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Sepsis initiates simultaneous pro- and anti-inflammatory processes, the pattern and intensity of which vary over time. The inability to evaluate the immune status of patients with sepsis in a rapid and quantifiable manner has undoubtedly been a major reason for the failure of many therapeutic trials. Although there has been considerable effort to immunophenotype septic patients, these methods have often not accurately assessed the functional state of host immunity, lack dynamic range, and are more reflective of molecular processes rather than host immunity. In contrast, ELISpot assay measures the number and intensity of cytokine-secreting cells and has excellent dynamic range with rapid turnaround. We investigated the ability of a (to our knowledge) novel whole blood ELISpot assay and compared it with a more traditional ELISpot assay using PBMCs in sepsis. IFN-γ and TNF-α ELISpot assays on whole blood and PBMCs were undertaken in control, critically ill nonseptic, and septic patients. Whole blood ELISpot was easy to perform, and results were generally comparable to PBMC-based ELISpot. However, the whole blood ELISpot assay revealed that nonmonocyte, myeloid populations are a significant source of ex vivo TNF-α production. Septic patients who died had early, profound, and sustained suppression of innate and adaptive immunity. A cohort of septic patients had increased cytokine production compared with controls consistent with either an appropriate or excessive immune response. IL-7 restored ex vivo IFN-γ production in septic patients. The whole blood ELISpot assay offers a significant advance in the ability to immunophenotype patients with sepsis and to guide potential new immunotherapies.

Hematopoietic stem/progenitor cells (HSPCs) reside in localized microenvironments, or niches, in the bone marrow that provide key signals regulating their activity. A fundamental property of hematopoiesis is the ability to respond to environmental cues such as inflammation. How these cues are transmitted to HSPCs within hematopoietic niches is not well established. Here, we show that perivascular bone marrow dendritic cells (DCs) express a high basal level of Toll-like receptor-1 (TLR1) and TLR2. Systemic treatment with a TLR1/2 agonist induces HSPC expansion and mobilization. It also induces marked alterations in the bone marrow microenvironment, including a decrease in osteoblast activity and sinusoidal endothelial cell numbers. TLR1/2 agonist treatment of mice in which Myd88 is deleted specifically in DCs using Zbtb46-Cre show that the TLR1/2-induced expansion of multipotent HPSCs, but not HSPC mobilization or alterations in the bone marrow microenvironment, is dependent on TLR1/2 signaling in DCs. Interleukin-1β (IL-1β) is constitutively expressed in both murine and human DCs and is further induced after TLR1/2 stimulation. Systemic TLR1/2 agonist treatment of Il1r1−/− mice show that TLR1/2-induced HSPC expansion is dependent on IL-1β signaling. Single-cell RNA-sequencing of low-risk myelodysplastic syndrome bone marrow revealed that IL1B and TLR1 expression is increased in DCs. Collectively, these data suggest a model in which TLR1/2 stimulation of DCs induces secretion of IL-1β and other inflammatory cytokines into the perivascular niche, which in turn, regulates multipotent HSPCs. Increased DC TLR1/2 signaling may contribute to altered HSPC function in myelodysplastic syndrome by increasing local IL-1β expression.

Objective Our study aims to provide a paradigm when it is ethical to perform cardiopulmonary resuscitation (CPR) on patients during the COVID-19 pandemic. Summary background data Hospitals around the nation are enacting systems to limit CPR in caring for COVID+ patients for a variety of legitimate reasons and based on concepts of medical futility and allocation of scarce resources. No ethical framework, however, has been proposed as a standard to guide care in this crucial matter. Methods Our analysis begins with definitions of ethically relevant terms. We then cycle an illustrative clinical vignette through the mathematically permissible possibilities to account for all conceivable scenarios. Scenarios with ethical tension are examined. Results Patients have the negative right to refuse care including CPR, but they do not have the positive right to demand it. Our detailed ethical analysis and recommendations support CPR if and only if 1) CPR is judged medically beneficial, and in line with the patient's and values and goals, 2) allocations or scarce resources follow a just and transparent triage system, and 3) providers are protected from contracting the disease. Conclusions CPR is an intervention like any other, with attendant risks and benefits and with responsibility for the utilization of limited resources. Our ethical analysis advocates for a systematic approach to codes that respects the important ethical considerations in caring for the critically ill and facilitates patient-centered, evidence-based, and fair treatment to all.

BACKGROUND:. Immunotherapy treatment for coronavirus disease 2019 combined with antiviral therapy and supportive care remains under intense investigation. However, the capacity to distinguish patients who would benefit from immunosuppressive or immune stimulatory therapies remains insufficient. Here, we present a patient with severe coronavirus disease 2019 with a defective immune response, treated successfully with interleukin-7 on compassionate basis with resultant improved adaptive immune function. CASE SUMMARY:. A previously healthy 43-year-old male developed severe acute respiratory distress syndrome due to the severe acute respiratory syndrome coronavirus 2 virus with acute hypoxemic respiratory failure and persistent, profound lymphopenia. Functional analysis demonstrated depressed lymphocyte function and few antigen-specific T cells. Interleukin-7 administration resulted in reversal of lymphopenia and improved T-cell function. Respiratory function and clinical status rapidly improved, and he was discharged home. Whole exome sequencing identified a deleterious autosomal dominant mutation in TICAM1, associated with a dysfunctional type I interferon antiviral response with increased severity of coronavirus disease 2019 disease. CONCLUSIONS:. Immunoadjuvant therapies to boost host immunity may be efficacious in life-threatening severe coronavirus disease 2019 infections, particularly by applying a precision medicine approach in selecting patients expressing an immunosuppressive phenotype.

