Molecular control of PDPN^hi macrophage subset induction by ADAP as a host defense in sepsis

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Abstract

Induction of podoplanin (PDPN) expression is a critical response of macrophages to LPS stimulation or bacterial infection in sepsis, but how this key process of TLR4-stimulated PDPN upregulation is regulated and the effect of PDPN expression on macrophage function remain elusive. Here, we determined how this process is regulated in vitro and in vivo. PDPN failed to be upregulated in TLR4-stimulated macrophages deficient in adhesion and degranulation-promoting adapter protein (ADAP), which could be rescued by the reconstitution of ADAP. A distinct PDPNhi peritoneal macrophage (PM) subset, which exhibited an M2-like phenotype and enhanced phagocytic activity, was generated in WT but not in ADAP-deficient septic mice. The blockade of PDPNhi PMs mimicked the effect of ADAP deficiency, which exacerbated sepsis. Mechanistically, Bruton’s tyrosine kinase–mediated (BTK-mediated) tyrosine phosphorylation of ADAP at Y571 worked together with mTOR to converge on STAT3 activation for the transactivation of the PDPN promoter. Moreover, agonist activation of STAT3 profoundly potentiated the PDPNhi PM subset generation and alleviated sepsis severity in mice. Together, our findings reveal a mechanism whereby ADAP resets macrophage function by controlling the TLR4-induced upregulation of PDPN as a host innate immune defense during sepsis.

Authors

Pengchao Zhang, Xinning Wang, Xiaodong Yang, Hebin Liu

Characterization of the vaginal microbiome of postmenopausal patients receiving chemoradiation for locally advanced cervical cancer

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Abstract

The standard-of-care treatment of locally advanced cervical cancer includes pelvic radiation therapy with concurrent cisplatin-based chemotherapy and is associated with a 30%–50% failure rate. New prognostic and therapeutic targets are needed to improve clinical outcomes. The vaginal microbiome has been linked to the pathogenesis of cervical cancer, but little is known about the vaginal microbiome in locally advanced cervical cancer as it relates to chemoradiation. In this pilot study, we utilized 16S rRNA gene community profiling to characterize the vaginal microbiomes of 26 postmenopausal women with locally advanced cervical cancer receiving chemoradiation. Our analysis revealed diverse anaerobe-dominated communities whose taxonomic composition, diversity, or bacterial abundance did not change with treatment. We hypothesized that characteristics of the microbiome might correlate with treatment response. Pretreatment microbial diversity and bacterial abundance were not associated with disease recurrence. We observed a greater relative abundance of Fusobacterium in patients who later had cancer recurrence, suggesting that Fusobacterium could play a role in modifying treatment response. Taken together, this hypothesis-generating pilot study provides insight into the composition and dynamics of the vaginal microbiome, offering proof of concept for the future study of the microbiome and its relationship with treatment outcomes in locally advanced cervical cancer.

Authors

Brett A. Tortelli, Jessika Contreras, Stephanie Markovina, Li Ding, Kristine M. Wylie, Julie K. Schwarz

GADD45α is a direct target of TFEB and contributes to tacrolimus-induced chronic nephrotoxicity

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Abstract

Tacrolimus-induced chronic nephrotoxicity (TICN) hinders long-term use of tacrolimus, but its mechanism remains unclear. Tacrolimus exerts its pharmacological effect by inhibiting calcineurin and its substrate nuclear factor of activated T cells. Whether the inhibition of other calcineurin substrates is related to TICN remains to be explored. Transcription factor EB (TFEB), a substrate of calcineurin, plays a crucial role in homeostasis. Herein, we found that tacrolimus inhibited TFEB nuclear translocation and activity in mouse kidneys and HK-2 cells. Then, TFEB gain and loss of function rescued and exacerbated, respectively, the effect of tacrolimus in HK-2 cells. Furthermore, TFEB activation by both phosphorylation site mutation and agonist rescued TICN in mice. To elucidate the mechanism of TFEB, we analyzed ChIP-Seq data. We identified growth arrest and DNA damage-inducible 45α (GADD45α) as a transcriptional target of TFEB via ChIP and dual-luciferase reporter assays. Then we revealed that GADD45α overexpression rescued DNA damage and kidney injury caused by tacrolimus or TFEB knockdown in vitro and vice versa. The protective effect of GADD45α against TICN and DNA damage was further demonstrated by overexpressing it in mice. In conclusion, the persistent inhibition of the TFEB/GADD45α pathway by tacrolimus contributes to TICN. This study identifies a specific target for intervention in TICN.

