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Regulatory T (Treg) cells are essential for maternal immune tolerance of the fetus and placenta. In preeclampsia, aberrant Treg cell tolerance is implicated, but how Treg cells affect the uterine vascular dysfunction thought to precede placental impairment and maternal vasculopathy is unclear. We used Foxp3-diphtheria toxin receptor mice to test the hypothesis that Treg cells are essential regulators of decidual spiral artery adaptation to pregnancy. Transient Treg cell depletion during early placental morphogenesis caused impaired remodeling of decidual spiral arteries, altered uterine artery function, and fewer Dolichos biflorus agglutinin+ uterine natural killer (uNK) cells, resulting in late-gestation fetal loss and fetal growth restriction. Replacing the Treg cells by transfer from wild-type donors mitigated the impact on uNK cells, vascular remodeling, and fetal loss. RNA sequencing of decidua revealed genes associated with NK cell function and placental extravillous trophoblasts were dysregulated after Treg cell depletion and normalized by Treg cell replacement. These data implicate Treg cells as essential upstream drivers of uterine vascular adaptation to pregnancy, through a mechanism likely involving phenotypic regulation of uNK cells and trophoblast invasion. The findings provide insight into mechanisms linking impaired adaptive immune tolerance and altered spiral artery remodeling, 2 hallmark features of preeclampsia.

Mechanisms underpinning signals from genome-wide association studies remain poorly understood, particularly for noncoding variation and for complex diseases such as type 2 diabetes mellitus (T2D) where pathogenic mechanisms in multiple different tissues may be disease driving. One approach is to study relevant endophenotypes, a strategy we applied to the UBE2E2 locus where noncoding single nucleotide variants (SNVs) are associated with both T2D and visceral adiposity (a pathologic endophenotype). We integrated CRISPR targeting of SNV-containing regions and unbiased CRISPR interference (CRISPRi) screening to establish candidate cis-regulatory regions, complemented by genetic loss of function in murine diet-induced obesity or ex vivo adipogenesis assays. Nomination of a single causal gene was complicated, however, because targeting of multiple genes near UBE2E2 attenuated adipogenesis in vitro; CRISPR excision of SNV-containing noncoding regions and a CRISPRi regulatory screen across the locus suggested concomitant regulation of UBE2E2, the more distant UBE2E1, and other neighborhood genes; and compound heterozygous loss of function of both Ube2e2 and Ube2e1 better replicated pathological adiposity and metabolic phenotypes compared with homozygous loss of either gene in isolation. This study advances a model whereby regulatory effects of noncoding variation not only extend beyond the nearest gene but may also drive complex diseases through polygenic regulatory effects.

Due to the limitations of available in vitro systems and animal models, we lack a detailed understanding of the pathogenetic mechanisms of and have minimal treatment options for liver fibrosis. Therefore, we engineered a live-cell imaging system that assessed fibrosis in a human multilineage hepatic organoid in a microwell (i.e., microHOs). Transcriptomic analysis revealed that TGFB converted mesenchymal cells in microHOs into myofibroblast-like cells resembling those in fibrotic human liver tissue. When pro-fibrotic intracellular signaling pathways were examined, the antifibrotic effect of receptor-specific tyrosine kinase inhibitors was limited to the fibrosis induced by the corresponding growth factor, which indicates their antifibrotic efficacy would be limited to fibrotic diseases solely mediated by that growth factor. Based upon transcriptomic and transcription factor activation analyses in microHOs, glycogen synthase kinase 3β and p38 MAPK inhibitors were identified as potential new broad-spectrum therapies for liver fibrosis. Other new therapies could subsequently be identified using the microHO system.

