Immunology
Abstract
IL-17A plays a critical role in the pathogenesis of steroid-resistant neutrophilic airway inflammation, which is a hallmark of severe asthma and chronic obstructive pulmonary disease (COPD). Through RNA sequencing analysis of transcriptomes of human airway smooth muscle cells treated with IL-17A, dexamethasone (DEX, a synthetic glucocorticoid drug), alone or in combination, we identified a group of genes that are synergistically induced by IL-17A and DEX, including the neutrophil-promoting cytokine CSF3. In type-17 (Th17/IL-17Ahi) preclinical models of neutrophilic severe asthma (acute and chronic) and COPD, although DEX treatment was able to reduce the expression of neutrophil-mobilizing CXCL1 and CXCL2 in lung tissue, CSF3 expression was upregulated by DEX treatment. We found that DEX treatment alone failed to alleviate neutrophilic airway inflammation and pathology, and even exacerbated the disease phenotype when CSF3 was highly induced. Disruption of the IL-17A/DEX synergy by IL-17A inhibition with anti–IL-17A mAb or cyanidin-3-glucoside (C3G, a small-molecule IL-17A blocker) or depletion of CSF3 effectively rendered DEX sensitivity in type-17 preclinical models of neutrophilic airway diseases. Our study elucidates what we believe is a novel mechanism of steroid resistance in type-17 neutrophilic airway inflammation and offers an effective steroid-sparing therapeutic strategy (combined low-dose DEX and C3G) for treating neutrophilic airway diseases.
Authors
Suidong Ouyang, Caini Liu, Jianxin Xiao, Xing Chen, Andy C. Lui, Xiaoxia Li
Abstract
Altered bone marrow hematopoiesis and immune suppression is a hallmark of myelodysplastic syndrome (MDS). While the bone marrow microenvironment influences malignant hematopoiesis, the mechanism leading to MDS-associated immune suppression is unknown. We tested whether mesenchymal stromal cells (MSCs) contribute to this process. Here, we developed a model to study cultured MSCs from MDS patients compared to similar aged matched normal controls for regulation of immune function. MSCs from MDS patients (MDS-MSC) and healthy donor MSC (HD-MSC) exhibited a similar in vitro phenotype and neither had a direct effect on NK cell function. However, when MDS and HD-MSCs were cultured with monocytes, only the MDS-MSCs acquired phenotypic and metabolic properties of myeloid-derived suppressor cells (MDSCs), with resulting suppression of NK cell function, along with T cell proliferation. A unique MSC transcriptome was observed in MDS-MSCs compared to HD-MSCs, including increased expression of the reactive oxygen species (ROS) regulator, ENC1. High ENC1 expression in MDS-MSC induced suppressive monocytes with increased INHBA, a gene that encodes for a member of the TGFβ superfamily of proteins. These monocytes also had reduced expression of the TGFβ transcriptional repressor MAB21L2, further adding to their immune suppressive function. Silencing ENC1 or inhibiting ROS production in MDS-MSCs abrogated the suppressive function of MDS-MSC conditioned monocytes. In addition, silencing MAB21L2 in healthy MSC conditioned monocytes mimicked the MDS-MSC suppressive transformation of monocytes. Our data demonstrate that MDS-MSCs are responsible for inducing an immune suppressive microenvironment in MDS through an indirect mechanism involving monocytes.
