Loss of NADPH oxidase (NOX2) exacerbates systemic lupus erythematosus (SLE) in mice and humans, but the mechanisms underlying this effect remain unclear. To identify the cell lineages in which NOX2 deficiency drives SLE, we employed conditional knockout (KO) and chimera approaches to delete Cybb in several hematopoietic cell lineages of MRL.Faslpr lupus-prone mice. Deletion of Cybb in macrophages/monocytes exacerbated lupus nephritis, though not to the degree observed in the Cybb global KOs. Unexpectedly, the absence of Cybb in B cells resulted in profound glomerulonephritis and interstitial nephritis, rivaling that seen with global deletion. Further, we identified that NOX2 is a key regulator of TLR7, a driver of SLE pathology, both globally and specifically in B cells. This is mediated in part through suppression of TLR7-mediated NF-kB signaling in B cells. Thus, NOX2's immunomodulatory effect in SLE is orchestrated not only by its function in the myeloid compartment, but through a pivotal role in B cells by selectively inhibiting TLR7 signaling.
Rachael A. Gordon, Haylee A. Cosgrove, Anthony Marinov, Sebastien Gingras, Jeremy S. Tilstra, Allison M. Campbell, Sheldon I. Bastacky, Michael Kashgarian, Andras Perl, Kevin M. Nickerson, Mark J. Shlomchik
Alveolar macrophages (AMs) act as gatekeepers of the lung’s immune responses, serving essential roles in recognizing and eliminating pathogens. The transcription factor (TF) Early Growth Response 2 (EGR2) has been recently described as required for mature AMs in mice; however, its mechanisms of action have not been explored. Here, we identified EGR2 as an epigenomic regulator and likely direct proximal transcriptional activator in AMs using epigenomic approaches (RNA-sequencing, ATAC-sequencing, and CUT&RUN). The predicted direct proximal targets of EGR2 included a subset of AM identity genes, and ones related to pathogen recognition, phagosome maturation, and adhesion, such as Clec7a, Atp6v0d2, Itgb2, Rhoc, and Tmsb10. We provided evidence that EGR2 deficiency led to impaired zymosan internalization and reduced the capacity to respond to Aspergillus fumigatus. Mechanistically, the lack of EGR2 altered the transcriptional response, secreted cytokines (i.e., CXCL11), and inflammation-resolving lipid mediators (i.e., RvE1) of AMs during in vivo zymosan-induced inflammation, which manifested in impaired resolution. Our findings demonstrated that EGR2 is a key proximal transcriptional activator and epigenomic bookmarker in AMs responsible for select, distinct components of cell identity and a protective transcriptional and epigenomic program against fungi.
Zsuzsanna Kolostyak, Dora Bojcsuk, Viktoria Baksa, Zsuzsa Mathene Szigeti, Krisztian Bene, Zsolt Czimmerer, Pal Boto, Lina Fadel, Szilard Poliska, Laszlo Halasz, Petros Tzerpos, Wilhelm K. Berger, Andres Villabona-Rueda, Zsofia Varga, Tunde Kovacs, Andreas Patsalos, Attila Pap, György Vámosi, Peter Bai, Balazs Dezso, Matthew Spite, Franco R. D'Alessio, Istvan Szatmari, Laszlo Nagy
The goal of this study was to determine if transplantation of enteric neural stem cells (ENSCs) can rescue the enteric nervous system (ENS), restore gut motility, reduce colonic inflammation, and improve survival in the Ednrb knock-out (KO) mouse model of Hirschsprung disease (HSCR). ENSCs were isolated from mouse intestine, expanded to form neurospheres, and microinjected into the colon of recipient Ednrb KO mice. Transplanted ENSCs were identified in recipient colons as cell clusters in “neo-ganglia”. Immunohistochemical evaluation demonstrated extensive cell migration away from the sites of cell delivery and across the muscle layers. Electrical field stimulation and optogenetics showed significantly enhanced contractile activity of aganglionic colonic smooth muscle following ENSC transplantation and confirmed functional neuromuscular integration of the transplanted ENSC-derived neurons. ENSC injection also partially restored the colonic migrating motor complex. Histological examination revealed a significant reduction in inflammation in ENSC-transplanted aganglionic recipient colon compared to sham-operated mice. Interestingly, mice that received cell transplant also had prolonged survival compared with controls. This study demonstrates that ENSC transplantation can improve outcomes in HSCR by restoring gut motility and reducing the severity of Hirschsprung-associated enterocolitis, the leading cause of death in human HSCR.
