Foxp3+ CD4 Tregs are central regulators of inflammation, including allergic inflammation in the lung. There is increasing evidence that inflammatory factors undermine adequate Treg functions and homeostasis, resulting in prolonged and exacerbated inflammation. Therefore, identifying the factors is of the utmost important. IL-27 is an antiinflammatory cytokine implicated in immune regulation and tolerance. However, the cellular mechanisms underlying IL-27–mediated immune regulation in vivo remain largely unknown. Utilizing a cockroach antigen–induced allergic inflammation model in mice, we sought to test the roles of Tregs during IL-27–mediated regulation of allergic inflammation. Intranasally delivered IL-27 significantly reduced the development of airway inflammation. Unexpectedly, the IL-27–induced reduction occurred only in the presence of Tregs. Il27ra–/– and Treg-specific Il27ra–/– mice developed severe airway inflammation, and IL-27 treatment had little impact on diminishing the inflammatory responses. IL-27–induced treatment was restored following transfer of WT Tregs but not of Tregs deficient in Lag3, a molecule induced by IL-27 in Tregs. Finally, Tregs from asthmatic patients exhibited blunted STAT1 phosphorylation following IL-27 stimulation. Taken together, our results uncover that Tregs are the primary target cells of IL-27 in vivo to mediate its antiinflammatory functions, suggesting that altered IL-27 responsiveness in Tregs may underlie inadequate Treg functions and perpetuation of inflammation.
Quang Tam Nguyen, Eunjung Jang, Hongnga T. Le, Sohee Kim, Dongkyun Kim, Nina Dvorina, Mark A. Aronica, William M. Baldwin III, Kewal Asosingh, Suzy Comhair, Booki Min
BACKGROUND. Human cytomegalovirus (CMV) reactivation is a common occurrence early after transplant and is associated with heterogeneous NK cell subset expansion. These adaptive NK cell expansions are highly variable between recipients, with respect to magnitude and relative frequencies of adaptive NK cell subsets. METHODS. To gain insight into the factors that influence adaptive NK cell expansion from a CMV naive graft source, we performed a high-resolution NK cell and CD8+ T cell phenotypic analysis of 215 patients with hematological malignancies that were transplanted with 2 partially HLA matched CMV negative umbilical cord blood units. RESULTS. We found that adaptive NK cells were significantly higher in recipients who received nonmyeloablative conditioning (NMAC) relative to myeloablative conditioning (MAC), and high CMV neutralizing antibody titers correlated with the degree of adaptive NK cell expansion. The frequencies of adaptive NK cell subsets (defined by NKG2C, FcεRγ, EAT-2, and SYK expression) that reconstitute from donor hematopoietic progenitor cells largely matched the frequencies observed in the NK cell compartment of the recipient prior to conditioning, suggesting that host — as well as viral reactivation factors — may determine the phenotypic diversification after transplant. Additionally, multivariable analyses show that higher adaptive NK cell expansion associated with better disease-free survival. CONCLUSIONS. Our findings provide important insights into adaptive NK cell reconstitution after transplant and support a role for adaptive NK cells in promoting better clinical outcomes. FUNDING. The NIH and the National Marrow Donor Program.
Frank Cichocki, Emily Taras, Flavia Chiuppesi, John E. Wagner, Bruce R. Blazar, Claudio Brunstein, Xianghua Luo, Don J. Diamond, Sarah Cooley, Daniel J. Weisdorf, Jeffrey S. Miller
Systemic lupus erythematosus (SLE) is a highly variable autoimmune disease that can involve severe organ-threatening symptoms, such as lupus nephritis. Certain drugs, such as mycophenolate mofetil (MMF), are effective at reducing morbidity associated with nephritis; however, the immune pathways associated with disease suppression are poorly defined. Here, we provide evidence that MMF inhibits phosphorylation of STAT3 and other associated immune pathways. Using mass cytometry and bead-based or ELISA assays, the systemic phenotype of SLE patients not taking (MMF–) or taking (MMF+) MMF were studied. MMF+ SLE patients had significant reductions in total numbers of transitional B cells, plasmablasts, and T cells, specifically CD4+ Th17-type and CD4+ Treg-type cells, compared with MMF– patients. Plasma soluble mediators were decreased in MMF+ patients including chemokines (MIG/CXCL9 and SDF-1α/CXCL12) and growth factors (VEGF-A and PDGF-BB). Soluble mediators and cell subsets grouped by functional properties revealed significant modifications associated with STAT3 and B cell pathways. Further, healthy PBMCs treated with IL-6 revealed a reduction in p-STAT3 following the addition of mycophenolic acid (the active metabolite of MMF). In conclusion, the inhibition of STAT3 phosphorylation by MMF may explain the effectiveness of this treatment in SLE patients, since increased levels of p-STAT3 are associated with disease pathology.
