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Asthma is a common respiratory disease currently affecting more than 300 million worldwide and is characterized by airway inflammation, hyperreactivity, and remodeling. It is a heterogeneous disease consisting of corticosteroid-sensitive T-helper cell type 2-driven eosinophilic and corticosteroid-resistant, T-helper cell type 17-driven neutrophilic phenotypes. One pathway recently described to regulate asthma pathogenesis is cholesterol trafficking. Scavenger receptors, in particular SR-BI (scavenger receptor class B type I), are known to direct cellular cholesterol uptake and efflux. We recently defined SR-BI functions in pulmonary host defense; however, the function of SR-BI in asthma pathogenesis is unknown. To elucidate the role of SR-BI in allergic asthma, SR-BI-sufficient (SR-BI

The intensity and longevity of inflammatory responses to inhaled allergens is determined largely by the balance between effector and regulatory immune responses, but the mechanisms that determine the relative magnitudes of these opposing forces remain poorly understood. We have found that the type of adjuvant used during allergic sensitization has a profound effect on both the nature and longevity of the pulmonary inflammation triggered by subsequent reexposure to that same provoking allergen. TLR ligand adjuvants and house dust extracts primed immune responses characterized by a mixed neutrophilic and eosinophilic inflammation that was suppressed by multiple daily allergen challenges. During TLR ligand-mediated allergic sensitization, mice displayed transient airway neutrophilia, which triggered the release of TGF-β into the airway. This neutrophil-dependent production of TGF-β during sensitization had a delayed, suppressive effect on eosinophilic responses to subsequent allergen challenge. Neutrophil depletion during sensitization did not affect numbers of Foxp3+ Tregs but increased proportions of Gata3+CD4+ T cells, which, upon their transfer to recipient mice, triggered stronger eosinophilic inflammation. Thus, a neutrophil/TGF-β axis acts during TLR-mediated allergic sensitization to fine-tune the phenotype of developing allergen-specific CD4+ T cells and limit their pathogenicity, suggesting a novel immunotherapeutic approach to control eosinophilia in asthma.

Purpose Left Ventricular Assist Devices (LVADs) are a leading treatment for advanced heart failure. However, it is primarily major medical centers that have the resources to support an LVAD program. Many patients live far from an implant center, which makes it imperative that LVAD programs can provide quality care and support patients at a distance. The purpose of this project is to assess if distance to an implant center has an effect on patient outcomes and quality of life. We hypothesized that due to decreased accessibility to the hospital, increased distance to LVAD center would have an adverse effect on outcome and quality of life. Methods We performed a retrospective analysis of LVAD patients implanted at our institution from January 2012 to September 2020. Patients’ residential distance to LVAD center, outcome (transplant, explant, or death) and quality of life data (from EuroQol EQ-5D and KCCQ questionnaires) were analyzed. Descriptive statistics and logistic regression modeling were performed using STATA/ SE 15.1. Results 228 patients were included in the study (82% male, mean age of 57). The median distance from the hospital was 44.2 miles, with a range of 0.7 - 2458.0 miles. Distance was not associated with INTERMACS score, age at implant, race, gender, discharge destination, number of rehospitalizations within the first-year post-implant, or outcome. Distance was not associated with confidence in heart failure management, stress from health issues, or enjoyment of life (measured by EuroQol EQ-5D and KCCQ). Additionally, change in QOL scores from pre-implant to 6 months post-implant were analyzed and no significant associations were found based on distance to LVAD center. The mean distance to implant center was much higher in patients who had a stroke, however this difference was not statistically significant (94.8 mi vs 211.6 mi, p = 0.082). Conclusion Patient distance from LVAD center was not associated with outcomes including stroke and readmission or quality of life in this study. This indicates that patients do not need to reside near their LVAD center to feel supported and confident in their ability to manage their LVAD and that regional programs can provide quality care at a distance. There is a signal that of an increased risk of stroke in patients who are farther away which needs further investigation.

T helper 17 (TH17) cells are critically involved in host defence, inflammation, and autoimmunity. Transforming growth factor β (TGFβ) is instrumental in TH17 cell differentiation by cooperating with interleukin-6 (refs 6, 7). Yet, the mechanism by which TGFβ enables TH17 cell differentiation remains elusive. Here we reveal that TGFβ enables TH17 cell differentiation by reversing SKI-SMAD4-mediated suppression of the expression of the retinoic acid receptor (RAR)-related orphan receptor γt (RORγt). We found that, unlike wild-type T cells, SMAD4-deficient T cells differentiate into TH17 cells in the absence of TGFβ signalling in a RORγt-dependent manner. Ectopic SMAD4 expression suppresses RORγt expression and TH17 cell differentiation of SMAD4-deficient T cells. However, TGFβ neutralizes SMAD4-mediated suppression without affecting SMAD4 binding to the Rorc locus. Proteomic analysis revealed that SMAD4 interacts with SKI, a transcriptional repressor that is degraded upon TGFβ stimulation. SKI controls histone acetylation and deacetylation of the Rorc locus and TH17 cell differentiation via SMAD4: ectopic SKI expression inhibits H3K9 acetylation of the Rorc locus, Rorc expression, and TH17 cell differentiation in a SMAD4-dependent manner. Therefore, TGFβ-induced disruption of SKI reverses SKI-SMAD4-mediated suppression of RORγt to enable TH17 cell differentiation. This study reveals a critical mechanism by which TGFβ controls TH17 cell differentiation and uncovers the SKI-SMAD4 axis as a potential therapeutic target for treating TH17-related diseases.

Sensitization to inhaled allergens is dependent on activation of conventional dendritic cells (cDCs) and on the adaptor molecule, MyD88. However, many cell types in the lung express Myd88, and it is unclear how signaling in these different cell types reprograms cDCs and leads to allergic inflammation of the airway. By combining ATAC-seq with RNA profiling, we found that MyD88 signaling in cDCs maintained open chromatin at select loci even at steady state, allowing genes to be rapidly induced during allergic sensitization. A distinct set of genes related to metabolism was indirectly controlled in cDCs through MyD88 signaling in airway epithelial cells (ECs). In mouse models of asthma, Myd88 expression in ECs was critical for eosinophilic inflammation, whereas Myd88 expression in cDCs was required for Th17 cell differentiation and consequent airway neutrophilia. Thus, both cell-intrinsic and cell-extrinsic MyD88 signaling controls gene expression in cDCs and orchestrates immune responses to inhaled allergens.

