Publications by authors named "Amrita Joshi"

30 Publications

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Facilitator lessons from online psychoeducational group for relational well-being in India during COVID-19 pandemic.

J Fam Ther 2021 Apr 15;43(2):314-328. Epub 2021 Feb 15.

Research officer, Sukoon, Tata Institute of Social Sciences Mumbai India.

The COVID-19 pandemic has affected the mental health of individuals, along with their couple and familial relationships, necessitating an effective response. Teletherapy offers an option to address these relationship concerns amidst pandemic-specific mobility restriction. Against this setting, Sukoon, a project of Tata Institute of Social Sciences, India, initiated a five-session online psychoeducational group series on relational wellbeing. This paper explores facilitator's reflections and learnings based on session documentation and facilitator notes. Preparing well and selecting participants carefully for online psychoeducational groups was critical to success. Effectiveness was enhanced by flexibly adapting the therapy process (didactic and interactive elements) to fit online delivery and the cultural context. Identifying the potential of online psychoeducational groups for relational wellbeing could make it a valuable addition to the COVID-19 pandemic mental health response toolkit.

Practitioner Points: Effective preparation and careful selection of group members is key to the success of therapist facilitated online psychoeducational groups.Psychoeducational groups comprising didactic and interactive elements are more suitable for effective online group processes.Use of co-facilitators managing various channels of communication (audio, chat) is important. Group facilitators need to be cognisant of challenges of online medium and address them in an ongoing manner.
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http://dx.doi.org/10.1111/1467-6427.12337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8013498PMC
April 2021

Inhibition of macrophage histone demethylase JMJD3 protects against abdominal aortic aneurysms.

J Exp Med 2021 Jun;218(6)

Section of Vascular Surgery, Department of Surgery, University of Michigan, Ann Arbor, MI.

Abdominal aortic aneurysms (AAAs) are a life-threatening disease for which there is a lack of effective therapy preventing aortic rupture. During AAA formation, pathological vascular remodeling is driven by macrophage infiltration, and the mechanisms regulating macrophage-mediated inflammation remain undefined. Recent evidence suggests that an epigenetic enzyme, JMJD3, plays a critical role in establishing macrophage phenotype. Using single-cell RNA sequencing of human AAA tissues, we identified increased JMJD3 in aortic monocyte/macrophages resulting in up-regulation of an inflammatory immune response. Mechanistically, we report that interferon-β regulates Jmjd3 expression via JAK/STAT and that JMJD3 induces NF-κB-mediated inflammatory gene transcription in infiltrating aortic macrophages. In vivo targeted inhibition of JMJD3 with myeloid-specific genetic depletion (JMJD3f/fLyz2Cre+) or pharmacological inhibition in the elastase or angiotensin II-induced AAA model preserved the repressive H3K27me3 on inflammatory gene promoters and markedly reduced AAA expansion and attenuated macrophage-mediated inflammation. Together, our findings suggest that cell-specific pharmacologic therapy targeting JMJD3 may be an effective intervention for AAA expansion.
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http://dx.doi.org/10.1084/jem.20201839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008365PMC
June 2021

Epigenetic regulation of the PGE2 pathway modulates macrophage phenotype in normal and pathologic wound repair.

JCI Insight 2020 09 3;5(17). Epub 2020 Sep 3.

Section of Vascular Surgery, Department of Surgery.

Macrophages are a primary immune cell involved in inflammation, and their cell plasticity allows for transition from an inflammatory to a reparative phenotype and is critical for normal tissue repair following injury. Evidence suggests that epigenetic alterations play a critical role in establishing macrophage phenotype and function during normal and pathologic wound repair. Here, we find in human and murine wound macrophages that cyclooxygenase 2/prostaglandin E2 (COX-2/PGE2) is elevated in diabetes and regulates downstream macrophage-mediated inflammation and host defense. Using single-cell RNA sequencing of human wound tissue, we identify increased NF-κB-mediated inflammation in diabetic wounds and show increased COX-2/PGE2 in diabetic macrophages. Further, we identify that COX-2/PGE2 production in wound macrophages requires epigenetic regulation of 2 key enzymes in the cytosolic phospholipase A2/COX-2/PGE2 (cPLA2/COX-2/PGE2) pathway. We demonstrate that TGF-β-induced miRNA29b increases COX-2/PGE2 production via inhibition of DNA methyltransferase 3b-mediated hypermethylation of the Cox-2 promoter. Further, we find mixed-lineage leukemia 1 (MLL1) upregulates cPLA2 expression and drives COX-2/PGE2. Inhibition of the COX-2/PGE2 pathway genetically (Cox2fl/fl Lyz2Cre+) or with a macrophage-specific nanotherapy targeting COX-2 in tissue macrophages reverses the inflammatory macrophage phenotype and improves diabetic tissue repair. Our results indicate the epigenetically regulated PGE2 pathway controls wound macrophage function, and cell-targeted manipulation of this pathway is feasible to improve diabetic wound repair.
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http://dx.doi.org/10.1172/jci.insight.138443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526451PMC
September 2020

Palmitate-TLR4 signaling regulates the histone demethylase, JMJD3, in macrophages and impairs diabetic wound healing.

Eur J Immunol 2020 12 20;50(12):1929-1940. Epub 2020 Jul 20.

Section of Vascular Surgery, Department of Surgery, University of Michigan, Ann Arbor, MI, USA.

Chronic macrophage inflammation is a hallmark of type 2 diabetes (T2D) and linked to the development of secondary diabetic complications. T2D is characterized by excess concentrations of saturated fatty acids (SFA) that activate innate immune inflammatory responses, however, mechanism(s) by which SFAs control inflammation is unknown. Using monocyte-macrophages isolated from human blood and murine models, we demonstrate that palmitate (C16:0), the most abundant circulating SFA in T2D, increases expression of the histone demethylase, Jmjd3. Upregulation of Jmjd3 results in removal of the repressive histone methylation (H3K27me3) mark on NFκB-mediated inflammatory gene promoters driving macrophage-mediated inflammation. We identify that the effects of palmitate are fatty acid specific, as laurate (C12:0) does not regulate Jmjd3 and the associated inflammatory profile. Further, palmitate-induced Jmjd3 expression is controlled via TLR4/MyD88-dependent signaling mechanism, where genetic depletion of TLR4 (Tlr4 ) or MyD88 (MyD88 ) negated the palmitate-induced changes in Jmjd3 and downstream NFκB-induced inflammation. Pharmacological inhibition of Jmjd3 using a small molecule inhibitor (GSK-J4) reduced macrophage inflammation and improved diabetic wound healing. Together, we conclude that palmitate contributes to the chronic Jmjd3-mediated activation of macrophages in diabetic peripheral tissue and a histone demethylase inhibitor-based therapy may represent a novel treatment for nonhealing diabetic wounds.
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http://dx.doi.org/10.1002/eji.202048651DOI Listing
December 2020

Epigenetic Regulation of TLR4 in Diabetic Macrophages Modulates Immunometabolism and Wound Repair.