Authors
Dhifaf Sarhan, Jinhua Wang, Upasana Sunil Arvindam, Caroline Hallstrom, Michael R. Verneris, Bartosz Grzywacz, Erica Warlick, Bruce R. Blazar, Jeffrey S. Miller
Abstract
Extracellular cold-inducible RNA-binding protein (eCIRP) is a recently-discovered damage-associated molecular pattern. Understanding the precise mechanism by which it exacerbates inflammation is essential. Here we identified that eCIRP is a new biologically active endogenous ligand of triggering receptor expressed on myeloid cells-1 (TREM-1), fueling inflammation in sepsis. Surface plasmon resonance revealed a strong binding affinity between eCIRP and TREM-1, and FRET assay confirmed eCIRP’s interaction with TREM-1 in macrophages. Targeting TREM-1 by its siRNA or a decoy peptide LP17 or by using TREM-1-/- mice dramatically reduced eCIRP-induced inflammation. We developed a novel 7-aa peptide derived from human eCIRP, M3, which blocked the interaction of TREM-1 and eCIRP. M3 suppressed inflammation induced by eCIRP or agonist TREM-1 Ab crosslinking in murine macrophages or human peripheral blood monocytes. M3 also inhibited eCIRP-induced systemic inflammation and tissue injury. Treatment with M3 further protected mice from sepsis, improved acute lung injury, and increased survival. Thus, we have discovered a novel TREM-1 ligand and developed a new peptide M3 to block the eCIRP-TREM-1 interaction and improve the outcomes in sepsis.
Authors
Naomi-Liza Denning, Monowar Aziz, Atsushi Murao, Steven D. Gurien, Mahendar Ochani, Jose M. Prince, Ping Wang
Abstract
Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern, whose effect on macrophages is not entirely elucidated. Here we identified that eCIRP promotes macrophage endotoxin tolerance. Septic mice had higher serum levels of eCIRP; this was associated with a reduced ex vivo immune response of their splenocytes to LPS. Pretreatment of macrophages with recombinant murine (rm) CIRP resulted in a tolerance to LPS stimulation as demonstrated by a significant reduction of TNF-α production. We found that eCIRP increased phosphorylation of STAT3 (pSTAT3) in macrophages. A STAT3 inhibitor, Stattic, rescued macrophages from rmCIRP-induced tolerance by restoring the release of TNF-α in response to LPS stimulation. We discovered strong binding affinity between eCIRP and IL-6R as revealed by Biacore, FRET, and their co-localization in macrophages by immunostaining assays. Blockade of IL-6R with its neutralizing Ab significantly inhibited eCIRP-induced pSTAT3 and restored LPS-stimulated TNF-α release in macrophages. Incubation of macrophages with rmCIRP skewed them towards a M2 phenotype, while treatment with anti-IL-6R Ab prevented rmCIRP-induced M2 polarization. Thus, we have demonstrated that eCIRP activates pSTAT3 via a novel receptor IL-6R to promote macrophage endotoxin tolerance. Targeting eCIRP appears to be a new therapeutic option to correct immune-tolerance in sepsis.
Authors
Mian Zhou, Monowar Aziz, Naomi-Liza Denning, Hao-Ting Yen, Gaifeng Ma, Ping Wang
Abstract
The HIV latent reservoir in resting memory CD4+ T cells prevents cure. Therapeutics to reactivate and eliminate this reservoir are in clinical trials in adults, but not in pediatric populations. We determined, ex vivo, the inducibility and size of the latent reservoir in perinatal compared with adult infections using the Tat/rev Induced Limiting Dilution Assay (TILDA), in which a single-round (12hr) of CD4+ T cell stimulation with PMA/ionomycin maximally activates T cells and leads to proviral expression with multiply-spliced HIV RNA production. Markers of immune activation and exhaustion were measured to assess interactions with inducibility. Despite similar rates of T cell activation with PMA/ionomycin, the latent reservoir in perinatal infection is slower to reactivate and of lower magnitude compared to adult infection, independent of proviral load. An enhanced TILDA with the addition of phytohemagglutin and for 18 hours augmented proviral expression in perinatal but not adult infection. Baseline HLA–DR+ CD4+ T cells was significantly lower in perinatal compared with adult infections, but not correlated with induced reservoir size. These data support differences in baseline immune activation and kinetics of latency reversal in perinatal compared with adult infections, with implications for latency reversal strategies towards reservoir clearance and remission.