Purpose Patients in end stage heart failure with a BMI >35 are considered unacceptable candidates for heart transplantation due to increased morbidity and mortality after transplant. Those with a left ventricular assist device (LVAD) may struggle losing weight (and often gain weight) and may be inactivated on the waiting list (or denied listing) until weight loss is achieved. To assist these patients in weight loss with a goal of reactivation on the transplant list, we created clinical guidelines for laparoscopic bariatric surgery. Methods A multidisciplinary collaboration to develop clinical guidelines was conducted with members from cardiology, cardiac surgery, bariatric surgery, nutrition, nursing, anesthesia, perfusion and anticoagulation management. Patients were admitted preoperatively for Heparin bridge (aspirin was not held) and to adjust antihypertensives and diuretics to prevent post-operative complications. Postoperatively antihypertensives were resumed based on MAP, diuretics were held, intravenous fluids given until patient was euvolemic on exam, and warfarin was resumed 24 hours after surgery. Results A clinical protocol was implemented, and 4 male patients underwent successful bariatric surgery between April 1, 2019 and September 30, 2019. The median age was 47 (range 31-61) with a median BMI of 40.27 (range 36.03 to 50.09). Patients were scheduled for laparoscopic sleeve gastrectomy with 3 successful surgeries performed. One surgery was aborted secondary to the HeartMate II (HMII) obstructing the gastrosplenic ligament, however, the patient successfully underwent laparoscopic magenstrassa mill procedure days later. The median preoperative length of stay was 3 days with a median post-operative length of stay of 7 days. There were no complications or re-admissions associated with the bariatric surgery and all patients lost weight after surgery (median 12.5kg; range 4.9 to 25.3kg at 1-5 months). Two of the four patients were able to be reactivated on the heart transplant waiting list at 1 and 4 months after surgery. Conclusion Our experience demonstrates that bariatric surgery can be safely performed on patients with an LVAD. Consideration may be required for patients with a HeartMate II related to device positioning. Multidisciplinary collaboration in establishing a clinical protocol ensures that VAD management strategies are addressed.

Alveolar macrophages (AM) play a central role in initiation and resolution of lung inflammation, but the integration of these opposing core functions is poorly understood. AM expression of cholesterol 25-hydroxylase (CH25H), the primary biosynthetic enzyme for 25-hydroxycholesterol (25HC), far exceeds the expression of macrophages in other tissues, but no role for CH25H has been defined in lung biology. As 25HC is an agonist for the antiinflammatory nuclear receptor, liver X receptor (LXR), we speculated that CH25H might regulate inflammatory homeostasis in the lung. Here, we show that, of natural oxysterols or sterols, 25HC is induced in the inflamed lung of mice and humans. Ch25h(–/–) mice fail to induce 25HC and LXR target genes in the lung after LPS inhalation and exhibit delayed resolution of airway neutrophilia, which can be rescued by systemic treatment with either 25HC or synthetic LXR agonists. LXR-null mice also display delayed resolution, suggesting that native oxysterols promote resolution. During resolution, Ch25h is induced in macrophages upon their encounter with apoptotic cells and is required for LXR-dependent prevention of AM lipid overload, induction of Mertk, efferocytic resolution of airway neutrophilia, and induction of TGF-β. CH25H/25HC/LXR is, thus, an inducible metabolic axis that programs AMs for efferocytic resolution of inflammation.

Airway neutrophilia occurs in approximately 50% of patients with asthma and is associated with particularly severe disease. Unfortunately, this form of asthma is usually refractory to corticosteroid treatment, and there is an unmet need for new therapies. Pulmonary neutrophilic inflammation is associated with Th17 cells, whose differentiation is controlled by the nuclear receptor retinoic acid–related orphan receptor γt (RORγt). Here, we tested whether VTP-938, a selective inverse agonist of this receptor, can reduce disease parameters in animal models of neutrophilic asthma. When administered before allergic sensitization through the airway, the RORγt inverse agonist blunted allergen-specific Th17 cell development in lung-draining lymph nodes and attenuated allergen-induced production of IL-17. VTP-938 also reduced pulmonary production of IL-17 and airway neutrophilia when given during the allergen challenge of the model. Finally, in an environmentally relevant model of allergic responses to house dust extracts, VTP-938 suppressed production of IL-17 and neutrophilic inflammation and also markedly diminished airway hyperresponsiveness. Together, these findings suggest that orally available inverse agonists of RORγt might provide an effective therapy to treat glucocorticoid-resistant neutrophilic asthma.

Purpose All DT-LVAD patients must receive a palliative care consultation prior to implant. No guidelines exist for post-LVAD palliative care follow up to help patients cope with increasing illness burden. We describe our institution's experience with our health system's home-based palliative care (HBPalC) program. Methods We performed a chart review of DT-LVAD implants at our institution. The MGB HBPalC Program is staffed by an MD Medical Director, 3 nurse practitioners and a social worker. All HBPalC clinicians are certified in palliative care. Patients referred to the program are seen at home. The NP performs an initial assessment and collaborates with the LVAD team in developing a comprehensive care plan, with special attention to symptom management, documentation of hopes and worries, anticipatory planning and recommendations to mitigate unnecessary hospitalizations. Follow up visits are scheduled based on clinical and psychosocial needs. Results In 2018-20, 4 LVAD patients were referred to the HBPalC Program. They were elderly, met the criteria for frailty, and could no longer travel to clinic. The HBPalC team, working closely with LVAD team, managed 3 patients’ symptoms and transitioned them to hospice when appropriate. These patients died peacefully at home or at a hospice residence and none presented to the ED or were hospitalized while enrolled in the HBPalC Program. The 4th patient presented to the emergency room with somnolence and was hypotensive and tachypneic. The HBPalC provider and VAD coordinator collaborated to support the patient's daughter through the difficult decision to deactivate the LVAD, easing anxiety for the patient and her family, as well as preventing a readmission. Conclusion HBPalC is a valuable resource that can help to minimize symptom burden for patients as well as support patients and their families with the complicated transition to hospice, allowing them to die in the location of their choosing, which is often at home. As the number of older adults receiving DT-LVADs grows, the need for closer collaboration between LVAD programs and HBPalC programs will also continue to grow. At our institution, we are exploring workflows to expand this collaboration and are additionally working to develop a Geriatric Cardiology Clinic to better assess for and manage geriatric syndromes pre-implant with the goal of further improving outcomes for this patient population.

Purpose The purpose of this study is to determine pre-operative indicators associated with longer hospital stays in the first year post-LVAD implant impact on quality of life. We hypothesized that patients with more high risk features pre-implant would require longer and more frequent hospitalizations and that their quality of life with be lower post-implant. Methods We performed a retrospective analysis of LVAD patients from our institution from 2017 to 2019 who survived the initial implant hospital stay and to one year on support. Descriptive statistics and logistic regression modeling were performed with STATA 15.1. Results 95 patients were included in the study population (78% men, average age 59 years). Patients’ burden of hospitalization was analyzed two ways: 1) total number of days spent inpatient in the first year post-LVAD implant, including both the initial implant and readmissions (mean = 37.1, SD = 25.3) and 2) total days readmitted in the first year post-implant from initial discharge (mean = 14.3 days, SD = 18.1). Pre-implant BMI was higher in patients who were readmitted for more days (coefficient = 0.825, p = 0.019). Pre-implant creatinine was higher in patients who had a greater number of days readmitted (coefficient = 9.13, p = 0.023) and greater total number of days inpatient (coefficient = 11.4, p = 0.048). Patients who spent greater total time in the hospital post-implant also had lower pre-operative INTERMACS scores (coefficient = -7.37, p = 0.008). Patients also completed the EuroQoL EQ-5D at 1 year, where they were asked about their satisfaction with the outcome of their VAD surgery. Patients with higher total numbers of days inpatient during their first year post-implant reported lower satisfaction on this question (coefficient = -0.065, p = 0.004). Other components of the EuroQoL were not significantly different. Conclusion Pre-operative creatinine and INTERMACS profile are associated with higher total number of days spent in the hospital in the first year post-LVAD implant (initial implant and readmission) and higher BMI is associated with greater readmission days. There is also an association between greater number of days spent in the hospital and decreased satisfaction post-implant. This highlights the need to establish greater supports for the high risk LVAD patient populations to improve post-implant quality of life.

