Journal of Clinical Investigation最新文献

Osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) has been recognized as the principal mechanism underlying vascular calcification (VC). Runt-related transcription factor 2 (RUNX2) in VSMCs plays a pivotal role because it constitutes an essential osteogenic transcription factor for bone formation. As a key DNA demethylation enzyme, ten-eleven translocation 2 (TET2) is crucial in maintaining the VSMC phenotype. However, whether TET2 involves in VC progression remains elusive. Here we identified a substantial downregulation of TET2 in calcified human and mouse arteries, as well as human primary VSMCs. In vitro gain- and loss-of function experiments demonstrated TET2 regulated VC. Subsequently, in vivo knockdown of TET2 significantly exacerbated VC in both vitamin D3 and adenine-diet-induced chronic kidney disease (CKD) mice models. Mechanistically, TET2 binds to and suppresses the activity of the P2 promoter within the RUNX2 gene, whereas an enzymatic loss-of-function mutation of TET2 has a comparable effect. Furthermore, TET2 forms a complex with histone deacetylases 1/2 (HDAC1/2 ) to deacetylate H3K27ac on the P2 promoter, thereby inhibiting its transcription. Moreover, SNIP1 is indispensable for TET2 to interact with HDAC1/2 to exert inhibitory effect on VC, and knockdown of SNIP1 accelerated VC in mice. Collectively, our findings imply that TET2 might serve as a potential therapeutic target for VC.

Phosphorylation of Smad3 is a critical mediator of TGF-β signaling, which plays an important role in regulating innate immune responses. However, whether Smad3 activation can be regulated in innate immune cells in TGF-β-independent contexts remains poorly understood. Here, we show that Smad3 is activated through the phosphorylation of its C-terminal residues (pSmad3C) in murine and human macrophages in response to bacterial and viral ligands, which is mediated by Activin A in a TGF-β independent manner. Specifically, infectious ligands, such as LPS, induced secretion of Activin A through the transcription factor STAT5 in macrophages, and Activin A signaling in turn activated pSmad3C. This Activin A-Smad3 axis controlled the mitochondrial ATP production and ATP conversion into adenosine by CD73 in macrophages, enforcing an anti-inflammatory mechanism. Consequently, mice with a deletion of Activin A receptor 1b specifically in macrophages (Acvr1bf/f-Lyz2cre) succumbed more to sepsis due to uncontrolled inflammation and exhibited exacerbated skin disease in a mouse model of imiquimod-induced psoriasis. Thus, we have revealed a previously unrecognized natural brake to inflammation in macrophages that occurs through the activation of Smad3 in an Activin A-dependent manner.

Biological targeting is crucial for effective cancer treatment with reduced toxicity but is limited by the availability of tumor surface markers. To overcome this, we developed a nanoparticle-based, Tumor-specific suRfACE maRker-independent (TRACER) targeting approach. Utilizing the unique biodistribution properties of nanoparticles, we encapsulated Ac4ManNAz to selectively label tumors with azide reactive groups. Surprisingly, while NP-delivered Ac4ManNAz was cleared by the liver, it did not label macrophages, potentially reducing off-target effects. To exploit this tumor-specific labeling, we functionalized anti-4-1BB antibodies with dibenzocyclooctyne (DBCO) to target azide-labeled tumor cells and activate the immune response. In syngeneic B16F10 melanoma and orthotopic 4T1 breast cancer models, TRACER enhanced anti-4-1BB's therapeutic efficacy, increasing median survival time. Immunofluorescence analyses revealed increased tumor infiltration of CD8+ T and NK cells with TRACER. Importantly, TRACER reduced hepatotoxicity associated with anti-4-1BB, resulting in normal serum ALT and AST levels and decreased CD8+ T cell infiltration in the liver. Quantitative analysis confirmed a 4.5-fold higher tumor-to-liver ratio of anti-4-1BB accumulation with TRACER compared to conventional anti-4-1BB antibodies. Our work provides a promising approach for developing targeted cancer therapies that circumvent limitations imposed by the paucity of tumor-specific markers, potentially improving efficacy and reducing off-target effects to overcome liver toxicity associated with anti-4-1BB.

