Allergens have been identified as potential triggers in patients with atopic dermatitis (AD). Patients with AD are highly sensitive to cockroach allergen. The underlying mechanism, however, remains undetermined. Here, we established a cockroach allergen–induced AD-like mouse model, and we demonstrate that repeated exposure to cockroach allergen led to aggravated mouse skin inflammation, characterized by increased type 2 immunity, type 2 innate lymphoid cells (ILC2s), and mast cells. Increased mast cells were also observed in patients with AD. Mast cell–deficient mice (KitW-sh/W-sh) showed diminished skin inflammation, suggesting that mast cells are required in allergen-induced skin inflammation. Furthermore, DC immunoreceptor (DCIR) is upregulated in skin mast cells of patients with AD and mediates allergen binding and uptake. DCIR–/– mice or reconstituted KitW-sh/W-sh mice with DCIR–/– mast cells showed a significant reduction in AD-like inflammation. Both in vitro and in vivo analyses demonstrate that DCIR–/– mast cells had reduced IgE-mediated mast cell activation and passive cutaneous anaphylaxis. Mechanistically, DCIR regulates allergen-induced IgE-mediated mast cell ROS generation and oxidation of calmodulin kinase II (ox-CaMKII). ROS-resistant CaMKII (MM-VVδ) prevents allergen-induced mast cell activation and inflammatory mediator release. Our study reveals a DCIR/ROS/CaMKII axis that controls allergen-induced mast cell activation and AD-like inflammation.
Xiaoyan Luo, Jingsi Chen, Huan Yang, Xinyue Hu, Martin P. Alphonse, Yingchun Shen, Yuko Kawakami, Xiaoying Zhou, Wei Tu, Toshiaki Kawakami, Mei Wan, Nathan K. Archer, Hua Wang, Peisong Gao
Diabetic nephropathy (DN) arises from systemic and local changes in glucose metabolism and hemodynamics. We have reported that many glycolytic and mitochondrial enzymes, such as pyruvate kinase M2 (PKM2), were elevated in renal glomeruli of DN-protected patients with type 1 and type 2 diabetes. Here, mice with PKM2 overexpression specifically in podocytes (PPKM2Tg) were generated to uncover the renal protective function of PPKM2Tg as a potential therapeutic target that prevented elevated albumin/creatinine ratio (ACR), mesangial expansion, basement membrane thickness, and podocyte foot process effacement after 7 months of streptozotocin-induced (STZ-induced) diabetes. Furthermore, diabetes-induced impairments of glycolytic rate and mitochondrial function were normalized in diabetic PPKM2Tg glomeruli, in concordance with elevated Ppargc1a and Vegf expressions. Restored VEGF expression improved glomerular maximal mitochondrial function in diabetic PPKM2Tg and WT mice. Elevated VEGF levels were observed in the glomeruli of DN-protected patients with chronic type 1 diabetes and clinically correlated with estimated glomerular filtration (GFR) — but not glycemic control. Mechanistically, the preservations of mitochondrial function and VEGF expression were dependent on tetrameric structure and enzymatic activities of PKM2 in podocytes. These findings demonstrate that PKM2 structure and enzymatic activation in podocytes can preserve the entire glomerular mitochondrial function against toxicity of hyperglycemia via paracrine factors such as VEGF and prevent DN progression.
Jialin Fu, Takanori Shinjo, Qian Li, Ronald St-Louis, Kyoungmin Park, Marc G. Yu, Hisashi Yokomizo, Fabricio Simao, Qian Huang, I-Hsien Wu, George L. King
Calcific aortic valve disease (CAVD) is heritable, as revealed by recent GWAS. While polymorphisms linked to increased expression of CACNA1C — encoding the CaV1.2 L-type voltage-gated Ca2+ channel — and increased Ca2+ signaling are associated with CAVD, whether increased Ca2+ influx through the druggable CaV1.2 causes CAVD is unknown. We confirmed the association between increased CaV1.2 expression and CAVD in surgically removed aortic valves from patients. We extended our studies with a transgenic mouse model that mimics increased CaV1.2 expression within aortic valve interstitial cells (VICs). In young mice maintained on normal chow, we observed dystrophic valve lesions that mimic changes found in presymptomatic CAVD and showed activation of chondrogenic and osteogenic transcriptional regulators within these valve lesions. Chronic administration of verapamil, a CaV1.2 antagonist used clinically, slowed the progression of lesion development in vivo. Exploiting VIC cultures, we demonstrated that increased Ca2+ influx through CaV1.2 drives signaling programs that lead to myofibroblast activation of VICs and upregulation of genes associated with aortic valve calcification. Our data support a causal role for Ca2+ influx through CaV1.2 in CAVD and suggest that early treatment with Ca2+ channel blockers is an effective therapeutic strategy.