Allergic airway inflammation (typified by atopic asthma) is a classic example of type 2 immunity, which can be triggered by non-microbial environmental stimuli, such as allergens. Importantly, type 2 immunity engages the entire affected tissue and can involve the activity of not only immune cells, like Th2 cells and eosinophils, but also non-hematopoietic cells, like epithelial cells. Often, type 2 immunity exists in competition with type 1 immunity, classically defined as the activity of phagocytic cells, such as classically activated “M1” macrophages, and IFNg-producing adaptive immune cells, like CD8+ T cells and Th1 cells. Our past studies have demonstrated that deficiency in LC3-associated phagocytosis (LAP), a non-canonical form of autophagy requiring Rubcn, results in increased type 1 inflammation across a variety of stimuli, suggesting that LAP represents an important mechanism to regulate the inflammatory response. How Rubcn deficiency affects the onset and severity of type 2 immunity, such as allergic airway inflammation, remains unknown. Here, we demonstrate that, in response to house dust extract (HDE)/OVA exposure, lungs from Rubcn−/− mice contain increased levels of Th1 and Tr17 cells (potent regulatory CD4+ T cells that co-express FOXP3 and RORg), decreased Th2 cells, and decreased neutrophils, resulting in overall protection from allergic airway pathology. Rubcn-deficiency was required in non-hematopoietic cells, presumably airway epithelial cells, to exert this protective effect. As approximately 50% of asthmatics suffer from corticosteroid-resistant, neutrophil-mediated inflammation, inhibition of RUBCN could represent a novel therapy for steroid-resistant asthmatics.

Purpose The early post-heart transplantation (HT) period entails frequent biopsies, which poses a unique challenge to administration of therapeutic anticoagulation when indicated. Heightened sensitivity to enoxaparin has been described primarily in lung transplant recipients but has not been assessed in relation to anti-Xa levels specifically in HT recipients. Herein we report our institutional experience with a range of doses of enoxaparin relative to anti-Xa levels. Methods We examined HT recipients who received therapeutic anticoagulation with enoxaparin and included patients with steady state anti-Xa levels at appropriate post-dosing time intervals (i.e. 4 hours) and GFR > 30. Based on suspected heightened sensitivity to enoxaparin, we initiated doses below 1mg/kg every 12 hours (Q12). Spearman's rank correlation was used to relate non-parametric data. Results A total of 14 patients (2016-2019) were included. Average age at the time of enoxaparin exposure was 62 years, 71% were male. Four patients (29%) had a GFR 30-60 while 10 patients (71%) had a GFR > 60. Of 39 anti-Xa levels collected, levels correlated with dose of enoxaparin in mg/kg (rho=0.48, p=0.002, Figure) but not GFR (rho=0.04, p=0.80). Dosing of 1 mg/kg Q12 resulted in average anti-Xa levels above the therapeutic range (mean ± SEM: 1.13±0.23 IU/mL), whereas doses of 0.6mg/kg Q12 resulted in average levels of 0.75±0.07 IU/mL, the midpoint of the desired therapeutic anticoagulation range. Lower doses of enoxaparin (between 0.2-0.5 mg/kg Q12, including those used for prophylaxis) often resulted in anti-Xa levels approaching the therapeutic range (Figure). Conclusion In our small population of HT recipients requiring therapeutic anticoagulation, use of enoxaparin 0.6 mg/kg BID results in average anti-Xa levels at the midpoint of the therapeutic range (0.75 IU/mL) and represents a preferable dosing strategy to FDA-label dosing of 1mg/kg Q12.

Pulmonary dendritic cells (DCs) are potent antigen-presenting cells that can activate both naïve and memory/effector T cells. However, very little is known of how movements and localization of DCs contribute to these events. To study this, we have developed new procedures that combine precision cut lung slices with cell staining using fluorescently-tagged antibodies to detect individual cell types. In this chapter, we describe these methods in detail and show how they can be used to study the localization of not only DCs, but also other leukocytes of interest, as well as structural cells within the lung.

The induction of allergen-specific T helper 2 (Th2) cells by lung dendritic cells (DCs) is a critical step in allergic asthma development. Airway delivery of purified allergens or microbial products can promote Th2 priming by lung DCs, but how environmentally relevant quantities and combinations of these factors affect lung DC function is unclear. Here, we investigated the ability of house dust extract (HDE), which contains a mixture of environmental adjuvants, to prime Th2 responses against an innocuous inhaled antigen. Inhalational exposure to HDE conditioned lung conventional DCs, but not monocyte-derived DCs, to induce antigen-specific Th2 differentiation. Conditioning of DCs by HDE was independent of Toll-like receptor 4 signaling, indicating that environmental endotoxin is dispensable for programming DCs to induce Th2 responses. DCs directly treated with HDE underwent maturation but were poor stimulators of Th2 differentiation. In contrast, DCs treated with bronchoalveolar lavage fluid (BALF) from HDE-exposed mice induced robust Th2 differentiation. DC conditioning by BALF was independent of the proallergic cytokines IL-25, IL-33, and thymic stromal lymphopoietin. BALF treatment of DCs resulted in upregulation of CD80 but low expression of CD40, CD86, and IL-12p40, which was associated with Th2 induction. These findings support a model whereby environmental adjuvants in house dust indirectly program DCs to prime Th2 responses by triggering the release of endogenous soluble factor(s) by airway cells. Identifying these factors could lead to novel therapeutic targets for allergic asthma.

Allergic asthma is thought to stem largely from maladaptive T helper 2 (Th2) responses to inhaled allergens, which in turn lead to airway eosinophilia and airway hyperresponsiveness (AHR). However, many individuals with asthma have airway inflammation that is predominantly neutrophilic and resistant to treatment with inhaled glucocorticoids. An improved understanding of the molecular basis of this form of asthma might lead to improved strategies for its treatment. Here, we identify novel roles of the adaptor protein, TRIF (TIR-domain-containing adapter-inducing interferon-β), in neutrophilic responses to inhaled allergens. In different mouse models of asthma, Trif-deficient animals had marked reductions in interleukin (IL)-17, airway neutrophils, and AHR compared with wild-type (WT) mice, whereas airway eosinophils were generally similar in these two strains. Compared with lung dendritic cells (DCs) from WT mice, lung DCs from Trif-deficient mice displayed impaired lipopolysaccharide (LPS)-induced migration to regional lymph nodes, lower levels of the costimulatory molecule, CD40, and produced smaller amounts of the T helper 17 (Th17)-promoting cytokines, IL-6, and IL-1β. When cultured with allergen-specific, naive T cells, Trif-deficient lung DCs stimulated robust Th2 cell differentiation but very weak Th1 and Th17 cell differentiation. Together, these findings reveal a TRIF-CD40-Th17 axis in the development of IL-17-associated neutrophilic asthma.