BACKGROUND Forty percent of critically ill trauma patients will develop an infectious complication. Pneumonia is the most common cause of death of trauma patients surviving their initial insult. We previously demonstrated that polytrauma (PT), defined as two or more severe injuries in at least two areas of the body, induces emergency hematopoiesis characterized by accelerated myelopoiesis in the bone marrow and increased myeloid cell frequency in the peripheral tissues. We hypothesized that PT alone induces priming of neutrophils, resulting in hyperactivation upon secondary exposure to bacteria and causing acute lung injury and increased susceptibility to secondary exposure to Pseudomonas aeruginosa pneumonia. METHODS C57BL/6 mice were subjected to PT consisting of a lower extremity pseudofracture, liver crush injury, and 15% blood-volume hemorrhage. Pneumonia was induced by intratracheal injection of 5 × 106 CFU live P. aeruginosa or 1 × 107 of heat-killed P. aeruginosa (HKPA). For reactive oxygen species (ROS), studies polymorphonuclear neutrophils (PMNs) were isolated by immunomagnetic bead negative selection and stimulated ex-vivo with HKPA. Reactive oxygen species production was measured by immunofluorescence. For histology, lung sections were stained by hematoxylin-eosin and analyzed by a blinded grader. RESULTS Polytrauma induced persistent changes in immune function at baseline and to secondary infection. Pneumonia after injury resulted in increased mortality (60% vs. 5% p < 0.01). Blood neutrophils from PT mice had higher resting (unstimulated) ROS production than in naive animals (p < 0.02) demonstrating priming of the neutrophils following PT. After intratracheal HKPA injection, bronchoalveolar lavage PMNs from injured mice had higher ROS production compared with naive mice (p < 0.01), demonstrating an overexuberant immunopathologic response of neutrophils following PT. CONCLUSION Polytrauma primes neutrophils and causes immunopathologic PMN ROS production, increased lung injury and susceptibility to secondary bacterial pneumonia. These results suggest that trauma-induced immune dysfunction can cause immunopathologic response to secondary infection and suggests neutrophil-mediated pulmonary damage as a therapeutic target for posttrauma pneumonia.

Background Sepsis induces gut barrier dysfunction characterized by increased gut epithelial apoptosis and increased intestinal permeability. The cytokine IL-22 has been demonstrated to regulate gut barrier function. Type-3 innate lymphoid cells (ILC3) are the predominate source of IL-22 in the GI tract. We hypothesized that sepsis may cause changes to the gut ILC3/IL-22 axis. Materials and Methods Sepsis was induced in WT and IL-22 KO mice by Pseudomonas aeruginosa pneumonia. Changes in gut-associated leukocyte populations were determined by flow-cytometry and ILC-associated transcripts were measured by RT-PCR. The effect of sepsis on gut permeability, pulmonary microbial burden, gut epithelial apoptosis, and survival was compared between WT and IL-22-/- mice. Results Sepsis resulted in a significant decrease in the number of ILC3 in the gut, with a reciprocal increase in type-1 ILC (ILC1). Consistent with prior reports, sepsis was associated with increased gut permeability; however there was no difference in gut permeability, gut epithelial apoptosis, pulmonary microbial burden, or survival between WT and IL-22-/- mice. Conclusions Septic pneumonia causes a decrease in gut-associated ILC3 and an associated reciprocal increase in ILC1. This may reflect inflammation-induced conversion of ILC3 to ILC1. Constitutive systemic IL-22 deficiency does not alter sepsis-induced gut barrier dysfunction.

A nonimmunocompromised patient developed life-threatening soft tissue infection with Trichosporon asahii, Fusarium, and Saksenaea that progressed despite maximum antifungal therapies and aggressive debridement. Interleukin-7 immunotherapy resulted in clinical improvement, fungal clearance, reversal of lymphopenia, and improved T-cell function. Immunoadjuvant therapies to boost host immunity may be efficacious in life-threatening fungal infections.

Objectives: Corticosteroid therapy has become standard of care therapy for hospitalized patients infected with the severe acute respiratory syndrome coronavirus-2 global pandemic-causing virus Whereas systemic inflammation is a notably important feature in coronavirus disease 2019 pathogenesis, adaptive immune suppression and the inability to eradicate effectively the virus remain significant factors as well We sought to evaluate the in vitro effects of dexamethasone phosphate on T cell function in peripheral blood mononuclear cells derived from patients with acute, severe, and moderate coronavirus disease 2019 Design: Prospective in vitro laboratory study Setting: Coronavirus disease 2019-specific medical wards and ICUs at a single-center, quaternary-care academic hospital between October 1, 2020, and November 15, 2020 Patients: Eleven patients diagnosed with coronavirus disease 2019 admitted to either the ICU or hospital coronavirus disease 2019 unit Three patients had received at least one dose of dexamethasone prior to enrollment Interventions: Fresh whole blood was collected, and peripheral blood mononuclear cells were immediately isolated and plated onto precoated enzyme-linked immunospot plates for detection of interferon-gamma production Samples were incubated with CD3/CD28 antibodies alone and with three concentrations of dexamethasone These conditions were also stimulated with recombinant human interleukin-7 Following overnight incubation, the plates were washed and stained for analysis using Cellular Technology Limited ImmunoSpot S6 universal analyzer (ImmunoSpot by Cellular Technology Limited, Cleveland, OH) Measurements AND MAIN RESULTS: Functional cytokine production was assessed by quantitation of cell spot number and total well intensity after calculation for each enzyme-linked immunospot well using the Cellular Technology Limited ImmunoSpot Version 7 0 professional software (CTL Analyzers, Shaker Heights, OH) Comparisons were made using t test and using a nonparametric analysis of variance Friedman test The number of functional T cells producing interferon-gamma and the intensity of the response decrease significantly with exposure to 1 2-microg/mL dexamethasone About 0 12 microg/mL does not significantly affect the functional immune response on enzyme-linked immunospot Interleukin-7 increases the overall number of activated T cells, including those exposed to dexamethasone Conclusions: Further evaluation of the effect of immunomodulatory therapies is warranted in coronavirus disease 2019 A refined functional, precision medicine approach that evaluates the cellular immune function of individual patients with coronavirus disease 2019 is needed to better define which therapies could have benefit or cause harm for specific patients

COVID-19–associated morbidity and mortality have been attributed to a pathologic host response. Two divergent hypotheses have been proposed: hyperinflammatory cytokine storm; and failure of host protective immunity that results in unrestrained viral dissemination and organ injury. A key explanation for the inability to address this controversy has been the lack of diagnostic tools to evaluate immune function in COVID-19 infections. ELISpot, a highly sensitive, functional immunoassay, was employed in 27 patients with COVID-19, 51 patients with sepsis, 18 critically ill nonseptic (CINS) patients, and 27 healthy control volunteers to evaluate adaptive and innate immune status by quantitating T cell IFN-ɣ and monocyte TFN-α production. Circulating T cell subsets were profoundly reduced in COVID-19 patients. Additionally, stimulated blood mononuclear cells produced less than 40%–50% of the IFN-ɣ and TNF-α observed in septic and CINS patients, consistent with markedly impaired immune effector cell function. Approximately 25% of COVID-19 patients had increased IL-6 levels that were not associated with elevations in other canonical proinflammatory cytokines. Collectively, these findings support the hypothesis that COVID-19 suppresses host functional adaptive and innate immunity. Importantly, IL-7 administered ex vivo restored T cell IFN-ɣ production in COVID-19 patients. Thus, ELISpot may functionally characterize host immunity in COVID-19 and inform prospective therapies., ELISpot, a highly sensitive, functional immunoassay, suggests that COVID-19 is immunosuppressive and lacks substantial cytokine storm.