Authors

Ping Gao, Xinwei Cheng, Maochang Liu, Hui Peng, Guodong Li, Tianze Shang, Jianqiao Wang, Qianyan Gao, Chenglong Zhu, Zhenpeng Qiu, Chengliang Zhang

Quinolinic acid potentially links kidney injury to brain toxicity

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Abstract

Kidney dysfunction often leads to neurological impairment, yet the complex kidney-brain relationship remains elusive. We employed spatial and bulk metabolomics to investigate a mouse model of rapid kidney failure induced by mouse double minute 2 (Mdm2) conditional deletion in the kidney tubules to interrogate kidney and brain metabolism. Pathway enrichment analysis of a focused plasma metabolomics panel pinpointed tryptophan metabolism as the most altered pathway with kidney failure. Spatial metabolomics showed toxic tryptophan metabolites in the kidneys and brains, revealing a connection between advanced kidney disease and accelerated kynurenine degradation. In particular, the excitotoxic metabolite quinolinic acid was localized in ependymal cells in the setting of kidney failure. These findings were associated with brain inflammation and cell death. Separate mouse models of ischemia-induced acute kidney injury and adenine-induced chronic kidney disease also exhibited systemic inflammation and accumulating toxic tryptophan metabolites. Patients with advanced chronic kidney disease (stage 3b-4 and stage 5) similarly demonstrated elevated plasma kynurenine metabolites, and quinolinic acid was uniquely correlated with fatigue and reduced quality of life. Overall, our study identifies the kynurenine pathway as a bridge between kidney decline, systemic inflammation, and brain toxicity, offering potential avenues for diagnosis and treatment of neurological issues in kidney disease.

Authors

Afaf Saliba, Subrata Debnath, Ian Tamayo, Hak Joo Lee, Nagarjunachary Ragi, Falguni Das, Richard Montellano, Jana Tumova, Meyer Maddox, Esmeralda Trevino, Pragya Singh, Caitlyn Fastenau, Soumya Maity, Guanshi Zhang, Leila Hejazi, Manjeri A. Venkatachalam, Jason C. O’Connor, Bernard Fongang, Sarah C. Hopp, Kevin F. Bieniek, James D. Lechleiter, Kumar Sharma

Short-term disruption of TGF-β signaling in adult mice renders the aorta vulnerable to hypertension-induced dissection

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Abstract

Hypertension and transient increases in blood pressure from extreme exertion are risk factors for aortic dissection in patients with age-related vascular degeneration or inherited connective tissue disorders. Yet, a common experimental model of angiotensin II–induced aortopathy in mice appears independent of high blood pressure, as lesions do not occur in response to an alternative vasoconstrictor, norepinephrine, and are not prevented by cotreatment with a vasodilator, hydralazine. We investigated vasoconstrictor administration to adult mice following 1 week of disrupted TGF-β signaling in smooth muscle cells (SMCs). Norepinephrine increased blood pressure and induced aortic dissection by 7 days and even within 30 minutes (as did angiotensin II) that was prevented by hydralazine. Initial medial injury manifested as blood extravasation among SMCs and fibrillar matrix, progressive delamination from accumulation of blood, and stretched or ruptured SMCs with persistent attachments to elastic fibers. Altered regulatory contractile molecule expression was not of pathological importance. Rather, reduced synthesis of extracellular matrix yielded a vulnerable aortic phenotype by decreasing medial collagen, most dynamically basement membrane–associated multiplexin collagen, and impairing cell-matrix adhesion. We conclude that transient and sustained increases in blood pressure can cause dissection in aortas rendered vulnerable by inhibition of TGF-β–driven extracellular matrix production by SMCs.