Ahmed A. Rahman, Takahiro Ohkura, Sukhada Bhave, Weikang Pan, Kensuke Ohishi, Leah Ott, Christopher Han, Abigail Leavitt, Rhian Stavely, Alan J. Burns, Allan M. Goldstein, Ryo Hotta
Primary ciliary dyskinesia (PCD) is a genetic condition that results in dysmotile cilia. The repercussions of cilia dysmotility and gene variants on the multiciliated cell remain poorly understood. We used single-cell RNA sequencing, proteomics, and advanced microscopy to compare primary culture epithelial cells from patients with PCD, their heterozygous mothers, healthy individuals, and induced pluripotent stem (iPS) cells generated from a PCD patient. Transcriptomic analysis revealed unique signatures in PCD airway cells compared to their mothers and healthy individuals. Gene expression in heterozygous mothers’ cells diverged from both control and PCD cells, marked by increased inflammatory and cellular stress signatures. Primary and iPS-derived PCD multiciliated cells had increased expression of glutathione-S-transferases, GSTA2 and GSTA1, as well as NRF2 target genes, accompanied by elevated levels of reactive oxygen species (ROS). Immunogold labeling in human cilia and proteomic analysis of the ciliated organism, Chlamydomonas reinhardtii, demonstrated that GSTA2 localizes to motile cilia. Loss of human GSTA2 and C. reinhardtii GSTA resulted in slowed cilia motility pointing to local cilia regulatory roles. Our findings identify cellular responses unique to PCD variants and independent of environmental stress and uncover a dedicated ciliary GSTA2 pathway essential for normal motility that may be a therapeutic target.
Jeffrey R. Koenitzer, Deepesh Kumar Gupta, Wang Kyaw Twan, Huihui Xu, Nicholas Hadas, Finn J. Hawkins, Mary Lou Beermann, Gervette M. Penny, Nathan T. Wamsley, Andrew Berical, Michael B. Major, Susan K. Dutcher, Steven L. Brody, Amjad Horani
Anal squamous cell carcinoma (ASCC) is a rare gastrointestinal malignancy linked to high-risk Human papillomavirus (HPV) infection, which develops from precursor lesions like Low-Grade Squamous Intraepithelial Lesions (LGSIL) and High-Grade Squamous Intraepithelial Lesions (HGSIL). ASCC incidence varies across populations, posing increased risk for People Living with HIV (PLWH). Our investigation focused on transcriptomic and metatranscriptomic changes from Squamous Intraepithelial Lesions (SILs) to ASCC. Metatranscriptomic analysis highlighted specific bacterial species (e.g., Fusobacterium nucleatum, Bacteroides fragilis) more prevalent in ASCC than precancerous lesions. These species correlated with gene encoding enzymes (Acca, glyQ, eno, pgk, por) and oncoproteins (FadA, dnaK), presenting potential diagnostic or treatment markers. Unsupervised transcriptome analysis identified distinct sample clusters reflecting histological diagnosis, immune infiltrate, HIV/HPV status, and pathway activities, recapitulating anal cancer progression's natural history. Our study unveiled molecular mechanisms in anal cancer progression, aiding in stratifying HGSIL cases based on low- or high-risk progression to malignancy.
