Maegan L. Capitano

NADPH Oxidase Limits Innate Immune Responses in the Lungs in Mice

Background Chronic granulomatous disease (CGD), an inherited disorder of the NADPH oxidase in which phagocytes are defective in generating superoxide anion and downstream reactive oxidant intermediates (ROIs), is characterized by recurrent bacterial and fungal infections and by excessive inflammation (e.g., inflammatory bowel disease). The mechanisms by which NADPH oxidase regulates inflammation are not well understood. Methodology/Principal Findings We found that NADPH oxidase restrains inflammation by modulating redox-sensitive innate immune pathways. When challenged with either intratracheal zymosan or LPS, NADPH oxidase-deficient p47phox−/− mice and gp91phox-deficient mice developed exaggerated and progressive lung inflammation, augmented NF-κB activation, and elevated downstream pro-inflammatory cytokines (TNF-α, IL-17, and G-CSF) compared to wildtype mice. Replacement of functional NADPH oxidase in bone marrow-derived cells restored the normal lung inflammatory response. Studies in vivo and in isolated macrophages demonstrated that in the absence of functional NADPH oxidase, zymosan failed to activate Nrf2, a key redox-sensitive anti-inflammatory regulator. The triterpenoid, CDDO-Im, activated Nrf2 independently of NADPH oxidase and reduced zymosan-induced lung inflammation in CGD mice. Consistent with these findings, zymosan-treated peripheral blood mononuclear cells from X-linked CGD patients showed impaired Nrf2 activity and increased NF-κB activation. Conclusions/Significance These studies support a model in which NADPH oxidase-dependent, redox-mediated signaling is critical for termination of lung inflammation and suggest new potential therapeutic targets for CGD.

Baseline tumor growth and immune control in laboratory mice are significantly influenced by subthermoneutral housing temperature.

Significance We show that the mandated, subthermoneutral laboratory housing temperature, which is known to cause chronic, metabolic cold stress, induces suppression of the antitumor immune response and promotes tumor growth and metastasis. When mice are housed at thermoneutrality, there are fewer immunosuppressive cells with significantly enhanced CD8+ T cell-dependent control of tumor growth. These findings underscore the fact that investigating mouse models under a single set of environmental temperature conditions may lead to a misunderstanding of the antitumor immune potential. These data also highlight the need for additional study to determine how systemic metabolic stress modulates the functions of immune effector cells, particularly in tumor-bearing mice, and whether cancer therapies, including immunotherapy, are impacted by housing temperature. We show here that fundamental aspects of antitumor immunity in mice are significantly influenced by ambient housing temperature. Standard housing temperature for laboratory mice in research facilities is mandated to be between 20–26 °C; however, these subthermoneutral temperatures cause mild chronic cold stress, activating thermogenesis to maintain normal body temperature. When stress is alleviated by housing at thermoneutral ambient temperature (30–31 °C), we observe a striking reduction in tumor formation, growth rate and metastasis. This improved control of tumor growth is dependent upon the adaptive immune system. We observe significantly increased numbers of antigen-specific CD8+ T lymphocytes and CD8+ T cells with an activated phenotype in the tumor microenvironment at thermoneutrality. At the same time there is a significant reduction in numbers of immunosuppressive MDSCs and regulatory T lymphocytes. Notably, in temperature preference studies, tumor-bearing mice select a higher ambient temperature than non-tumor-bearing mice, suggesting that tumor-bearing mice experience a greater degree of cold-stress. Overall, our data raise the hypothesis that suppression of antitumor immunity is an outcome of cold stress-induced thermogenesis. Therefore, the common approach of studying immunity against tumors in mice housed only at standard room temperature may be limiting our understanding of the full potential of the antitumor immune response.