Authors

Bo Jiang, Pengwei Ren, Changshun He, Mo Wang, Sae-Il Murtada, María Jesús Ruiz-Rodríguez, Yu Chen, Abhay B. Ramachandra, Guangxin Li, Lingfeng Qin, Roland Assi, Martin A. Schwartz, Jay D. Humphrey, George Tellides

The administration of exogenous HSP47 as a collagen-specific therapeutic approach

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Abstract

The proof of principle of the therapeutic potential of heat shock protein 47 (HSP47) for diseases characterized by defects in collagen I synthesis is here demonstrated in osteogenesis imperfecta (OI), a prototype of collagen disorders. Most of the OI mutations delay collagen I chain folding, increasing their exposure to posttranslational modifications that affect collagen secretion and impact extracellular matrix fibril assembly. As a model, we used primary fibroblasts from OI individuals with a defect in the collagen prolyl 3-hydroxylation complex, since they are characterized by the synthesis of homogeneously overmodified collagen molecules. We demonstrated that exogenous recombinant HSP47 (rHSP47) is taken up by the cells and localizes at the ER exit sites and ER-Golgi intermediate compartment. rHSP47 treatment increased collagen secretion, reduced collagen posttranslational modifications and intracellular collagen retention, and ameliorated general ER proteostasis, leading to improved cellular homeostasis and vitality. These positive changes were also mirrored by an increased collagen content in the OI matrix. A mutation-dependent effect was found in fibroblasts from 3 probands with collagen I mutations, for which rHSP47 was effective only in cells with the most N-terminal defect. A beneficial effect on bone mineralization was demonstrated in vivo in the zebrafish p3h1–/– OI model.

Authors

Roberta Besio, Nadia Garibaldi, Alessandra Sala, Francesca Tonelli, Carla Aresi, Elisa Maffioli, Claudio Casali, Camilla Torriani, Marco Biggiogera, Simona Villani, Antonio Rossi, Gabriella Tedeschi, Antonella Forlino

Complement activation at the interface between adipocytes and cancer cells drives tumor progression

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Abstract

The omentum is the primary site of metastasis for ovarian cancer (OC). Interactions between cancer cells and adipocytes drive an invasive and prometastatic phenotype. Here we studied cancer cell–adipocyte crosstalk by using a direct coculture model with immortalized human visceral nondiabetic pre-adipocytes (VNPADs) and OC cells. We demonstrated increased proliferation, invasiveness, and resistance to cisplatin of cocultured compared with monocultured OC cells. RNA sequencing of OC cells from coculture versus monoculture revealed significant transcriptomic changes, identifying over 200 differentially expressed genes common to OVCAR5 and OVCAR8 cell lines. Enriched pathways included PI3K/AKT and complement activation. Lipid transfer into OC cells from adipocytes induced upregulation of complement C3 and C5 proteins. Inhibiting C3 or C5 reversed the invasive phenotype and C3 knockdown reduced tumor progression in vivo. Increased C3 expression was observed in omental implants compared with primary ovarian tumors and C3 secretion was higher in OC ascites from high-BMI versus low-BMI patients. C3 upregulation in OC cells involved activation of the ATF4-mediated integrated stress response (ISR). Overall, adipocyte–cancer cell interactions promoted invasiveness and tumorigenesis via lipid transfer, activating the ISR, and upregulating complement proteins C3 and C5.

Authors

Andres Valdivia, Ana Maria Isac, Horacio Cardenas, Guangyuan Zhao, Yaqi Zhang, Hao Huang, Jian-Jun Wei, Mauricio Cuello-Fredes, Sumie Kato, Fernán Gómez-Valenzuela, Francoise Gourronc, Aloysius Klingelhutz, Daniela Matei

Targeting fibroblast–endothelial cell interactions in LAM pathogenesis using 3D spheroid models and spatial transcriptomics

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Abstract

Lymphangioleiomyomatosis (LAM) is a progressive lung disease with limited treatments, largely because of an incomplete understanding of its pathogenesis. Lymphatic endothelial cells (LECs) invade LAM cell clusters, which include human melanoma black-45–positive epithelioid cells and smooth muscle α-actin–expressing LAM-associated fibroblasts (LAMFs). Recent evidence shows that LAMFs resemble cancer-associated fibroblasts, with LAMF-LEC interactions contributing to disease progression. To explore these mechanisms, we used spatial transcriptomics on LAM lung tissues and identified a gene cluster enriched in kinase signaling pathways linked to myofibroblasts and coexpressed with LEC markers. Kinase arrays revealed elevated PDGFR and FGFR in LAMFs. Using a 3D coculture spheroid model of primary LAMFs and LECs, we observed increased invasion in LAMF-LEC spheroids compared with non-LAM fibroblasts. Treatment with sorafenib, a multikinase inhibitor, significantly reduced invasion, outperforming rapamycin. We also verified tuberous sclerosis complex 2–deficient renal angiomyolipoma (TSC2-null AML) cells as key VEGF-A secretors; VEGF-A was suppressed by sorafenib in both TSC2-null AML cells and LAMFs. These findings highlight VEGF-A and basic FGF as potential therapeutic targets and suggest multikinase inhibition as a promising strategy for LAM.