Functional recovery in the end target muscle is a determinant of outcome after peripheral nerve injury. The neuromuscular junction (NMJ) provides the interface between nerve and muscle and includes non-myelinating terminal Schwann cells (tSCs). After nerve injury, tSCs extend cytoplasmic processes between NMJs to guide axon growth and NMJ reinnervation. The mechanisms related to NMJ reinnervation are not known. We used multiple mouse models to investigate the mechanisms of NMJ reinnervation in both sexes, specifically whether macrophage-derived vascular endothelial growth factor-A (Vegf-A) is crucial to establishing NMJ reinnervation at the end target muscle. Both macrophage number and Vegf-A expression increased in end target muscles after nerve injury and repair. In mice with impaired recruitment of macrophages and monocytes (Ccr2−/− mice), the absence of CD68+ cells (macrophages) in the muscle resulted in diminished muscle function. Using a Vegf-receptor 2 (VegfR2) inhibitor (cabozantinib; CBZ) via oral gavage in wild-type (WT) mice resulted in reduced tSC cytoplasmic process extension and decreased NMJ reinnervation compared with saline controls. Mice with Vegf-A conditionally knocked out in macrophages (Vegf-Afl/fl; LysMCremice) demonstrated a more prolonged detrimental effect on NMJ reinnervation and worse functional muscle recovery. Together, these results show that contributions of the immune system are integral for NMJ reinnervation and functional muscle recovery after nerve injury.SIGNIFICANCE STATEMENTThis work demonstrates beneficial contributions of a macrophage-mediated response for neuromuscular junction (NMJ) reinnervation following nerve injury and repair. Macrophage recruitment occurred at the NMJ, distant from the nerve injury site, to support functional recovery at the muscle. We have shown hindered terminal Schwann cell (tSC) injury response and NMJ recovery with inhibition of: (1) macrophage recruitment after injury; (2) vascular endothelial growth factor receptor 2 (VegfR2) signaling; and (3) Vegf secretion from macrophages. We conclude that macrophage-derived Vegf is a key component of NMJ recovery after injury. Determining the mechanisms active at the end target muscle after motor nerve injury reveals new therapeutic targets that may translate to improve motor recovery following nerve injury.

The global COVID-19 pandemic has claimed the lives of more than 450,000 US citizens. Dysregulation of the immune system underlies the pathogenesis of COVID-19, with inflammation mediated local tissue injury to the lung in the setting of suppressed systemic immune function. To define the molecular mechanisms of immune dysfunction in COVID-19 we utilized a systems immunology approach centered on the circulating leukocyte phosphoproteome measured by mass cytometry. COVID-19 is associated with wholesale activation of a broad set of signaling pathways across myeloid and lymphoid cell populations. STAT3 phosphorylation predominated in both monocytes and T cells and was tightly correlated with circulating IL-6 levels. High levels of STAT3 phosphorylation was associated with decreased markers of myeloid cell maturation/activation and decreased ex-vivo T cell IFN-gamma production, demonstrating that during COVID-19 dysregulated cellular activation is associated with suppression of immune effector cell function. Collectively, these data reconcile the systemic inflammatory response and functional immunosuppression induced by COVID-19 and suggest STAT3 signaling may be the central pathophysiologic mechanism driving immune dysfunction in COVID-19.

Antibiotic-impregnated central venous catheters (CVCs) decrease the incidence of infection in high-risk patients. However, use of these catheters carries the hypothetical risk of inducing antibiotic resistance. We hypothesized that routine use of minocycline and rifampin-impregnated catheters (MR-CVC) in a single intensive care unit (ICU) would change the resistance profile for Staphylococcus aureus.We reviewed antibiotic susceptibilities of S. aureus isolates obtained from blood cultures in a large urban teaching hospital from 2002-2015. Resistance patterns were compared before and after implementation of MR-CVC use in the surgical ICU (SICU) in August 2006. We also compared resistance patterns of S. aureus obtained in other ICUs and in non-ICU patients, in whom MR-CVCs were not used.Data for rifampin, oxacillin, and clindamycin were available for 9,703 cultures; tetracycline resistance data were available for 4,627 cultures. After implementation of MR-CVC use in the SICU, rifampin resistance remained unchanged, with rates the same as in other ICU and non-ICU populations (3%). After six years of use of MR-CVCs in the SICU, the rate of tetracycline resistance was unchanged in all facilities (1%-3%). The use of MR-CVCs was not associated with any change in S. aureus oxacillin-resistance rates in the SICU (66% vs. 60%). However, there was a significant decrease in S. aureus clindamycin resistance (59% vs. 34%; p 0.05) in SICU patients.Routine use of rifampin-minocycline-impregnated CVCs in the SICU was not associated with increased resistance of S. aureus isolates to rifampin or tetracyclines.

Long-term survival after lung transplantation remains profoundly limited by graft rejection. Recent work has shown that bronchus-associated lymphoid tissue (BALT), characterized by the development of peripheral nodal addressin (PNAd)-expressing high endothelial venules and enriched in B and Foxp3+ T cells, is important for the maintenance of allograft tolerance. Mechanisms underlying BALT induction in tolerant pulmonary allografts, however, remain poorly understood. Here, we show that the development of PNAd-expressing high endothelial venules within intragraft lymphoid follicles and the recruitment of B cells, but not Foxp3+ cells depends on IL-22. We identify graft-infiltrating gamma-delta (γδ) T cells and Type 3 innate lymphoid cells (ILC3s) as important producers of IL-22. Reconstitution of IL-22 at late time points through retransplantation into wildtype hosts mediates B cell recruitment into lymphoid follicles within the allograft, resulting in a significant increase in their size, but does not induce PNAd expression. Our work has identified cellular and molecular requirements for the induction of BALT in pulmonary allografts during tolerance induction and may provide a platform for the development of new therapies for lung transplant patients.