Samantha Slight-Webb, Joel M. Guthridge, Eliza F. Chakravarty, Hua Chen, Rufei Lu, Susan Macwana, Krista Bean, Holden T. Maecker, Paul J. Utz, Judith A. James
Acute kidney injury (AKI) is a devastating clinical condition affecting at least two-thirds of critically ill patients, and, among these patients, it is associated with a greater than 60% risk of mortality. Kidney mononuclear phagocytes (MPs) are implicated in pathogenesis and healing in mouse models of AKI and, thus, have been the subject of investigation as potential targets for clinical intervention. We have determined that, after injury, F4/80hi-expressing kidney-resident macrophages (KRMs) are a distinct cellular subpopulation that does not differentiate from nonresident infiltrating MPs. However, if KRMs are depleted using polyinosinic/polycytidylic acid (poly I:C), they can be reconstituted from bone marrow–derived precursors. Further, KRMs lack major histocompatibility complex class II (MHCII) expression before P7 but upregulate it over the next 14 days. This MHCII– KRM phenotype reappears after injury. RNA sequencing shows that injury causes transcriptional reprogramming of KRMs such that they more closely resemble that found at P7. KRMs after injury are also enriched in Wingless-type MMTV integration site family (Wnt) signaling, indicating that a pathway vital for mouse and human kidney development is active. These data indicate that mechanisms involved in kidney development may be functioning after injury in KRMs.
Jeremie M. Lever, Travis D. Hull, Ravindra Boddu, Mark E. Pepin, Laurence M. Black, Oreoluwa O. Adedoyin, Zhengqin Yang, Amie M. Traylor, Yanlin Jiang, Zhang Li, Jacelyn E. Peabody, Hannah E. Eckenrode, David K. Crossman, Michael R. Crowley, Subhashini Bolisetty, Kurt A. Zimmerman, Adam R. Wende, Michal Mrug, Bradley K. Yoder, Anupam Agarwal, James F. George
Osteoarthritis (OA) is a leading cause of disability, globally. Despite an emerging role for synovial inflammation in OA pathogenesis, attempts to target inflammation therapeutically have had limited success. A better understanding of the cellular and molecular processes occurring in the OA synovium is needed to develop novel therapeutics. We investigated macrophage phenotype and gene expression in synovial tissue of OA and inflammatory-arthritis (IA) patients. Compared with IA, OA synovial tissue contained higher but variable proportions of macrophages (P < 0.001). These macrophages exhibited an activated phenotype, expressing folate receptor-2 and CD86, and displayed high phagocytic capacity. RNA sequencing of synovial macrophages revealed 2 OA subgroups. Inflammatory-like OA (iOA) macrophages are closely aligned to IA macrophages and are characterized by a cell proliferation signature. In contrast, classical OA (cOA) macrophages display cartilage remodeling features. Supporting these findings, when compared with cOA, iOA synovial tissue contained higher proportions of macrophages (P < 0.01), expressing higher levels of the proliferation marker Ki67 (P < 0.01). These data provide new insight into the heterogeneity of OA synovial tissue and suggest distinct roles of macrophages in pathogenesis. Our findings could lead to the stratification of OA patients for suitable disease-modifying treatments and the identification of novel therapeutic targets.