Authors

Sinem Koc-Gunel, Emily C. Liu, Lalit K. Gautam, Ben A. Calvert, Shubha Murthy, Noa C. Harriott, Janna C. Nawroth, Beiyun Zhou, Vera P. Krymskaya, Amy L. Ryan

CD103⁺ dendritic cell–fibroblast crosstalk via TLR9, TDO2, and AHR signaling drives lung fibrogenesis

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Abstract

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive scarring and loss of lung function. With limited treatment options, patients die from the disease within 2–5 years. The molecular pathogenesis underlying the immunologic changes that occur in IPF is poorly understood. We characterize noncanonical aryl-hydrocarbon receptor (ncAHR) signaling in DCs as playing a role in the production of IL-6 and increased IL-17+ cells, promoting fibrosis. TLR9 signaling in myofibroblasts is shown to regulate production of TDO2, which converts tryptophan into the endogenous AHR ligand kynurenine. Mice with augmented ncAHR signaling were created by crossing mice harboring a floxed AHR exon 2 deletion (AHRΔex2) with mice harboring a CD11c-Cre. Bleomycin (blm) was used to study fibrotic pathogenesis. Isolated CD11c+ cells and primary fibroblasts were treated ex vivo with relevant TLR agonists and AHR-modulating compounds to study how AHR signaling influenced inflammatory cytokine production. Human datasets were also interrogated. Inhibition of all AHR signaling rescued fibrosis; however, AHRΔex2 mice treated with blm developed more fibrosis, and DCs from these mice were hyperinflammatory and profibrotic upon adoptive transfer. Treatment of fibrotic fibroblasts with TLR9 agonist increased expression of TDO2, and fibrotic fibroblasts activated IL-6 production in CD103+ DCs. Study of human samples corroborated the relevance of these findings in patients with IPF. We also show, for the first time to our knowledge, that AHR exon 2 floxed mice retain the capacity for ncAHR signaling.

Authors

Hannah Carter, Rita Medina Costa, Taylor S. Adams, Talon M. Gilchrist, Claire E. Emch, Monica Bame, Justin M. Oldham, Steven K. Huang, Angela L. Linderholm, Imre Noth, Naftali Kaminski, Bethany B. Moore, Stephen J. Gurczynski

Effect of aging on pulmonary cellular responses during mechanical ventilation

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Abstract

Acute respiratory distress syndrome (ARDS) results in substantial morbidity and mortality, especially in elderly people. Mechanical ventilation, a common supportive treatment for ARDS, is necessary for maintaining gas exchange but can also propagate injury. We hypothesized that aging leads to alterations in surfactant function, inflammatory signaling, and microvascular permeability within the lung during mechanical ventilation. Young and aged male mice were mechanically ventilated, and surfactant function, inflammation, and vascular permeability were assessed. Additionally, single-cell RNA-Seq was used to delineate cell-specific transcriptional changes. The results showed that, in aged mice, surfactant dysfunction and vascular permeability were significantly augmented, while inflammation was less pronounced. Differential gene expression and pathway analyses revealed that alveolar macrophages in aged mice showed a blunted inflammatory response, while aged endothelial cells exhibited altered cell-cell junction formation. In vitro functional analysis revealed that aged endothelial cells had an impaired ability to form a barrier. These results highlight the complex interplay between aging and mechanical ventilation, including an age-related predisposition to endothelial barrier dysfunction, due to altered cell-cell junction formation, and decreased inflammation, potentially due to immune exhaustion. It is concluded that age-related vascular changes may underlie the increased susceptibility to injury during mechanical ventilation in elderly patients.

Authors

Aminmohamed Manji, Lefeng Wang, Cynthia M. Pape, Lynda A. McCaig, Alexandra Troitskaya, Onon Batnyam, Leah J.J. McDonald, C. Thomas Appleton, Ruud A.W. Veldhuizen, Sean E. Gill