Authors
Adit Dhummakupt, Jessica H. Rubens, Thuy Anderson, Laura Powell, Bareng A.S. Nonyane, Lilly V. Siems, Aleisha Collinson-Streng, Tricia L. Nilles, R. Brad Jones, Vicki Tepper, Allison Agwu, Deborah Persaud
Abstract
Group-2 innate lymphoid cells (ILC2s) are a critical innate source of type-2 cytokines in allergic inflammation. Although ILC2s are recognized as a critical cell population in the allergic inflammation, the regulatory mechanism(s) of ILC2s are less well understood. Here, we show that Regnase-1, an immune-regulatory RNase that degrades inflammatory mRNAs, negatively regulates ILC2 function, and that IkB kinase (IKK) complex-mediated Regnase-1 degradation is essential for IL-33– and IL-25–induced ILC2 activation. ILC2s from Regnase-1AA/AA mice expressing a Regnase-1 S435A/S439A mutant resistant to IKK complex-mediated degradation, accumulated Regnase-1 protein in response to IL-33 and IL-25. IL-33– and IL-25–stimulated Regnase-1AA/AA ILC2s showed reduced cell proliferation and type-2 cytokine (IL-5, IL-9, and IL-13) production and increased cell death. In addition, Il2ra and Il1rl1, but not Il5, Il9, or Il13, mRNAs were destabilized in IL-33–stimulated Regnase-1AA/AA ILC2s. In vivo, Regnase-1AA/AA mice showed attenuated acute type-2 pulmonary inflammation induced by the instillation of IL-33, IL-25, or papain. Furthermore, the expulsion of Nippostrongylus brasiliensis was significantly delayed in Regnase-1AA/AA mice. These results demonstrate that IKK complex-mediated Regnase-1 degradation is essential for ILC2s-mediated type-2 responses both in vitro and in vivo. Therefore, controlling Regnase-1 degradation is a potential therapeutic target for ILC2-contributed allergic disorders.
Authors
Kazufumi Matsushita, Hiroki Tanaka, Koubun Yasuda, Takumi Adachi, Ayumi Fukuoka, Shoko Akasaki, Atsuhide Koida, Etsushi Kuroda, Shizuo Akira, Tomohiro Yoshimoto
Abstract
Pulmonary Langerhans cell histiocytosis (PLCH) is a rare, smoking-related, lung disease characterized by dendritic cell (DC) accumulation, bronchiolocentric nodule formation, and cystic lung remodeling. Approximately 50% of PLCH patients harbor somatic BRAF-V600E mutations in cells of the myeloid/monocyte lineage. However, the rarity of the disease and lack of animal models has impeded the study of PLCH pathogenesis. Here, we established a cigarette smoke (CS)-exposed, BRAF-V600E mutant mouse model that recapitulates many hallmark characteristics of PLCH. We show that CD11c-targeted expression of BRAF-V600E increases DC responsiveness to stimuli, including the chemokine CCL20, and that mutant DC accumulation in the lungs of CS-exposed mice is due to both increased cellular viability and enhanced recruitment. Moreover, we report that the chemokine CCL7 is secreted from DCs and human peripheral blood monocytes in a BRAF-V600E-dependent manner, suggesting a possible mechanism for recruitment of cells known to dominate PLCH lesions. Inflammatory lesions and airspace dilation in BRAF-V600E mice in response to CS are attenuated by transitioning animals to filtered air and treatment with a BRAF-V600E inhibitor, PLX4720. Collectively, this model provides mechanistic insights into the role of DCs, the BRAF-V600E mutation and CS exposure in PLCH pathogenesis, and provides a platform to develop therapeutic targets.
Authors
Huan Liu, Andrew R. Osterburg, Jennifer Flury, Zulma Swank, Dennis W. McGraw, Nishant Gupta, Kathryn A. Wikenheiser-Brokamp, Ashish Kumar, Abdellatif Tazi, Yoshikazu Inoue, Masaki Hirose, Francis X. McCormack, Michael Borchers
Abstract
Current models of B lymphocyte biology posit that B cells continuously recirculate between lymphoid organs without accumulating in peripheral healthy tissues. Nevertheless, B lymphocytes are one of the most prevalent leukocyte populations in the naive murine heart. To investigate this apparent inconsistency in the literature, we conducted a systematic analysis of myocardial B cell ontogeny, trafficking dynamics, histology, and gene expression patterns. We found that myocardial B cells represent a subpopulation of circulating B cells that make close contact with the microvascular endothelium of the heart and arrest their transit as they pass through the heart. The vast majority (> 95%) of myocardial B cells remain intravascular, whereas few (< 5%) myocardial B cells cross the endothelium into myocardial tissue. Analyses of mice with B cell deficiency or depletion indicated that B cells modulate the myocardial leukocyte pool composition. Analysis of B cell deficient animals suggested that B cells modulate myocardial growth and contractility. These results transform our current understanding of B cell recirculation in the naive state and reveal a previously unknown relationship between B cells and myocardial physiology. Further work will be needed to assess the relevance of these findings to other organs.