Maiko Matsui, Rihab Bouchareb, Mara Storto, Yasin Hussain, Andrew Gregg, Steven O. Marx, Geoffrey S. Pitt
Benchmarks for protective immunity from infection or severe disease after SARS-CoV-2 vaccination are still being defined. Here, we characterized virus neutralizing and ELISA antibody levels, cellular immune responses, and viral variants in 4 separate groups: healthy controls (HCs) weeks (early) or months (late) following vaccination in comparison with symptomatic patients with SARS-CoV-2 after partial or full mRNA vaccination. During the period of the study, most symptomatic breakthrough infections were caused by the SARS-CoV-2 Alpha variant. Neutralizing antibody levels in the HCs were sustained over time against the vaccine parent virus but decreased against the Alpha variant, whereas IgG titers and T cell responses against the parent virus and Alpha variant declined over time. Both partially and fully vaccinated patients with symptomatic infections had lower virus neutralizing antibody levels against the parent virus than the HCs, similar IgG antibody titers, and similar virus-specific T cell responses measured by IFN-γ. Compared with HCs, neutralization activity against the Alpha variant was lower in the partially vaccinated infected patients and tended to be lower in the fully vaccinated infected patients. In this cohort of breakthrough infections, parent virus neutralization was the superior predictor of breakthrough infections with the Alpha variant of SARS-CoV-2.
Han-Sol Park, Janna R. Shapiro, Ioannis Sitaras, Bezawit A. Woldemeskel, Caroline C. Garliss, Amanda Dziedzic, Jaiprasath Sachithanandham, Anne E. Jedlicka, Christopher A. Caputo, Kimberly E. Rousseau, Manjusha Thakar, San Suwanmanee, Pricila Hauk, Lateef Aliyu, Natalia I. Majewska, Sushmita Koley, Bela Patel, Patrick Broderick, Giselle Mosnaim, Sonya L. Heath, Emily S. Spivak, Aarthi Shenoy, Evan M. Bloch, Thomas J. Gniadek, Shmuel Shoham, Arturo Casadevall, Daniel Hanley, Andrea L. Cox, Oliver Laeyendecker, Michael J. Betenbaugh, Steven M. Cramer, Heba H. Mostafa, Andrew Pekosz, Joel N. Blankson, Sabra L. Klein, Aaron A.R. Tobian, David Sullivan, Kelly A. Gebo
Infants born prematurely worldwide have up to a 50% chance of developing bronchopulmonary dysplasia (BPD), a clinical morbidity characterized by dysregulated lung alveolarization and microvascular development. It is known that PDGFR alpha–positive (PDGFRA+) fibroblasts are critical for alveolarization and that PDGFRA+ fibroblasts are reduced in BPD. A better understanding of fibroblast heterogeneity and functional activation status during pathogenesis is required to develop mesenchymal population–targeted therapies for BPD. In this study, we utilized a neonatal hyperoxia mouse model (90% O2 postnatal days 0–7, PN0–PN7) and performed studies on sorted PDGFRA+ cells during injury and room air recovery. After hyperoxia injury, PDGFRA+ matrix and myofibroblasts decreased and PDGFRA+ lipofibroblasts increased by transcriptional signature and population size. PDGFRA+ matrix and myofibroblasts recovered during repair (PN10). After 7 days of in vivo hyperoxia, PDGFRA+ sorted fibroblasts had reduced contractility in vitro, reflecting loss of myofibroblast commitment. Organoids made with PN7 PDGFRA+ fibroblasts from hyperoxia in mice exhibited reduced alveolar type 1 cell differentiation, suggesting reduced alveolar niche-supporting PDGFRA+ matrix fibroblast function. Pathway analysis predicted reduced WNT signaling in hyperoxia fibroblasts. In alveolar organoids from hyperoxia-exposed fibroblasts, WNT activation by CHIR increased the size and number of alveolar organoids and enhanced alveolar type 2 cell differentiation.
