The Deubiquitylase Otub1 Regulates the Chemotactic Response of Splenic B Cells by Modulating the Stability of the γ-Subunit Gng2.

The ubiquitin proteasome system performs the covalent attachment of lysine 48-linked polyubiquitin chains to substrate proteins, thereby targeting them for degradation, while deubiquitylating enzymes (DUBs) reverse this process. This posttranslational modification regulates key features both of innate and adaptative immunity, including antigen presentation, protein homeostasis and signal transduction. Here we show that loss of one of the most highly expressed DUBs, Otub1, results in changes in murine splenic B cell subsets, leading to a significant increase in marginal zone and transitional B cells and a concomitant decrease in follicular B cells. We demonstrate that Otub1 interacts with the γ-subunit of the heterotrimeric G protein, Gng2, and modulates its ubiquitylation status, thereby controlling Gng2 stability. Proximal mapping of Gng2 revealed an enrichment in partners associated with chemokine signaling, actin cytoskeleton and cell migration. In line with these findings, we show that Otub1-deficient B cells exhibit greater Ca2+ mobilization, F-actin polymerization and chemotactic responsiveness to Cxcl12, Cxcl13 and S1P in vitro, which manifests in vivo as altered localization of B cells within the spleen. Together, our data establishes Otub1 as a novel regulator of G-protein coupled receptor signaling in B cells, regulating their differentiation and positioning in the spleen.

Nuclear segmentation facilitates neutrophil migration.

Neutrophils are among the fastest-moving immune cells. Their speed is critical to their function as 'first responder' cells at sites of damage or infection, and it has been postulated that the unique segmented nucleus of neutrophils functions to assist their rapid migration. Here, we tested this hypothesis by imaging primary human neutrophils traversing narrow channels in custom-designed microfluidic devices. Individuals were given an intravenous low dose of endotoxin to elicit recruitment of neutrophils into the blood with a high diversity of nuclear phenotypes, ranging from hypo- to hyper-segmented. Both by cell sorting of neutrophils from the blood using markers that correlate with lobularity and by directly quantifying the migration of neutrophils with distinct lobe numbers, we found that neutrophils with one or two nuclear lobes were significantly slower to traverse narrower channels, compared to neutrophils with more than two nuclear lobes. Thus, our data show that nuclear segmentation in primary human neutrophils provides a speed advantage during migration through confined spaces.

Pre-existing chromatin accessibility and gene expression differences among naive CD4+ T cells influence effector potential.

CD4+ T cells have a remarkable potential to differentiate into diverse effector lineages following activation. Here, we probe the heterogeneity present among naive CD4+ T cells before encountering their cognate antigen to ask whether their effector potential is modulated by pre-existing transcriptional and chromatin landscape differences. Single-cell RNA sequencing shows that key drivers of variability are genes involved in T cell receptor (TCR) signaling. Using CD5 expression as a readout of the strength of tonic TCR interactions with self-peptide MHC, and sorting on the ends of this self-reactivity spectrum, we find that pre-existing transcriptional differences among naive CD4+ T cells impact follicular helper T (TFH) cell versus non-TFH effector lineage choice. Moreover, our data implicate TCR signal strength during thymic development in establishing differences in naive CD4+ T cell chromatin landscapes that ultimately shape their effector potential.

Migration-induced cell shattering due to DOCK8 deficiency causes a type 2-biased helper T cell response.

Mutations that impact immune cell migration and result in immune deficiency illustrate the importance of cell movement in host defense. In humans, loss-of-function mutations in DOCK8, a guanine exchange factor involved in hematopoietic cell migration, lead to immunodeficiency and, paradoxically, allergic disease. Here, we demonstrate that, like humans, Dock8-/- mice have a profound type 2 CD4+ helper T (TH2) cell bias upon pulmonary infection with Cryptococcus neoformans and other non-TH2 stimuli. We found that recruited Dock8-/-CX3CR1+ mononuclear phagocytes are exquisitely sensitive to migration-induced cell shattering, releasing interleukin (IL)-1ß that drives granulocyte-macrophage colony-stimulating factor (GM-CSF) production by CD4+ T cells. Blocking IL-1ß, GM-CSF or caspase activation eliminated the type-2 skew in mice lacking Dock8. Notably, treatment of infected wild-type mice with apoptotic cells significantly increased GM-CSF production and TH2 cell differentiation. This reveals an important role for cell death in driving type 2 signals during infection, which may have implications for understanding the etiology of type 2 CD4+ T cell responses in allergic disease.

Regulatory T cells differ from conventional CD4+ T cells in their recirculatory behavior and lymph node transit times.

Regulatory T cells (Tregs) continuously suppress autoreactive immune responses within tissues to prevent autoimmunity, yet the recirculatory behavior of Tregs between and within tissues enabling the maintenance of peripheral tolerance remains incompletely defined. Here, we quantified homing efficiency to and the dwell time of Tregs within secondary lymphoid organs (SLOs) and used intravital two-photon microscopy to measure Treg surveillance behavior of dendritic cells. Tregs homed substantially less efficiently to SLOs compared with conventional CD4+ T cells (Tconvs), despite similar expression of homing receptors. Tregs remained on average 2-3 times longer within the LN than Tconvs before exiting, and retained Tregs differed from recirculating Tregs in phenotype, motility and interaction duration with dendritic cells. Taken together, these data revealed fundamental differences in Treg versus conventional T cell in vivo recirculation and migration behaviors, identified a Treg population with prolonged LN dwell time, and provided quantitative insight into their spatiotemporal behavior within LNs.

Disparities in access to emergency general surgery care in the United States.

BACKGROUND: As fewer surgeons take emergency general surgery call and hospitals decrease emergency services, a crisis in access looms in the United States. We examined national emergency general surgery capacity and county-level determinants of access to emergency general surgery care with special attention to disparities. METHODS: To identify potential emergency general surgery hospitals, we queried the database of the American Hospital Association for "acute care general hospital," with "surgical services," and "emergency department," and ≥1 "operating room." Internet search and direct contact confirmed emergency general surgery services that covered the emergency room 7 days a week, 24 hours a day. Geographic and population-level emergency general surgery access was derived from Geographic Information Systems and US Census. RESULTS: Of the 6,356 hospitals in the 2013 American Hospital Association database, only 2,811 were emergency general surgery hospitals. Counties with greater percentages of black, Hispanic, uninsured, and low-education individuals and rural counties disproportionately lacked access to emergency general surgery care. For example, counties above the 75th percentile of African American population (10.2%) had >80% odds of not having an emergency general surgery hospital compared with counties below the 25th percentile of African American population (0.6%). CONCLUSION: Gaps in access to emergency general surgery services exist across the United States, disproportionately affecting underserved, rural communities. Policy initiatives need to increase emergency general surgery capacity nationwide.