Background: Arsenic exposure via drinking water impacts millions of people worldwide. Although arsenic has been associated epidemiologically with increased lung infections, the identity of the lung cell types targeted by peroral arsenic and the associated immune mechanisms remain poorly defined. Objectives: We aimed to determine the impact of peroral arsenic on pulmonary antibacterial host defense. Methods: Female C57BL/6 mice were administered drinking water with 0, 250 ppb, or 25 ppm sodium arsenite for 5 wk and then challenged intratracheally with Klebsiella pneumoniae, Streptococcus pneumoniae, or lipopolysaccharide. Bacterial clearance and immune responses were profiled. Results: Arsenic had no effect on bacterial clearance in the lung or on the intrapulmonary innate immune response to bacteria or lipopolysaccharide, as assessed by neutrophil recruitment to, and cytokine induction in, the airspace. Alveolar macrophage TNFα production was unaltered. By contrast, arsenic-exposed mice had significantly reduced plasma TNFα in response to systemic lipopolysaccharide challenge, together suggesting that the local airway innate immune response may be relatively preserved from arsenic intoxication. Despite intact intrapulmonary bacterial clearance during pneumonia, arsenic-exposed mice suffered dramatically increased bacterial dissemination to the bloodstream. Mechanistically, this was linked to increased respiratory epithelial permeability, as revealed by intratracheal FITC-dextran tracking, serum Club Cell protein 16 measurement, and other approaches. Consistent with barrier disruption at the alveolar level, arsenic-exposed mice had evidence for alveolar epithelial type 1 cell injury. Conclusions: Peroral arsenic has little effect on local airway immune responses to bacteria but compromises respiratory epithelial barrier integrity, increasing systemic translocation of inhaled pathogens and small molecules. https://doi.org/10.1289/EHP1878

Endometriosis is a gynecological disease that negatively affects the health of 1 in 10 women. Although more information is known about late stage disease, the early initiation of endometriosis and lesion development is poorly understood. Herein, we use a uterine tissue transfer mouse model of endometriosis to examine early disease development and its dependence on estradiol (E2) and estrogen receptor (ER) α within 72 hours of disease initiation. Using wild-type and ERα knockout mice as hosts or donors, we find substantial infiltration of neutrophils and macrophages into the peritoneal cavity. Examining cell infiltration, lesion gene expression, and peritoneal fluid, we find that E2/ERα plays a minor role in early lesion development. Immune-mediated signaling predominates E2-mediated signaling, but 48 hours after the initiation of disease, a blunted interleukin (IL)-6-mediated response is found in developing lesions lacking ERα. Our data provide evidence that the early initiation of endometriosis is predominantly dependent on the immune system, whereas E2/ERα/IL-6-mediated cross-talk plays a partial role. These findings suggest there are two phases of endometriosis—an immune-dependent phase and a hormone-dependent phase, and that targeting the innate immune system could prevent lesion attachment in this susceptible population of women., A mouse model of endometriosis, used to examine the early initiation of disease, revealed that two phases of disease exist—an immune-predominant phase and hormone-predominant phase.

Mechanisms that elicit mucosal TWe sought to understand whether maintenance of lung TAnimals were exposed repeatedly to aspiration of ovalbumin alone or together with environmental adjuvants, including common house dust extract (HDE), to test their role in maintaining lung inflammation. Alternatively, antigen-specific effector/memory TTT

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly, chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

Allergic asthma is largely driven by the actions of allergen-specific CD4+T helper cells. Following allergen inhalation, these cells are induced by the interaction of naïve CD4+ T cells with antigen-bearing dendritic cells (DCs) in lung-draining lymph nodes. The molecular mechanisms underlying the induction of T helper cells have been well studied, and include the migration of allergen-bearing DCs to lung-draining lymph nodes in a manner dependent on the chemokine receptor, CCR7. However, comparatively little is known regarding how these effector T cells become activated following their subsequent encounter with allergen in the lung. This knowledge would be useful in the design of novel therapies for asthma, especially for neutrophilic asthma, which is refractory to inhaled glucocorticoids. We recently noticed that lung DCs migrating to lymph nodes originate from the large airways, whereas DCs associated with small airways, parenchyma and subpleural areas are non-migratory. Video confocal analysis of precision cut lung slices revealed that these non-migratory DCs form tight interactions with OVA-specific T cells and stimulate T cell receptor signaling, as measured by activation of a Nur77-gfpreporter gene. Furthermore, Ccl19, which encodes a CCR7-ligand, is highly expressed by type 1 alveolar epithelial cells, and in mouse model of asthma, airway inflammation was significantly diminished in CCL19-deficient mice compared with WT mice. Together, our findings suggest that CCL19 produced by type I epithelial cells acts to position lung DCs in the distal area of the lung where they interact with effector T cells, thereby promoting allergic airway inflammation.

Background: Humans with asthma display considerable heterogeneity with regard to T helper (Th) 2–associated eosinophilic and Th17-associated neutrophilic inflammation, but the impact of the environment on these different forms of asthma is poorly understood. Objective: We studied the nature and longevity of asthma-like responses triggered by inhalation of allergen together with environmentally relevant doses of inhaled lipopolysaccharide (LPS). Methods: Ovalbumin (OVA) was instilled into the airways of mice together with a wide range of LPS doses. Following a single OVA challenge, or multiple challenges, animals were assessed for pulmonary cytokine production, airway inflammation, and airway hyperresponsiveness (AHR). Results: Mice instilled with OVA together with very low doses (≤ 10–3 μg) of LPS displayed modest amounts of Th2 cytokines, with associated airway eosinophilia and AHR after a single challenge, and these responses were sustained after multiple OVA challenges. When the higher but still environmentally relevant dose of 10–1 μg LPS was used, mice initially displayed similar Th2 responses, as well as Th17-associated neutrophilia. After multiple OVA challenges, however, the 10–1 μg LPS animals also accumulated large numbers of allergen-specific T regulatory (Treg) cells with high levels of inducible co-stimulatory molecule (ICOS). As a result, asthma-like features in these mice were shorter-lived than in mice sensitized using lower doses of LPS. Conclusions: The nature and longevity of Th2, Th17, and Treg immune responses to inhaled allergen are dependent on the quantity of LPS inhaled at the time of allergic sensitization. These findings might account in part for the heterogeneity of inflammatory infiltrates seen in lungs of asthmatics. Citation: Whitehead GS, Thomas SY, Cook DN. 2014. Modulation of distinct asthmatic phenotypes in mice by dose-dependent inhalation of microbial products. Environ Health Perspect 122:34–42; http://dx.doi.org/10.1289/ehp.1307280

Asthma is associated with exposure to a wide variety of allergens and adjuvants. The extent to which overlap exists between the cellular and molecular mechanisms triggered by these various agents is poorly understood, but it might explain the differential responsiveness of patients to specific therapies. In particular, it is unclear why some, but not all, patients benefit from blockade of TNF. Here, we characterized signaling pathways triggered by distinct types of adjuvants during allergic sensitization. Mice sensitized to an innocuous protein using TLR ligands or house dust extracts as adjuvants developed mixed eosinophilic and neutrophilic airway inflammation and airway hyperresponsiveness (AHR) following allergen challenge, whereas mice sensitized using proteases as adjuvants developed predominantly eosinophilic inflammation and AHR. TLR ligands, but not proteases, induced TNF during allergic sensitization. TNF signaled through airway epithelial cells to reprogram them and promote Th2, but not Th17, development in lymph nodes. TNF was also required during the allergen challenge phase for neutrophilic and eosinophilic inflammation. In contrast, TNF was dispensable for allergic airway disease in a protease-mediated model of asthma. These findings might help to explain why TNF blockade improves lung function in only some patients with asthma.