Matthew R. Riccetti, Mereena George Ushakumary, Marion Waltamath, Jenna Green, John Snowball, Sydney E. Dautel, Mehari Endale, Bonny Lami, Jason Woods, Shawn K. Ahlfeld, Anne-Karina T. Perl
Severe acute lung injury has few treatment options and a high mortality rate. Upon injury, neutrophils infiltrate the lungs and form neutrophil extracellular traps (NETs), damaging the lungs and driving an exacerbated immune response. Unfortunately, no drug preventing NET formation has completed clinical development. Here, we report that disulfiram — an FDA-approved drug for alcohol use disorder — dramatically reduced NETs, increased survival, improved blood oxygenation, and reduced lung edema in a transfusion-related acute lung injury (TRALI) mouse model. We then tested whether disulfiram could confer protection in the context of SARS-CoV-2 infection, as NETs are elevated in patients with severe COVID-19. In SARS-CoV-2–infected golden hamsters, disulfiram reduced NETs and perivascular fibrosis in the lungs, and it downregulated innate immune and complement/coagulation pathways, suggesting that it could be beneficial for patients with COVID-19. In conclusion, an existing FDA-approved drug can block NET formation and improve disease course in 2 rodent models of lung injury for which treatment options are limited.
Jose M. Adrover, Lucia Carrau, Juliane Daßler-Plenker, Yaron Bram, Vasuretha Chandar, Sean Houghton, David Redmond, Joseph R. Merrill, Margaret Shevik, Benjamin R. tenOever, Scott K. Lyons, Robert E. Schwartz, Mikala Egeblad
Therapeutic IL-12 has demonstrated the ability to reduce local immune suppression in preclinical models, but clinical development has been limited by severe inflammation-related adverse events with systemic administration. Here, we show that potent immunologic tumor control of established syngeneic carcinomas can be achieved by i.t. administration of a tumor-targeted IL-12 antibody fusion protein (NHS–rmIL-12) using sufficiently low doses to avoid systemic toxicity. Single-cell transcriptomic analysis and ex vivo functional assays of NHS–rmIL-12–treated tumors revealed reinvigoration and enhanced proliferation of exhausted CD8+ T lymphocytes, induction of Th1 immunity, and a decrease in Treg number and suppressive capacity. Similarly, myeloid cells transitioned toward inflammatory phenotypes and displayed reduced suppressive capacity. Cell type–specific IL-12 receptor–KO BM chimera studies revealed that therapeutic modulation of both lymphoid and myeloid cells is required for maximum treatment effect and tumor cure. Study of single-cell data sets from human head and neck carcinomas revealed IL-12 receptor expression patterns similar to those observed in murine tumors. These results describing the diverse mechanisms underlying tumor-directed IL-12–induced antitumor immunity provide the preclinical rationale for the clinical study of i.t. NHS–IL-12.
Youji Hong, Yvette Robbins, Xinping Yang, Wojciech K. Mydlarz, Anastasia Sowers, James B. Mitchell, James L. Gulley, Jeffrey Schlom, Sofia R. Gameiro, Cem Sievers, Clint T. Allen
Understanding the endogenous mechanisms regulating resolution of pain may identify novel targets for treatment of chronic pain. Resolution of chemotherapy-induced peripheral neuropathy (CIPN) after treatment completion depends on CD8+ T cells and on IL-10 produced by other cells. Using Rag2–/– mice lacking T and B cells and adoptive transfer of Il13–/– CD8+ T cells, we showed that CD8+ T cells producing IL-13 were required for resolution of CIPN. Intrathecal administration of anti–IL-13 delayed resolution of CIPN and reduced IL-10 production by dorsal root ganglion macrophages. Depleting local CD206+ macrophages also delayed resolution of CIPN. In vitro, TIM3+CD8+ T cells cultured with cisplatin, apoptotic cells, or phosphatidylserine liposomes produced IL-13, which induced IL-10 in macrophages. In vivo, resolution of CIPN was delayed by intrathecal administration of anti-TIM3. Resolution was also delayed in Rag2–/– mice reconstituted with Havcr2 (TIM3)–/– CD8+ T cells. Our data indicated that cell damage induced by cisplatin activated TIM3 on CD8+ T cells, leading to increased IL-13 production, which in turn induced macrophage IL-10 production and resolution of CIPN. Development of exogenous activators of the IL-13/IL-10 pain resolution pathway may provide a way to treat the underlying cause of chronic pain.