Diabetes mellitus (DM) is acknowledged as an independent risk factor for acute kidney injury. Ras guanine nucleotide-releasing protein-4 (RasGRP4) exerts a notable role in modulating immune-inflammatory responses and kidney disease progression in diabetes. Herein, we delved into the specific role and mechanism of RasGRP4 in diabetic renal ischemia-reperfusion injury. Diabetes was induced by a high-fat diet and streptozocin (STZ) injections, followed by creating an ischemia-reperfusion kidney injury via renal pedicle clamping and reperfusion. In vitro, a high glucose and hypoxia-reoxygenation modeled cellular inflammatory injury. We found RasGRP4-KO mice, compared with C57BL/6J (WT) mice, showed markedly less renal dysfunction and fibrosis in diabetic ischemia-reperfusion injury. There was a significant decrease in the renal infiltration of M1 macrophages and Th17 cells, along with downregulated IL-17 pathway proteins and effectors. In vitro, RasGRP4 deletion restrained M1 macrophage polarization and Th17 cell differentiation, inhibiting the IL-17 signaling pathway in HK-2 cells. Hyperglycemia intensified renal inflammation state. Together, RasGRP4, through the regulation of interactions among M1 macrophages, CD4+ T cells, and HK-2 cells, formed a cascade that intensified the inflammatory storm activity, ultimately exacerbating the inflammatory injury of diabetic ischemia-reperfusion kidneys. DM intensified this inflammatory injury mechanism, worsening the injury from renal ischemia-reperfusion.

Peptidoglycans (PGNs) are structural polymers of the bacterial cell wall and a common microbial molecular pattern encountered by the immune system daily. Low levels of PGNs are constitutively present in the systemic circulation in humans and rise during inflammatory pathologies. Since all known PGN sensors are intracellular, PGN internalization is a prerequisite for the initiation of cellular immune responses. Here, we report the mechanisms controlling the recognition and uptake of polymeric PGNs by circulating human mononuclear phagocytes. We found that complement C3 and C4 opsonins govern PGN recognition and internalization, but no single opsonin is indispensable because of multiple uptake redundancies. We observed a bimodal internalization of polymeric PGNs with distinct requirements for complement C4. At low PGN concentrations, C3 mediated PGN recognition by surface receptors while the efficient internalization of PGN polymers critically required C4. Supraphysiologic PGN concentrations triggered a secondary uptake modality that was insensitive to C4 and mediated instead by C3 engagement of complement receptors 1 and 3. To our knowledge, this is the first description of nonoverlapping C3 and C4 opsonophagocytoses working in parallel. Controlling these uptake mechanisms has the potential to modulate PGN clearance or the dysregulated immune responses during bacterial infections.

Ocular hypertension, believed to result partly from increased contractile activity, cell adhesive interactions, and stiffness within the trabecular meshwork (TM), is a major risk factor for glaucoma, a leading cause of blindness. However, the identity of molecular mechanisms governing organization of actomyosin and cell adhesive interactions in the TM remains limited. Based on our previous findings, in which proteomics analyses revealed elevated levels of septins, including septin-9 in human TM cells treated with the ocular hypertensive agent dexamethasone, here, we evaluated the effects of septin-9 overexpression, deficiency, and pharmacological targeting in TM cells. These studies demonstrated a profound impact on actomyosin organization, cell adhesion, contraction, and phagocytosis. Overexpression raised intraocular pressure (IOP) in mice, while inhibition increased cell permeability. In addition, we replicated a significant association between a common variant (rs9038) in SEPT9 with IOP in the Genetic Epidemiology Research on Adult Healthy and Aging (GERA) cohort. Collectively, these data reveal a link between dysregulated septin cytoskeletal organization in the TM and increased IOP, likely due to enhanced cell contraction, adhesive interactions, and fibrotic activity. This suggests that targeting the septin cytoskeleton could offer a novel approach for lowering IOP in patients with glaucoma.

African green monkeys (AGMs) are natural hosts of SIV whose infection does not progress to AIDS. Since early events of infection may be critical to pathogenesis in nonnatural hosts, we investigated early SIV infection in 29 adult male AGMs intrarectally inoculated with SIVsab92018 (SIVsab) and serially sacrificed throughout acute into early chronic infection to understand patterns of viral establishment, dissemination, and their effect on disease progression. Using this model, we showed that foci of virus replication could be detected at the site of inoculation and in the draining lymphatics as early as 1-3 days postinfection (dpi). Furthermore, testing with ultrasensitive assays showed rapid onset of viremia (2-4 dpi). After systemic spread, virus was detected in all tissues surveyed. Multiple transmitted/founder viruses were identified, confirming an optimal challenge dose, while demonstrating a moderate mucosal genetic bottleneck. Resident CD4+ T cells were the initial target cells; other immune cell populations were not significantly altered at the site of entry. Thus, intrarectal SIVsab infection is characterized by swift dissemination of the virus, a lack of major target cell recruitment, and no window of opportunity for interventions to prevent virus dissemination during the earliest stages of infection, similar to intrarectal transmission but different from vaginal transmission in macaques.