Mild Elevation of Body Temperature Reduces Tumor Interstitial Fluid Pressure and Hypoxia and Enhances Efficacy of Radiotherapy in Murine Tumor Models

Human and rodent solid tumors often exhibit elevated interstitial fluid pressure (IFP). This condition is recognized as a prognostic indicator for reduced responses to therapy and decreased disease-free survival rate. In the present study, we tested whether induction of a thermoregulatory-mediated increase in tissue blood flow, induced by exposure of mice to mild environmental heat stress, could influence IFP and other vascular parameters within tumors. Using several murine tumor models, we found that heating results in a sustained reduction in tumor IFP correlating with increased tumor vascular perfusion (measured by fluorescent imaging of perfused vessels, laser Doppler flowmetry, and MRI) as well as a sustained reduction in tumor hypoxia. Furthermore, when radiation therapy was administered 24 hours postheating, we observed a significant improvement in efficacy that may be a result of the sustained reduction in tumor hypoxia. These data suggest, for the first time, that environmental manipulation of normal vasomotor function is capable of achieving therapeutically beneficial changes in IFP and microvascular function in the tumor microenvironment.

The HMGB1-RAGE axis mediates traumatic brain injury–induced pulmonary dysfunction in lung transplantation

Traumatic brain injury induces acute lung injury that negatively impacts the physiology of the donor lung before and after lung transplantation. Sounding the Alarm for RAGE Only 20% of lungs are transplantable because traumatic brain injury, a major cause of death in organ doors, may induce acute lung injury. High-mobility group box-1 (HMGB1) release from the injured brain likely contributes to acute lung injury in donors by preferentially interacting with receptor for advanced glycation end products (RAGE) in the lung. Blocking the HMGB1-RAGE axis improves lung function in murine donors with traumatic brain injury and after transplant. In translational studies, lungs sourced from donors with high HMGB1 levels had worse pulmonary function after transplant. Targeting the HMGB1-RAGE axis may increase the number of lungs available for transplantation and improve patient outcomes. Traumatic brain injury (TBI) results in systemic inflammatory responses that affect the lung. This is especially critical in the setting of lung transplantation, where more than half of donor allografts are obtained postmortem from individuals with TBI. The mechanism by which TBI causes pulmonary dysfunction remains unclear but may involve the interaction of high-mobility group box-1 (HMGB1) protein with the receptor for advanced glycation end products (RAGE). To investigate the role of HMGB1 and RAGE in TBI-induced lung dysfunction, RAGE-sufficient (wild-type) or RAGE-deficient (RAGE−/−) C57BL/6 mice were subjected to TBI through controlled cortical impact and studied for cardiopulmonary injury. Compared to control animals, TBI induced systemic hypoxia, acute lung injury, pulmonary neutrophilia, and decreased compliance (a measure of the lungs’ ability to expand), all of which were attenuated in RAGE−/− mice. Neutralizing systemic HMGB1 induced by TBI reversed hypoxia and improved lung compliance. Compared to wild-type donors, lungs from RAGE−/− TBI donors did not develop acute lung injury after transplantation. In a study of clinical transplantation, elevated systemic HMGB1 in donors correlated with impaired systemic oxygenation of the donor lung before transplantation and predicted impaired oxygenation after transplantation. These data suggest that the HMGB1-RAGE axis plays a role in the mechanism by which TBI induces lung dysfunction and that targeting this pathway before transplant may improve recipient outcomes after lung transplantation.

Differentiation of CD8+ T cells into effector cells is enhanced by physiological range hyperthermia