Ezequiel Lacunza, Valeria Fink, María E. Salas, Ana M. Gun, Jorge A. Basiletti, María Alejandra Picconi, Mariano Golubicki, Juan Robbio, Mirta Kujaruk, Soledad Iseas, Sion L. Williams, María Figueroa, Omar Coso, Pedro Cahn, Juan C. Ramos, Martin C. Abba
HLA-B*27 was one of the first HLA alleles associated with an autoimmune disease, i.e., axial spondyloarthritis (axSpA) and acute anterior uveitis (B27AAU), which cause joint and eye inflammation, respectively. Gastrointestinal inflammation has been suggested as a trigger of axSpA. We recently identified a bacterial peptide (YeiH) that can be presented by HLA-B*27 to expanded public T cell receptors (TCRs) in the joint in axSpA and the eye in B27AAU. While YeiH is present in enteric microbiota and pathogens, additional evidence that pathogenic T cells in HLA-B*27-associated autoimmunity may have had a prior antigenic encounter within the gastrointestinal tract remains lacking. Here, we analyze ocular, synovial, and blood T cells in B27AAU and axSpA, showing that YeiH-specific CD8 T cells express a mucosal gene set and surface proteins consistent with intestinal differentiation, including CD161, integrin α4β7, and CCR6. In addition, we find an expansion of YeiH-specific CD8 T cells in the blood of axSpA and B27AAU over healthy controls, whereas influenza-specific CD8 T cells were equivalent across groups. Lastly, we demonstrate the dispensability of TRBV9 for antigen recognition. Collectively, our data suggest that, in HLA-B27-associated autoimmunity, early antigen exposure and differentiation of pathogenic CD8 T cells may occur in enteric organs.
Michael A. Paley, Xinbo Yang, Lynn M. Hassman, Frank Penkava, Lee I. Garner, Grace L. Paley, Nicole Linskey, Ryan Agnew, Paulo Henrique Arantes de Faria, Annie Feng, Sophia Y. Li, Davide Simone, Elisha D.O. Roberson, Philip A. Ruzycki, Ekaterina Esaulova, Jennifer Laurent, Lacey Feigl-Lenzen, Luke E. Springer, Chang Liu, Geraldine M. Gillespie, Paul Bowness, K. Christopher Garcia, Wayne M. Yokoyama
Gain-of-function mutations in the dsDNA sensing adaptor STING lead to a severe autoinflammatory syndrome known as STING-associated vasculopathy with onset in Infancy (SAVI). SAVI patients develop interstitial lung disease (ILD) and produce autoantibodies that are commonly associated with systemic autoimmune diseases. Mice expressing the most common SAVI mutation STING V154M (VM) similarly develop ILD, but exhibit severe T and B cell lymphopenia, low serum Ig titers, and lack autoantibodies. Importantly, lethally irradiated VM hosts reconstituted with wildtype (WT) stem cells (WT→VM) still develop ILD. In this study, we find that WT→VM chimeras had restored B cell function, produced autoantibodies, and thereby recapitulated the loss of tolerance seen in SAVI patients. Lymphocytes derived from both WT and BCR or TCR transgenic (Tg) donors accumulated in the extravascular lung tissue of WT+Tg→VM mixed chimeras, but lymphocyte activation and germinal center formation required WT cells with a diverse repertoire. Furthermore, when T cells isolated from the WTVM chimeras were adoptively transferred to naïve Rag1-deficient 2º hosts, they trafficked to the lung and recruited neutrophils. Overall, these findings indicated that VM expression by radioresistant cells promoted the activation of autoreactive B cells and T cells that then differentiated into potentially pathogenic effector subsets.
