did not receive the scavenger molecule. Furthermore, in vivo results demonstrate sepsis-induced mice treated with CAPE have better survival outcomes compared with the control cohort. We believe CAPE is indeed a promising therapeutic approach in treating a variety of inflammatory disease caused by reactive oxygen species, as supported by the elegant combination of in vitro and in vivo evidence presented by Kassim et al. Liang et al. (12) report that muramyl dipeptide exacerbates the pathophysiology of thermal injury through the use of animal modeling. Muramyl dipeptide is a peptidoglycan, which is a key constituent of both gram-positive and gram-negative bacteria that is recognized by the NOD-like receptors. Upon recognition by NOD-like receptors, the inflammasome is activated, and a myriad of proinflammatory response ensues. By infusing purified muramyl dipeptide into thermally injured rats, these animals show heightened inflammation and poorer survival outcomes. This observation is in line with the clinical observation that opportunistic infection after thermal injury can be detrimental to the patient. To conclude the sepsis-themed marathon, Ariga et al. (13) show mice primed with LPS before induction of sepsis by CLP display an advantageous outlook in survival compared with the control groups. Also known as “endotoxin tolerance,” mice receiving continuous subcutaneous LPS challenge (5 days) followed by CLP express higher levels of adhesion molecules in the peritoneum compared with the control group. Furthermore, a significantly lower level of pulmonary adhesion molecule expression is detected in the endotoxin tolerant cohort relative to the nontolerant group. These data explain why endotoxin-tolerant mice lack neutrophil recruitment into lung and in turn traffic to the peritoneum, where the bacteria are accumulating after CLP. Albeit an interesting study, further investigation is required to conclude that the survival advantage is indeed attributed to the redirection of neutrophils and not due to the prepriming of the immune system of these animals by the repeated dosing of LPS, thus leading to a more efficient eradication of the microbe. The August 2014 issue of Shock concludes with the editorial comment by Roesner and Thiemermann (14), deliberating whether the off-pump CABG approach is indeed a more practical and safer surgical approach when compared with the on-pump CABG, echoing the same issue raised by Jongman et al. (6). The authors summarized the findings presented by Jongman and colleagues and presented a rather comprehensive list of “missing links,” which if present may help provide a clearer perception of the pros and cons between the on- and off-pump maneuver. Items on this list include an unknown source of the inflammatory stimuli, the inability to survey tissue samples in patients, and the source of endothelial biomarkers (e.g., heart, lungs, etc.) cannot be tracked. Lastly, Roesner et al. suggest to incorporate more subjects into the study described by Jongman and colleagues’ work (6), as the magnitude of their report, albeit interesting, is still far from conclusive and still possesses huge potential for future in-depth studies. With the numerous exciting articles featured in this issue of Shock, it will undoubtedly stimulate novel ideas leading to great experiments and scientific progression.

Infections with HIV, hepatitis B virus, and hepatitis C virus can turn into chronic infections, which currently affect more than 500 million patients worldwide. It is generally thought that virus-mediated T-cell exhaustion limits T-cell function, thus promoting chronic disease. Here we demonstrate that natural killer (NK) cells have a negative impact on the development of T-cell immunity by using the murine lymphocytic choriomeningitis virus. NK cell-deficient (Nfil3−/−, E4BP4−/−) mice exhibited a higher virus-specific T-cell response. In addition, NK cell depletion caused enhanced T-cell immunity in WT mice, which led to rapid virus control and prevented chronic infection in lymphocytic choriomeningitis virus clone 13- and reduced viral load in DOCILE-infected animals. Further experiments showed that NKG2D triggered regulatory NK cell functions, which were mediated by perforin, and limited T-cell responses. Therefore, we identified an important role of regulatory NK cells in limiting T-cell immunity during virus infection.

Monoclonal antibodies (mAbs) specific for cell surface antigens are an invaluable tool to study immune receptor expression and function. Here, we outline a generalized reporter cell-based approach to the generation and high-throughput screening of mAbs specific for cell surface antigens. Termed CELLISA, this technology hinges upon the capture of hybridoma supernatants in mAb arrays that facilitate ligation of an antigen of interest displayed on BWZ reporter cells in the form of a CD3ζ-fusion chimeric antigen receptor (zCAR); in turn, specific mAb-mediated cross-linking of zCAR on BWZ cells results in the production of β-galactosidase enzyme (β-gal), which can be assayed colorimetrically. Importantly, the BWZ reporter cells bearing the zCAR of interest may be used for immunization as well as screening. In addition, serial immunizations employing additional zCAR- or native antigen-bearing cell lines can be used to increase the frequency of the desired antigen-specific hybridomas. Finally, the use of a cohort of epitope-tagged zCAR (e.g., zCAR(FLAG)) variants allows visualization of the cell surface antigen prior to immunization, and coimmunization using these variants can be used to enhance the immunogenicity of the target antigen. Employing the CELLISA strategy, we herein describe the generation of mAb directed against an uncharacterized natural killer cell receptor protein.

The Nkrp1 (Klrb) family of NK cell receptors and their genetically linked Clr (Clec2) ligands are conserved between rodents and humans. Nonetheless, certain mouse and rat Nkrp1 genes exhibit significant allelic polymorphism between inbred strains. We previously demonstrated that the Nkrp1–Clr recognition system is genetically and functionally conserved between the B6 and BALB/c strains, with focused sequence divergence evident in certain genes (e.g., Nkrp1b,c). Here, we extend this finding by mapping the 129-strain Nkrp1–Clr gene cluster, which is structurally conserved yet displays significant sequence divergence relative to the B6 haplotype. In addition, we show that 129-strain NK cells possess comparable Nkrp1 and Clr transcript expression, and characterize several NKR-P1:Clr interactions that are functionally conserved between the B6 and 129 strains, including documented and novel receptor–ligand pairs. Thus, despite significant allelic polymorphism observed in the Nkrp1–Clr region, the overall genetic organization and functional repertoire appear to be conserved among mouse strains, in contrast to the striking variation observed in the corresponding Ly49 region. These data extend our knowledge of the complex genetically linked Nkrp1–Clr NK recognition system in mice.