The acute inflammatory response to infection or injury dramatically increases the hematopoietic demand on the bone marrow to replace effector leukocytes consumed in the inflammatory response. In the setting of infection, pathogen-associated molecular patterns induce emergency hematopoiesis, activating hematopoietic stem and progenitor cells to proliferate, thereby providing progeny for accelerated myelopoiesis. Sterile tissue injury due to trauma also increases leukocyte demand; however, the effect of sterile tissue injury on hematopoiesis is not well described. We used a mouse model of multisystem injury to investigate the effects of these injuries on bone marrow progenitor frequencies and phenotypes. We find that tissue injury alone induces emergency hematopoiesis in mice subjected to polytrauma. This process is driven by IL-1/MyD88-dependent production of G-CSF. G-CSF induces expansion of hematopoietic progenitors including hematopoietic stem cells and multipotent progenitors and increases the frequency of myeloid-skewed progenitors. These data provide the first comprehensive description of injury-induced emergency hematopoiesis and identify an IL-1/MyD88/G-CSF dependent pathway as the key regulator of emergency hematopoiesis after injury.

Background Burnout is a complex syndrome of emotional distress that can disproportionately affect individuals who work in health care professions. Study Design For a national survey of burnout in US general surgery residents, we asked all ACGME-accredited general surgery program directors to email their general surgery residents an invitation to complete an anonymous, online survey. Burnout was assessed with the Maslach Burnout Inventory; total scores for Emotional Exhaustion (EE), Depersonalization (DP), and Personal Accomplishment (PA) subscales were calculated. Burnout was defined as having a score in the highest tertile for EE or DP or lowest tertile for PA. Chi-square tests and one-way ANOVA were used to test associations between burnout tertiles for each subscale and various resident and training-program characteristics as appropriate. Results From April to December 2014, six hundred and sixty-five residents actively engaged in clinical training had data for analysis; 69% met the criterion for burnout on at least one subscale. Higher burnout on each subscale was reported by residents planning private practice careers compared with academic careers. A greater proportion of women than men reported burnout on EE and PA. Higher burnout on EE and DP was associated with greater work hours per week. Having a structured mentoring program was associated with lower burnout on each subscale. Conclusions The high rates of burnout among general surgery residents are concerning, given the potential impact of burnout on the quality of patient care. Efforts to identify at-risk populations and to design targeted interventions to mitigate burnout in surgical trainees are warranted.

Previous studies have suggested that percutaneous drainage and interval appendectomy is an effective treatment for appendicitis with associated abscess. Few studies to date have analyzed risk factors for failed drain management. We hypothesized that older patients with more co-morbidities would be at higher risk for failing conservative treatment.The 2010-2014 editions of the National Inpatient Sample (NIS) were queried for patients with diagnoses of peri-appendiceal abscesses. Minors and elective admissions were excluded. We identified patients who underwent percutaneous drainage and defined drain failure as undergoing a surgical operation after drainage but during the same inpatient visit to assess for factors associated with failure of drainage alone as a treatment. After univariable analysis, binomial logistic regression was used to assess for independent risk factors. Frequencies were analyzed by χA total of 2,209 patients with appendiceal abscesses received drains; 561 patients (25.4%) failed conservative management and underwent operative intervention. On univariable analysis, patients who failed conservative management were younger, more likely to be Hispanic, have more inpatient diagnoses, and to have undergone drainage earlier in the hospital course. Multivariable regression demonstrated that the number of diagnoses, female sex, and Hispanic race were predictive of failure of drainage alone. Older age, West and Midwest census regions, and later drain placement were predictive of successful treatment with drainage alone. Failure was associated with more charges and longer hospital stay but not with a higher mortality rate.Approximately a quarter of patients will fail management of appendiceal abscess with percutaneous drain placement alone. Risk factors for failure are patient complexity, female sex, earlier drainage, and Hispanic race. Failure of drainage is associated with higher total charges and longer hospital stay; however, no change in the mortality rate was noted.

Sepsis is life-threatening organ dysfunction caused by a dysregulated host response to infection. Early recognition and treatment are the cornerstones of management.Review of the English-language literature.For both sepsis and septic shock "antimicrobials [should be] be initiated as soon as possible and within one hour" (Surviving Sepsis Campaign). The risk of progression from severe sepsis to septic shock increases 8% for each hour before antibiotics are started. Selection of antimicrobial agents is based on a combination of patient factors, predicted infecting organism(s), and local microbial resistance patterns. The initial drugs should have activity against typical gram-positive and gram-negative causative micro-organisms. Anaerobic coverage should be provided for intra-abdominal infections or others where anaerobes are significant pathogens. Empiric antifungal or antiviral therapy may be warranted. For patients with healthcare-associated infections, resistant micro-organisms will further complicate the choice of empiric antimicrobials. Recommendations are given for specific infections.Early administration of broad-spectrum antimicrobial drugs is one of the most important, if not the most important, treatment for patients with sepsis or septic shock. Drugs should be initiated as soon as possible, and the choice of should take into account patient factors, common local pathogens, hospital antibiograms and resistance patterns, and the suspected source of infection. Antimicrobial agent therapy should be de-escalated as soon as possible.

For more than two decades, sepsis was defined as a microbial infection that produces fever (or hypothermia), tachycardia, tachypnoea and blood leukocyte changes. Sepsis is now increasingly being considered a dysregulated systemic inflammatory and immune response to microbial invasion that produces organ injury for which mortality rates are declining to 15-25%. Septic shock remains defined as sepsis with hyperlactataemia and concurrent hypotension requiring vasopressor therapy, with in-hospital mortality rates approaching 30-50%. With earlier recognition and more compliance to best practices, sepsis has become less of an immediate life-threatening disorder and more of a long-term chronic critical illness, often associated with prolonged inflammation, immune suppression, organ injury and lean tissue wasting. Furthermore, patients who survive sepsis have continuing risk of mortality after discharge, as well as long-term cognitive and functional deficits. Earlier recognition and improved implementation of best practices have reduced in-hospital mortality, but results from the use of immunomodulatory agents to date have been disappointing. Similarly, no biomarker can definitely diagnose sepsis or predict its clinical outcome. Because of its complexity, improvements in sepsis outcomes are likely to continue to be slow and incremental.