J Immunol 2020 05 23;204(9):2503-2513. Epub 2020 Mar 23.

Section of Vascular Surgery, Department of Surgery, University of Michigan, Ann Arbor, MI 48109;

Macrophages are critical for the initiation and resolution of the inflammatory phase of wound healing. In diabetes, macrophages display a prolonged inflammatory phenotype preventing tissue repair. TLRs, particularly TLR4, have been shown to regulate myeloid-mediated inflammation in wounds. We examined macrophages isolated from wounds of patients afflicted with diabetes and healthy controls as well as a murine diabetic model demonstrating dynamic expression of TLR4 results in altered metabolic pathways in diabetic macrophages. Further, using a myeloid-specific mixed-lineage leukemia 1 (MLL1) knockout ( ), we determined that MLL1 drives expression in diabetic macrophages by regulating levels of histone H3 lysine 4 trimethylation on the promoter. Mechanistically, MLL1-mediated epigenetic alterations influence diabetic macrophage responsiveness to TLR4 stimulation and inhibit tissue repair. Pharmacological inhibition of the TLR4 pathway using a small molecule inhibitor (TAK-242) as well as genetic depletion of either ( ) or myeloid-specific resulted in improved diabetic wound healing. These results define an important role for MLL1-mediated epigenetic regulation of TLR4 in pathologic diabetic wound repair and suggest a target for therapeutic manipulation.
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http://dx.doi.org/10.4049/jimmunol.1901263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443363PMC
May 2020

TNF-α regulates diabetic macrophage function through the histone acetyltransferase MOF.

JCI Insight 2020 03 12;5(5). Epub 2020 Mar 12.

Department of Surgery.

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 TNF-α. Previous studies have shown that an altered epigenetic program directs diabetic macrophages toward a proinflammatory phenotype, contributing to a sustained inflammatory phase. Males absent on the first (MOF) is a histone acetyltransferase (HAT) that has been shown be a coactivator of TNF-α signaling and promote NF-κB-mediated gene transcription in prostate cancer cell lines. Based on MOF's role in TNF-α/NF-κB-mediated gene expression, we hypothesized that MOF influences macrophage-mediated inflammation during wound repair. We used myeloid-specific Mof-knockout (Lyz2Cre Moffl/fl) 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, MOF's primary substrate of HAT activity, on the promoters of inflammatory genes. We further identified that MOF expression could be stimulated by TNF-α and that treatment with etanercept, an FDA-approved TNF-α inhibitor, reduced MOF levels and improved wound healing in DIO mice. This report is the first to our knowledge to define an important role for MOF in regulating macrophage-mediated inflammation in wound repair and identifies TNF-α inhibition as a potential therapy for the treatment of chronic inflammation in diabetic wounds.
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http://dx.doi.org/10.1172/jci.insight.132306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141388PMC
March 2020

Sepsis Induces Prolonged Epigenetic Modifications in Bone Marrow and Peripheral Macrophages Impairing Inflammation and Wound Healing.

Arterioscler Thromb Vasc Biol 2019 11 5;39(11):2353-2366. Epub 2019 Sep 5.

From the Section of Vascular Surgery, Department of Surgery (F.M.D., A.D., A.D.J., A.S.K., A.T.O., W.J.M., K.A.G.), University of Michigan, Ann Arbor.

Objective: Sepsis represents an acute life-threatening disorder resulting from a dysregulated host response. For patients who survive sepsis, there remains long-term consequences, including impaired inflammation, as a result of profound immunosuppression. The mechanisms involved in this long-lasting deficient immune response are poorly defined. Approach and Results: Sepsis was induced using the murine model of cecal ligation and puncture. Following a full recovery period from sepsis physiology, mice were subjected to our wound healing model and wound macrophages (CD11b+, CD3-, CD19-, Ly6G-) were sorted. Post-sepsis mice demonstrated impaired wound healing and decreased reepithelization in comparison to controls. Further, post-sepsis bone marrow-derived macrophages and wound macrophages exhibited decreased expression of inflammatory cytokines vital for wound repair (IL [interleukin]-1β, IL-12, and IL-23). To evaluate if decreased inflammatory gene expression was secondary to epigenetic modification, we conducted chromatin immunoprecipitation on post-sepsis bone marrow-derived macrophages and wound macrophages. This demonstrated decreased expression of , an epigenetic enzyme, and impaired histone 3 lysine 4 trimethylation (activation mark) at NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells)-binding sites on inflammatory gene promoters in bone marrow-derived macrophages and wound macrophages from postcecal ligation and puncture mice. Bone marrow transplantation studies demonstrated epigenetic modifications initiate in bone marrow progenitor/stem cells following sepsis resulting in lasting impairment in peripheral macrophage function. Importantly, human peripheral blood leukocytes from post-septic patients demonstrate a significant reduction in compared with nonseptic controls.

Conclusions: These data demonstrate that severe sepsis induces stable mixed-lineage leukemia 1-mediated epigenetic modifications in the bone marrow, which are passed to peripheral macrophages resulting in impaired macrophage function and deficient wound healing persisting long after sepsis recovery.
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http://dx.doi.org/10.1161/ATVBAHA.119.312754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6818743PMC
November 2019

The Histone Methyltransferase Setdb2 Modulates Macrophage Phenotype and Uric Acid Production in Diabetic Wound Repair.

Immunity 2019 08 23;51(2):258-271.e5. Epub 2019 Jul 23.

Department of Surgery, University of Michigan, Ann Arbor, MI, USA; Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI, USA. Electronic address:

Macrophage plasticity is critical for normal tissue repair to ensure transition from the inflammatory to the proliferative phase of healing. We examined macrophages isolated from wounds of patients afflicted with diabetes and of healthy controls and found differential expression of the methyltransferase Setdb2. Myeloid-specific deletion of Setdb2 impaired the transition of macrophages from an inflammatory phenotype to a reparative one in normal wound healing. Mechanistically, Setdb2 trimethylated histone 3 at NF-κB binding sites on inflammatory cytokine gene promoters to suppress transcription. Setdb2 expression in wound macrophages was regulated by interferon (IFN) β, and under diabetic conditions, this IFNβ-Setdb2 axis was impaired, leading to a persistent inflammatory macrophage phenotype in diabetic wounds. Setdb2 regulated the expression of xanthine oxidase and thereby the uric acid (UA) pathway of purine catabolism in macrophages, and pharmacologic targeting of Setdb2 or the UA pathway improved healing. Thus, Setdb2 regulates macrophage plasticity during normal and pathologic wound repair and is a target for therapeutic manipulation.
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http://dx.doi.org/10.1016/j.immuni.2019.06.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6703945PMC
August 2019

SIRT3 Regulates Macrophage-Mediated Inflammation in Diabetic Wound Repair.