Matthew J. Wood, Adam Leckenby, Gary Reynolds, Rachel Spiering, Arthur G. Pratt, Kenneth S. Rankin, John D. Isaacs, Muzlifah A. Haniffa, Simon Milling, Catharien M.U. Hilkens
Allergic eosinophilic asthma is a chronic condition causing airway remodeling resulting in lung dysfunction. We observed that expression of Sirtuin 2 (Sirt2), a histone deacetylase, regulates the recruitment of eosinophils after sensitization and challenge with a triple-antigen: dust mite, ragweed and Aspergillus fumigatus (DRA). Our data demonstrate that IL-4 regulates the expression of Sirt2 isoform 3/5. Pharmacological inhibition of Sirt2 by AGK2 resulted in diminished cellular recruitment, decreased CCL17/TARC, and reduced goblet cell hyperplasia. YM1 and Fizz1 expression was reduced in AGK2-treated, IL-4-stimulated lung macrophages in vitro as well as in lung macrophages from AGK2-DRA challenged mice. Conversely, overexpression of Sirt2 resulted in increased cellular recruitment, CCL17 production, and goblet cell hyperplasia following DRA challenge. Sirt2 isoform 3/5 was upregulated in primary human alveolar macrophages following IL-4 and AGK2 treatment resulted in reduced CCL17 and markers of alternative activation. These gain-of-function and loss-of-function studies indicate that Sirt2 could be developed as a treatment for eosinophilic asthma.
Yong Gyu Lee, Brenda F. Reader, Derrick Herman, Adam Streicher, Joshua A. Englert, Mathias Ziegler, Sangwoon Chung, Manjula Karpurapu, Gye Young Park, John W. Christman, Megan N. Ballinger
Ricin toxin (RT) ranks at the top of the list of bioweapons of concern to civilian and military personnel alike, due to its high potential for morbidity and mortality after inhalation. In nonhuman primates, aerosolized ricin triggers severe acute respiratory distress characterized by perivascular and alveolar edema, neutrophilic infiltration, and severe necrotizing bronchiolitis and alveolitis. There are currently no approved countermeasures for ricin intoxication. Here, we report the therapeutic potential of a humanized mAb against an immunodominant epitope on ricin’s enzymatic A chain (RTA). Rhesus macaques that received i.v. huPB10 4 hours after a lethal dose of ricin aerosol exposure survived toxin challenge, whereas control animals succumbed to ricin intoxication within 30 hours. Antibody intervention at 12 hours resulted in the survival of 1 of 5 monkeys. Changes in proinflammatory cytokine, chemokine, and growth factor profiles in bronchial alveolar lavage fluids before and after toxin challenge successfully clustered animals by treatment group and survival, indicating a relationship between local tissue damage and experimental outcome. This study represents the first demonstration, to our knowledge, in nonhuman primates that the lethal effects of inhalational ricin exposure can be negated by a drug candidate, and it opens up a path forward for product development.
Chad J. Roy, Dylan J. Ehrbar, Natasha Bohorova, Ognian Bohorov, Do Kim, Michael Pauly, Kevin Whaley, Yinghui Rong, Fernando J. Torres-Velez, Ellen S. Vitetta, Peter J. Didier, Lara Doyle-Meyers, Larry Zeitlin, Nicholas J. Mantis
Psoralen plus UVA (PUVA) is an effective therapy for mycosis fungoides (MF), the skin-limited variant of cutaneous T cell lymphoma (CTCL). In low-burden patients, PUVA reduced or eradicated malignant T cells and induced clonal expansion of CD8+ T cells associated with malignant T cell depletion. High-burden patients appeared to clinically improve but large numbers of malignant T cells persisted in skin. Clinical improvement was linked to turnover of benign T cell clones but not to malignant T cell reduction. Benign T cells were associated with the Th2-recruiting chemokine CCL18 before therapy and with the Th1-recruiting chemokines CXCL9, CXCL10, and CXCL11 after therapy, suggesting a switch from Th2 to Th1. Inflammation was correlated with OX40L and CD40L gene expression; immunostaining localized these receptors to CCL18-expressing c-Kit+ dendritic cells that clustered together with CD40+OX40+ benign and CD40+CD40L+ malignant T cells, creating a proinflammatory synapse in skin. Our data suggest that visible inflammation in CTCL results from the recruitment and activation of benign T cells by c-Kit+OX40L+CD40L+ dendritic cells and that this activation may provide tumorigenic signals. Targeting c-Kit, OX40, and CD40 signaling may be novel therapeutic avenues for the treatment of MF.
