Krüppel-like factor 3 (KLF3) suppresses NF-κB-driven inflammation in mice.

Bacterial products such as lipopolysaccharides (or endotoxin) cause systemic inflammation, resulting in a substantial global health burden. The onset, progression, and resolution of the inflammatory response to endotoxin are usually tightly controlled to avoid chronic inflammation. Members of the NF-κB family of transcription factors are key drivers of inflammation that activate sets of genes in response to inflammatory signals. Such responses are typically short-lived and can be suppressed by proteins that act post-translationally, such as the SOCS (suppressor of cytokine signaling) family. Less is known about direct transcriptional regulation of these responses, however. Here, using a combination of in vitro approaches and in vivo animal models, we show that endotoxin treatment induced expression of the well-characterized transcriptional repressor Krüppel-like factor 3 (KLF3), which, in turn, directly repressed the expression of the NF-κB family member RELA/p65. We also observed that KLF3-deficient mice were hypersensitive to endotoxin and exhibited elevated levels of circulating Ly6C+ monocytes and macrophage-derived inflammatory cytokines. These findings reveal that KLF3 is a fundamental suppressor that operates as a feedback inhibitor of RELA/p65 and may be important in facilitating the resolution of inflammation.

A Negative Feedback Loop Regulates Integrin Inactivation and Promotes Neutrophil Recruitment to Inflammatory Sites.

Neutrophils are abundant circulating leukocytes that are rapidly recruited to sites of inflammation in an integrin-dependent fashion. Contrasting with the well-characterized regulation of integrin activation, mechanisms regulating integrin inactivation remain largely obscure. Using mouse neutrophils, we demonstrate in this study that the GTPase activating protein ARAP3 is a critical regulator of integrin inactivation; experiments with Chinese hamster ovary cells indicate that this is not restricted to neutrophils. Specifically, ARAP3 acts in a negative feedback loop downstream of PI3K to regulate integrin inactivation. Integrin ligand binding drives the activation of PI3K and of its effectors, including ARAP3, by outside-in signaling. ARAP3, in turn, promotes localized integrin inactivation by negative inside-out signaling. This negative feedback loop reduces integrin-mediated PI3K activity, with ARAP3 effectively switching off its own activator, while promoting turnover of substrate adhesions. In vitro, ARAP3-deficient neutrophils display defective PIP3 polarization, adhesion turnover, and transendothelial migration. In vivo, ARAP3-deficient neutrophils are characterized by a neutrophil-autonomous recruitment defect to sites of inflammation.

First insights into the vertical habitat use of the whitespotted eagle ray Aetobatus narinari revealed by pop-up satellite archival tags.

The whitespotted eagle ray Aetobatus narinari is a tropical to warm-temperate benthopelagic batoid that ranges widely throughout the western Atlantic Ocean. Despite conservation concerns for the species, its vertical habitat use and diving behaviour remain unknown. Patterns and drivers in the depth distribution of A. narinari were investigated at two separate locations, the western North Atlantic (Islands of Bermuda) and the eastern Gulf of Mexico (Sarasota, Florida, U.S.A.). Between 2010 and 2014, seven pop-up satellite archival tags were attached to A. narinari using three methods: a through-tail suture, an external tail-band and through-wing attachment. Retention time ranged from 0 to 180 days, with tags attached via the through-tail method retained longest. Tagged rays spent the majority of time (82.85 ± 12.17% S.D.) within the upper 10 m of the water column and, with one exception, no rays travelled deeper than ~26 m. One Bermuda ray recorded a maximum depth of 50.5 m, suggesting that these animals make excursions off the fore-reef slope of the Bermuda Platform. Individuals occupied deeper depths (7.42 ± 3.99 m S.D.) during the day versus night (4.90 ± 2.89 m S.D.), which may be explained by foraging and/or predator avoidance. Each individual experienced a significant difference in depth and temperature distributions over the diel cycle. There was evidence that mean hourly depth was best described by location and individual variation using a generalized additive mixed model approach. This is the first study to compare depth distributions of A. narinari from different locations and describe the thermal habitat for this species. Our study highlights the importance of region in describing A. narinari depth use, which may be relevant when developing management plans, whilst demonstrating that diel patterns appear to hold across individuals.

