Neutrophils are the most abundant inflammatory cells at the earliest stages of wound healing and play important roles in wound repair and fibrosis. Formyl peptide receptor 1 (FPR-1) is abundantly expressed on neutrophils and has been shown to regulate their function, yet the importance of FPR-1 in fibrosis remains ill defined. FPR-1–deficient (fpr1–/–) mice were protected from bleomycin-induced pulmonary fibrosis but developed renal and hepatic fibrosis normally. Mechanistically, we observed a failure to effectively recruit neutrophils to the lungs of fpr1–/– mice, whereas neutrophil recruitment was unaffected in the liver and kidney. Using an adoptive transfer model we demonstrated that the defect in neutrophil recruitment to the lung was intrinsic to the fpr1–/– neutrophils, as C57BL/6 neutrophils were recruited normally to the damaged lung in fpr1–/– mice. Finally, C57BL/6 mice in which neutrophils had been depleted were protected from pulmonary fibrosis. In conclusion, FPR-1 and FPR-1 ligands are required for effective neutrophil recruitment to the damaged lung. Failure to recruit neutrophils or depletion of neutrophils protects from pulmonary fibrosis.
Jack Leslie, Ben J.M. Millar, Alicia del Carpio Pons, Rachel A. Burgoyne, Joseph D. Frost, Ben S. Barksby, Saimir Luli, Jon Scott, A. John Simpson, Jack Gauldie, Lynne A. Murray, Donna K. Finch, Alan M. Carruthers, John Ferguson, Matthew A. Sleeman, David Rider, Rachel Howarth, Christopher Fox, Fiona Oakley, Andrew J. Fisher, Derek A. Mann, Lee A. Borthwick
Adjuvant chemotherapy in breast cancer patients causes immune cell depletion at an age when the regenerative capacity is compromised. Successful regeneration requires the recovery of both quantity and quality of immune cell subsets. Although immune cell numbers rebound within a year after treatment, it is unclear whether overall compositional diversity is recovered. We investigated the regeneration of immune cell complexity by comparing peripheral blood mononuclear cells from breast cancer patients ranging from 1–5 years after chemotherapy with those of age-matched healthy controls using mass cytometry and T cell receptor sequencing. These data reveal universal changes in patients’ CD4+ T cells that persisted for years and consisted of expansion of Th17-like CD4 memory populations with incomplete recovery of CD4+ naive T cells. Conversely, CD8+ T cells fully recovered within a year. Mechanisms of T cell regeneration, however, were unbiased, as CD4+ and CD8+ T cell receptor diversity remained high. Likewise, terminal differentiated effector memory cells were not expanded, indicating that regeneration was not driven by recognition of latent viruses. These data suggest that, while CD8+ T cell immunity is successfully regenerated, the CD4 compartment may be irreversibly affected. Moreover, the bias of CD4 memory toward inflammatory effector cells may impact responses to vaccination and infection.
Claire E. Gustafson, Rohit Jadhav, Wenqiang Cao, Qian Qi, Mark Pegram, Lu Tian, Cornelia M. Weyand, Jorg J. Goronzy
IL-4 is a pleiotropic antiinflammatory cytokine, which can be neuroprotective after nervous system injury. The beneficial actions of IL-4 are thought to result from the blunting of action of inflammatory mediators, such as proinflammatory cytokines. Here, we demonstrate that IL-4 induces M2 macrophages to continuously produce opioid peptides and ameliorate pain. IL-4 application at injured nerves in mice shifted F4/80+ macrophages from the proinflammatory M1 to the antiinflammatory M2 phenotype, which synthesized opioid peptides (Met-enkephalin, β-endorphin, and dynorphin A 1-17). These effects were accompanied by a long-lasting attenuation of neuropathy-induced mechanical hypersensitivity, beyond the IL-4 treatment. This IL-4-induced analgesia was decreased by opioid peptide antibodies and opioid receptor (δ, μ, κ) antagonists applied at injured nerves, which confirms the involvement of the local opioid system. The participation of M2 macrophages was supported by analgesia in recipient mice injected at injured nerves with F4/80+ macrophages from IL-4–treated donors. Together, IL-4–induced M2 macrophages at injured nerves produced opioid peptides, which activated peripheral opioid receptors to diminish pain. Fostering the opioid-mediated actions of intrinsic M2 macrophages may be a strategy to tackle pathological pain.