In this study, we asked whether exposure to different physiologically relevant temperatures (33°C, 37°C, and 39.5°C) could affect subsequent antigen‐specific, activation‐related events of naive CD8+ T cells. We observed that temporary exposure of CD62LhiCD44lo Pmel‐1 CD8+ cells to 39.5°C prior to their antigen‐dependent activation with gp10025–33 peptide‐pulsed C57BL/6 splenocytes resulted in a greater percentage of cells, which eventually differentiated into CD62LloCD44hi effector cells compared with cells incubated at 33°C and 37°C. However, the proliferation rate of naive CD8+ T cells was not affected by mild heating. While exploring these effects further, we observed that mild heating of CD8+ T cells resulted in the reversible clustering of GM1+ CD‐microdomains in the plasma membrane. This could be attributable to a decrease in line tension in the plasma membrane, as we also observed an increase in membrane fluidity at higher temperatures. Importantly, this same clustering phenomenon was observed in CD8+ T cells isolated from spleen, LNs, and peripheral blood following mild whole‐body heating of mice. Further, we observed that mild heating also resulted in the clustering of TCRβ and the CD8 coreceptor but not CD71R. Finally, we observed an enhanced rate of antigen‐specific conjugate formation with APCs following mild heating, which could account for the difference in the extent of differentiation. Overall, these novel findings may help us to further understand the impact of physiologically relevant temperature shifts on the regulation of antigen‐specific CD8+ T cell activation and the subsequent generation of effector cells.

Mild Heat Treatment Primes Human CD34+ Cord Blood Cells for Migration Toward SDF-1α and Enhances Engraftment in an NSG Mouse Model

Simple efforts are needed to enhance cord blood (CB) transplantation. We hypothesized that short‐term exposure of CD34+ CB cells to 39.5°C would enhance their response to stromal‐derived factor‐1 (SDF‐1), by increasing lipid raft aggregation and CXCR4 expression, thus leading to enhanced engraftment. Mild hyperthermia (39.5°C) significantly increased the percent of CD34+ CB that migrated toward SDF‐1. This was associated with increased expression of CXCR4 on the cells. Mechanistically, mild heating increased the percent of CD34+ cells with aggregated lipid rafts and enhanced colocalization of CXCR4 within lipid raft domains. Using methyl‐β‐cyclodextrin (MβCD), an agent that blocks lipid raft aggregation, it was determined that this enhancement in chemotaxis was dependent upon lipid raft aggregation. Colocalization of Rac1, a GTPase crucial for cell migration and adhesion, with CXCR4 to the lipid raft was essential for the effects of heat on chemotaxis, as determined with an inhibitor of Rac1 activation, NSC23766. Application‐wise, mild heat treatment significantly increased the percent chimerism as well as homing and engraftment of CD34+ CB cells in sublethally irradiated non‐obese diabetic severe combined immunodeficiency IL‐2 receptor gamma chain d (NSG) mice. Mild heating may be a simple and inexpensive means to enhance engraftment following CB transplantation in patients. Stem Cells Stem Cells 2015;33:1975–1984

Granzyme B–Mediated Damage of CD8+ T Cells Impairs Graft-versus-Tumor Effect

Allogeneic hematopoietic cell transplantation is an established treatment for hematologic and other malignancies. Donor-derived immune cells can identify and attack host tumor cells, producing a graft-versus-tumor (GVT) effect that is crucial to the treatment. Using multiple tumor models and diverse donor–host combinations, we have studied the role of granzyme B (GzmB) in GVT effect. We first confirmed previous findings that GzmB deficiency diminished the ability of a high dose of CD8+ T cells to cause lethal graft-versus-host disease. However, when GVT studies were performed using a moderate cell dose that the hosts could tolerate, GzmB−/− CD8+ T cells demonstrated a significantly enhanced GVT effect. GzmB-mediated, activation-induced cell death in wild-type CD8+ T cells was found responsible for their reduced GVT activity. Conversely, GzmB−/− CD8+ T cells exhibited enhanced expansion, skewed toward an effector or effector memory phenotype, and produced higher amounts of IFN-γ and Fas ligand that might contribute to GzmB-independent tumor control. These findings demonstrate for the first time, to our knowledge, that GzmB-mediated damage of CD8+ T cells impairs the desired GVT effect. This study suggests that inhibiting donor-derived GzmB function may represent a promising strategy to improve GVT effect without exacerbating graft-versus-host disease.