Pneumonia is the most common complication observed in patients with severe injuries. Although the average age of injured patients is 47 years, existing studies of the effect of injury on the susceptibility to infectious complications have focused on young animals, equivalent to a late adolescent human. We hypothesized that mature adult animals are more susceptible to infection after injury than younger counterparts. To test this hypothesis, we challenged 6 to 8-month-old mature mice to a polytrauma injury followed by Pseudomonas aeruginosa pneumonia and compared them to young (8-10-week-old) animals. We demonstrate that polytrauma injury increases mortality from pneumonia in mature animals (sham-pneumonia 21% vs. polytrauma-pneumonia 62%) but not younger counterparts. After polytrauma, pneumonia in mature mice is associated with higher bacterial burden in lung, increased incidence of bacteremia, and elevated levels of bacteria in the blood, demonstrating that injury decreases the ability to control the infectious challenge. We further find that polytrauma did not induce elevations in circulating cytokine levels (TNF-alpha, IL-6, KC, and IL-10) 24 h after injury. However, mature mice subjected to polytrauma demonstrated an exaggerated circulating inflammatory cytokine response to subsequent Pseudomonas pneumonia. Additionally, whereas prior injury increases LPS-stimulated IL-6 production by peripheral blood leukocytes from young (8-10-week-old) mice, injury does not prime IL-6 production by cell from mature adult mice. We conclude that in mature mice polytrauma results in increased susceptibility to Pseudomonas pneumonia while priming an exaggerated but ineffective inflammatory response.

Objective Pathogens that cause pneumonia may be treated in a targeted fashion by antibiotics, but if this therapy fails, then treatment involves only nonspecific supportive measures, independent of the inciting infection. The purpose of this study was to determine whether host response is similar after disparate infections with similar mortalities. Design Prospective, randomized controlled study. Setting Animal laboratory in a university medical center. Interventions Pneumonia was induced in FVB/N mice by either Streptococcus pneumoniae or two different concentrations of Pseudomonas aeruginosa. Plasma and bronchoalveolar lavage fluid from septic animals was assayed by a microarray immunoassay measuring 18 inflammatory mediators at multiple time points. Measurements and main results The host response was dependent on the causative organism as well as kinetics of mortality, but the pro-inflammatory and anti-inflammatory responses were independent of inoculum concentration or degree of bacteremia. Pneumonia caused by different concentrations of the same bacteria, Pseudomonas aeruginosa, also yielded distinct inflammatory responses; however, inflammatory mediator expression did not directly track the severity of infection. For all infections, the host response was compartmentalized, with markedly different concentrations of inflammatory mediators in the systemic circulation and the lungs. Hierarchical clustering analysis resulted in the identification of five distinct clusters of the host response to bacterial infection. Principal components analysis correlated pulmonary macrophage inflammatory peptide-2 and interleukin-10 with progression of infection, whereas elevated plasma tumor necrosis factor sr2 and macrophage chemotactic peptide-1 were indicative of fulminant disease with >90% mortality within 48 hrs. Conclusions Septic mice have distinct local and systemic responses to Streptococcus pneumoniae and Pseudomonas aeruginosa pneumonia. Targeting specific host inflammatory responses induced by distinct bacterial infections could represent a potential therapeutic approach in the treatment of sepsis.

Lymphocytes help determine whether gut epithelial cells proliferate or differentiate but are not known to affect whether they live or die. Here, we report that lymphocytes play a controlling role in mediating gut epithelial apoptosis in sepsis but not under basal conditions. Gut epithelial apoptosis is similar in unmanipulated Rag-1−/− and wild-type (WT) mice. However, Rag-1−/− animals have a 5-fold augmentation in gut epithelial apoptosis following cecal ligation and puncture (CLP) compared to septic WT mice. Reconstitution of lymphocytes in Rag-1−/− mice via adoptive transfer decreases intestinal apoptosis to levels seen in WT animals. Subset analysis indicates that CD4+ but not CD8+, γδ, or B cells are responsible for the antiapoptotic effect of lymphocytes on the gut epithelium. Gut-specific overexpression of Bcl-2 in transgenic mice decreases mortality following CLP. This survival benefit is lymphocyte dependent since gut-specific overexpression of Bcl-2 fails to alter survival when the transgene is overexpressed in Rag-1−/− mice. Further, adoptively transferring lymphocytes to Rag-1−/− mice that simultaneously overexpress gut-specific Bcl-2 results in improved mortality following sepsis. Thus, sepsis unmasks CD4+ lymphocyte control of gut apoptosis that is not present under homeostatic conditions, which acts as a key determinant of both cellular survival and host mortality.—Stromberg, P. E., Woolsey, C. A., Clark, A. T., Clark, J. A., Turnbull, I. R., McConnell, K. W., Chang, K. C., Chung, C.-S., Ayala, A., Buchman, T. G., Hotchkiss, R. S., Coopersmith, C. M. CD4+ lymphocytes control gut epithelial apoptosis and mediate survival in sepsis.

The PA200 proteasome activator is a broadly expressed nuclear protein. Although how PA200 normally functions is not fully understood, it has been suggested to be involved in the repair of DNA double-strand breaks (DSBs). The PA200 gene (Psme4) is composed of 45 coding exons spanning 108 kb on mouse chromosome 11. We generated a PA200 null allele (PA200(Delta)) through Cre-loxP-mediated interchromosomal recombination after targeting loxP sites at either end of the locus. PA200(Delta/Delta) mice are viable and have no obvious developmental abnormalities. Both lymphocyte development and immunoglobulin class switching, which rely on the generation and repair of DNA DSBs, are unperturbed in PA200(Delta/Delta) mice. Additionally, PA200(Delta/Delta) embryonic stem cells do not exhibit increased sensitivity to either ionizing radiation or bleomycin. Thus, PA200 is not essential for the repair of DNA DSBs generated in these settings. Notably, loss of PA200 led to a marked reduction in male, but not female, fertility. This was due to defects in spermatogenesis observed in meiotic spermatocytes and during the maturation of postmeiotic haploid spermatids. Thus, PA200 serves an important nonredundant function during spermatogenesis, suggesting that the efficient generation of male gametes has distinct protein metabolic requirements.