J Invest Dermatol 2019 12 15;139(12):2528-2537.e2. Epub 2019 Jun 15.

Department of Surgery, University of Michigan, Ann Arbor, Michigan, USA; Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA. Electronic address:

Control of inflammation is critical for the treatment of nonhealing wounds, but a delicate balance exists between early inflammation that is essential for normal tissue repair and the pathologic inflammation that can occur later in the repair process. This necessitates the development of novel therapies that can target inflammation at the appropriate time during repair. Here, we found that SIRT3 is essential for normal healing and regulates inflammation in wound macrophages after injury. Under prediabetic conditions, SIRT3 was decreased in wound macrophages and resulted in dysregulated inflammation. In addition, we found that FABP4 regulates SIRT3 in human blood monocytes, and inhibition of FABP4 in wound macrophages decreases inflammatory cytokine expression, making FABP4 a viable target for the regulation of excess inflammation and wound repair in diabetes. Using a series of ex vivo and in vivo studies with genetically engineered mouse models and diabetic human monocytes, we showed that FABP4 expression is epigenetically upregulated in diabetic wound macrophages and, in turn, diminishes SIRT3 expression, thereby promoting inflammation. These findings have significant implications for controlling inflammation and promoting tissue repair in diabetic wounds.
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http://dx.doi.org/10.1016/j.jid.2019.05.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7185380PMC
December 2019

Histone Methylation Directs Myeloid TLR4 Expression and Regulates Wound Healing following Cutaneous Tissue Injury.

J Immunol 2019 03 1;202(6):1777-1785. Epub 2019 Feb 1.

Section of Vascular Surgery, Department of Surgery, University of Michigan, Ann Arbor, MI 48109;

Myeloid cells are critical for orchestrating regulated inflammation during wound healing. TLRs, particularly TLR4, and its downstream-signaling MyD88 pathway play an important role in regulating myeloid-mediated inflammation. Because an initial inflammatory phase is vital for tissue repair, we investigated the role of TLR4-regulated, myeloid-mediated inflammation in wound healing. In a cutaneous tissue injury murine model, we found that TLR4 expression is dynamic in wound myeloid cells during the course of normal wound healing. We identified that changes in myeloid TLR4 during tissue repair correlated with increased expression of the histone methyltransferase, mixed-lineage leukemia 1 (MLL1), which specifically trimethylates the histone 3 lysine 4 (H3K4me3) position of the TLR4 promoter. Furthermore, we used a myeloid-specific Mll1 knockout ( ) to determine MLL1 drives expression during wound healing. To understand the critical role of myeloid-specific TLR4 signaling, we used mice deficient in ( ), (), and myeloid-specific to demonstrate delayed wound healing at early time points postinjury. Furthermore, in vivo wound myeloid cells isolated from and wounds demonstrated decreased inflammatory cytokine production. Importantly, adoptive transfer of monocyte/macrophages from wild-type mice trafficked to wounds with restoration of normal healing and myeloid cell function in -deficient mice. These results define a role for myeloid-specific, MyD88-dependent TLR4 signaling in the inflammatory response following cutaneous tissue injury and suggest that MLL1 regulates TLR4 expression in wound myeloid cells.
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http://dx.doi.org/10.4049/jimmunol.1801258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6401313PMC
March 2019

Murine macrophage chemokine receptor CCR2 plays a crucial role in macrophage recruitment and regulated inflammation in wound healing.

Eur J Immunol 2018 09 26;48(9):1445-1455. Epub 2018 Jun 26.

Department of Surgery, University of Michigan, Ann Arbor, MI, USA.

Macrophages play a critical role in the establishment of a regulated inflammatory response following tissue injury. Following injury, CCR2 monocytes are recruited from peripheral blood to wound tissue, and direct the initiation and resolution of inflammation that is essential for tissue repair. In pathologic states where chronic inflammation prevents healing, macrophages fail to transition to a reparative phenotype. Using a murine model of cutaneous wound healing, we found that CCR2-deficient mice (CCR2 ) demonstrate significantly impaired wound healing at all time points postinjury. Flow cytometry analysis of wounds from CCR2 and WT mice revealed a significant decrease in inflammatory, Ly6C recruited monocyte/macrophages in CCR2 wounds. We further show that wound macrophage inflammatory cytokine production is decreased in CCR2 wounds. Adoptive transfer of mT/mG monocyte/macrophages into CCR2 and CCR2 mice demonstrated that labeled cells on days 2 and 4 traveled to wounds in both CCR2 and CCR2 mice. Further, adoptive transfer of monocyte/macrophages from WT mice restored normal healing, likely through a restored inflammatory response in the CCR2-deficient mice. Taken together, these data suggest that CCR2 plays a critical role in the recruitment and inflammatory response following injury, and that wound repair may be therapeutically manipulated through modulation of CCR2.
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http://dx.doi.org/10.1002/eji.201747400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6371802PMC
September 2018

Ly6C Blood Monocyte/Macrophage Drive Chronic Inflammation and Impair Wound Healing in Diabetes Mellitus.

Arterioscler Thromb Vasc Biol 2018 05 1;38(5):1102-1114. Epub 2018 Mar 1.

From the Department of Surgery (A.K., A.J., F.M.D., A.D., A.B., A.O., P.K.H., K.A.G.)

Objective: Wound monocyte-derived macrophage plasticity controls the initiation and resolution of inflammation that is critical for proper healing, however, in diabetes mellitus, the resolution of inflammation fails to occur. In diabetic wounds, the kinetics of blood monocyte recruitment and the mechanisms that control in vivo monocyte/macrophage differentiation remain unknown.