Susmita K. Singh, Karen Krukowski, Geoffroy O. Laumet, Drew Weis, Jenolyn F. Alexander, Cobi J. Heijnen, Annemieke Kavelaars
Phosphopeptides derived from dysregulated protein phosphorylation in cancer cells can be processed and presented by MHC class I and class II molecules and, therefore, represent an untapped class of tumor-specific antigens that could be used as widely expressed “public” cancer neoantigens (NeoAgs). We generated a TCR mimic (TCRm) mAb, 6B1, specific for a phosphopeptide derived from insulin receptor substrate 2 (pIRS2) presented by HLA-A*02:01. The pIRS2 epitope’s presentation by HLA-A*02:01 was confirmed by mass spectrometry. The TCRm 6B1 specifically bound to pIRS2/HLA-A2 complex on tumor cell lines that expressed pIRS2 in the context of HLA-A*02:01. Bispecific mAbs engaging CD3 of T cells were able to kill tumor cell lines in a pIRS2- and HLA-A*02:01–restricted manner. Structure modeling shows a prerequisite for an arginine or lysine at the first position to bind mAb. Therefore, 6B1 could recognize phosphopeptides derived from various phosphorylated proteins with similar amino acid compositions. This raised the possibility that a TCRm specific for the pIRS2/HLA-A2 complex could target a range of phosphopeptides presented by HLA-A*02:01 in various tumor cells. This is the first TCRm mAb to our knowledge targeting a phosphopeptide/MHC class I complex; the potential of this class of agents for clinical applications warrants further investigation.
Tao Dao, Sung Soo Mun, Zaki Molvi, Tatyana Korontsvit, Martin G. Klatt, Abdul G. Khan, Elisabeth K. Nyakatura, Mary Ann Pohl, Thomas E. White, Paul J. Balderes, Ivo C. Lorenz, Richard J. O’Reilly, David A. Scheinberg
The intermittent fasting (IF) diet has profound benefits for diabetes prevention. However, the precise mechanisms underlying IF’s beneficial effects remain poorly defined. Here, we show that the expression levels of cyclooxygenase-2 (COX-2), an enzyme that produces prostaglandins, are suppressed in white adipose tissue (WAT) of obese humans. In addition, the expression of COX-2 in WAT is markedly upregulated by IF in obese mice. Adipocyte-specific depletion of COX-2 led to reduced fractions of CD4+Foxp3+ Tregs and a substantial decrease in the frequency of CD206+ macrophages, an increase in the abundance of γδT cells in WAT under normal chow diet conditions, and attenuation of IF-induced antiinflammatory and insulin-sensitizing effects, despite a similar antiobesity effect in obese mice. Mechanistically, adipocyte-derived prostaglandin E2 (PGE2) promoted Treg proliferation through the CaMKII pathway in vitro and rescued Treg populations in adipose tissue in COX-2–deficient mice. Ultimately, inactivation of Tregs by neutralizing anti-CD25 diminished IF-elicited antiinflammatory and insulin-sensitizing effects, and PGE2 restored the beneficial effects of IF in COX-2–KO mice. Collectively, our study reveals that adipocyte COX-2 is a key regulator of Treg proliferation and that adipocyte-derived PGE2 is essential for IF-elicited type 2 immune response and metabolic benefits.
Chunqing Wang, Xing Zhang, Liping Luo, Yan Luo, Xin Yang, Xiaofeng Ding, Lu Wang, Huyen Le, Lily Elizabeth R. Feldman, Xuebo Men, Cen Yan, Wendong Huang, Yingmei Feng, Feng Liu, Xuexian O. Yang, Meilian Liu
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