Transcription factors IRF8 and PU.1 are required for follicular B cell development and BCL6-driven germinal center responses.

The IRF and Ets families of transcription factors regulate the expression of a range of genes involved in immune cell development and function. However, the understanding of the molecular mechanisms of each family member has been limited due to their redundancy and broad effects on multiple lineages of cells. Here, we report that double deletion of floxed Irf8 and Spi1 (encoding PU.1) by Mb1-Cre (designated DKO mice) in the B cell lineage resulted in severe defects in the development of follicular and germinal center (GC) B cells. Class-switch recombination and antibody affinity maturation were also compromised in DKO mice. RNA-seq (sequencing) and ChIP-seq analyses revealed distinct IRF8 and PU.1 target genes in follicular and activated B cells. DKO B cells had diminished expression of target genes vital for maintaining follicular B cell identity and GC development. Moreover, our findings reveal that expression of B-cell lymphoma protein 6 (BCL6), which is critical for development of germinal center B cells, is dependent on IRF8 and PU.1 in vivo, providing a mechanism for the critical role for IRF8 and PU.1 in the development of GC B cells.

Outcomes of a Resident-Led Early Hospital Discharge Intervention.

BACKGROUND: Early morning patient discharge from the hospital is increasingly being recognized as a key dimension of quality of care. At our institution, there is a significantly lower early discharge rate on the teaching hospitalist teams in comparison with the non-teaching teams. OBJECTIVE: To implement a resident-driven intervention in the teaching medical services to increase overall discharge order rate before 11 am (DOB-11) and assess the effect of this intervention on hospital length of stay (LOS), 30-day readmission rates (RR), and resident perception. DESIGN: Interrupted time series as well as controlled before-after designs. PARTICIPANTS: All inpatients discharged from general medicine units. INTERVENTIONS: We implemented an educational didactic in conjunction with resident-attending daily walk rounds followed by resident-led multidisciplinary discharge huddles to identify next-day discharges. MAIN MEASURES: The primary outcome was DOB-11 rates 18 months pre- and 12 months post-intervention. SECONDARY OUTCOMES: LOS and RR. Additionally, we assessed residents' perception of the early discharge protocol. KEY RESULTS: The DOB-11 rate increased from 12 to 29% (p < 0.001), LOS increased by 1.47 days (P < 0.001), and RR increased by 0.32% (P = 0.84), respectively, on the teaching teams. Compared with the non-teaching (control) teams, the teaching teams registered a greater increase in DOB-11 rate (by 17%, p < 0.001; ratio of adjusted ORs 2.16; 95% CI, 1.65, 2.85; p value < 0.001), small increase in LOS (by 0.74 day, p = 0.39; ratio of adjusted post-/pre-intervention ratio [teaching] and post-/pre- intervention ratio [non-teaching] = 1.05, 95% CI, 0.97, 1.14, p = 0.23), and relative increase in RR (by 3.98%, p = 0.07, and ratio of ORs = 1.35, 95% CI, 1.03, 1.8), p = 0.03). Approximately 55% (16/29) of the residents agreed that the early discharge initiative helped in understanding the importance of prioritizing patients for early discharge. Additionally, 55% (20/36) of the residents "agreed" that the early discharge initiative compromised their learning during teaching rounds. CONCLUSION: Our study demonstrates that DOB-11 is an achievable goal, not only for non-teaching teams but also for resident-run teaching teams.

Cutting Edge: Expression of IRF8 in Gastric Epithelial Cells Confers Protective Innate Immunity against Helicobacter pylori Infection.

IFN regulatory factor 8 (IRF8) is expressed in many types of blood cells and plays critical roles in cellular differentiation and function. However, the role of IRF8 in nonhematopoietic systems remains poorly understood. In this study, we provide evidence that IRF8 is a transcriptional modulator of the gastric mucosa necessary for limiting Helicobacter pylori colonization. H. pylori infection significantly upregulated expression of IRF8, which, in turn, promoted IFN-γ expression by gastric epithelial cells. Mice deficient in IRF8 exhibited increased H. pylori colonization and aborted induction of mucosal IFN-γ. Genome-wide analyses of IFN-γ-treated gastric epithelial cells by chromatin immunoprecipitation sequencing and RNA sequencing led to the identification of IRF8 target genes, with many belonging to the IFN-regulated gene family that was observed previously in immune cells. Our results identify the IRF8-IFN-γ circuit as a novel gastric innate immune mechanism in the host defense against infection with H. pylori.

Plasma cell alloantigen ENPP1 is expressed by a subset of human B cells with potential regulatory functions.