Approach And Results: Here, we characterized the kinetics and function of Ly6C [Lin (CD3CD19NK1.1Ter-119) Ly6GCD11b] and Ly6C [Lin (CD3CD19NK1.1Ter-119) Ly6GCD11b] monocyte/macrophage subsets in normal and diabetic wounds. Using flow-sorted -labeled Ly6C monocyte/macrophages, we show Ly6C cells transition to a Ly6C phenotype in normal wounds, whereas in diabetic wounds, there is a late, second influx of Ly6C cells that fail transition to Ly6C. The second wave of Ly6C cells in diabetic wounds corresponded to a spike in MCP-1 (monocyte chemoattractant protein-1) and selective administration of anti-MCP-1 reversed the second Ly6C influx and improved wound healing. To examine the in vivo phenotype of wound monocyte/macrophages, RNA-seq-based transcriptome profiling was performed on flow-sorted Ly6C [LinLy6GCD11b] and Ly6C [LinLy6GCD11b] cells from normal and diabetic wounds. Gene transcriptome profiling of diabetic wound Ly6C cells demonstrated differences in proinflammatory and profibrotic genes compared with controls.

Conclusions: Collectively, these data identify kinetic and functional differences in diabetic wound monocyte/macrophages and demonstrate that selective targeting of CD11bLy6C monocyte/macrophages is a viable therapeutic strategy for inflammation in diabetic wounds.
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http://dx.doi.org/10.1161/ATVBAHA.118.310703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5920725PMC
May 2018

The STAT4/MLL1 Epigenetic Axis Regulates the Antimicrobial Functions of Murine Macrophages.

J Immunol 2017 09 21;199(5):1865-1874. Epub 2017 Jul 21.

Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109.

Macrophages are critical immune cells for the clearance of microbial pathogens and cellular debris from peripheral tissues. Macrophage inflammatory responses are governed by gene expression patterns, and these patterns are often subject to epigenetic control. Chromatin modifications, such as histone methylation, regulate gene accessibility in macrophages, and macrophage polarization is governed in part by the expression and function of chromatin-modifying enzymes. The histone methyltransferase mixed-lineage leukemia 1 (MLL1) preferentially modifies lysine residue 4 on the unstructured protein tail of histone H3. MLL1 expression and function have been shown to be governed by signal transduction pathways that are activated by inflammatory stimuli, such as NF-κB. Therefore, we sought to investigate the role of MLL1 in mediating macrophage inflammatory responses. Bone marrow-derived macrophages from mice with a targeted MLL1 gene knockout (Lys2-Cre MLL1) exhibited decreased proinflammatory gene expression with concurrent decreases in activating histone methylation. However, MLL1-deficient macrophages also exhibited increased phagocytic and bacterial killing activity in vitro. RNA profiling of MLL1-knockout macrophages identified numerous genes involved with inflammatory responses whose expression was altered in response to TLR ligands or proinflammatory cytokines, including STAT4. STAT4-dependent cytokines, such as type I IFNs were able to drive MLL1 expression in macrophages, and MLL1-knockout macrophages exhibited decreased activating histone methylation in the STAT4 promoter. These results implicate an important role for MLL1-dependent epigenetic regulation of macrophage antimicrobial functions.
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http://dx.doi.org/10.4049/jimmunol.1601272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568492PMC
September 2017

The Histone Methyltransferase MLL1 Directs Macrophage-Mediated Inflammation in Wound Healing and Is Altered in a Murine Model of Obesity and Type 2 Diabetes.

Diabetes 2017 09 29;66(9):2459-2471. Epub 2017 Jun 29.

Department of Surgery, University of Michigan, Ann Arbor, MI

Macrophages are critical for the initiation and resolution of the inflammatory phase of wound repair. In diabetes, macrophages display a prolonged inflammatory phenotype in late wound healing. Mixed-lineage leukemia-1 (MLL1) has been shown to direct gene expression by regulating nuclear factor-κB (NF-κB)-mediated inflammatory gene transcription. Thus, we hypothesized that MLL1 influences macrophage-mediated inflammation in wound repair. We used a myeloid-specific knockout ( ) to determine the function of MLL1 in wound healing. mice display delayed wound healing and decreased wound macrophage inflammatory cytokine production compared with control animals. Furthermore, wound macrophages from mice demonstrated decreased histone H3 lysine 4 trimethylation (H3K4me3) (activation mark) at NF-κB binding sites on inflammatory gene promoters. Of note, early wound macrophages from prediabetic mice displayed similarly decreased MLL1, H3K4me3 at inflammatory gene promoters, and inflammatory cytokines compared with controls. Late wound macrophages from prediabetic mice demonstrated an increase in MLL1, H3K4me3 at inflammatory gene promoters, and inflammatory cytokines. Prediabetic macrophages treated with an MLL1 inhibitor demonstrated reduced inflammation. Finally, monocytes from patients with type 2 diabetes had increased compared with control subjects without diabetes. These results define an important role for MLL1 in regulating macrophage-mediated inflammation in wound repair and identify a potential target for the treatment of chronic inflammation in diabetic wounds.
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http://dx.doi.org/10.2337/db17-0194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566299PMC
September 2017

Notch Regulates Macrophage-Mediated Inflammation in Diabetic Wound Healing.

Front Immunol 2017 1;8:635. Epub 2017 Jun 1.

Department of Surgery, University of Michigan, Ann Arbor, MI, United States.

Macrophages are essential immune cells necessary for regulated inflammation during wound healing. Recent studies have identified that Notch plays a role in macrophage-mediated inflammation. Thus, we investigated the role of Notch signaling on wound macrophage phenotype and function during normal and diabetic wound healing. We found that Notch receptor and ligand expression are dynamic in wound macrophages during normal healing. Mice with a myeloid-specific Notch signaling defect ( ) demonstrated delayed early healing (days 1-3) and wound macrophages had decreased inflammatory gene expression. In our physiologic murine model of type 2 diabetes (T2D), Notch receptor expression was significantly increased in wound macrophages on day 6, following the initial inflammatory phase of wound healing, corresponding to increased inflammatory cytokine expression. This increase in and was also observed in human monocytes from patients with T2D. Further, in prediabetic mice with a genetic Notch signaling defect ( on a high-fat diet), improved wound healing was seen at late time points (days 6-7). These findings suggest that Notch is critical for the early inflammatory phase of wound healing and directs production of macrophage-dependent inflammatory mediators. These results identify that canonical Notch signaling is important in directing macrophage function in wound repair and define a translational target for the treatment of non-healing diabetic wounds.
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http://dx.doi.org/10.3389/fimmu.2017.00635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451506PMC
June 2017

Enhancement of macrophage inflammatory responses by CCL2 is correlated with increased miR-9 expression and downregulation of the ERK1/2 phosphatase Dusp6.

Cell Immunol 2017 04 22;314:63-72. Epub 2017 Feb 22.