Melih Ö. Celik, Dominika Labuz, Jacqueline Keye, Rainer Glauben, Halina Machelska
A terminally differentiated cellular phenotype is thought to be maintained, at least in part, by both active and repressive histone marks. However, it is unclear whether regenerating cells after injury need to replicate such epigenetic marks to recover. To test whether renal epithelial cell regeneration is dependent on histone H3K4 methylation, we generated a mouse model that deleted the Paxip1 gene in mature renal proximal tubules. Paxip1 encodes PTIP, an essential protein in the Mll3/4 histone H3K4 methyltransferase complex. Mice with PTIP deletions in the adult kidney proximal tubules were viable and fertile. Upon acute kidney injury, such mice failed to regenerate damaged tubules, leading to scarring and interstitial fibrosis. The inability to repair damage was likely due to a failure to reenter mitosis and reactivate regulatory genes such as Sox9. PTIP deletion reduced histone H3K4 methylation in uninjured adult kidneys but did not significantly affect function or the expression of epithelial specific markers. Strikingly, cell lineage tracing revealed that surviving PTIP mutant cells could alter their phenotype and lose epithelial markers. These data demonstrate that PTIP and associated MLL3/4-mediated histone methylation are needed for regenerating proximal tubules and to maintain or reestablish the cellular epithelial phenotype.
Abdul Soofi, Ana P. Kutschat, Mohammad Azam, Ann M. Laszczyk, Gregory R. Dressler
Dengue virus (DENV) and Zika virus (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 the dermis and emigration of infected cells out of skin. Heterotypic immune sera increased viral RNA in the dermis almost 10-fold and reduced the amount of virus required to infect a majority of myeloid cells by 100- to 1000-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
Idiopathic inflammatory myopathies (IIM) are characterized by muscle inflammation and weakness, myositis-specific autoantibodies (MSAs), and extramuscular organ damage. The role of neutrophil dysregulation and neutrophil extracellular traps (NETs) in IIM is unclear. We assessed whether pathogenic neutrophil subsets (low-density granulocytes [LDGs]) and NETs were elevated in IIM, associated with clinical presentation and MSAs, and their effect on skeletal myoblasts and myotubes. Circulating NETs and LDGs were quantified and correlated with clinical measures. Specific MSAs were tested for their ability to induce NETs. NETs and neutrophil gene expression were measured in IIM biopsies. Whether NETs damage skeletal myoblasts and myotubes was tested. Circulating LDGs and NETs were increased in IIM. IIM LDGs had an enhanced ability to form NETs. LDGs and NETs correlated with IIM disease activity and muscle damage. The serum MSA anti-MDA5 correlated with circulating and tissue NETs and directly enhanced NET formation. An enhanced neutrophil gene signature was present in IIM muscle and associated with muscle injury and tissue IFN gene signatures. IIM NETs decreased the viability of myotubes in a citrullinated histone-dependent manner. Dysregulated neutrophil pathways may play pathogenic roles in IIM through their ability to directly injure muscle cells and other affected tissues.
Nickie Seto, Jose Jiram Torres-Ruiz, Carmelo Carmona-Rivera, Iago Pinal-Fernandez, Katherine Pak, Monica M. Purmalek, Yuji Hosono, Catia Fernandes-Cerqueira, Prateek Gowda, Nathan Arnett, Alexander Gorbach, Olivier Benveniste, Diana Gómez-Martín, Albert Selva-O’Callaghan, José C. Milisenda, Josep M. Grau-Junyent, Lisa Christopher-Stine, Frederick W. Miller, Ingrid E. Lundberg, J. Michelle Kahlenberg, Adam I. Schiffenbauer, Andrew Mammen, Lisa G. Rider, Mariana J. Kaplan
Interleukin-3 (IL-3) receptor α (IL-3Rα) is the α subunit of the ligand-specific IL-3R and initiates intracellular signaling in response to IL-3. IL-3 amplifies proinflammatory signaling and cytokine storm in murine sepsis models. Here we found that RNFT2 (RING finger transmembrane-domain containing protein 2, also TMEM118), a previously uncharacterized RING finger ubiquitin E3 ligase, negatively regulated IL-3–dependent cellular responses through IL-3Rα ubiquitination and degradation in the proteasome. In vitro, IL-3 stimulation promoted IL-3Rα proteasomal degradation dependent on RNFT2, and we identified IL-3Rα lysine 357 as a ubiquitin acceptor site. We determined that LPS priming reduces RNFT2 abundance, extends IL-3Rα half-life, and sensitizes cells to the effects of IL-3, acting synergistically to increase proinflammatory signaling. In vivo, IL-3 synergized with LPS to exacerbate lung inflammation in LPS and Pseudomonas aeruginosa–challenged mice; conversely, IL-3 neutralization reduced LPS-induced lung injury. Further, RNFT2 overexpression reduced lung inflammation and injury, whereas Rnft2 knockdown exacerbated inflammatory responses in LPS-induced murine lung injury. Last, we examined RNFT2 and IL-3Rα in human lung explants from patients with cystic fibrosis and also showed that IL-3 is elevated in mechanically ventilated critically ill humans at risk for acute respiratory distress syndrome. These results identify RNFT2 as a negative regulator of IL-3Rα and show a potential role for the RNFT2/IL-3Rα/IL-3 axis in regulating innate immune responses in the lung.