Concise Review: Role of DEK in Stem/Progenitor Cell Biology

Understanding the factors that regulate hematopoiesis opens up the possibility of modifying these factors and their actions for clinical benefit. DEK, a non‐histone nuclear phosphoprotein initially identified as a putative proto‐oncogene, has recently been linked to regulate hematopoiesis. DEK has myelosuppressive activity in vitro on proliferation of human and mouse hematopoietic progenitor cells and enhancing activity on engraftment of long‐term marrow repopulating mouse stem cells, has been linked in coordinate regulation with the transcription factor C/EBPα, for differentiation of myeloid cells, and apparently targets a long‐term repopulating hematopoietic stem cell for leukemic transformation. This review covers the uniqueness of DEK, what is known about how it now functions as a nuclear protein and also as a secreted molecule that can act in paracrine fashion, and how it may be regulated in part by dipeptidylpeptidase 4, an enzyme known to truncate and modify a number of proteins involved in activities on hematopoietic cells. Examples are provided of possible future areas of investigation needed to better understand how DEK may be regulated and function as a regulator of hematopoiesis, information possibly translatable to other normal and diseased immature cell systems. STEM Cells 2013;31:1447–1453

A Role for DEK in Stem/Progenitor Cell Biology

Understanding the factors that regulate hematopoiesis opens up the possibility of modifying these factors and their actions for clinical benefit. DEK, a non‐histone nuclear phosphoprotein initially identified as a putative proto‐oncogene, has recently been linked to regulate hematopoiesis. DEK has myelosuppressive activity in vitro on proliferation of human and mouse hematopoietic progenitor cells and enhancing activity on engraftment of long‐term marrow repopulating mouse stem cells, has been linked in coordinate regulation with the transcription factor C/EBPα, for differentiation of myeloid cells, and apparently targets a long‐term repopulating hematopoietic stem cell for leukemic transformation. This review covers the uniqueness of DEK, what is known about how it now functions as a nuclear protein and also as a secreted molecule that can act in paracrine fashion, and how it may be regulated in part by dipeptidylpeptidase 4, an enzyme known to truncate and modify a number of proteins involved in activities on hematopoietic cells. Examples are provided of possible future areas of investigation needed to better understand how DEK may be regulated and function as a regulator of hematopoiesis, information possibly translatable to other normal and diseased immature cell systems. STEM Cells 2013;31:1447–1453

Winner of the 2007 Society for Thermal Medicine Young Investigator Award. Fever-range whole body hyperthermia prevents the onset of type 1 diabetes in non-obese diabetic mice.

Purpose: Type 1 diabetes (T1D) is an autoimmune disease in which the insulin producing β cells of the pancreatic islets are destroyed by cytotoxic T lymphocytes (CTLs). It has been demonstrated that the injection of complete Freunds adjuvant (CFA) can prevent disease onset in non-obese diabetic (NOD) mice. This effect has been attributed to CFA-enhanced natural killer (NK) cell mediated control of autoimmune CTLs. Fever-range whole body hyperthermia (FR-WBH) has also been shown to stimulate NK cell cytotoxicity. This led to the hypothesis that FR-WBH can prevent disease onset in NOD mice by a thermally regulated mechanism. Methods: FR-WBH or mock treatment was administered weekly until the NOD mice reached 32 weeks of age. Blood glucose levels were monitored weekly, with measurements ≥33.5 mM indicating onset of diabetes, at which time the mice were euthanized for histological and cellular analyses. Results: Weekly FR-WBH prevented the onset of T1D in NOD mice and this effect correlated with increased NK cell cytotoxicity and control of blood glucose concentration. Histological analysis revealed significantly fewer lymphocytes infiltrating the pancreatic islets of FR-WBH treated mice than those of untreated mice, suggesting a relationship between thermally induced protection of β cells and their ability to regulate blood glucose concentrations. Conclusions: These studies show, for the first time, that mild systemic hyperthermia can prevent the generation of T1D in a clinically relevant mouse model. Further study of the thermally sensitive aspects of immunoregulation could lead to the development of heat-based therapies for the prevention or treatment of autoimmune diseases.