Resident intravascular NKT cells exacerbate airway hyperreactivity in mice dependent on dendritic cell co-presentation of glycolipid and peptide antigens., Airborne exposure to microbial cell wall lipids such as lipopolysaccharide triggers innate immune responses that regulate susceptibility to allergic airway inflammation. α-Glycosylceramides represent another widespread class of microbial lipids that directly stimulate innate-like, IL-4– and IL-13–producing, CD1d-restricted NKT cells. In this study, we demonstrate that NKT cells constitutively accumulate and reside in the microvasculature of the mouse lung. After a single airborne exposure to lipid antigen, they promptly extravasate to orchestrate the formation of peribronchiolar and interstitial lymphohistiocytic granulomas containing numerous eosinophils. Concomitant airborne exposure to ovalbumin (OVA) induces the priming of OVA-specific Th2 cells and IgE antibodies by the same dendritic cell coexpressing CD1d and MHC class II. Although NKT cell activation remains confined to the lipid-exposed lung and draining lymph nodes, Th2 cells recirculate and seed the lung of a parabiotic partner, conferring susceptibility to OVA challenge months after the initial exposure, in a manner independent of NKT cells and CD1d. Thus, transient recruitment and activation of lung-resident intravascular NKT cells can trigger long-term susceptibility to allergic airway inflammation.

PLZF-expressing NKT cells establish residence at intravascular locations, failing to enter the circulation because of constitutive interactions with LFA-1 and ICAM-1., Innate-like NKT cells conspicuously accumulate within the liver microvasculature of healthy mice, crawling on the luminal side of endothelial cells, but their general recirculation pattern and the mechanism of their intravascular behavior have not been elucidated. Using parabiotic mice, we demonstrated that, despite their intravascular location, most liver NKT cells failed to recirculate. Antibody blocking experiments established that they were retained locally through constitutive LFA-1–intercellular adhesion molecule (ICAM) 1 interactions. This unprecedented lifelong intravascular residence could be induced in conventional CD4 T cells by the sole expression of promyelocytic leukemia zinc finger (PLZF), a transcription factor specifically expressed in the NKT lineage. These findings reveal the unique genetic and biochemical pathway that underlies the innate intravascular surveillance program of NKT cells.

Background Mechanisms that elicit mucosal T H 17 cell responses have been described, yet how these cells are sustained in chronically inflamed tissues remains unclear. Objective We sought to understand whether maintenance of lung T H 17 inflammation requires environmental agents in addition to antigen and to identify the lung antigen-presenting cell (APC) types that sustain the self-renewal of T H 17 cells. Methods Animals were exposed repeatedly to aspiration of ovalbumin alone or together with environmental adjuvants, including common house dust extract (HDE), to test their role in maintaining lung inflammation. Alternatively, antigen-specific effector/memory T H 17 cells, generated in culture with CD4 + T cells from Il17a fate-mapping mice, were adoptively transferred to assess their persistence in genetically modified animals lacking distinct lung APC subsets or cell-specific Toll-like receptor (TLR) 4 signaling. T H 17 cells were also cocultured with lung APC subsets to determine which of these could revive their expansion and activation. Results T H 17 cells and the consequent neutrophilic inflammation were poorly sustained by inhaled antigen alone but were augmented by inhalation of antigen together with HDE. This was associated with weight loss and changes in lung physiology consistent with interstitial lung disease. The effect of HDE required TLR4 signaling predominantly in lung hematopoietic cells, including CD11c + cells. CD103 + and CD11b + conventional dendritic cells interacted directly with T H 17 cells in situ and revived the clonal expansion of T H 17 cells both ex vivo and in vivo , whereas lung macrophages and B cells could not. Conclusion T H 17-dependent inflammation in the lungs can be sustained by persistent TLR4-mediated activation of lung conventional dendritic cells.

Asthma is a multifactorial disease of the airways characterized by airway inflammation, mucus hypersecretion, and airway hyperresponsiveness. Conventional MHC class II–restricted CD4 + T cells are considered a key cell in asthma pathogenesis because they have a broad T-cell receptor repertoire, providing specificity and reactivity to diverse protein allergens. This notion was challenged when a study found that invariant Natural Killer (NK) T cells were the predominant T cells in the lung and bronchoalveolar lavage fluid of all asthmatic subjects studied. This finding was provocative because invariant NKT cells have a very limited T-cell receptor repertoire and are specific for a restricted set of lipid antigens that bind to CD1d, a nonpolymorphic MHC-like molecule. However, multiple subsequent studies failed to replicate the initial study and instead found that invariant NKT cells are present as a small fraction of the total T cells in the asthmatic lung. Thus, we believe that although CD1d-restricted NKT cells might play a role in modulating the asthmatic phenotype, they are not the critical drivers of the asthmatic response, a role we believe is still held by conventional MHC class II–restricted CD4 + T cells.

STAT6-mediated chemokine production in the lung is required for Th2 lymphocyte and eosinophil homing into the airways in allergic pulmonary inflammation, and thus is a potential therapeutic target in asthma. However, the critical cellular source of STAT6-mediated chemokine production has not been defined. In this study, we demonstrate that STAT6 in bone marrow-derived myeloid cells was sufficient for the production of CCL17, CCL22, CCL11, and CCL24 and for Th2 lymphocyte and eosinophil recruitment into the allergic airway. In contrast, STAT6 in airway-lining cells did not mediate chemokine production or support cellular recruitment. Selective depletion of CD11b+ myeloid cells in the lung identified these cells as the critical cellular source for the chemokines CCL17 and CCL22. These data reveal that CD11b+ myeloid cells in the lung help orchestrate the adaptive immune response in asthma, in part, through the production of STAT6-inducible chemokines and the recruitment of Th2 lymphocytes into the airway.