Krüppel-like Factor 3 (KLF3/BKLF) Is Required for Widespread Repression of the Inflammatory Modulator Galectin-3 (Lgals3).

The Lgals3 gene encodes a multifunctional ß-galactoside-binding protein, galectin-3. Galectin-3 has been implicated in a broad range of biological processes from chemotaxis and inflammation to fibrosis and apoptosis. The role of galectin-3 as a modulator of inflammation has been studied intensively, and recent evidence suggests that it may serve as a protective factor in obesity and other metabolic disorders. Despite considerable interest in galectin-3, little is known about its physiological regulation at the transcriptional level. Here, using knockout mice, chromatin immunoprecipitations, and cellular and molecular analyses, we show that the zinc finger transcription factor Krüppel-like factor 3 (KLF3) directly represses galectin-3 transcription. We find that galectin-3 is broadly up-regulated in KLF3-deficient mouse tissues, that KLF3 occupies regulatory regions of the Lgals3 gene, and that KLF3 directly binds its cognate elements (CACCC boxes) in the galectin-3 promoter and represses its activation in cellular assays. We also provide mechanistic insights into the regulation of Lgals3, demonstrating that C-terminal binding protein (CtBP) is required to drive optimal KLF3-mediated silencing. These findings help to enhance our understanding of how expression of the inflammatory modulator galectin-3 is controlled, opening up avenues for potential therapeutic interventions in the future.

PLD1 rather than PLD2 regulates phorbol-ester-, adhesion-dependent and Fc{gamma}-receptor-stimulated ROS production in neutrophils.

The signalling lipid phosphatidic acid (PA) is generated by the hydrolysis of phosphatidylcholine (PC), which is catalysed by phospholipase D (PLD) enzymes. Neutrophils, important cells of the innate immune system, maintain the body's defence against infection. Previous studies have implicated PLD-generated PA in neutrophil function; these have relied heavily on the use of primary alcohols to act as inhibitors of PA production. The recent development of isoform-selective small molecule inhibitors and the generation of a knockout mouse model provide us with accurate tools to study the role of PLDs in neutrophil responses. We show that PLD1 is a regulator of phorbol-ester-, chemoattractant, adhesion-dependent and Fcγ-receptor-stimulated production of reactive oxygen species (ROS) in neutrophils. Significantly we found that this role of PLD is isoform specific: the absence of PLD2 does not negatively affect these processes. Contrary to expectation, other functions required for an efficient immune response operate effectively in Pld2-deficient neutrophils or when both isoforms are inhibited pharmacologically. We conclude that although PLD1 does have important regulatory roles in neutrophils, the field has been confused by the use of primary alcohols; now that gold standard Pld-knockout mouse models are available, previous work might need to be reassessed.

The GTPase-activating protein ARAP3 regulates chemotaxis and adhesion-dependent processes in neutrophils.

Neutrophils form a vital part of the innate immune response, but at the same time their inappropriate activation contributes to autoimmune diseases. Many molecular components are involved in fine-tuning neutrophil function. We report here the first characterization of the role of ARAP3, a PI3K and Rap-regulated GTPase-activating protein for RhoA and Arf6 in murine neutrophils. We show that neutrophils lacking ARAP3 are preactivated in vitro and in vivo, exhibiting increased ß2 integrin affinity and avidity. ARAP3-deficient neutrophils are hyperresponsive in several adhesion-dependent situations in vitro, including the formation of reactive oxygen species, adhesion, spreading, and granule release. ARAP3-deficient cells adhere more firmly under flow conditions in vitro and to the vessel wall in vivo. Finally, loss of ARAP3 interferes with integrin-dependent neutrophil chemotaxis. The results of the present study suggest an important function of ARAP3 downstream of Rap. By modulating ß2 integrin activity, ARAP3 guards neutrophils in their quiescent state unless activated.