Plasma cell alloantigen 1 (PC1), also known as ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), is an enzyme involved primarily in hydrolysis of adenosine triphosphate at the cell surface. Although the expression pattern of PC1 is relatively broad, its expression in B cells is found at significant levels only in terminally differentiated germinal center B cells, plasma cells and a subset of B-1a cells in mice. Here we describe studies designed to determine whether expression of PC1 might define novel populations of human B cells with similarities to mouse B cells. We found that PC1 is expressed in small populations of human B lineage cells in peripheral blood, cord blood, tonsils, bone marrow and pediatric peritoneal fluid, with the highest levels in plasma cells. The characteristics of human PC1(+) B cells differ from mouse peritoneal B-1a subsets and from features of the human CD20(+)CD27(+)CD43(+)CD70(-) B-cell subset proposed to be human B-1 cells. Expression of PC1 was greatly increased in B cells stimulated with the combination of CD40 ligand, interleukin (IL)-4 and IL-21. In addition, PC1(+) B cells activated CD4(+) T regulatory cells. ENPP1 thus defines a subset of human B cells that differs significantly from mouse peritoneal B-1a and proposed human B-1 cells.

Expression of plasma cell alloantigen 1 defines layered development of B-1a B-cell subsets with distinct innate-like functions.

Innate-like B-1a cells contribute significantly to circulating natural antibodies and mucosal immunity as well as to immunoregulation. Here we show that these classic functions of B-1a cells segregate between two unique subsets defined by expression of plasma cell alloantigen 1 (PC1), also known as ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1). These subsets, designated B-1a.PC1(lo) and B-1a.PC1(hi), differ significantly in IgH chain utilization. Adoptively transferred PC1(lo) cells secreted significantly more circulating natural IgM and intestinal IgA than PC1(hi) cells. In contrast, PC1(hi) cells produced more IL-10 than PC1(lo) cells when stimulated with LPS and phorbol 12-myristate 13-acetate (PMA). PC1(hi) cells were also more efficient than PC1(lo) cells in regulating Th1 cell differentiation, even though both B-1a subsets were comparably active in stimulating T-cell proliferation. Furthermore, PC1(lo) cells generated antigen-specific IgM responses to pneumococcal polysaccharide antigens, whereas PC1(hi) cells do not. We found that PC1(lo) cells develop from an early wave of B-1a progenitors in fetal life, whereas PC1(hi) cells are generated from a later wave after birth. We conclude that identification of B-1a.PC1(lo) and B-1a.PC1(hi) cells extends the concept of a layered immune system with important implications for developing effective vaccines and promoting the generation of immunoregulatory B cells.

Capecitabine-Induced Enterocolitis.

Capecitabine is an oral fluoropyrimidine carbamate chemotherapy agent approved by the United States Food and Drug Administration (FDA) for the treatment of metastatic colorectal and breast cancer. The common side effects associated with it include gastrointestinal (GI) upset, abdominal pain, palmar-plantar erythrodysesthesia, fatigue, alopecia, leukopenia, neutropenia, thrombocytopenia, anemia, and hyperbilirubinemia. Although GI symptoms are relatively common, enterocolitis is one of the rare side effects of this drug. We present a case of 53-year-old female who developed severe enterocolitis leading to ileus secondary to capecitabine chemotherapy for metastatic breast cancer. She was treated successfully via conservative management.

IFN regulatory factor 8 regulates MDM2 in germinal center B cells.

IFN regulatory factor 8 (IRF8) is a transcription factor that affects the differentiation and function of myeloid, dendritic, and B cells. Herein we report that IRF8 regulates the expression of Mdm2, a suppressor of p53-dependent and -independent apoptosis pathways, in germinal center (GC) B cells. In GC B cells of IRF8-deficient mice, Mdm2 transcripts were greatly down-regulated, and MDM2 protein was poorly expressed in GC of Irf8(-/-) mice. Small interfering RNA-induced repression of IRF8 in a GC-derived B cell line resulted in decreased expression of MDM2 at the protein level but increased expression of p53 and p21. We found that IRF8 binds to the Mdm2 P2 promoter, and that cotransfection of an IRF8 expression vector with an Mdm2 reporter construct stimulated significant increases in reporter activity. Additionally, transcripts of the p53 target Pmaip1 (Noxa) were significantly increased in IRF8-deficient GC B cells as well as in the IRF8 knockdown B cell line. Finally, cells deficient in IRF8 exhibited growth suppression and increased sensitivity to apoptosis induced by etoposide or IL-21. These results suggest that by regulating MDM2, IRF8 might allow GC B cells to tolerate physiological DNA breaks that otherwise would trigger apoptosis.

IRF8 regulates B-cell lineage specification, commitment, and differentiation.