Cerebral malaria is a significant cause of global mortality, causing an estimated two million deaths per year, mainly in children. The pathogenesis of this disease remains incompletely understood. Chemokines have been implicated in the development of cerebral malaria, and the IFN-inducible CXCR3 chemokine ligand IP-10 (CXCL10) was recently found to be the only serum biomarker that predicted cerebral malaria mortality in Ghanaian children. We show that the CXCR3 chemokine ligands IP-10 and Mig (CXCL9) were highly induced in the brains of mice with murine cerebral malaria caused by Plasmodium berghei ANKA. Mice deficient in CXCR3 were markedly protected against cerebral malaria and had far fewer T cells in the brain compared with wild-type mice. In competitive transfer experiments, CXCR3-deficient CD8 + T cells were 7-fold less efficient at migrating into the infected brains than wild-type CD8 + T cells. Adoptive transfer of wild-type CD8 + effector T cells restored susceptibility of CXCR3-deficient mice to cerebral malaria and also restored brain proinflammatory cytokine and chemokine production and recruitment of T cells, independent of CXCR3. Mice deficient in IP-10 or Mig were both partially protected against cerebral malaria mortality when infected with P. berghei ANKA. Brain immunohistochemistry revealed Mig staining of endothelial cells, whereas IP-10 staining was mainly found in neurons. These data demonstrate that CXCR3 on CD8 + T cells is required for T cell recruitment into the brain and the development of murine cerebral malaria and suggest that the CXCR3 ligands Mig and IP-10 play distinct, nonredundant roles in the pathogenesis of this disease.

Human allergic asthma is a chronic inflammatory disease of the airways thought to be driven by allergen-specific Th2 cells, which are recruited into the lung in response to inhaled allergen. To identify chemoattractant receptors that control this homing pattern, we used endobronchial segmental allergen challenge in human atopic asthmatics to define the pattern of chemoattractant receptor expression on recruited T cells as well as the numbers of recruited CD1d-restricted NKT cells and levels of chemokines in the bronchoalveolar (BAL) fluid. CD1d-restricted NKT cells comprised only a small minority of BAL T cells before or after Ag challenge. BAL T cells were enriched in their expression of specific chemoattractant receptors compared with peripheral blood T cells prechallenge, including CCR5, CCR6, CXCR3, CXCR4, and BLT1. Surprisingly, following segmental allergen challenge, no chemoattractant receptor was specifically increased. However, CCR6 and CXCR3, which were expressed on virtually all CD4+ BAL T cells prechallenge, were markedly decreased on all recruited BAL T cells following Ag challenge, suggesting that these receptors were internalized following encounter with ligand in the airway. Our data therefore suggests a role for CCR6 and CXCR3, in conjunction with other chemoattractant receptors, in the recruitment of inflammatory T cells into the BAL during the allergic asthmatic response.

Th1 and Th2 effector CD4+ T cells orchestrate distinct counterregulatory biological responses. To deliver effective tissue Th1- and Th2-type responses, Th1 and Th2 cell recruitment into tissue must be differentially regulated. We show that tissue-derived STAT1 controls the trafficking of adoptively transferred, Ag-specific, wild-type Th1 cells into the lung. Trafficking of Th1 and Th2 cells is differentially regulated as STAT6, which regulates Th2 cell trafficking, had no effect on the trafficking of Th1 cells and STAT1 deficiency did not alter Th2 cell trafficking. We demonstrate that STAT1 control of Th1 cell trafficking is not mediated through T-bet. STAT1 controls the recruitment of Th1cells through the induction of CXCL9, CXCL10, CXCL11, and CXCL16, whose expression levels in the lung were markedly decreased in STAT1−/− mice. CXCL10 replacement partially restored Th1 cell trafficking in STAT1-deficient mice in vivo, and deficiency in CXCR3, the receptor for CXCL9, CXCL10, and CXCL11, impaired the trafficking of adoptively transferred Th1 cells in wild-type mice. Our work identifies that STAT1 in peripheral tissue regulates the homing of Ag-specific Th1 cells through the induction of a distinct subset of chemokines and establishes that Th1 and Th2 cell trafficking is differentially controlled in vivo by STAT1 and STAT6, respectively.

Chemokine receptor CCR7 guides T cell exit from peripheral tissues and entry into afferent lymphatics

Leukotriene B4 is a lipid mediator that recently has been shown to have potent chemotactic activity for effector T lymphocytes mediated through its receptor, BLT1. Here, we developed a novel murine model of acute lung rejection to demonstrate that BLT1 controls effector CD8+ T cell trafficking into the lung and that disruption of BLT1 signaling in CD8+ T cells reduces lung inflammation and mortality in the model. In addition, we used BLT1-deficient mice and a BLT1 antagonist in two tracheal transplant models of lung transplantation to demonstrate the importance of BLT1 for the recruitment of T cells into tracheal allografts. We also show that BLT1-mediated CD8+ T cell recruitment plays an important role in the development of airway fibroproliferation and obliteration. Finally, in human studies of lung transplant recipients, we found that BLT1 is up-regulated on T lymphocytes isolated from the airways of patients with obliterative bronchiolitis. These data demonstrate that BLT1 contributes to the development of lung rejection and obliterative bronchiolitis by mediating effector T lymphocyte trafficking into the lung. This is the first report that describes a pathologic role for BLT1-mediated T lymphocyte recruitment in disease and identifies BLT1 as a potential therapeutic target after lung transplantation.

Microbial products can prime antigen-specific T helper 17 cell responses to inhaled allergens. However, once established, it is unclear how pathogenic lung Th17 responses are sustained. To study this, we sensitized mice to inhaled ovalbumin (OVA)together with various environmental adjuvants, including house dust extract(HDE). Animals were then chronically challenged with OVA alone, or OVA plus the adjuvant previously used during sensitization. Challenge with OVA plus HDE elicited greater airway neutrophilia, lymphocytosis and IL-17A than did challenge with OVA alone. HDE exposure with OVA also promoted greater expansion of adoptively transferred, OVA-specific, IL-17A-fate mapping Th17 cells. These effects were independent of enzymatic activity in HDE, but required TLR4 in recipient mice. Analysis of Tlr4 bone marrow chimeric mice and conditional TLR4 knockout mice indicated that Th17 proliferation is sustained by HDE via TLR4 signaling predominantly in hematopoietic cells, including CD11c+ cells, rather than in airway epithelial cells. IL-23 contributes to the maintenance of these lung Th17 cells. Use of genetically modified mice lacking distinct lung myeloid cell subsets, and co-culture of various lung antigen-presenting cell types with mature Th17 cells, revealed that conventional dendritic cells (either CD103+ or CD11b+), rather than lung macrophages or B cells, support the revival of Th17 clonal expansion and activation. Thus, maintenance of Th17-dependent inflammation in the lung requires persistent innate immune responses, TLR4, and the presence of conventional dendritic cells.