Type 2 diabetes (T2D) arises when pancreatic β cells fail to produce sufficient insulin to control blood glucose appropriately. Aberrant nutrient sensing by O-GlcNAcylation and mTORC1 is linked to T2D and the failure of insulin-producing β cells. However, the nature of their crosstalk in β cells remains unexplored. Recently, O-GlcNAcylation, a posttranslation modification controlled by enzymes O-GlcNAc transferase/O-GlcNAcase (OGT/OGA), emerged as a pivotal regulator for β cell health; deficiency in either enzyme causes β cell failure. The present study investigates the previously unidentified connection between nutrient sensor OGT and mTORC1 crosstalk to regulate β cell mass and function in vivo. We show reduced OGT and mTORC1 activity in islets of a preclinical β cell dysfunction model and islets from humans with obesity. Using loss or gain of function of OGT, we identified that O-GlcNAcylation positively regulated mTORC1 signaling in β cells. O-GlcNAcylation negatively modulated autophagy, as the removal of OGT increased autophagy, while the deletion of OGA decreased it. Increasing mTORC1 signaling, via deletion of TSC2, alleviated the diabetic phenotypes by increasing β cell mass but not β cell function in OGT-deficient mice. Downstream phospho-protein signaling analyses revealed diverging effects on MKK4 and calmodulin signaling between islets with OGT, TSC2, or combined deletion. These data provide evidence of OGT's significance as an upstream regulator of mTORC1 and autophagy, crucial for the regulation of β cell function and glucose homeostasis.

Lineage plasticity mediates resistance to androgen receptor pathway inhibitors (ARPIs) and progression from adenocarcinoma to neuroendocrine prostate cancer (NEPC), a highly aggressive and poorly understood subtype. Neuronal transcription factor ASCL1 has emerged as a central regulator of the lineage plasticity driving neuroendocrine differentiation. Here, we showed that ASCL1 was reprogrammed in ARPI-induced transition to terminal NEPC and identified that the ASCL1 binding pattern tailored the expression of lineage-determinant transcription factor combinations that underlie discrete terminal NEPC identity. Notably, we identified FOXA2 as a major cofactor of ASCL1 in terminal NEPC, which is highly expressed in ASCL1-driven NEPC. Mechanistically, FOXA2 and ASCL1 interacted and worked in concert to orchestrate terminal neuronal differentiation. We identified that prospero homeobox 1 was a target of ASCL1 and FOXA2. Targeting prospero homeobox 1 abrogated neuroendocrine characteristics and led to a decrease in cell proliferation in vitro and tumor growth in vivo. Our findings provide insights into the molecular conduit underlying the interplay between different lineage-determinant transcription factors to support the neuroendocrine identity and nominate prospero homeobox 1 as a potential target in ASCL1-high NEPC.

Mechanical loading, essential for bone health, promotes bone formation and remodeling. However, the positive response diminishes in cases of disuse and aging, leading to bone loss and an increased fracture risk. This study demonstrates that activating hemichannels (HCs) using a connexin 43 (Cx43) antibody, Cx43(M2), in bone osteocytes revitalizes aging and disused bones. Using a hindlimb suspension (HLS) disuse model and a tibial mechanical loading model, we found that Cx43(M2) inhibited bone loss and osteocyte apoptosis induced by unloading in 16-week-old adult mice. Additionally, it enhanced bone mass in response to tibial loading in 22-month-old aged mice. The HC opening released bone anabolic factor prostaglandin E2 (PGE2) and suppressed catabolic factor sclerostin (SOST). This suppressed the increase of cortical bone formation and reduction of bone resorption during unloading and promoted trabecular and cortical bone formation during loading. Cx43(M2)-induced HC opening, coupled with PGE2 release, effectively rescued unloading-induced bone loss and restored the diminished anabolic response of aged bones to mechanical loading. Activating HCs with the Cx43 antibody holds promise as a de novo therapeutic approach, as it can overcome the limitations of existing treatment regimens for treating bone loss and osteoporosis associated with aging and disuse.