PU.1, IKAROS, E2A, EBF, and PAX5 comprise a transcriptional network that orchestrates B-cell lineage specification, commitment, and differentiation. Here we identify interferon regulatory factor 8 (IRF8) as another component of this complex, and show that it also modulates lineage choice by hematopoietic stem cells (HSCs). IRF8 binds directly to an IRF8/Ets consensus sequence located in promoter regions of Sfpi1 and Ebf1, which encode PU.1 and EBF, respectively, and is associated with transcriptional repression of Sfpi1 and transcriptional activation of Ebf1. Bone marrows of IRF8 knockout mice (IRF8(-/-)) had significantly reduced numbers of pre-pro-B cells and increased numbers of myeloid cells. Although HSCs of IRF8(-/-) mice failed to differentiate to B220(+) B-lineage cells in vitro, the defect could be rescued by transfecting HSCs with wild-type but not with a signaling-deficient IRF8 mutant. In contrast, overexpression of IRF8 in HSC-differentiated progenitor cells resulted in growth inhibition and apoptosis. We also found that IRF8 was expressed at higher levels in pre-pro-B cells than more mature B cells in wild-type mice. Together, these results indicate that IRF8 modulates lineage choice by HSCs and is part of the transcriptional network governing B-cell lineage specification, commitment, and differentiation.

The transformation of hospital medicine to tackle the COVID-19 pandemic crisis.

The coronavirus disease 2019 (COVID-19) pandemic is placing extraordinary strains not only on hospital-wide systems but most especially on hospital medicine across the nation. The specific challenges faced by our hospitalist services are unfathomable. Hospitalist leaders are tasked to rapidly restructure clinical operations to accommodate the large surge in COVID-19 patients. In this perspective, we focus on the management strategies conducted by the Division of Hospital Medicine to tackle the major crisis that specifically impacted the general medicine services.

A Process Approach to Decreasing Hospital Onset Clostridium difficile Infections.

BACKGROUND: Health care facility-onset Clostridium difficile infections (HO-CDI) contribute to prolonged hospital stays, inappropriate antimicrobial use, increased readmissions, and excess expenditures for health care institutions. National guidelines define appropriate C. difficile testing criteria as ≥ 3 unformed stools within a 24-hour period and the absence of laxative administration within 48 hours, criteria developed to reduce inappropriate reporting. METHODS: Stony Brook University Hospital (SBUH) quality improvement team implemented a process approach aimed at decreasing HO-CDI events. Through a number of improvement cycles in 2016, SBUH implemented (1) Information Technology hard stops and alert systems to enforce appropriate specimen collection and laboratory testing, (2) incorporation of an antimicrobial stewardship program, (3) heightened room turnover monitoring, and (4) an extensive educational module. Outcome measures included HO-CDI rate per 10,000 patient days and testing volume. RESULTS: The analysis timelines were divided into three periods: baseline (January 2014 - November 2014), Rejection of Formed Stools/Electronic Alert (December 2014 - September 2015) and Laxative Rule (October 2015 - July 2018). The average monthly HO-CDI rate at baseline of 11.94 (SD: 2.86) per 10,000 patient days had fallen to 7.35 (SD: 2.91) for the Laxative Rule period (p < 0.0001). Baseline average lab testing volume decreased from 290.27 tests per month (SD: 22.61) to 177.21 (SD: 33.24) in the Laxative Rule period (p < 0.0001). Hospital surveillance systems confirmed no undiagnosed missed cases within the postimplementation period. CONCLUSIONS: By reducing inappropriate testing and hardwiring best-practice guidelines into a system with real-time monitoring, a sustainable decrease in hospitalwide HO-CDI rates was observed.

ATP-degrading ENPP1 is required for survival (or persistence) of long-lived plasma cells.

Survival of antibody-secreting plasma cells (PCs) is vital for sustained antibody production. However, it remains poorly understood how long-lived PCs (LLPCs) are generated and maintained. Here we report that ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is preferentially upregulated in bone marrow LLPCs compared with their splenic short-lived counterparts (SLPCs). We studied ENPP1-deficient mice (Enpp1 -/- ) to determine how the enzyme affects PC biology. Although Enpp1 -/- mice generated normal levels of germinal center B cells and plasmablasts in periphery, they produced significantly reduced numbers of LLPCs following immunization with T-dependent antigens or infection with plasmodium C. chabaudi. Bone marrow chimeric mice showed B cell intrinsic effect of ENPP1 selectively on generation of bone marrow as well as splenic LLPCs. Moreover, Enpp1 -/- PCs took up less glucose and had lower levels of glycolysis than those of wild-type controls. Thus, ENPP1 deficiency confers an energetic disadvantage to PCs for long-term survival and antibody production.

Characterization of monoclonal antibodies to the plasma cell alloantigen ENPP1.

The ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) has documented roles in mineralization, nucleotide recycling, and insulin resistance. While ENPP1 was first identified as an alloantigen on mouse plasma cells (PCs), later studies revealed expression in many tissues. Previously described monoclonal antibodies against ENPP1 expressed at the cell surface recognized cells only from mice bearing the a allotype, ENPP1(a), precluding studies of mice bearing the alternative allele, ENPP1(b). Here, we characterize a novel anti-ENPP1 monoclonal antibody that recognizes both alleles and can be used for flow cytometry.