Impaired B cell development in the absence of Krüppel-like factor 3.

Krüppel-like factor 3 (Klf3) is a member of the Klf family of transcription factors. Klfs are widely expressed and have diverse roles in development and differentiation. In this study, we examine the function of Klf3 in B cell development by studying B lymphopoiesis in a Klf3 knockout mouse model. We show that B cell differentiation is significantly impaired in the bone marrow, spleen, and peritoneal cavity of Klf3 null mice and confirm that the defects are cell autonomous. In the bone marrow, there is a reduction in immature B cells, whereas recirculating mature cells are noticeably increased. Immunohistology of the spleen reveals a poorly structured marginal zone (MZ) that may in part be caused by deregulation of adhesion molecules on MZ B cells. In the peritoneal cavity, there are significant defects in B1 B cell development. We also report that the loss of Klf3 in MZ B cells is associated with reduced BCR signaling strength and an impaired ability to respond to LPS stimulation. Finally, we show increased expression of a number of Klf genes in Klf3 null B cells, suggesting that a Klf regulatory network may exist in B cells.

Effect Modification of Racial Differences in Pediatric COVID-19 Inpatient Admissions in a Large Healthcare Database.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has been more severe in racial and ethnic minorities relative to non-Hispanic White populations. Here, we investigate how these disparities vary across effect modifiers in a pediatric population. METHODS: Using the TriNetX Dataworks Network from April 2020 to September 2021, we compared inpatient rates between non-Hispanic Black and non-Hispanic White patients among pediatric COVID-19 cases. Following propensity score matching, comparisons were performed within subgroups of 4 potential effect modifiers: age group (0-2, 3-5, 6-11 and 12-18 years), presence of complex comorbidities, quarter of the year (from 2020 Q2 to 2021 Q3) and geographic regions of the United States. RESULTS: The cohort included 47,487 COVID-19 cases, of which 13,130 were Black patients. Among most subgroups of effect modifiers, inpatient rates were higher among the Black patients. The largest variation in disparities was observed across age groups and the presence of complex comorbidities. Twelve to 18 years old Black children had a 1.7% point [confidence interval (CI): 0.8-2.4] higher inpatient rate than the matched White children, whereas 0-2 years old Black children had a 2.5% point (CI: 0.9-4.1) lower rate than the matched White children. Among children with complex comorbidities, inpatient rates for Black children was 6.2 (CI: 3.4-8.9) percentage points higher than the White children; however, among kids without complex comorbidities, inpatient rates were comparable. CONCLUSIONS: Among pediatric patients experiencing COVID-19, higher inpatient rates for Black compared with White patients were observed among older children and those with complex comorbidities. These findings can spur discussions of potential root causes of these disparities, including structural racism.

ESCAPE the Boring Lecture: Tips and Tricks on Building Puzzles for Medical Education Escape Rooms.

Escape rooms in medical education are a novel, game-based learning approach for teaching medical topics. In these escape rooms, learners complete a sequential series of medical-themed puzzles leading them to "escape" a specific story. Designing puzzles can be anxiety-provoking and may be the gatekeeper for educators in medicine to create their own escape rooms. Though there have been publications on the importance and methods of building a healthcare-themed-escape room, there is a gap in the literature on designing puzzles to teach specific learning objectives successfully. In this Scholarly Perspective, the authors share puzzle ideas and support tools and use Bloom's taxonomy as the framework to teach educators how to design challenging and engaging escape room puzzles.

Inpatient and Outpatient Differences in Pediatric Patients with Laboratory-confirmed COVID-19.

Among 30,286 pediatric inpatient and outpatient encounters with laboratory-confirmed COVID-19 seen at one of 40 US healthcare organizations, 1586 (5.2%) were inpatient. Encounter types varied by age and sex; the proportion of Black/African American inpatients was significantly higher than outpatients, and Hispanic/Latinx children made up nearly one-fourth of patients.

