Research
Abstract
A critical component of wound healing is the transition from the inflammatory phase to the proliferation phase to initiate healing and remodeling of the wound. Macrophages are critical for the initiation and resolution of the inflammatory phase during wound repair. In diabetes, macrophages display a sustained inflammatory phenotype in late wound healing characterized by elevated production of inflammatory cytokines such as TNFα. Previous studies have shown that an altered epigenetic program directs diabetic macrophages towards a pro-inflammatory phenotype contributing to a sustained inflammatory phase. Males absent on the first (MOF) is a histone acetyl-transferase (HAT) that has been shown be a co-activator of TNFα-signaling and promote NFκB-mediated gene transcription in prostate cancer cell lines. Based on MOFs role in TNFα/NFκB-mediated gene expression, we hypothesized that MOF influences macrophage-mediated inflammation during wound repair. We used a myeloid-specific Mof knockout (Lyz2Cre Moff/f) and diet-induced obese (DIO) mice, to determine the function of MOF in diabetic wound healing. MOF deficient mice exhibited reduced inflammatory cytokine gene expression. Furthermore, we found that wound macrophages from DIO mice had elevated MOF levels and higher levels of acetylated histone H4K16, MOFs primary substrate of HAT activity, on the promoters of inflammatory genes. We further identified that MOF expression could by stimulated by TNFα and that treatment with Etanercept, an FDA-approved TNFα inhibitor, reduced MOF levels and improved wound healing in DIO mice. This report is the first to define an important role for MOF in regulating macrophage-mediated inflammation in wound repair and identifies TNFα-inhibition as a potential therapy for the treatment of chronic inflammation in diabetic wounds.
Authors
Aaron D. denDekker, Frank M. Davis, Amrita D. Joshi, Sonya J. Wolf, Ronald Allen, Jay Lipinski, Brenda Nguyen, Joseph Kirma, Dylan Nycz, Jennifer R. Bermick, Bethany B. Moore, Johann E. Gudjonsson, Steven L. Kunkel, Katherine A. Gallagher
Abstract
Maintaining cellular proteostasis is essential for oligodendrocyte viability and function; however, its underlying mechanisms remain unexplored. The UPR, comprising three parallel branches IRE1, PERK, and ATF6α, is a major mechanism that maintains cellular proteostasis by facilitating protein folding, attenuating protein translation, and enhancing autophagy and ERAD. Here we reported that impaired UPR in oligodendrocytes via deletion of PERK and ATF6α did not affect developmental myelination, but caused late-onset mature oligodendrocyte dysfunction and death in young adult mice. The detrimental effects of the impaired UPR on mature oligodendrocytes were accompanied by autophagy impairment and intracellular PLP accumulation, and were rescued by PLP deletion. Data indicate that PLP is degraded by autophagy and that intracellular PLP accumulation is cytotoxic to oligodendrocytes. Thus, these findings imply that the UPR is required for maintaining cellular proteostasis and the viability and function of mature oligodendrocytes in adults by regulating autophagy of PLP.
Authors
Sarrabeth Stone, Shuangchan Wu, Klaus-Armin Nave, Wensheng Lin
Abstract
Development of gastric cancer is often preceded by chronic inflammation, but the immune cellular mechanisms underlying this process are unclear. Here we demonstrated that an inflammasome molecule, absent in melanoma 2 (Aim2), was upregulated in gastric cancer patients, and in spasmolytic polypeptide-expressing metaplasia (SPEM) of chronically Helicobacter felis (H. felis)-infected stomachs in mice. However, we found that Aim2 was not necessary for inflammasome function during gastritis. In contrast, Aim2 deficiency led to an increase in gastric CD8+ T cell frequency, which exacerbated metaplasia. These gastric CD8+ T cells from Aim2-/- mice were found to have lost their homing receptor expression (S1pr1 and CD62l), a feature of tissue resident memory T cells (TRM). The process was not mediated by Aim2-dependent regulation of IFN-β, or by dendritic cell-intrinsic Aim2. Rather, Aim2 deficiency contributed to an increased production of Cxcl16 by B cells, which could suppress S1pr1 and CD62l in CD8+ T cells. The study describes a novel function of Aim2 that regulates CD8+ T cell infiltration and retention within chronically inflamed solid organ tissue. This function operates independently of the inflammasome, IFN-β or dendritic cells. We provide evidence that B cells can contribute to this mechanism via Cxcl16.