Department of Pathology, University of Michigan Medical School, 109 Zina Pitcher Place, Ann Arbor, MI 48109, USA. Electronic address:

Macrophage polarization plays a central role in both protective immunity and immunopathology. While the role of cytokines in driving macrophage polarization is well characterized, less is understood about the role of chemokines. The purpose of this study was to determine if CC chemokine 2 (CCL2/MCP1) could influence macrophage polarization in response to subsequent activation with cytokines and microbial products. Treatment of bone marrow-derived macrophages with CCL2 alone did not result in increased expression of either classical or alternatively-activated macrophage genes as compared to standard skewing cytokines or Toll-like receptor agonists. However, subsequent stimulation of CCL2 pre-treated macrophages with classical activation stimuli resulted in enhanced expression of genes associated with classical activation. This enhancement correlated with increased phosphorylation of ERK1/2 kinases, a decrease in expression of the ERK phosphatase Dusp6 and enhanced expression of miR-9. These results indicate that CCL2 supports the classical activation of macrophages, with miR-9 mediated down-regulation of Dusp6 and enhanced ERK-mediated signal transduction possibly mediating this enhanced pro-inflammatory gene expression.
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http://dx.doi.org/10.1016/j.cellimm.2017.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5425952PMC
April 2017

Epigenetic changes in bone marrow progenitor cells influence the inflammatory phenotype and alter wound healing in type 2 diabetes.

Diabetes 2015 Apr 3;64(4):1420-30. Epub 2014 Nov 3.

Department of Pathology, University of Michigan, Ann Arbor, MI.

Classically activated (M1) macrophages are known to play a role in the development of chronic inflammation associated with impaired wound healing in type 2 diabetes (T2D); however, the mechanism responsible for the dominant proinflammatory (M1) macrophage phenotype in T2D wounds is unknown. Since epigenetic enzymes can direct macrophage phenotypes, we assessed the role of histone methylation in bone marrow (BM) stem/progenitor cells in the programming of macrophages toward a proinflammatory phenotype. We have found that a repressive histone methylation mark, H3K27me3, is decreased at the promoter of the IL-12 gene in BM progenitors and this epigenetic signature is passed down to wound macrophages in a murine model of glucose intolerance (diet-induced obese). These epigenetically "preprogrammed" macrophages result in poised macrophages in peripheral tissue and negatively impact wound repair. We found that in diabetic conditions the H3K27 demethylase Jmjd3 drives IL-12 production in macrophages and that IL-12 production can be modulated by inhibiting Jmjd3. Using human T2D tissue and murine models, we have identified a previously unrecognized mechanism by which macrophages are programmed toward a proinflammatory phenotype, establishing a pattern of unrestrained inflammation associated with nonhealing wounds. Hence, histone demethylase inhibitor-based therapy may represent a novel treatment option for diabetic wounds.
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http://dx.doi.org/10.2337/db14-0872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4375075PMC
April 2015

Inflammasome components coordinate autophagy and pyroptosis as macrophage responses to infection.

mBio 2013 Feb 12;4(1):e00620-12. Epub 2013 Feb 12.

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Unlabelled: When microbes contaminate the macrophage cytoplasm, leukocytes undergo a proinflammatory death that is initiated by nucleotide-binding-domain-, leucine-rich-repeat-containing proteins (NLR proteins) that bind and activate caspase-1. We report that these inflammasome components also regulate autophagy, a vesicular pathway to eliminate cytosolic debris. In response to infection with flagellate Legionella pneumophila, C57BL/6J mouse macrophages equipped with caspase-1 and the NLR proteins NAIP5 and NLRC4 stimulated autophagosome turnover. A second trigger of inflammasome assembly, K(+) efflux, also rapidly activated autophagy in macrophages that produced caspase-1. Autophagy protects infected macrophages from pyroptosis, since caspase-1-dependent cell death occurred more frequently when autophagy was dampened pharmacologically by either 3-methyladenine or an inhibitor of the Atg4 protease. Accordingly, in addition to coordinating pyroptosis, both (pro-) caspase-1 protein and NLR components of inflammasomes equip macrophages to recruit autophagy, a disposal pathway that raises the threshold of contaminants necessary to trigger proinflammatory leukocyte death.

Importance: An exciting development in the innate-immunity field is the recognition that macrophages enlist autophagy to protect their cytoplasm from infection. Nutrient deprivation has long been known to induce autophagy; how infection triggers this disposal pathway is an active area of research. Autophagy is encountered by many of the intracellular pathogens that are known to trigger pyroptosis, an inflammatory cell death initiated when nucleotide-binding-domain-, leucine-rich-repeat-containing proteins (NLR proteins) activate caspase-1 within inflammasome complexes. Therefore, we tested the hypothesis that NLR proteins and caspase-1 also coordinate autophagy as a barrier to cytosolic infection. By exploiting classical bacterial and mouse genetics and kinetic assays of autophagy, we demonstrate for the first time that, when confronted with cytosolic contamination, primary mouse macrophages rely not only on the NLR proteins NAIP5 and NLRC4 but also on (pro-)caspase-1 protein to mount a rapid autophagic response that wards off proinflammatory cell death.
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http://dx.doi.org/10.1128/mBio.00620-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3573666PMC
February 2013

Secrets of a successful pathogen: legionella resistance to progression along the autophagic pathway.

Front Microbiol 2011 28;2:138. Epub 2011 Jun 28.

Department of Microbiology and Immunology, University of Michigan Medical School Ann Arbor, MI, USA.

To proliferate within phagocytes, Legionella pneumophila relies on Type IV secretion to modulate host cellular pathways. Autophagy is an evolutionarily conserved degradative pathway that captures and transfers a variety of microbes to lysosomes. Biogenesis of L. pneumophila-containing vacuoles and autophagosomes share several features, including endoplasmic reticulum (ER)-derived membranes, contributions by the host GTPases Rab1, Arf1 and Sar1, and a final destiny in lysosomes. We discuss morphological, molecular genetic, and immunological data that support the model that, although A/J mouse macrophages efficiently engulf L. pneumophila within autophagosomal membranes, the Type IV effector proteins DrrA/SidM, LidA, and RalF prolong association with the ER. By inhibiting immediately delivery to lysosomes, the bacteria persist in immature autophagosomal vacuoles for a period sufficient to differentiate into an acid-resistant, replicative form. Subsequent secretion of the Type IV effector LepB releases the block to autophagosome maturation, and the adapted progeny continue to replicate within autophagolysosomes. Accordingly, L. pneumophila can be exploited as a genetic tool to analyze the recruitment and function of the macrophage autophagy pathway.
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http://dx.doi.org/10.3389/fmicb.2011.00138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3127087PMC
July 2011

A micro RNA processing defect in rapidly progressing idiopathic pulmonary fibrosis.