Improving management of ventilator associated tracheitis in a level IV NICU.

BACKGROUND: There are no published guidelines regarding the diagnosis and treatment of ventilator-associated tracheitis (VAT) in the neonatal intensive care unit (NICU). VAT is likely over-diagnosed and over-treated, increasing antibiotic burden and cost. LOCAL PROBLEM: Diagnosis and treatment of VAT were entirely NICU provider dependent. METHODS: Retrospective pre- and post-intervention chart reviews were performed. INTERVENTIONS: A VAT diagnosis and treatment algorithm was created for use in the care of intubated patients without tracheostomies. 3 plan-do-study-act (PDSA) cycles were used to implement change. RESULTS: Intubated patients treated for VAT with <25 PMNs on Gram stain decreased from 79% to 35% following the quality improvement (QI) initiative. Treatment of VAT with >7 days of antibiotic therapy decreased from 42% to 10%. CONCLUSION: Implementing a QI initiative to improve the diagnosis and treatment of VAT in the NICU decreased the percent of patients treated inappropriately for VAT.

The treatment of streptococcal tonsillitis/pharyngitis in young children.

Pharyngitis is common in children, accounting for nearly 12 million visits annually in the United States. Streptococcus pyogenes or group A streptococcus (GAS) is the most common bacterial cause of pharyngitis for which antibiotics are indicated. Antibiotic treatment of streptococcal pharyngitis virtually eliminates the presence of bacteria from the pharynx and thus removes the risk of subsequent rheumatic fever. GAS is spread from person to person via respiratory droplets with a short incubation period of 2∼5 days. GAS pharyngitis peaks in the late winter and early spring months when children are predominately indoors for school and sports. Colonization is also higher in winter months, and while up to 20% of school age children are colonized with GAS in their throat during this time, colonization has not been shown to contribute to the spread of disease. In low- and middle-income countries and other situations in which crowding is common (e.g., schools), outbreaks of pharyngitis are common. GAS pharyngitis can occur at all ages and it is most common in school-aged children with a peak at 7∼8 years of age. Pharyngitis caused by GAS is rare in children <3 years of age and becomes much less common in late adolescence through adulthood.

Implementation of a Pharmacist-Driven Penicillin and Cephalosporin Allergy Assessment Tool: A Pilot Evaluation.

OBJECTIVE: Penicillin is the most commonly reported drug allergy despite the low incidence of true immune-mediated reactions. Penicillin allergy labels have been shown to lead to significant patient, institutional, and public health care consequences. This project's purpose was to improve quality of care for patients with penicillin and cephalosporin allergies, admitted to a pediatric institution, by implementation of a pharmacist-driven allergy assessment tool. METHODS: A group of physicians, pharmacists, and a nurse collaborated for process development. The process was standardized, and a tool was created to assist with assessments. Pharmacists were educated on the importance of this quality improvement project and trained on the process and tool used. Implementation occurred on March 2, 2020. RESULTS: During the 3-month implementation period, 40 patients were admitted with a documented penicillin or cephalosporin allergy. Of these, 11 patients (27.5%) received an allergy assessment. Most were identified as having low or moderate risk of recurrent reaction with future use of a penicillin or cephalosporin agent (81.8%), and 2 patients (18.2%) were de-labeled from their documented allergy. CONCLUSIONS: Penicillin and cephalosporin allergy assessment implementation at a pediatric hospital was successfully implemented and allowed for identification and initiation of future quality improvement projects including implementation of penicillin skin testing and direct oral amoxicillin challenges.

Eosinophil function in adipose tissue is regulated by Krüppel-like factor 3 (KLF3).