Objectives The aim of this study was to determine the effects of acute lung injury on the gut epithelium and examine mechanisms underlying changes in crypt proliferation and apoptosis. The relationship between severity and timing of lung injury to intestinal pathology was also examined. Design Randomized, controlled study. Setting University research laboratory. Subjects Genetically inbred mice. Interventions Following induction of acute lung injury, gut epithelial proliferation and apoptosis were assessed in a) C3H/HeN wild-type and C3H/HeJ mice, which lack functional Toll-like receptor 4 (n = 17); b) C57Bl/6 mice that received monoclonal anti-tumor necrosis factor-alpha or control antibody (n = 22); and c) C57Bl/6 wild-type and transgenic mice that overexpress Bcl-2 in their gut epithelium (n = 21). Intestinal epithelial proliferation and death were also examined in animals with differing degrees of lung inflammation (n = 24) as well as in a time course analysis following a fixed injury (n = 18). Measurements and main results Acute lung injury caused decreased proliferation and increased apoptosis in crypt epithelial cells in all animals studied. C3H/HeJ mice had higher levels of proliferation than C3H/HeN animals without additional changes in apoptosis. Anti-tumor necrosis factor-alpha antibody had no effect on gut epithelial proliferation or death. Overexpression of Bcl-2 did not change proliferation despite decreasing gut apoptosis. Proliferation and apoptosis were not correlated to severity of lung injury, as gut alterations were lost in mice with more severe acute lung injury. Changes in both gut epithelial proliferation and death were apparent within 12 hrs, but proliferation was decreased 36 hrs following acute lung injury while apoptosis returned to normal. Conclusions Acute lung injury causes disparate effects on crypt proliferation and apoptosis, which occur, at least in part, through differing mechanisms involving Toll-like receptor 4 and Bcl-2. Severity of lung injury does not correlate with perturbations in proliferation or death in the gut epithelium, and acute lung injury-induced changes in intestinal epithelial proliferation persist longer than those in apoptosis.

Background Sepsis, iron loading, and aging cause independent increases in gut epithelial and splenic apoptosis. It is unknown how their combination will affect apoptosis and systemic cytokine levels. Materials and Methods Hfe−/− mice (a murine homologue of hemochromatosis) abnormally accumulate iron in their tissues. Aged (24–26 months) or mature (16–18 months) Hfe−/− mice and wild type (WT) littermates were subjected to cecal ligation and puncture (CLP) or sham laparotomy. Intestine, spleen, and blood were harvested 24 h later and assessed for apoptosis and cytokine levels. Results Gut epithelial and splenic apoptosis were low in both aged septic and sham Hfe−/− mice, regardless of the amount of iron in their diet. Mature septic WT mice had increased apoptosis compared to age-matched sham WT mice. Mature septic Hfe−/− mice had similar levels of intestinal cell death to age-matched septic WT mice but higher levels of splenic apoptosis. Apoptosis was significantly lower in septic aged Hfe−/− mice than septic mature Hfe−/− animals. Interleukin-6 was elevated in septic aged Hfe−/− mice compared to sham mice. Conclusions Although sepsis, chronic iron dysregulation, and aging each increase gut and splenic apoptosis, their combination yields cell death levels similar to sham animals despite the fact that aged Hfe−/− mice are able to mount an inflammatory response following CLP and mature Hfe−/− mice have elevated sepsis-induced apoptosis. Combining sepsis with two risk factors that ordinarily increase cell death and increase mortality in CLP yields an apoptotic response that could not have been predicted based upon each element in isolation.

DAP12 (KARAP) is a transmembrane signaling adaptor for a family of innate immunoreceptors that have been shown to activate granulocytes and monocytes/macrophages, amplifying production of inflammatory cytokines. Contrasting with these data, recent studies suggest that DAP12 signaling has an inhibitory role in the macrophage response to microbial products (Hamerman, J.A., N.K. Tchao, C.A. Lowell, and L.L. Lanier. 2005. Nat. Immunol. 6:579–586). To determine the in vivo role for DAP12 signaling in inflammation, we measured the response of wild-type (WT) and DAP12−/− mice to septic shock. We show that DAP12−/− mice have improved survival from both endotoxemia and cecal ligation and puncture–induced septic shock. As compared with WT mice, DAP12−/− mice have decreased plasma cytokine levels and a decreased acute phase response during sepsis, but no defect in the recruitment of cells or bacterial control. In cells isolated after sepsis and stimulated ex vivo, DAP12 signaling augments lipopolysaccharide-mediated cytokine production. These data demonstrate that, during sepsis, DAP12 signaling augments the response to microbial products, amplifying inflammation and contributing to mortality.

Both aging and sepsis independently increase splenic and gut epithelial apoptosis. Sepsis-induced apoptosis in either cell type is also associated with increased mortality in young mice. We sought to determine whether age alters sepsis-induced splenic and gut epithelial cell death. Young (2 months) and aged (22 months) male ND4 mice were subjected to either single-puncture cecal ligation and puncture (CLP) with a 23-gauge needle or sham laparotomy. Apoptosis was assessed 24 hours later in the spleen and gut epithelium by active caspase 3 and hematoxylin and eosin staining. Aged septic mice had increased splenic apoptosis compared with either young septic animals or aged sham animals (15 vs. 7 vs. 5 apoptotic cells/high-powered field, P < 0.05). Similarly, aged septic animals had an elevation in gut epithelial cell death compared with either young septic or aged sham mice (33 vs. 16 vs. 6 apoptotic cells/100 contiguous crypts, P < 0.05). Elevated intestinal cell death was not associated with changes in either gut proliferation or cell division. To verify that the increase in splenic apoptosis seen in septic aged animals was not strain specific, double-puncture CLP with a 25-gauge needle or sham laparotomy was performed on young (4 months) or aged (24 months) C57BL/6 male mice. Similar to results seen in outbred animals, aged septic animals in this inbred strain had increased splenic apoptosis compared with either young septic animals or aged sham animals (23 vs. 7 vs. 4 apoptotic cells/ high powered field, P < 0.05). These results indicate that although infection and aging each independently cause an increase in splenic and gut epithelial apoptosis, their combination leads to a disproportionate increase in cell death in these rapidly dividing cell populations,and potentially plays a role in the marked increase in mortality seen with aging in sepsis.