PLoS One 2011 21;6(6):e21253. Epub 2011 Jun 21.

Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.

Background: Idiopathic pulmonary fibrosis exhibits differential progression from the time of diagnosis but the molecular basis for varying progression rates is poorly understood. The aim of the present study was to ascertain whether differential miRNA expression might provide one explanation for rapidly versus slowly progressing forms of IPF.

Methodology And Principal Findings: miRNA and mRNA were isolated from surgical lung biopsies from IPF patients with a clinically documented rapid or slow course of disease over the first year after diagnosis. A quantitative PCR miRNA array containing 88 of the most abundant miRNA in the human genome was used to profile lung biopsies from 9 patients with rapidly progressing IPF, 6 patients with slowly progressing IPF, and 10 normal lung biopsies. Using this approach, 11 miRNA were significantly increased and 36 were significantly decreased in rapid biopsies compared with normal biopsies. Slowly progressive biopsies exhibited 4 significantly increased miRNA and 36 significantly decreased miRNA compared with normal lung. Among the miRNA present in IPF with validated mRNA targets were those with regulatory effects on epithelial-mesenchymal transition (EMT). Five miRNA (miR-302c, miR-423-5p, miR-210, miR-376c, and miR-185) were significantly increased in rapid compared with slow IPF lung biopsies. Additional analyses of rapid biopsies and fibroblasts grown from the same biopsies revealed that the expression of AGO1 and AGO2 (essential components of the miRNA processing RISC complex) were lower compared with either slow or normal lung biopsies and fibroblasts.

Conclusion: These findings suggest that the development and/or clinical progression of IPF might be the consequence of aberrant miRNA processing.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021253PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3119674PMC
November 2011

TLR9 differentiates rapidly from slowly progressing forms of idiopathic pulmonary fibrosis.

Sci Transl Med 2010 Nov;2(57):57ra82

Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109-2200, USA.

Idiopathic pulmonary fibrosis is characterized by diffuse alveolar damage and severe fibrosis, resulting in a steady worsening of lung function and gas exchange. Because idiopathic pulmonary fibrosis is a generally progressive disorder with highly heterogeneous disease progression, we classified affected patients as either rapid or slow progressors over the first year of follow-up and then identified differences between the two groups to investigate the mechanism governing rapid progression. Previous work from our laboratory has demonstrated that Toll-like receptor 9 (TLR9), a pathogen recognition receptor that recognizes unmethylated CpG motifs in bacterial and viral DNA, promotes myofibroblast differentiation in lung fibroblasts cultured from biopsies of patients with idiopathic pulmonary fibrosis. Therefore, we hypothesized that TLR9 functions as both a sensor of pathogenic molecules and a profibrotic signal in rapidly progressive idiopathic pulmonary fibrosis. Indeed, TLR9 was present at higher concentrations in surgical lung biopsies from rapidly progressive patients than in tissue from slowly progressing patients. Moreover, fibroblasts from rapid progressors were more responsive to the TLR9 agonist, CpG DNA, than were fibroblasts from slowly progressing patients. Using a humanized severe combined immunodeficient mouse, we then demonstrated increased fibrosis in murine lungs receiving human lung fibroblasts from rapid progressors compared with mice receiving fibroblasts from slowly progressing patients. This fibrosis was exacerbated by intranasal CpG challenges. Furthermore, CpG induced the differentiation of blood monocytes into fibrocytes and the epithelial-to-mesenchymal transition of A549 lung epithelial cells. These data suggest that TLR9 may drive the pathogenesis of rapidly progressive idiopathic pulmonary fibrosis and may serve as a potential indicator for this subset of the disease.
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http://dx.doi.org/10.1126/scitranslmed.3001510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3235647PMC
November 2010

Interleukin-33 contributes to both M1 and M2 chemokine marker expression in human macrophages.

BMC Immunol 2010 Oct 19;11:52. Epub 2010 Oct 19.

Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, USA.

Background: Interleukin-33 is a member of the IL-1 cytokine family whose functions are mediated and modulated by the ST2 receptor. IL-33-ST2 expression and interactions have been explored in mouse macrophages but little is known about the effect of IL-33 on human macrophages. The expression of ST2 transcript and protein levels, and IL-33-mediated effects on M1 (i.e. classical activation) and M2 (i.e. alternative activation) chemokine marker expression in human bone marrow-derived macrophages were examined.

Results: Human macrophages constitutively expressed the membrane-associated (i.e. ST2L) and the soluble (i.e. sST2) ST2 receptors. M2 (IL-4 + IL-13) skewing stimuli markedly increased the expression of ST2L, but neither polarizing cytokine treatment promoted the release of sST2 from these cells. When added to naïve macrophages alone, IL-33 directly enhanced the expression of CCL3. In combination with LPS, IL-33 blocked the expression of the M2 chemokine marker CCL18, but did not alter CCL3 expression in these naive cells. The addition of IL-33 to M1 macrophages markedly increased the expression of CCL18 above that detected in untreated M1 macrophages. Similarly, alternatively activated human macrophages treated with IL-33 exhibited enhanced expression of CCL18 and the M2 marker mannose receptor above that detected in M2 macrophages alone.

Conclusions: Together, these data suggest that primary responses to IL-33 in bone marrow derived human macrophages favors M1 chemokine generation while its addition to polarized human macrophages promotes or amplifies M2 chemokine expression.
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http://dx.doi.org/10.1186/1471-2172-11-52DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2967528PMC
October 2010

Serum amyloid P therapeutically attenuates murine bleomycin-induced pulmonary fibrosis via its effects on macrophages.

PLoS One 2010 Mar 12;5(3):e9683. Epub 2010 Mar 12.

Promedior, Inc, Malvern, Pennsylvania, United States of America.

Macrophages promote tissue remodeling but few mechanisms exist to modulate their activity during tissue fibrosis. Serum amyloid P (SAP), a member of the pentraxin family of proteins, signals through Fcgamma receptors which are known to affect macrophage activation. We determined that IPF/UIP patients have increased protein levels of several alternatively activated pro-fibrotic (M2) macrophage-associated proteins in the lung and monocytes from these patients show skewing towards an M2 macrophage phenotype. SAP therapeutically inhibits established bleomycin-induced pulmonary fibrosis, when administered systemically or locally to the lungs. The reduction in aberrant collagen deposition was associated with a reduction in M2 macrophages in the lung and increased IP10/CXCL10. These data highlight the role of macrophages in fibrotic lung disease, and demonstrate a therapeutic action of SAP on macrophages which may extend to many fibrotic indications caused by over-exuberant pro-fibrotic macrophage responses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0009683PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2837381PMC
March 2010

Toll-like receptor 6 drives interleukin-17A expression during experimental hypersensitivity pneumonitis.