The conversion of white adipocytes to thermogenic beige adipocytes represents a potential mechanism to treat obesity and related metabolic disorders. However, the mechanisms involved in converting white to beige adipose tissue remain incompletely understood. Here we show profound beiging in a genetic mouse model lacking the transcriptional repressor Krüppel-like factor 3 (KLF3). Bone marrow transplants from these animals confer the beige phenotype on wild type recipients. Analysis of the cellular and molecular changes reveal an accumulation of eosinophils in adipose tissue. We examine the transcriptomic profile of adipose-resident eosinophils and posit that KLF3 regulates adipose tissue function via transcriptional control of secreted molecules linked to beiging. Furthermore, we provide evidence that eosinophils may directly act on adipocytes to drive beiging and highlight the critical role of these little-understood immune cells in thermogenesis.

Reducing Streptococcal Testing in Patients <3 Years Old in an Emergency Department.

BACKGROUND: Although pharyngitis is common, group A Streptococcus is an uncommon etiology, and sequelae are rare in patients <3 years old. Inappropriate testing leads to increased cost of health care and unnecessary exposure to antibiotics. Rapid streptococcal tests (RSTs) for group A Streptococcus pharyngitis are not routinely indicated in this age group. At our urban, tertiary pediatric emergency department (ED), on average, 20 RSTs were performed each month for patients <3 years of age. Our objective was to reduce RSTs in the ED in patients aged <3 years by 50% in 18 months. METHODS: We initiated this project in October 2016 at an urban, tertiary pediatric ED. We surveyed pertinent multidisciplinary stakeholders to identify factors leading to RSTs in children <3 years of age. We conducted multiple interventions and collected weekly data on the number of RSTs in children aged <3 years (outcome measure) and the number of family complaints and return visits for complications of pharyngitis (balancing measure). We used statistical process control for analysis. RESULTS: The mean number of RSTs ordered per month in patients aged <3 years declined by 52% in 10 months. The majority of tests during the study phase were ordered by nurse practitioners (62%) for patients aged 25 to 36 months (66%). There has been 1 family grievance and no patient complications attributable to the project. CONCLUSIONS: Our interventions led to a successful and sustained reduction of RSTs in patients aged <3 years. A local clinical practice guideline was developed, and the project was expanded to other acute care settings.

The effect of dedicated breast surgeons on the short-term outcomes in breast cancer.

OBJECTIVE: The impact of breast surgeons on short-term outcomes in breast cancer care was compared at a single institution. SUMMARY BACKGROUND DATA: Many studies have demonstrated a correlation between high procedural volume and lower mortality in technically challenging procedures. Breast cancer treatment has significant impact on patient behavior, psychology, and appearance. Therefore, evaluation of outcomes cannot be limited to only operative mortality and morbidity. We sought to determine the effect of dedicated breast cancer surgeons on short-term outcomes at a single institution. METHODS: Wishard Memorial Hospital is the county hospital affiliated with the Indiana University School of Medicine. A retrospective review was performed of all patients from January 1, 1997, to February 28, 2006. On July 1, 2003, coverage for the Breast Clinic was changed from general surgeons (G) to breast surgeons (B). There were 596 patients included in the study period. RESULTS: There were no significant differences in patient demographics or disease characteristics between the 2 time periods. For early stage (stage I and II) breast cancer, a higher percentage of patients underwent breast conservation in the breast surgeon period than in the general surgeon period (P = 0.04). Lumpectomy margins in breast conserving operations during the G period were more often positive (P = 0.025) or close (<1 mm) (P = 0.01). Similarly, the rates of re-excision lumpectomy were also significantly lower during the B period (21% vs. 39%, respectively, P = 0.01). Breast surgeons were more likely to perform the sentinel node procedure (P = 0.001). There were no differences in the use of adjuvant chemotherapy and radiation therapy. The use of hormonal manipulation, however, was significantly higher in the B group than in the G group (P < 0.0002). CONCLUSIONS: Surgeons specialized in diseases of the breast demonstrate significant improvement in short-term outcomes associated with breast cancer treatment at a single institution. The differences identified cannot be attributed to differences in institutional function, patient population, surgeon case volume, or on the influence of nonsurgeon physicians.

Improving Guideline-Based Streptococcal Pharyngitis Testing: A Quality Improvement Initiative.