Sterile acute kidney injury (AKI) is common in the clinic and frequently associated with unexplained hypoxemia that does not improve with dialysis. AKI induces remote lung inflammation with neutrophil recruitment in mice and humans, but which cellular cues establish neutrophilic inflammation and how it contributes to hypoxemia is not known. Here we report that AKI induces rapid intravascular neutrophil retention in lung alveolar capillaries without extravasation into tissue or alveoli, causing hypoxemia by reducing lung capillary blood flow in the absence of substantial lung interstitial or alveolar edema. In contrast to direct ischemic lung injury, lung neutrophil recruitment during remote lung inflammation did not require cues from intravascular non-classical monocytes or tissue-resident alveolar macrophages. Instead, lung neutrophil retention depended on neutrophil chemoattractant CXCL2 released by activated classical monocytes. Comparative single-cell RNA-sequencing analysis of direct and remote lung inflammation revealed that alveolar macrophages are highly activated and produce CXCL2 only in direct lung inflammation. Establishing a CXCL2 gradient into the alveolus by intratracheal CXCL2 administration during AKI-induced remote lung inflammation enabled neutrophils to extravasate. We thus discovered important differences in lung neutrophil recruitment in direct versus remote lung inflammation and identified lung capillary neutrophil retention that negatively affects oxygenation by causing a ventilation-perfusion mismatch as a driver of AKI-induced hypoxemia.

Background: Naïve cells comprise 90% of the CD4+ T-cell population in neonates and exhibit distinct age-specific capacities for proliferation and activation. We hypothesized that HIV-infected naïve CD4+ T-cell populations in children on long-term antiretroviral therapy (ART) would thus be distinct from infected memory cells.

Methods: Peripheral blood naïve and memory CD4+ T cells from 8 children with perinatal HIV on ART initiated at age 1.7-17 months were isolated by FACS. DNA was extracted from sorted cells and HIV proviruses counted, evaluated for intactness, and subjected to integration site analysis.

Results: Naïve CD4+ T cells containing HIV proviruses were detected in children with 95% statistical confidence. A median of 4.7% of LTR-containing naïve CD4+ T cells also contained HIV genetic elements consistent with intactness. Full-length proviral sequencing confirmed intactness of one provirus. In the participant with the greatest level of naïve cell infection, ISA revealed infected expanded cell clones in both naïve and memory T cells with no common HIV integration sites detected between subsets. Divergent integration site profiles reflected differential gene expression patterns of naïve and memory T cells.

Conclusions: These results demonstrate that HIV persists in both naïve and memory CD4+ T cells that undergo clonal expansion and harbor intact proviruses, suggesting that infected memory T-cell clones do not frequently arise from naïve cell differentiation in children with perinatal HIV on long-term ART.

Funding: Center for Cancer Research, NCI and Office of AIDS Research funding to MFK, NCI FLEX funding to JWR. Children's and Emory JFF pilot to MM.

Postoperative atrial fibrillation (poAF) is AF occurring days after surgery with a prevalence of 33% among patients undergoing open-heart surgery. The degree of postoperative inflammation correlates with poAF risk, but less is known about the cellular and molecular mechanisms driving postoperative atrial arrhythmogenesis. We performed single-cell RNA sequencing comparing atrial non-myocytes from mice with versus without poAF, which revealed infiltrating CCR2+ macrophages to be the most altered cell type. Pseudotime trajectory analyses identified Il-6 as a top gene in macrophages, which we confirmed in pericardial fluid collected from human patients after cardiac surgery. Indeed, macrophage depletion and macrophage-specific Il6ra conditional knockout (cKO) prevented poAF in mice. Downstream STAT3 inhibition with TTI-101 and cardiomyocyte-specific Stat3 cKO rescued poAF, indicating a pro-arrhythmogenic role of STAT3 in poAF development. Confocal imaging in isolated atrial cardiomyocytes (ACMs) uncovered a novel link between STAT3 and CaMKII-mediated ryanodine receptor-2 (RyR2)-Ser(S)2814 phosphorylation. Indeed, non-phosphorylatable RyR2S2814A mice were protected from poAF, and CaMKII inhibition prevented arrhythmogenic Ca2+ mishandling in ACMs from mice with poAF. Altogether, we provide multiomic, biochemical, and functional evidence from mice and humans that IL-6-STAT3-CaMKII signaling driven by infiltrating atrial macrophages is a pivotal driver of poAF that portends therapeutic utility for poAF prevention.

Background: Despite growing preclinical evidence that glucagon-like peptide-1 receptor agonists (GLP-1RAs) could be repurposed to treat alcohol use disorder (AUD), clinical evidence is scarce. Additionally, the potential impact of dipeptidyl peptidase-4 inhibitors (DPP-4Is) on alcohol intake is largely unknown.

Methods: We conducted a large cohort study using 2008-2023 electronic health records data from the U.S. Department of Veterans Affairs. Changes in Alcohol Use Disorders Identification Test-Consumption (AUDIT-C) scores were compared between propensity-score-matched GLP-1RA recipients, DPP-4I recipients, and unexposed comparators. We further tested the effects of two DPP-4Is, linagliptin and omarigliptin, on binge-like alcohol drinking in mice and operant oral alcohol self-administration in alcohol-dependent rats, models previously used to show a significant effect of the GLP-1RA semaglutide in reducing alcohol intake.