Salts of the donor molecule, bis(ethylenedithio)tetrathiafulvalene (BEDT-TTF or ET), with pentafluorothiomethylsulfonate (SF{sub 5}CX{sub 2}SO{sub 3}{sup -}, X = H or F) anions have been prepared. Three phases, {beta}'-(ET){sub 2}SF{sub 5}CH{sub 2}SO{sub 3}, {beta}'-(ET){sub 2}SF{sub 5}CF{sub 2}SO{sub 3}, and {beta}'-(ET){sub 2}SF{sub 5}CHFSO{sub 3} were obtained by electrocrystallization with the corresponding LiSF{sub 5}CX{sub 2}SO{sub 3} electrolytes. The structures of these salts were determined by single-crystal X-ray diffraction, and their physical properties were examined by electrical resistivity measurements as well as by ESR and Raman spectroscopy. The {beta}'-(ET){sub 2}SF{sub 5}CH{sub 2}SO{sub 3}, {beta}'-(ET){sub 2}SF{sub 5}CHFSO{sub 3} and {beta}'-(ET){sub 2}SF{sub 5}CF{sub 2}SO{sub 3} salts are considerably different in their crystal structures, physical properties, and electronic structures despite the similarity in the structures of the SF{sub 5}CX{sub 2}SO{sub 3}{sup -} (X = H, F) anions. The {beta}'-(ET){sub 2}SF{sub 5}CH{sub 2}SO{sub 3} salt has two kinds of ET donor molecules with considerably different charge densities. The electronic structure of {beta}'-(ET){sub 2}SF{sub 5}CHFSO{sub 3} has both one-dimensional (1D) and two-dimensional (2D) Fermi surfaces which are similar to those found in the organic superconductor {beta}'-(ET){sub 2}SF{sub 5}CH{sub 2}CF{sub 2}SO{sub 3}. The ESR data for the {beta}'-(ET){sub 2}SF{sub 5}CF{sub 2}SO{sub 3} salt indicate that it opens a spin gapmore » below 45 K. The differences in the physical properties of the three salts were analyzed by calculating the HOMO-HOMO interaction energies between nearest-neighbor ET molecules in their donor molecule layers.« less

Electrocrystallization of ET in the presence of aromatic and aliphatic sulfonate anions has led to many new ET salts. These new ET based sulfonate complexes are characterized with use of x-ray diffraction, four-probe conductivity measurements, ESR and Raman spectroscopies. A new κ(4 × 4) donor packing motif was observed in (ET)2(C6H5CH2SO3)(H2O). Strong hydrogen bonding between the sulfonate anion S-O and the donor ethylene C-H provides the driving force for the ET salt formation. Many of these new sulfonate salts are highly conductive with some remaining metallic to ~ 4 K.

Tetraalkyl and dialkyl derivatives, where alkyl=ethyl and propyl, of the organic electron donor molecule bis(ethylenedithio)tetrathiafulvalene, BEDT-TTF or ET, have been synthesized via the Diels-Alder approach. Several cation-radical salts of these new donors have been prepared and structurally characterized, and found to contain donor molecules in nominally higher oxidation states (+1, +1.5 and +2) than the typically observed oxidation state of +0.5 in BEDT-TTF salts. The higher solubility of the tetraalkyl and dialkyl derivatives in solvents used for crystal growth is proposed as the principal reason for this finding. Surprisingly, X-ray crystallographic studies reveal that the alkyl groups in the neutral tetraethyl-ET as well as the oxidized tetraethyl-ET and diethyl-ET molecules in their cation-radical salts adopt axial configurations, rather than the expected equatorial configurations. Electrical properties of the cation-radical salts have been found to be either insulating or semiconducting, consistent with the higher oxidation states of the donor molecules in the salts and the crystal structures.

Allergic asthma is characterized by exaggerated lung T helper 2 (Th2) and Th17 responses which require signals that stimulate the innate immune system, provided by exposure to environmental adjuvants with an allergen. Once airway disease has developed, it is unclear as to whether the same mechanisms that initiated the disease remain important in sustaining it. We investigated the contribution of persistent innate danger signals in sustaining a chronic antigen-specific Th17 cell response. Animals were sensitized and challenged acutely by aspiration of ovalbumin (OVA) with different adjuvants, including house dust extract (HDE). They were then challenged for a further 5 weeks with OVA alone or OVA + adjuvant to test whether the adjuvant becomes redundant in sustaining airway inflammation once it has already been established. Removal of the adjuvant during the chronic challenges resulted in the animals displaying minimal airway inflammation, whereas continuous challenge with adjuvant and OVA caused significant inflammation. HDE was particularly potent at promoting airway neutrophilia, lymphocytosis and greater IL-17A production from lung explants restimulated ex vivo with OVA, suggesting that persistent exposure to this adjuvant amplified the antigen-specific Th17 response. Furthermore, CD4+ cells from OVA-receptor transgenic (OT-II) IL-17A-fate mapping mice were differentiated under Th17 polarizing conditions and adoptively transferred to recipients which were then challenged with either OVA alone, OVA + HDE or HDE alone. Persistent exposure to HDE and OVA, compared to either one alone, amplified the lung antigen-specific memory Th17 response, independently of the enzymatic activity of HDE but rather via TLR4 signaling.

T cells are critical mediators of the allergic airway inflammation seen in asthma. Pathogenic allergen-specific T cells are generated in regional lymph nodes and are then recruited into the airway by chemoattractants produced by the asthmatic lung. These recruited effector T cells and their products then mediate the cardinal features of asthma: airway eosinophilia, mucus hypersecretion, and airway hyperreactivity. There has been considerable progress in delineating the molecular mechanisms that control T cell trafficking into peripheral tissue, including the asthmatic lung. In this review, we summarize these advances and formulate them into a working model that proposes that T cell trafficking into and out of the allergic lung is controlled by several discrete regulatory pathways that involve the collaboration of innate and acquired immune cells.

Effector T cells significantly contribute to inflammatory diseases. These cells are recruited into tissue, where they orchestrate an inflammatory response that can either protect against infection or sometimes stimulate human disease. The recruitment of T cells into tissue from the blood and lymphoid compartments is an active process controlled by chemokines and the chemokine receptors expressed on distinct effector T-cell subsets. Thus, the chemokines secreted in the tissue will determine the specific types of T lymphocyte recruited into that tissue based on the chemokine receptors expressed on these cells. It follows that the chemokine receptor profile on T cells isolated from the lungs of patients with inflammatory pulmonary disease will define the subtype of pathogenic T lymphocytes mediating the disease process and will identify the mechanisms that recruit these cells into the lung. This article reviews data from both human and animal studies that define the chemokine receptors involved in the recruitment of T lymphocytes into the lung in various inflammatory pulmonary diseases, including asthma, obliterative bronchiolitis, sarcoidosis, and chronic eosinophilic pneumonia. We then speculate on the potential role of these chemokine receptors in the pathogenesis of these disorders and potential novel therapeutic approaches suggested by these data.

We have recently shown that the leukotriene B4 (LTB4)–BLT1 pathway is important in early effector T-cell recruitment in mouse models of inflammation. Here we characterize the phenotype and function of human peripheral blood BLT1+ T cells in health and illustrate their involvement in asthma and acute infection. In healthy individuals, BLT1+ T cells are a rare peripheral blood T-cell population enriched for the activation markers CD38 and HLA-DR. Compared with BLT1– T cells, a larger proportion of peripheral blood BLT1+ T cells express the effector cytokines IFNγ and IL-4 and inflammatory chemokine receptors, CCR1, CCR2, CCR6, and CXCR1. Consequently, in healthy individuals peripheral blood BLT1+ T cells are a rare antigen-primed T-cell subset with unique phenotypic, migratory, and functional properties. BLT1 expression on T cells is tightly regulated by inflammation and only transiently expressed after naive T-cell activation by dendritic cells. Although rare in the peripheral blood of healthy individuals, BLT1+ T cells are markedly increased in frequency in the peripheral blood in response to acute Epstein-Barr virus (EBV) infection and moderately increased in the airways of asymptomatic allergic asthmatics. Our studies provide novel insights into the LTB4-BLT1 lipid chemoattractant pathway in human T-cell responses, and how it may link innate and adaptive immunity.