BACKGROUND AND OBJECTIVES: Acute pharyngitis is a common diagnosis in ambulatory pediatrics. The Infectious Diseases Society of America (IDSA) clinical practice guideline for group A streptococcal (GAS) pharyngitis recommends strict criteria for GAS testing to avoid misdiagnosis and unnecessary treatment of children who are colonized with group A Streptococcus. We sought to improve adherence to the IDSA guideline for testing and treatment of GAS pharyngitis in a large community pediatrics practice. METHODS: The Model for Improvement was used, and iterative Plan-Do-Study-Act cycles were completed. The quality improvement project was approved for American Board of Pediatrics Part 4 Maintenance of Certification credit. Interventions included provider education, modification of existing office procedure, communication strategies, and patient and family education. Outcomes were assessed by using statistical process control charts. RESULTS: An absolute reduction in unnecessary GAS testing of 23.5% (from 64% to 40.5%) was observed during the project. Presence of viral symptoms was the primary reason for unnecessary testing. Appropriate antibiotic use for GAS pharyngitis did not significantly change during the project; although, inappropriate use was primarily related to unnecessary testing. At the end of the intervention period, the majority of providers perceived an improvement in their ability to communicate with families about the need for GAS pharyngitis testing and about antibiotic use. CONCLUSIONS: The majority of GAS pharyngitis testing in this practice before intervention was inconsistent with IDSA guideline recommendations. A quality improvement initiative, which was approved for Part 4 Maintenance of Certification credit, led to improvement in guideline-based testing for GAS pharyngitis.

Natural regulatory mutations elevate the fetal globin gene via disruption of BCL11A or ZBTB7A binding.

ß-hemoglobinopathies such as sickle cell disease (SCD) and ß-thalassemia result from mutations in the adult HBB (ß-globin) gene. Reactivating the developmentally silenced fetal HBG1 and HBG2 (γ-globin) genes is a therapeutic goal for treating SCD and ß-thalassemia 1 . Some forms of hereditary persistence of fetal hemoglobin (HPFH), a rare benign condition in which individuals express the γ-globin gene throughout adulthood, are caused by point mutations in the γ-globin gene promoter at regions residing ~115 and 200 bp upstream of the transcription start site. We found that the major fetal globin gene repressors BCL11A and ZBTB7A (also known as LRF) directly bound to the sites at -115 and -200 bp, respectively. Furthermore, introduction of naturally occurring HPFH-associated mutations into erythroid cells by CRISPR-Cas9 disrupted repressor binding and raised γ-globin gene expression. These findings clarify how these HPFH-associated mutations operate and demonstrate that BCL11A and ZBTB7A are major direct repressors of the fetal globin gene.

KLF1 directly activates expression of the novel fetal globin repressor ZBTB7A/LRF in erythroid cells.

Genes encoding the human ß-like hemoglobin proteins undergo a developmental switch from fetal γ-globin to adult ß-globin expression around the time of birth. ß-hemoglobinopathies, such as sickle-cell disease and ß-thalassemia, result from mutations affecting the adult ß-globin gene. The only treatment options currently available carry significant adverse effects. Analyses of heritable variations in fetal hemoglobin (HbF) levels have provided evidence that reactivation of the silenced fetal γ-globin genes in adult erythroid cells is a promising therapy. The γ-globin repressor BCL11A has become the major focus, with several studies investigating its regulation and function as a first step to inhibiting its expression or activity. However, a second repression mechanism was recently shown to be mediated by the transcription factor ZBTB7A/LRF, suggesting that understanding the regulation of ZBTB7A may also be useful. Here we show that Krüppel-like factor 1 (KLF1) directly drives expression of ZBTB7A in erythroid cells by binding to its proximal promoter. We have also uncovered an erythroid-specific regulation mechanism, leading to the upregulation of a novel ZBTB7A transcript in the erythroid compartment. The demonstration that ZBTB7A, like BCL11A, is a KLF1 target gene also fits with the observation that reduced KLF1 expression or activity is associated with HbF derepression.