Authors
Mohamad El-Zaatari, Shrinivas Bishu, Min Zhang, Helmut Grasberger, Guoqing Hou, Henry R. Haley, Brock A. Humphries, Li-Jyun Syu, Andrzej Dlugosz, Kathryn E. Luker, Gary Luker, Kathryn A. Eaton, Nobuhiko Kamada, Marilia Cascalho, John Y. Kao
Abstract
Small primary breast cancers can show surprisingly high potential for metastasis. Clinical decision making for tumor aggressiveness, including molecular profiling, relies primarily on analysis of the cancer cells. Here we show that this is insufficient; that the stromal microenvironment of the primary tumor plays a key role in tumor-cell dissemination and implantation at distant sites. We previously described two cancer-associated fibroblasts (CAFs) that either express (CD146pos) or lack (CD146neg) CD146 (official symbol MCAM; alias MUC18). We now find that when mixed with human breast cancer cells, each fibroblast subtype determines the fate of cancer-cells: CD146neg fibroblasts promote increased metastasis compared to CD146pos fibroblasts. Novel quantitative and qualitative proteomic analyses show that CD146pos CAFs produce an environment rich in basement membrane proteins, while CD146neg CAFs exhibit increases in FN1, LOX, and TNC; all over-expressed in aggressive disease. We also show clinically, that CD146neg CAFs predict for likelihood of lymph node involvement even in small primary tumors (<5 cm). Clearly small tumors enriched for CD146neg CAFs require aggressive treatments.
Authors
Heather M. Brechbuhl, Alexander S. Barrett, Etana Kopin, Jaime C. Hagen, Amy L. Han, Austin E. Gillen, Jessica Finlay-Schultz, Diana M. Cittelly, Philip Owens, Kathryn B. Horwitz, Carol A. Sartorius, Kirk C. Hansen, Peter Kabos
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
Recent studies have presented compelling evidence that it is not tissue-resident, but rather monocyte-derived alveolar macrophages (TR-AMs vs. Mo-AMs) are essential to development of experimental lung fibrosis. However, whether Apolipoprotein E (ApoE), which is produced abundantly by Mo-AMs in the lung, plays a role in the pathogenesis is unclear. In this study, we found that pulmonary ApoE was almost exclusively produced by Mo-AMs in mice with bleomycin induced lung fibrosis. We showed although ApoE was not necessary for developing maximal fibrosis in bleomycin injured lung, it was required for the resolution of this pathology. We found that ApoE directly bound to Collagen I and mediated Collagen I phagocytosis in vitro and in vivo, and this process was dependent on low density lipoprotein receptor-related protein 1 (LPR1). Furthermore, interference of ApoE/LRP1 interaction impaired the resolution of lung fibrosis in bleomycin treated wild-type mice. In contrast, supplementation of ApoE promoted this process in ApoE–/– animals. In conclusion, Mo-AM derived ApoE is beneficial to the resolution of lung fibrosis, supporting the notion that Mo-AMs may have distinct functions in different phases of lung fibrogenesis. The findings also suggest a novel therapeutic target for treating lung fibrosis, to which effective remedies remain scarce.
Authors
Huachun Cui, Dingyuan Jiang, Sami Banerjee, Na Xie, Tejaswini Kulkarni, Rui-Ming Liu, Steven R. Duncan, Gang Liu
Abstract
The mitochondrial calcium uniporter is widely accepted as the primary route of rapid calcium entry into mitochondria, where increases in matrix calcium contribute to bioenergetics but also mitochondrial permeability and cell death. Hence, regulation of uniporter activity is critical to mitochondrial homeostasis. The uniporter subunit EMRE is known to be an essential regulator of the channel-forming protein MCU in cell culture, but EMRE’s impact on organismal physiology is less understood. Here we characterize a novel mouse model of EMRE deletion and show that EMRE is indeed required for mitochondrial calcium uniporter function in vivo. EMRE–/– mice are born less frequently; however, the mice which are born are viable, healthy, and do not manifest overt metabolic impairment, at rest or with exercise. Finally, to investigate the role of EMRE in disease processes, we examine the effects of EMRE deletion in a muscular dystrophy model associated with mitochondrial calcium overload.
Authors
Julia C. Liu, Nicole C. Syder, Nima S. Ghorashi, Thomas B. Willingham, Randi J. Parks, Junhui Sun, Maria M. Fergusson, Jie Liu, Kira M. Holmström, Sara Menazza, Danielle A. Springer, Chengyu Liu, Brian Glancy, Toren Finkel, Elizabeth Murphy
Abstract
Decades ago, investigators reported that mice lacking DLX1 and DLX2, transcription factors expressed in the enteric nervous system (ENS), die with possible bowel motility problems. These problems were never fully elucidated. We found that mice lacking DLX1 and DLX2 (Dlx1/2-/- mice) had slower small bowel transit and reduced or absent neurally-mediated contraction complexes. In contrast, small bowel motility seemed normal in adult mice lacking DLX1 (Dlx1-/-). Even with detailed anatomic studies, we found no defects in ENS precursor migration, or neuron and glia density, in Dlx1/2-/- or Dlx1-/- mice. However, RNA sequencing of Dlx1/2-/- ENS revealed dysregulation of many genes, including vasoactive intestinal peptide (Vip). Our study reveals a novel connection between Dlx genes and Vip and highlights the observation that dangerous bowel motility problems can occur in the absence of easily identifiable ENS structural defects. These findings may be relevant for disorders like chronic intestinal pseudo-obstruction (CIPO) syndrome.
Authors
Christina M. Wright, James P. Garifallou, Sabine Schneider, Heather L. Mentch, Deepika R. Kothakapa, Beth A. Maguire, Robert O. Heuckeroth
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