Yao Tong, Travis B. Lear, John Evankovich, Yanwen Chen, James D. Londino, Michael M. Myerburg, Yingze Zhang, Iulia D. Popescu, John F. McDyer, Bryan J. McVerry, Karina C. Lockwood, Michael J. Jurczak, Yuan Liu, Bill B. Chen
Chronic sympathoexcitation is implicated in ventricular arrhythmogenesis (VAs) following myocardial infarction (MI), but the critical neural pathways involved are not well understood. Cardiac adrenergic function is partly regulated by sympathetic afferent reflexes, transduced by spinal afferent fibers expressing the transient receptor potential cation subfamily V member 1 (TRPV1) channel. The role of chronic TRPV1 afferent signaling in VAs is not known. We hypothesized that persistent TRPV1 afferent neurotransmission promotes VAs after MI. Using epicardial resiniferatoxin (RTX) to deplete cardiac TRPV1–expressing fibers, we dissected the role of this neural circuit in VAs after chronic MI in a porcine model. We examined the underlying mechanisms using molecular approaches, IHC, in vitro and in vivo cardiac electrophysiology, and simultaneous cardioneural mapping. Epicardial RTX depleted cardiac TRPV1 afferent fibers and abolished functional responses to TRPV1 agonists. Ventricular tachycardia/fibrillation (VT/VF) was readily inducible in MI subjects by programmed electrical stimulation or cesium chloride administration; however, TRPV1 afferent depletion prevented VT/VF induced by either method. Mechanistically, TRPV1 afferent depletion did not alter cardiomyocyte action potentials and calcium transients, the expression of ion channels, or calcium handling proteins. However, it attenuated fibrosis and mitigated electrical instability in the scar border zone. In vivo recordings of cardiovascular-related stellate ganglion neurons (SGNs) revealed that MI enhances SGN function and disrupts integrated neural processing. Depleting TRPV1 afferents normalized these processes. Taken together, these data indicate that, after MI, TRPV1 afferent–induced adrenergic dysfunction promotes fibrosis and adverse cardiac remodeling, and it worsens border zone electrical heterogeneity, resulting in electrically unstable ventricular myocardium. We propose targeting TRPV1-expressing afferent to reduce VT/VF following MI.
Koji Yoshie, Pradeep S. Rajendran, Louis Massoud, Janki Mistry, M. Amer Swid, Xiaohui Wu, Tamer Sallam, Rui Zhang, Joshua I. Goldhaber, Siamak Salavatian, Olujimi A. Ajijola
Recovery from measles results in life-long protective immunity. To understand induction of long-term immunity, rhesus macaques were studied for 6 months after infection with wild-type measles virus (MeV). Infection caused viremia and rash, with clearance of infectious virus by day 14. 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 T follicular helper cells for the study duration. ASCs in lymph nodes and PBMCs produced antibody against both H and N, with more H-specific ASCs in BM. During days 14–21, 20- to 100-fold more total ASCs than MeV-specific ASCs appeared in circulation, suggesting mobilization of preexisting 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 Mangus, Robert J. Adams, Debra Hauer, Victoria K. Baxter, Diane E. Griffin
Alternative polyadenylation (APA) is a widespread and important mechanism in regulation of gene expression. Dysregulation of the 3′ UTR cleavage and polyadenylation represents a common characteristic among many disease states, including lung fibrosis. In this study, we investigated the role of mammalian cleavage factor I–mediated (CFIm-mediated) APA in regulating extracellular matrix production in response to mechanical stimuli from stiffened matrix simulating the fibrotic lungs. We found that stiff matrix downregulated expression of CFIm68, CFIm59 and CFIm25 subunits and promoted APA in favor of the proximal poly(A) site usage in the 3′ UTRs of type I collagen (COL1A1) and fibronectin (FN1) in primary human lung fibroblasts. Knockdown and overexpression of each individual CFIm subunit demonstrated that CFIm68 and CFIm25 are indispensable attributes of stiff matrix–induced APA and overproduction of COL1A1, whereas CFIm did not appear to mediate stiffness-regulated FN1 APA. Furthermore, expression of the CFIm subunits was associated with matrix stiffness in vivo in a bleomycin-induced mouse model of pulmonary fibrosis. These data suggest that stiff matrix instigates type I collagen biogenesis by selectively targeting mRNA transcripts for 3′ UTR shortening. The current study uncovered a potential mechanism for regulation of the CFIm complex by mechanical cues under fibrotic conditions.
Zijing Zhou, Jing Qu, Li He, Yi Zhu, Shan-Zhong Yang, Feng Zhang, Ting Guo, Hong Peng, Ping Chen, Yong Zhou
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