Immunology 2010 May 11;130(1):125-36. Epub 2010 Jan 11.

Immunology Program, Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109-0602, USA.

Hypersensitivity pneumonitis (HP) is a T-cell-driven disease that is histologically characterized by diffuse mononuclear cell infiltrates and loosely formed granulomas in the lungs. We have previously reported that interleukin-17A (IL-17A) contributes to the development of experimental HP, and that the pattern recognition receptor Toll-like receptor 6 (TLR6) might be a factor in the initiation of this response. Using a well-established murine model of Saccharopolyspora rectivirgula-induced HP, we investigated the role of TLR6 in the immunopathogenesis of this disease. In the absence of TLR6 signalling, mice that received multiple challenges with S. rectivirgula-antigen (SR-Ag) had significantly less lung inflammation compared with C57BL/6 mice (wild-type; WT) similarly challenged with SR-Ag. Flow cytometric analysis of whole lung samples from SR-Ag-challenged mice showed that TLR6(-/-) mice had a decreased CD4(+) : CD8(+) T-cell ratio compared with WT mice. Cytokine analysis at various days after the final SR-Ag challenge revealed that whole lungs from TLR6(-/-) mice contained significantly less IL-17A than lungs from WT mice with HP. The IL-17A-driving cytokines IL-21 and IL-23 were also expressed at lower levels in SR-Ag-challenged TLR6(-/-) mice, when compared with SR-Ag-challenged WT mice. Other pro-inflammatory cytokines, namely interferon-gamma and RANTES, were also found to be regulated by TLR6 signalling. Anti-TLR6 neutralizing antibody treatment of dispersed lung cells significantly impaired SR-Ag-induced IL-17A and IL-6 generation. Together, these results indicate that TLR6 plays a pivotal role in the development and severity of HP via its role in IL-17A production.
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http://dx.doi.org/10.1111/j.1365-2567.2009.03219.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2855800PMC
May 2010

Toll-like receptor 9 activation is a key mechanism for the maintenance of chronic lung inflammation.

Am J Respir Crit Care Med 2009 Dec 24;180(12):1227-38. Epub 2009 Sep 24.

Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Rationale: Accumulating evidence supports the hypothesis that the continuous host response to a persistent challenge can polarize the cytokine environment toward a Th2 cytokine phenotype, but the mechanisms responsible for this skewing are not clear.

Objectives: We investigated the role of Toll-like receptor 9 (TLR9) in a Th2-driven pulmonary granulomatous response initiated via the embolization of Schistosoma mansoni eggs to the lungs of mice.

Methods: Mice were intravenously injected with S. mansoni eggs. Histological and flow cytometric analysis, cytokine measurement, adoptive transfer of bone marrow (BM)-derived dendritic cells (DCs), and in vitro T-cell treatments with antigen-presenting cells were examined.

Measurements And Main Results: In comparison to wild-type mice, TLR9(-/-) mice showed increased pulmonary granuloma size, augmented collagen deposition, increased Th2 cytokine phenotype, and impaired accumulation of DCs. BM-derived DCs, but not macrophages, recovered from animals with developed Th2-type lung granulomas promoted the production of type 2 cytokines from CD4(+) T cells. BM-derived DCs from TLR9(-/-) mice induced impaired Th1 cytokine and enhanced Th2 cytokine production by T cells, compared with DCs from WT mice. Macrophages from TLR9(-/-) mice expressed a significantly higher alternatively activated (M2) phenotype characterized by increased "found in inflammatory zone-1" (FIZZ1) and arginase-1 expression. The adoptive transfer of BM-derived DCs from syngeneic WT mice into TLR9(-/-) mice restored the granuloma phenotype seen in WT mice.

Conclusions: These studies suggest that TLR9 plays an important mechanistic role in the maintenance of the pulmonary granulomatous response.
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http://dx.doi.org/10.1164/rccm.200906-0892OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2796734PMC
December 2009

Interleukin-17-mediated immunopathogenesis in experimental hypersensitivity pneumonitis.

Am J Respir Crit Care Med 2009 Apr 16;179(8):705-16. Epub 2009 Jan 16.

Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109-0602, USA.

Rationale: T cells play a critical role in the development of Saccharopolyspora rectivirgula-induced hypersensitivity pneumonitis (HP) but little is known about the role of IL-17A in this disease.

Objectives: We examined the role of IL-17A in a murine model of S. rectivirgula antigen (SR-Ag)-induced HP.

Methods: Experimental HP was induced by oropharyngeal instillation of SR-Ag in wild-type and IL-17 gene-deficient mice.

Measurements And Main Results: SR-Ag-induced murine HP was characterized by increased transcript levels of IFN-gamma and IL-12p35 compared with saline-treated control mice. Furthermore, mice with HP showed increased IL-17 in lung homogenates, bronchoalveolar lavage fluid, and ex-vivo lung cultures compared with control mice. Flow cytometric analysis of SR-Ag-challenged lungs revealed increased Th17 and CD11c(+) cells. The role of IL-17 in SR-induced HP was examined in IL-17 deficient (IL17(-/-)) and in wild-type (IL-17(+/+)) mice immunodepleted of IL-17. Histological examination of IL17(-/-) mice challenged with SR-Ag revealed reduced inflammatory cell infiltration, decreased CD11c(+) cells, and reduced levels of inflammatory mediators such as IL-12p70, CCL3, and CXCL9 compared with similarly treated IL17(+/+) mice. Anti-IL-17 antibody treatment of IL-17(+/+) mice with HP resulted in reduced inflammation and a lower percentage of CD11c(+) cells compared with IgG-treated IL-17(+/+) mice with HP.

Conclusions: SR-Ag-induced IL-17 plays a pivotal role in the immunopathology of HP and targeting IL-17 is an attractive therapeutic option for this disease.
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http://dx.doi.org/10.1164/rccm.200811-1700OCDOI Listing
April 2009

TLR3 modulates immunopathology during a Schistosoma mansoni egg-driven Th2 response in the lung.

Eur J Immunol 2008 Dec;38(12):3436-49

Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109-0602, USA.