Authors
Luigi Adamo, Cibele Rocha-Resende, Chieh-Yu Lin, Sarah Evans, Jesse W. Williams, Hao Dun, Wenjun Li, Cedric Mpoy, Prabhakar Andhey, Buck E. Rogers, Kory Lavine, Daniel Kreisel, Maxim N. Artyomov, Gwendalyn J. Randolph, Douglas Mann
Abstract
Colitis, an inflammatory bowel disease, is caused by a variety of factors, but luminal microbiota are thought to play crucial roles in disease development and progression. Indole is produced by gut microbiota and is believed to protect the colon from inflammatory damage. In the current study, we investigated whether indole-3-carbinol (I3C), a naturally occurring plant product found in numerous cruciferous vegetables, can prevent colitis-associated microbial dysbiosis and attempted to identify the mechanisms. Treatment with I3C led to repressed colonic inflammation and prevention of microbial dysbiosis caused by colitis, increasing a subset of gram-positive bacteria known to produce butyrate. I3C was shown to increase production of butyrate, and when mice with colitis were treated with butyrate, there was reduced colonic inflammation accompanied by suppression of Th17 and induction of Tregs, protection of the mucus layer, and upregulation in Pparg expression. Additionally, IL-22 was increased only after I3C but not butyrate administration, and neutralization of IL-22 prevented the beneficial effects of I3C against colitis, as well as blocked I3C-mediated dysbiosis and butyrate induction. This study suggests that I3C attenuates colitis primarily through induction of IL-22, which leads to modulation of gut microbiota that promote antiinflammatory butyrate.
Authors
Philip B. Busbee, Lorenzo Menzel, Haider Alrafas, Nicholas Dopkins, William Becker, Kathryn Miranda, Chaunbing Tang, Saurabh Chatterjee, Udai Singh, Mitzi Nagarkatti, Prakash S. Nagarkatti
Abstract
Recovery from measles results in life-long protective immunity. To understand induction of long-term immunity, rhesus macaques were studied for six months after infection with WT measles virus (MeV). Infection caused viremia and rash with clearance of infectious virus by 14 days. MeV RNA persisted in PBMCs for 30-90 days and in lymphoid tissue for 6 months most often in B cells but was rarely detected in BM. Antibody with neutralizing activity and binding specificity for MeV nucleocapsid (N), hemagglutinin (H) and fusion proteins appeared with the rash and avidity matured over 3-4 months. Lymph nodes had increasing numbers of MeV-specific antibody-secreting cells (ASCs) and germinal centers with late hyalinization. ASCs appeared in circulation with the rash and continued to appear along with peripheral Tfh cells for the study duration. ASCs in lymph nodes and PBMCs produced antibody to both H and N, with more H-specific ASCs in BM. From 14-21 days 20-100-fold more total ASCs than MeV-specific ASCs appeared in circulation suggesting mobilization of pre-existing ASCs. Therefore, persistence of MeV RNA in lymphoid tissue was accompanied by continued germinal center formation, ASC production, avidity maturation and accumulation of H-specific ASCs in BM to sustain neutralizing antibody and protective immunity.
Authors
Ashley N. Nelson, Wen-Hsuan W. Lin, Rupak Shivakoti, Nicole E. Putnam, Lisa M. Mangus, Robert J. Adams, Debra Hauer, Victoria K. Baxter, Diane E. Griffin
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