Results: GLP-1RA recipients reported a greater reduction in AUDIT-C scores than unexposed individuals [difference-in-difference: 0.09(0.03,0.14), p=0.0025] and DPP-4I recipients [difference-in-difference: 0.11(0.05,0.17), p=0.0002]. Reductions in drinking were more pronounced among individuals with baseline AUD [GLP-1RA vs. unexposed: 0.51(0.29,0.72), p<0.0001; GLP-1RA vs. DPP-4I: 0.65(0.43,0.88), p<0.0001] and baseline hazardous drinking [GLP-1RA vs. unexposed: 1.38(1.07,1.69), p<0.0001; GLP-1RA vs. DPP-4I: 1.00(0.68,1.33), p<0.0001]. There were no differences between DPP-4I recipients and unexposed individuals. The latter results were confirmed via a reverse translational approach. Specifically, neither linagliptin nor omarigliptin reduced alcohol drinking in mice or rats. The rodent experiments also confirmed target engagement as both DPP-4Is reduced blood glucose levels.

Conclusion: Convergent findings across humans, mice, and rats indicate that GLP-1RAs but not DPP-4Is reduce alcohol consumption and may be efficacious in treating AUD.

Clostridioides difficile infection (CDI) recurs in one of five patients. Monoclonal antibodies targeting the virulence factor TcdB reduce disease recurrence, suggesting that an inadequate anti-TcdB response to CDI leads to recurrence. In patients with CDI, we discovered that IL33 measured at diagnosis predicts future recurrence, leading us to test the role of IL33 signaling in the induction of humoral immunity during CDI. Using a mouse recurrence model, IL33 was demonstrated to be integral for anti-TcdB antibody production. IL33 acted via ST2+ ILC2 cells, facilitating germinal center T follicular helper (GC-Tfh) cell generation of antibodies. IL33 protection from reinfection was antibody-dependent, as mMT KO mice and mice treated with anti-CD20 mAb were not protected. These findings demonstrate the critical role of IL33 in generating humoral immunity to prevent recurrent CDI.

The induction of durable protective immune responses is the main goal of prophylactic vaccines, and adjuvants play a role as drivers of such responses. Despite advances in vaccine strategies, a safe and effective HIV vaccine remains a significant challenge. The use of an appropriate adjuvant is crucial to the success of HIV vaccines. Here we assessed the saponin/MPLA nanoparticle (SMNP) adjuvant with an HIV envelope (Env) trimer, evaluating the safety and impact of multiple variables including adjuvant dose (16-fold dose range), immunization route, and adjuvant composition on the establishment of Env-specific memory T and B cell responses (TMem and BMem) and long-lived plasma cells in non-human primates (NHPs). Robust BMem were detected in all groups, but a 6-fold increase was observed in the highest SMNP dose group vs. the lowest dose group. Similarly, stronger vaccine responses were induced in the highest SMNP dose for CD40L+OX40+ CD4 TMem (11-fold), IFN-γ+ CD4 TMem (15-fold), IL21+ CD4 TMem (9-fold), circulating TFH (3.6-fold), bone marrow plasma cells (7-fold), and binding IgG (1.3-fold). Substantial tier-2 neutralizing antibodies were only observed in the higher SMNP dose groups. These investigations highlight the dose-dependent potency of SMNP in NHPs, which are relevant for human use and next-generation vaccines.

Umbilical cord blood (UCB) showcases substantial roles in hematopoietic stem cells (HSCs) transplantation and regenerative medicine. UCB is usually cryopreserved for years before use. Whether and how cryopreservation affects its function remain unclear. We constructed single-cell transcriptomic profile of CD34+ hematopoietic stem and progenitor cells (HSPCs) and mononuclear cells (MNCs) from fresh and cryopreserved UCB stored for 1-, 5-, 10-, and 19- years. Compared to fresh UCB, cryopreserved HSCs and multipotent progenitors (MPPs) exhibited more active cell cycle and lower HSC/MPP signature gene expressions. Hematopoietic reconstitution of cryopreserved HSPCs gradually decreased during the first 5 years but stabilized thereafter, aligning with the negative correlation between clinical neutrophil engraftment and cryopreservation duration of UCB. Cryopreserved HSPCs also showed reduced megakaryocyte generation. In contrast, cryopreserved natural killer (NK) cells and T cells maintained cytokine production and cytotoxic ability comparable to fresh cells. Mechanistically, cryopreserved HSPCs exhibited elevated reactive oxygen species, reduced ATP synthesis, and abnormal mitochondrial distribution, which collectively led to attenuated hematopoietic reconstitution. These effects could be ameliorated by sulforaphane. Together, we elucidated the negative impact of cryopreservation on UCB HSPCs and provided sulforaphane as a mitigation strategy, broadening the temporal window and scope for clinical applications of cryopreserved UCB.  .