We examined the role of TLR3 in Th2-driven pulmonary granulomatous disease, using wildtype (TLR3(+/+)) and TLR3 gene-deficient (TLR3(-/-)) mice in a well-established model of Schistosoma mansoni egg-induced pulmonary granuloma. The intravenous bolus injection of S. mansoni eggs into S. mansoni-sensitized TLR3(+/+) mice was associated with an increase in TLR3 transcript expression in alveolar macrophages and ex vivo spleen and lung cultures at day 8 after egg injection. Lungs from TLR3(-/-) mice showed an increase in granuloma size, greater collagen deposition around the granuloma, and increased Th2 cytokine and chemokine levels compared with similarly sensitized and challenged TLR3(+/+) mice. Macrophages from TLR3(-/-) mice exhibited an M2 phenotype characterized by increased arginase and CCL2 expression. Significantly greater numbers of CD4(+)CD25(+) T cells were present in the lungs of TLR3(-/-) mice compared with TLR3(+/+) mice at day 8 after egg embolization. Cells derived from granulomatous lung and lung draining lymph nodes of TLR3(-/-) mice released significantly higher levels of IL-17 levels relative to TLR3(+/+) cells. Thus, our data suggest that TLR3 has a major regulatory role during a Th2-driven granulomatous response as its absence enhanced immunopathology.
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http://dx.doi.org/10.1002/eji.200838629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5382867PMC
December 2008

Deleterious role of TLR3 during hyperoxia-induced acute lung injury.

Am J Respir Crit Care Med 2008 Dec 10;178(12):1227-37. Epub 2008 Oct 10.

Immunobiology Department, Centocor, Radnor, PA, USA.

Rationale: Acute respiratory distress syndrome (ARDS) manifests clinically as a consequence of septic and/or traumatic injury in the lung. Oxygen therapy remains a major therapeutic intervention in ARDS, but this can contribute further to lung damage. Patients with ARDS are highly susceptible to viral infection and it may be due to altered Toll-like receptor (TLR) expression.

Objectives: To evaluate the role of TLR3 in ARDS.

Methods: TLR3 expression and signaling was determined in airway epithelial cells after in vitro hyperoxia challenge. Using a murine model of hyperoxia-induced lung injury, the role of TLR3 was determined using either TLR3-gene deficient mice or a specific neutralizing antibody directed to TLR3.

Measurements And Main Results: Increased TLR3 expression was observed in airway epithelial cells from patients with ARDS. Further, hyperoxic conditions alone were a major stimulus for increased TLR3 expression and activation in cultured human epithelial cells. Interestingly, TLR3(-/-) mice exhibited less acute lung injury, activation of apoptotic cascades, and extracellular matrix deposition after 5 days of 80% oxygen compared with wild-type (TLR3(+/+)) mice under the same conditions. Administration of a monoclonal anti-TLR3 antibody to TLR3(+/+) mice exposed to hyperoxic conditions likewise protected these mice from lung injury and inflammation.

Conclusions: The potential for redundancy in function as well as cross-talk between distinct TLRs may indeed contribute to whether the inflammatory cascade can be effectively disrupted once signaling has been initiated. Together, these data show that TLR3 has a major role in the development of ARDS-like pathology in the absence of a viral pathogen.
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http://dx.doi.org/10.1164/rccm.200807-1020OCDOI Listing
December 2008

CC chemokine receptor 4 modulates Toll-like receptor 9-mediated innate immunity and signaling.

Eur J Immunol 2008 Aug;38(8):2290-302

Immunology Program, Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109-2200, USA.

The present study addressed the modulatory role of CC chemokine receptor 4 (CCR4) in Toll-like receptor (TLR) 9-mediated innate immunity and explored the underlying molecular mechanisms. Our results demonstrated that CCR4-deficient mice were resistant to both septic peritonitis induced by cecal ligation and puncture (CLP) and CpG DNA/D-galactosamine-induced shock. In bone marrow-derived macrophages (BMMPhi) from CLP-treated CCR4-deficient mice, TLR9-mediated pathways of MAPK/AP-1, PI3K/Akt, and IkappaB kinase (IKK)/NF-kappaB were impaired compared to wild-type (WT) cells. While TLR9 expression was not altered, the intensity of internalized CpG DNA was increased in CCR4-deficient macrophages when compared to WT macrophages. Pharmacological inhibitor studies revealed that impaired activation of JNK, PI3K/Akt, and/or IKK/NF-kappaB could be responsible for decreased proinflammatory cytokine expression in CCR4-deficient macrophages. Interestingly, the CCR4-deficient BMMPhi exhibited an alternatively activated (M2) phenotype and the impaired TLR9-mediated signal transduction responses in CCR4-deficient cells were similar to the signaling responses observed in WT BMMPhi skewed to an alternatively activated phenotype. These results indicate that macrophages deficient in CCR4 impart a regulatory influence on TLR9-mediated innate immunity.
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http://dx.doi.org/10.1002/eji.200838360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2925393PMC
August 2008

A systemic granulomatous response to Schistosoma mansoni eggs alters responsiveness of bone-marrow-derived macrophages to Toll-like receptor agonists.

J Leukoc Biol 2008 Feb 20;83(2):314-24. Epub 2007 Nov 20.

Department of Pathology, University of Michigan Medical School, 4057 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109-0602, USA.

Macrophages play a pivotal role in innate and acquired immune responses to Schistosoma mansoni. Classical (M1) or alternative (M2) activation states of these cells further delineate their roles in tissue damage through innate immunity or fibrotic remodeling, respectively. In the present study, we addressed the following question: Does systemic Th2-type cytokine polarization evoked by S. mansoni affect macrophage differentiation and activation? To this end, we analyzed bone marrow-derived macrophages from mice with S. mansoni egg-induced pulmonary granulomas and unchallenged (or naïve) mice to determine their activation state and their response to specific TLR agonists, including S. mansoni egg antigens. Unlike naïve macrophages, macrophages from Th2-polarized mice constitutively expressed significantly higher "found in inflammatory zone-1" (FIZZ1) and ST2 (M2 markers) and significantly lower NO synthase 2, CCL3, MIP-2, TNF-alpha, and IL-12 (M1 markers). Also, compared with naïve macrophages, Th2-polarized macrophages exhibited enhanced responses to the presence of specific TLR agonists, which consistently induced significantly higher levels of gene and protein levels for M2 and M1 markers in these cells. Together, these data show that signals received by bone marrow precursors during S. mansoni egg-induced granuloma responses dynamically alter the development of macrophages and enhance the TLR responsiveness of these cells, which may ultimately have a significant effect on the pulmonary granulomatous response.
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http://dx.doi.org/10.1189/jlb.1007689DOI Listing
February 2008