Kevin MingJie Gao, Kristy Chiang, Sharon Subramanian, Xihui Yin, Paul J. Utz, Kerstin Nündel, Kate A. Fitzgerald, Ann Marshak-Rothstein
Type 2 diabetes (T2D) is on the rise worldwide and is associated with various complications of the oral cavity. Using an adult-onset diabetes preclinical model, we demonstrated profound periodontal alterations in T2D mice, including inflamed gingiva, disintegrated periodontal ligaments (PDL), marked alveolar bone loss, and unbalanced bone remodeling due to decreased formation and increased resorption. Notably, we observed elevated levels of the Wnt signaling inhibitor sclerostin in the alveolar bone of T2D mice. Motivated by these findings, we investigated whether a sclerostin-neutralizing antibody (Scl-Ab) could rescue the compromised periodontium in T2D mice. Administering Scl-Ab subcutaneously once a week for four weeks, starting four weeks after T2D induction, led to substantial increases in bone mass. This effect was attributed to the inhibition of osteoclasts and promotion of osteoblasts in both control and T2D mice, effectively reversing the bone loss caused by T2D. Furthermore, Scl-Ab stimulated PDL cell proliferation, partially restored the PDL fibers, and mitigated inflammation in the periodontium. Our study thus established a T2D-induced periodontitis mouse model characterized by inflammation and tissue degeneration. Scl-Ab emerged as a promising intervention to counteract the detrimental effects of T2D on the periodontium, exhibiting limited side effects on other craniofacial hard tissues.
Hakan Turkkahraman, Shannan Flanagan, Tianli Zhu, Nisreen Akel, Silvia Marino, Dayane Ortega-Gonzalez, Xue Yuan, Teresita M. Bellido
Abdominal aortic aneurysm (AAA) is one of the most life-threatening cardiovascular diseases; however, effective drug treatments are still lacking. The formation of neutrophil extracellular traps (NETs) has been shown to be crucial trigger of AAA and identifying upstream regulatory targets is thus key to discovering therapeutic agents for AAA. We revealed that phosphoinositide 3-kinase gamma (PI3Kγ) acted as an upstream regulatory molecule, and that PI3Kγ inhibition reduced NET formation and aortic wall inflammation, thereby markedly ameliorating AAA. However, the mechanism of NET formation regulated by PI3Kγ remains unclear. In this study, we showed that PI3Kγ deficiency inactivated the noncanonical pyroptosis pathway, which suppressed downstream NET formation. In addition, PI3Kγ regulation of noncanonical pyroptosis was dependent on cAMP/protein kinase A (cAMP/PKA) signaling. These results clarify the molecular mechanism and crosstalk between PI3Kγ and NETosis in the development of AAA, potentially facilitating the discovery of therapeutic options for AAA.
Yacheng Xiong, Shuai Liu, Yu Liu, Jiani Zhao, Jinjian Sun, Yongqing Li, Baihong Pan, Wei Wang
To determine whether hyperlipidemia and chronic kidney disease (CKD) have a synergy in accelerating vascular inflammation via trained immunity (TI), we performed aortic pathological analysis and RNA-sequencing of high-fat diet (HFD)-fed 5/6 nephrectomy CKD (HFD+CKD) mice. We made the following findings: 1) HFD+CKD increased aortic cytosolic lipopolysaccharide (LPS) levels, caspase-11 (CASP11) activation, and 998 gene expressions of TI pathways in the aorta (first-tier TI mechanism); 2) CASP11–/– decreased aortic neointima hyperplasia, aortic recruitment of macrophages, and casp11-gasdermin D-mediated cytokine secretion; 3) CASP11–/– decreased N-terminal gasdermin D (N-GSDMD) membrane expression on aortic endothelial cells and aortic IL-1B levels; 4) LPS transfection into human aortic endothelial cells resulted in CASP4 (human)/CASP11 (mouse) activation and increased N-GSDMD membrane expression; 5) IL-1B served as the second-tier mechanism underlying HFD+CKD-promoted TI. Taken together, hyperlipidemia and CKD accelerated vascular inflammation by promoting two-tier trained immunity.
Yu Sun, Yifan Lu, Lu Liu, Fatma Saaoud, Ying Shao, Keman Xu, Charles Drummer IV, Ramon Cueto, Huimin Shan, Xiaohua Jiang, Huaqing Zhao, Hong Wang, Xiaofeng Yang
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