Primary effusion lymphomas (PEL) are uniformly infected with Kaposi's sarcoma-associated herpesvirus (KSHV), and thus likely present both tumor and viral antigens to the immune system. In order to grow unrestricted and cause disease, multiple immune evasion strategies may be utilized by PEL to evade immune surveillance. Using six well-established PEL cell lines and comparing these to Epstein-Barr virus-transformed B cell lines and peripheral blood B cells, significant differences were found in the surface expression of molecules involved in antigen presentation, T cell activation and cell-cell adhesion. Significantly reduced stimulation of cytotoxic T lymphocytes, lowered sensitivity to natural killer cell-mediated lysis and impaired function as antigen presenting cells in mixed leukocyte reactions were found for three PEL cell lines with particularly low CD54, CD58 and CD81 expression. Comparative microarray analysis demonstrated specific patterns of KSHV-encoded gene expression that were associated with the different immune functions of these cell lines. Thus, the present data suggest that distinct patterns of KSHV gene expression may be associated with particular phenotypic and functional characteristics of PEL cells, which may influence PEL pathogenesis.

Most people infected with HIV-1 cannot control viral replication despite the presence of virus-specific CD8+ T cells. It has been postulated that this inability is related to the failure of these cells to mature into fully differentiated effector cells. We tested this hypothesis by comparing the maturation phenotype of virus-specific CD8+ T cells in people who could control viral replication off anti-retroviral therapy with those who could not. In five patients with treated acute HIV-1-infection, structured treatment interruption (STI) induced control of viral replication was associated with expansion of virus-specific CD8+ T cells with a fully differentiated effector phenotype. These effector cells were also expanded in treatment-naive chronically infected individuals who spontaneously controlled viral replication, and augmented expression of perforin was noted in both settings. Our data show that full maturation of virus-specific CD8+ T cells is possible in the context of HIV-1-infection, and suggest that such maturation might be important in viral control.

CD1d-restricted T cells (NKT cells) are innate memory cells activated by lipid Ags and play important roles in the initiation and regulation of the immune response. However, little is known about the trafficking patterns of these cells or the tissue compartment in which they exert their regulatory activity. In this study, we determined the chemokine receptor profile expressed by CD1d-restricted T cells found in the peripheral blood of healthy volunteers as well as CD1d-restricted T cell clones. CD1d-restricted T cells were identified by Abs recognizing the invariant Vα24 TCR rearrangement or by binding to CD1d-Fc fusion tetramers loaded with α-GalCer. CD1d-restricted T cells in the peripheral blood and CD1d-restricted T cell clones expressed high levels of CXCR3, CCR5, and CCR6; intermediate levels of CXCR4 and CXCR6; and low levels of CXCR1, CCR1, CCR2, and CX3CR1, a receptor pattern often associated with tissue-infiltrating effector Th1 cells and CD8+ T cells. Very few of these cells expressed the lymphoid-homing receptors CCR7 or CXCR5. CCR4 was expressed predominantly on CD4+, but not on double-negative CD1d-restricted T cells, which may indicate differential trafficking patterns for these two functionally distinct subsets. CD1d-restricted T cell clones responded to chemokine ligands for CXCR1/2, CXCR3, CXCR4, CXCR6, CCR4, and CCR5 in calcium flux and/or chemotaxis assays. These data indicate that CD1d-restricted T cells express a chemokine receptor profile most similar to Th1 inflammatory homing cells and suggest that these cells perform their function in peripheral tissue sites rather than in secondary lymphoid organs.

The HIV-1 accessory proteins Vpr, Vpu, and Vif are essential for viral replication, and their cytoplasmic production suggests that they should be processed for recognition by CTLs. However, the extent to which these proteins are targeted in natural infection, as well as precise CTL epitopes within them, remains to be defined. In this study, CTL responses against HIV-1 Vpr, Vpu, and Vif were analyzed in 60 HIV-1-infected individuals and 10 HIV-1-negative controls using overlapping peptides spanning the entire proteins. Peptide-specific IFN-γ production was measured by ELISPOT assay and flow-based intracellular cytokine quantification. HLA class I restriction and cytotoxic activity were confirmed after isolation of peptide-specific CD8+ T cell lines. CD8+ T cell responses against Vpr, Vpu, and Vif were found in 45%, 2%, and 33% of HIV-1-infected individuals, respectively. Multiple CTL epitopes were identified in functionally important regions of HIV-1 Vpr and Vif. Moreover, in infected individuals in whom the breadth of HIV-1-specific responses was assessed comprehensively, Vpr and p17 were the most preferentially targeted proteins per unit length by CD8+ T cells. These data indicate that despite the small size of these proteins Vif and Vpr are frequently targeted by CTL in natural HIV-1 infection and contribute importantly to the total HIV-1-specific CD8+ T cell responses. These findings will be important in evaluating the specificity and breadth of immune responses during acute and chronic infection, and in the design and testing of candidate HIV vaccines.

The host response to intracellular pathogens requires the coordinated action of both the innate and acquired immune systems. Chemokines play a critical role in the trafficking of immune cells and transitioning an innate immune response into an acquired response. We analyzed the host response of mice deficient in the chemokine receptor CCR5 following infection with the intracellular protozoan parasite Toxoplasma gondii. We found that CCR5 controls recruitment of natural killer (NK) cells into infected tissues. Without this influx of NK cells, tissues from CCR5-deficient (CCR5−/−) mice were less able to generate an inflammatory response, had decreased chemokine and interferon γ production, and had higher parasite burden. As a result, CCR5−/− mice were more susceptible to infection with T. gondii but were less susceptible to the immune-mediated tissue injury seen in certain inbred strains. Adoptive transfer of CCR5+/+ NK cells into CCR5−/− mice restored their ability to survive lethal T. gondii infection and demonstrated that CCR5 is required for NK cell homing into infected liver and spleen. This study establishes CCR5 as a critical receptor guiding NK cell trafficking in host defense., Synopsis Toxoplasma gondii, an obligate intracellular parasite, evokes a strong cellular immune response, which is critical for host protection. Chemokines and their receptors control the trafficking of immune cells to sites of infection. In this study, the authors examined the immune response to T. gondii in mice deficient in the chemokine receptor CCR5. CCR5-deficient mice exhibited a decreased inflammatory response, which led to a higher parasite burden and ultimate mortality. The deficiency in the immune response in CCR5-deficient mice was the result of decreased migration of natural killer (NK) cells into infected tissues. Transfer of normal NK cells into CCR5-deficient mice restored NK cell homing into infected tissue and the normal immune response capable of controlling T. gondii infection. These findings demonstrate an important role for CCR5 in trafficking the NK cells during acute T. gondii infection. Furthermore, since CCR5 is the main co-receptor for HIV-1 entry into cells, the results of this study raise a concern about the potential complication of impaired NK cell function in the clinical use of CCR5 antagonists, currently being developed as therapeutics for HIV-1 and inflammatory diseases. This may be of particular concern for HIV-1-infected patients coinfected with T. gondii or for those who acquire an acute T. gondii infection while taking a CCR5 antagonist.