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.
Mian Zhou, Monowar Aziz, Naomi-Liza Denning, Hao-Ting Yen, Gaifeng Ma, Ping Wang
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.
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
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.
Kazufumi Matsushita, Hiroki Tanaka, Koubun Yasuda, Takumi Adachi, Ayumi Fukuoka, Shoko Akasaki, Atsuhide Koida, Etsushi Kuroda, Shizuo Akira, Tomohiro Yoshimoto
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.
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
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.
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
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.
Philip B. Busbee, Lorenzo Menzel, Haider Alrafas, Nicholas Dopkins, William Becker, Kathryn Miranda, Chaunbing Tang, Saurabh Chatterjee, Udai Singh, Mitzi Nagarkatti, Prakash S. Nagarkatti
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.
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
Dengue (DENV) and Zika viruses (ZIKV) are closely related mosquito-borne flaviviruses that co-circulate in tropical regions and constitute major threats to global human health. Whether preexisting immunity to one virus affects disease caused by the other during primary or secondary infections is unknown but is critical in preparing for future outbreaks and predicting vaccine safety. Using a human skin explant model, we show that DENV-3 immune sera increased recruitment and infection of Langerhans cells, macrophages and dermal dendritic cells following inoculation with DENV-2 or ZIKV. Similarly, ZIKV immune sera enhanced infection with DENV-2. Immune sera increased migration of infected Langerhans cells to dermis and emigration of infected cells out of skin. Heterotypic immune sera increased viral RNA in dermis almost tenfold and reduced the amount of virus required to infect a majority of myeloid cells by 100 to 1,000 fold. Enhancement was associated with cross-reactive IgG and induction of IL-10 expression and was mediated by both CD32 and CD64 Fcγ receptors. These findings reveal that preexisting heterotypic immunity greatly enhances DENV and ZIKV infection, replication and spread in human skin. This relevant tissue model will be valuable in assessing the efficacy and risk of dengue and Zika vaccines in humans.
Priscila M.S. Castanha, Geza Erdos, Simon C. Watkins, Louis D. Falo, Jr., Ernesto T.A. Marques, Simon M. Barratt-Boyes
Genetic variants within/near the interferon regulatory factor 5 (IRF5) locus associate with systemic lupus erythematosus (SLE) across ancestral groups. The major IRF5-SLE risk haplotype is common across populations, yet immune functions for the risk haplotype are undefined. We characterized the global immune-phenotype of healthy donors homozygous for the major risk and non-risk haplotypes and identified cell lineage-specific alterations that mimic pre-symptomatic SLE. Contrary to previous studies in B lymphoblastoid cell lines and SLE immune cells, IRF5 genetic variants had little effect on IRF5 protein levels in healthy donors. Instead, we detected basal IRF5 hyper-activation in the myeloid compartment of risk donors that drives the SLE immune-phenotype. Risk donors were ANA positive with anti-Ro and -MPO specificity, had increased circulating plasma cells and plasmacytoid dendritic cells, and enhanced spontaneous NETosis. The IRF5-SLE immune-phenotype was conserved over time and probed mechanistically by ex vivo co-culture, indicating that risk neutrophils are drivers of the global immune-phenotype. RNA-seq of risk neutrophils revealed increased IRF5 transcript expression, IFN pathway enrichment and decreased expression of ROS pathway genes. Altogether, data support that individuals carrying the IRF5-SLE risk haplotype are more susceptible to environmental/stochastic influences that trigger chronic immune activation, predisposing to the development of clinical SLE.
Dan Li, Bharati Matta, Su Song, Victoria Nelson, Kirsten Diggins, Kim R. Simpfendorfer, Peter K. Gregersen, Peter Linsley, Betsy J. Barnes
A recent study of AHSCT for active relapsing-remitting multiple sclerosis (RRMS) showed efficacy in preventing disease worsening. However, the immunologic basis for efficacy remains poorly defined. MS pathology is known to be driven by inflammatory T cells that infiltrate the central nervous system (CNS). Therefore, we hypothesized that the pre-existing T cell repertoire in the intrathecal compartment of active RRMS participants was ablated, and replaced with new clones following AHSCT. T cell repertoires were assessed using high-throughput TCRβ chain sequencing in paired cerebrospinal fluid (CSF) and peripheral blood CD4+ and CD8+ T cells from participants that underwent AHSCT, before and up to 4 years following transplantation. >90% of the pre-existing CSF repertoire in participants with active RRMS was removed following AHSCT, and replaced with clonotypes predominantly generated from engrafted autologous stem cells. Of the pre-existing clones in CSF, ~60% were also detected in pre-therapy blood, and concordant treatment effects were observed for clonotypes in both compartments following AHSCT. These results indicate that replacement of the pre-existing TCR repertoire in active RRMS is a mechanism for AHSCT efficacy, and suggest that peripheral blood could serve as a surrogate for CSF to define mechanisms associated with efficacy in future studies of AHSCT.
Kristina M. Harris, Noha Lim, Paul Lindau, Harlan Robins, Linda M. Griffith, Richard A. Nash, Laurence A. Turka, Paolo A. Muraro
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