Genetically identical mice have a heterogeneous response to antibiotic therapy in sepsis, with only a subset deriving therapeutic benefit. We sought to determine whether the severity of a septic insult correlates with the survival benefit conferred by antibiotics. We also sought to determine whether antibiotics given 12 h after injury alter survival in animals predicted to die based upon high interleukin (IL)-6 levels drawn 6 h earlier. Adult male ND4 mice (n = 363) were subjected to double-puncture cecal ligation and puncture (CLP) with a 19-, 21-, or 23-gauge needle. Animals were randomized to receive imipenem or 0.9% NaCl every 12 h after CLP for 5 days. Ten-day survival was 16%, 26%, and 52%, respectively, for untreated animals. Antibiotics decreased the absolute risk of death 17% to 23% regardless of injury severity. In a separate cohort, mice (n = 37) were subjected to single or double-puncture CLP with a 21-gauge needle. IL-6 levels were assayed 6 h postoperatively and mice were followed for survival. Levels greater than 14,000 pg/mL were identified as predicting a 100% mortality (7/7 animals dead). A third set of mice (n = 94) then underwent double-puncture CLP with either 21-, 23-, or 25-gauge needle and had IL-6 levels measured in a similar fashion. Animals were randomized to receive imipenem or 0.9% NaCl beginning 12 h postoperatively (6 h after IL-6 levels were drawn) and continued for 5 days or until death. Although antibiotics decreased mortality overall, all animals with IL-6 levels greater than 14,000 pg/mL (n = 13) died, regardless of whether they received antibiotics or the gauge of needle used. These results indicate that antibiotics improve outcome in murine sepsis, regardless of injury severity. Furthermore, there is a threshold IL-6 level that can be identified 6 h after sepsis above which animals are destined to die, and antibiotic treatment does not alter their outcome.

Iron metabolism is dysregulated in critically ill patients. A mouse model of dysregulated iron metabolism was used to examine the consequence of iron loading upon sepsis. Mice deleted in the hfe gene (hfe-/-) abnormally accumulate iron in tissue; defects in the human hfe gene are clinically expressed as hemochromatosis. Hfe-/- mice and wild-type counterparts were randomized to receive either high- or low-iron diets for 2 weeks. After iron loading, mice were subjected to cecal ligation and puncture (CLP), a clinically relevant animal model of intra-abdominal sepsis. A preliminary (but underpowered) study suggested that iron-loaded hfe-/- mice had increased mortality as compared with hfe-/- mice fed a low-iron diet. There was no difference between wild-type and hfe-/- mice fed a low-iron diet or between wild-type mice fed hihg- and low-iron diets. A subsequent, appropriately powered study showed that iron-loaded hfe-/- mice had significantly higher mortality from intra-abdominal sepsis than hfe-/- mice fed a low-iron diet. Iron loading was confirmed through chemical assay of iron concentration in hepatic tissue. Animals with dysregulated iron handling, loaded with iron and subjected to CLP, had double the mortality of animals with normal iron levels. Critical care patients often have altered iron metabolism. In clinical practice, critically ill patients may receive iron through direct administration and the transfusion of blood products. Iron therapy may adversely affect the clinical outcome from sepsis.

The incidence and mortality of sepsis increase with age, consequently, 80% of the clinical mortality from sepsis occurs in patients over age 65. Despite this aged clinical population, most research models of sepsis use 6- to 16-week-old mice as patient surrogates. This age range of mice corresponds to human ages 10 to 17 years. To assess the influence of age on rodent CLP and on antibiotic therapy, we studied young (4 month), mature (12 month), and aged (24 month) mice. Male C57BL/6 mice (n = 27-30 in each age group) were subjected to cecal ligation and puncture (CLP), two punctures with a 25-gauge needle. Mice were observed untreated for 10 days. Young mice had 20% mortality, mature mice had 70% mortality (P = 0.0013 vs. young), and aged mice had 75% mortality (P = 0.0001 vs. young). To assess the effects of age on antibiotic therapy, mice were subjected to CLP as above (n = 38-40 in each age group). Mice were then randomized to treatment with intraperitoneal injections of ceftriaxone and metronidazole or normal saline. Therapy was initiated 12 h after CLP, and injections were repeated every 12 h for 7 days. Young mice saw a 56% decrease in mortality from CLP with antibiotic therapy (P = 0.001), and mature mice had a 30% decrease in mortality (P = 0.06). Aged mice saw no benefit from antibiotic therapy. We also compared plasma cytokine levels between young and aged mice after CLP. When compared with young mice, aged mice had higher levels of IL-6 and TNF-alpha 24 h after CLP. However, high IL-6 was predictive of mortality at any age. Mice appear to have age-dependent responses to intra-abdominal sepsis and to appropriate therapy.

Homologous aquaporin water channels utilize different folding pathways to acquire their transmembrane (TM) topology in the endoplasmic reticulum (ER). AQP4 acquires each of its six TM segments via cotranslational translocation events, whereas AQP1 is initially synthesized with four TM segments and subsequently converted into a six membrane-spanning topology. To identify sequence determinants responsible for these pathways, peptide segments from AQP1 and AQP4 were systematically exchanged. Chimeric proteins were then truncated, fused to a C-terminal translocation reporter, and topology was analyzed by protease accessibility. In each chimeric context, TM1 initiated ER targeting and translocation. However, AQP4-TM2 cotranslationally terminated translocation, while AQP1-TM2 failed to terminate translocation and passed into the ER lumen. This difference in stop transfer activity was due to two residues that altered both the length and hydrophobicity of TM2 (Asn(49) and Lys(51) in AQP1 versus Met(48) and Leu(50) in AQP4). A second peptide region was identified within the TM3-4 peptide loop that enabled AQP4-TM3 but not AQP1-TM3 to reinitiate translocation and cotranslationally span the membrane. Based on these findings, it was possible to convert AQP1 into a cotranslational biogenesis mode similar to that of AQP4 by substituting just two peptide regions at the N terminus of TM2 and the C terminus of TM3. Interestingly, each of these substitutions disrupted water channel activity. These data thus establish the structural basis for different AQP folding pathways and provide evidence that variations in cotranslational folding enable polytopic proteins to acquire and/or maintain primary sequence determinants necessary for function.