Pablo Vieyra-Garcia, Jack D. Crouch, John T. O’Malley, Edward W. Seger, Chao H. Yang, Jessica E. Teague, Anna Maria Vromans, Ahmed Gehad, Thet Su Win, Zizi Yu, Elizabeth L. Lowry, Jung-Im Na, Alain H. Rook, Peter Wolf, Rachael A. Clark
CD4+ follicular helper T (Tfh) cells are specialized providers of T cell help to B cells and can function as pathogenic mediators of murine antibody-dependent chronic graft-versus-host disease (GvHD). Using a parent→F1 model of lupus-like chronic GvHD, in which Tfh cell and germinal center (GC) B cell differentiation occurs over 14 days, we demonstrate that absence of CD4+ T cell–expressed C5a receptor 1 (C5ar1) or pharmacological C5aR1 blockade abrogated generation/expansion of Tfh cells, GC B cells, and autoantibodies. In a Tfh cell–dependent model of chronic GvHD manifested by bronchiolitis obliterans syndrome (BOS), C5aR1 antagonism initiated in mice with established disease ameliorated BOS and abolished the associated differentiation of Tfh and GC B cells. Guided by RNA-sequencing data, mechanistic studies performed using murine and human T cells showed that C5aR1 signaling amplifies IL-6–dependent expression of the transcription factor c-MAF and the cytokine IL-21 via phosphorylating phosphokinase B (AKT) and activating the mammalian target of rapamycin (mTOR). In addition to linking C5aR1-initiated signaling to Tfh cell differentiation, our findings suggest that C5aR1 may be a useful therapeutic target for prevention and/or treatment of individuals with Tfh cell–dependent diseases, including those chronic GvHD patients who have anti-host reactive antibodies.
Divya A. Verghese, Nicholas Chun, Katelyn Paz, Miguel Fribourg, Trent M. Woodruff, Ryan Flynn, Yuan Hu, Huabao Xiong, Weijia Zhang, Zhengzi Yi, Jing Du, Bruce R. Blazar, Peter S. Heeger
Tregs are impaired in human systemic lupus erythematosus (SLE) and contribute to effector T cell activation. However, the mechanisms responsible for the Treg deficiency in SLE remain unclear. We hypothesized that the OX40L/OX40 axis is implicated in Treg and regulatory follicular helper T (Tfr) cell dysfunction in human SLE. OX40L/OX40 axis engagement on Tregs and Tfr cells not only specifically impaired their ability to regulate effector T cell proliferation, but also their ability to suppress T follicular helper (Tfh) cell–dependent B cell activation and immunoglobulin secretion. Antigen-presenting cells from patients with active SLE mediated Treg dysfunction in an OX40L-dependent manner, and OX40L-expressing cells colocalized with Foxp3+ cells in active SLE skin lesions. Engagement of the OX40L/OX40 axis resulted in Foxp3 downregulation in Tregs, and expression in SLE Tregs correlated with the proportion of circulating OX40L-expressing myeloid DCs. These data support that OX40L/OX40 signals are implicated in Treg dysfunction in human SLE. Thus, blocking the OX40L/OX40 axis appears to be a promising therapeutic strategy.
Clément Jacquemin, Jean-François Augusto, Marc Scherlinger, Noémie Gensous, Edouard Forcade, Isabelle Douchet, Emeline Levionnois, Christophe Richez, Estibaliz Lazaro, Pierre Duffau, Marie-Elise Truchetet, Julien Seneschal, Lionel Couzi, Jean-Luc Pellegrin, Jean-François Viallard, Thierry Schaeverbeke, Virginia Pascual, Cécile Contin-Bordes, Patrick Blanco
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