The topology of most eukaryotic polytopic membrane proteins is established cotranslationally in the endoplasmic reticulum (ER) through a series of coordinated translocation and membrane integration events. For the human aquaporin water channel AQP1, however, the initial four-segment-spanning topology at the ER membrane differs from the mature six-segment-spanning topology at the plasma membrane. Here we use epitope-tagged AQP1 constructs to follow the transmembrane (TM) orientation of key internal peptide loops in Xenopus oocyte and cell-free systems. This analysis revealed that AQP1 maturation in the ER involves a novel topological reorientation of three internal TM segments and two peptide loops. After the synthesis of TMs 4–6, TM3 underwent a 180-degree rotation in which TM3 C-terminal flanking residues were translocated from their initial cytosolic location into the ER lumen and N-terminal flanking residues underwent retrograde translocation from the ER lumen to the cytosol. These events convert TM3 from a type I to a type II topology and reposition TM2 and TM4 into transmembrane conformations consistent with the predicted six-segment-spanning AQP1 topology. AQP1 topological reorientation was also associated with maturation from a protease-sensitive conformation to a protease-resistant structure with water channel function. These studies demonstrate that initial protein topology established via cotranslational translocation events in the ER is dynamic and may be modified by subsequent steps of folding and/or maturation.

Aging is associated with increased inflammation following sepsis. The purpose of this study was to determine whether this represents a fundamental age-based difference in the host response or is secondary to the increased mortality seen in aged hosts.Prospective, randomized controlled study.Animal laboratory in a university medical center.Young (6-12 weeks) and aged (20-24 months) FVB/N mice.Mice were subjected to 2 x 25 or 1 x 30 cecal ligation and puncture (CLP).Survival was similar in young mice subjected to 2 x 25 CLP and aged mice subjected to 1 x 30 CLP (p = 0.15). Young mice subjected to 1 x 30 CLP had improved survival compared with the other groups (p0.05). When injury was held constant but mortality was greater, both systemic and peritoneal levels of tumor necrosis factor-alpha, interleukin (IL)-6, IL-10, and monocyte chemotactic protein-1 were elevated 24 hours after CLP in aged animals compared with young animals (p0.05). When mortality was similar but injury severity was different, there were no significant differences in systemic cytokines between aged mice and young mice. In contrast, peritoneal levels of tumor necrosis factor-alpha, IL-6, and IL-10 were higher in aged mice subjected to 1 x 30 CLP than young mice subjected to 2 x 25 CLP despite their similar mortalities (p0.05). There were no significant differences in either bacteremia or peritoneal cultures when animals of different ages sustained similar injuries or had different injuries with similar mortalities.Aged mice are more likely to die of sepsis than young mice when subjected to an equivalent insult, and this is associated with increases in both systemic and local inflammation. There is an exaggerated local but not systemic inflammatory response in aged mice compared with young mice when mortality is similar. This suggests that systemic processes that culminate in death may be age independent, but the local inflammatory response may be greater with aging.

Macrophage colony stimulating factor (MCSF) influences proliferation and survival of mononuclear phagocytes through the CSF-1 receptor. The DAP12 adaptor protein, which transduces signals emanating from various myeloid receptors, is critical for mononuclear phagocyte function. DAP12-mutant mice and humans show defects in osteoclasts and microglia and exhibit brain and bone abnormalities. Here, we demonstrated that DAP12 deficiency impairs MCSF-induced macrophage proliferation and survival in vitro. In addition, DAP12-deficient mice show fewer microglia in defined central nervous system areas, and DAP12-deficient progenitors regenerate myeloid cells inefficiently following BM transplantation. MCSF-CSF1-R signaling induced stabilization and nuclear translocation of β-catenin, which activates cell cycle genes. DAP12 was essential for phosphorylation and nuclear accumulation of β-catenin. These results outline a mechanistic explanation for the multiple defects in DAP12-deficient mononuclear phagocytes.

Phosphoinositide 3-kinases (PI-3Ks) are key enzymes for cell development, activation, and survival. Here we showed that PI-3K class IB and class IA catalytic subunits, p110gamma and p110delta, played a crucial role in the development and functions of murine NK cells. p110gamma deficiency and impairment of G protein-coupled receptor (GPRC) signaling prevented full NK cell maturation. Concomitant loss of p110gamma and p110delta exacerbated this defect, resulting in a very small population of NK cells with a highly immature phenotype in the bone marrow and periphery. Moreover, combined p110gamma and p110delta signals were required for cytotoxicity and activation of the kinase ERK during NK cell-target cell interaction. p110gamma played a major role in receptor-induced interferon-gamma (IFN-gamma) production through a pathway that involved the kinase ERK and 5-Lipoxigenase, which most likely generates lipid mediators activating GPRCs. Conversely, PI3Ks negatively regulated interleukin-12 (IL-12) and IL-18-induced IFN-gamma by modulating p38 kinase activation. Our data shed light on the multiple intersecting pathways through which PI3Ks control NK cell-mediated innate responses.

The triggering receptor expressed on myeloid cells 2 (TREM-2) delivers intracellular signals through the adaptor DAP12 to regulate myeloid cell function both within and outside the immune system. The role of TREM-2 in immunity has been obscured by the failure to detect expression of the TREM-2 protein in vivo. In this study, we show that TREM-2 is expressed on macrophages infiltrating the tissues from the circulation and that alternative activation with IL-4 can induce TREM-2. TREM-2 expression is abrogated by macrophage maturation with LPS of IFN-γ. Using TREM-2−/− mice, we find that TREM-2 functions to inhibit cytokine production by macrophages in response to the TLR ligands LPS, zymosan, and CpG. Furthermore, we find that TREM-2 completely accounts for the increased cytokine production previously reported by DAP12−/− macrophages. Taken together, these data show that TREM-2 is expressed on newly differentiated and alternatively activated macrophages and functions to restrain macrophage activation.